Técnicas de Imagen y Proteómicas Aplicadas Al Seguimiento de La Interacción Virus-Planta Huésped
Técnicas de Imagen y Proteómicas Aplicadas Al Seguimiento de La Interacción Virus-Planta Huésped
Técnicas de Imagen y Proteómicas Aplicadas Al Seguimiento de La Interacción Virus-Planta Huésped
del Zaidín
Tesis Doctoral
Granada 2007
Editor: Editorial de la Universidad de Granada
Autor: Mónica Pineda Dorado
D.L.: Gr. 550 - 2007
ISBN: 978-84-338-4267-1
Técnicas de imagen y proteómicas aplicadas al
seguimiento de la interacción virus-planta huésped
Granada, 2007
El trabajo que se presenta en esta memoria de Tesis Doctoral ha sido
realizado en el Departamento de Bioquímica, Biología Celular y Molecular de
Plantas de la Estación Experimental del Zaidín de Granada (Consejo Superior de
Investigaciones Científicas), con la ayuda de una beca predoctoral del Ministerio
de Educación y Ciencia y un contrato asociado a proyecto (BIO2004-04968-CO2-
02). El trabajo ha sido financiado por los proyectos del Programa Nacional de
Biotecnología BIO2001-1937-C02-02 y BIO2004-04968-CO2-02.
Parte de los resultados de esta Tesis Doctoral han sido expuestos por la
Licenciada M. Pineda en el seminario “Fluorescencia roja de la clorofila: Una
herramienta para la detección del estrés vegetal”, celebrado en la Estación
Experimental La Mayora (Algarrobo-Costa, Málaga) en marzo de 2006.
Algunos de los resultados de esta Tesis Doctoral han dado lugar a las
siguientes publicaciones:
- Sajnani, C., Pérez-Bueno, M.L., Pineda, M., García-Luque, I., Nedbal, L.,
Benedikty, Z., Roncel, M., Ortega, J.M., Ducruet, J.M., Römer, S. y Barón, M.
(2005) The chloroplast as target of biotic stress: damage and defence during viral
pathogenesis. En: Photosynthesis: Fundamental aspects to global perspectiva. Van
Der Est, A. y Bruce, D. eds. ACG Publishing, Lawrence, KS. pp 610-611.
- Chaerle, L., Pineda, M., Romero-Aranda, R., Van Der Straeten, D. y Barón, M.
(2006) Robotized thermal and chlorophyll fluorescence imaging of Pepper Mild
Mottle Virus infection in Nicotiana benthamiana. Plant Cell. Physiol. 47: 1323-
1336.
Durante todos estos años de realización de Tesis Doctoral, mucha gente ha
contribuido a que mi trabajo aquí en Granada o en los lugares donde he realizado
estancias, haya sido una de las mejores experiencias que me tenía reservada la vida.
Me gustaría dar las gracias a todos ellos, espero acordarme de todos y si alguno se
me queda en el tintero, ruego sea capaz de perdonarme, pues el olvido es fruto del
despiste y no de la falta de cariño. A todos: GRACIAS. Todas las cosas buenas que
se pueden encontrar en esta Tesis es consecuencia de vuestra ayuda, todos los
fallos son sólo míos.
Si estos agradecimientos tuvieran que seguir un orden cronológico, en primer
lugar me gustaría agradecerle a Cayo Ramos, buen profesor y mejor persona, el
que fuera el primero que “científicamente” confió en mí. Siempre trabajaré con la
esperanza de no defraudar esa confianza.
De la misma manera, me he esforzado por no defraudar la confianza de
aquella que a su vez confió en Cayo y me “adoptó” en su laboratorio, ofreciéndome
todo tipo de facilidades para que mi trabajo y mi vida en Granada fueran lo más
fáciles y agradables posible. Por eso, quiero agradecer a mi directora de Tesis,
Matilde Barón, todo el empeño que ha puesto para que este trabajo saliera adelante
contra viento y marea, por seguir pensando que podía sacar algo bueno de mi aun
cuando los resultados iniciales no fueron todo lo satisfactorios que yo hubiera
deseado, por darme la oportunidad de viajar y por seguir queriéndome en su
laboratorio.
Gracias a Carmen Lluch Plá, mi tutora de tercer ciclo porque su ayuda ha
sido enorme, haciendo fácil algo tan difícil como es superar con éxito toda la
burocracia que rodea a la Tesis.
Gracias a Isabel García Luque y Maite Serra, por recibirme con los brazos
abiertos en su laboratorio del CIB, por su gran apoyo logístico, por dar siempre
buenos consejos y por el material biológico cedido a nuestro laboratorio.
Gracias a Carlota y a Marisa, excelentes maestras en la poyata del
laboratorio. Su enorme calidad como científicas sólo se ve superada por su calidad
humana. Su amistad es uno de los tesoros que me llevo conmigo. Muchas gracias
por la paciencia, los consejos, las horas dedicadas a enseñarme, los ánimos, las
risas, por ser paño de lágrimas y por ¡la terapia de grupo!
Gracias a Abdel, por estar siempre dispuesto a regar mis plantas durante
vacaciones y fines de semana, por ayudarme con el medio de cultivo hidropónico y
por enseñarme tantas cosas de la cultura marroquí y de la religión musulmana.
Aunque no consiga convencerme de muchas de esas cosas, lo que sí ha conseguido
es que las respete aún más.
Gracias a Iratxe, porque aunque haya llegado hace poco tiempo, ha traído
frescura al laboratorio. Gracias por su compañía, por su ayuda y por estar siempre
dispuesta a darla.
Gracias a Juan José Lázaro, por su habitual buen humor y carácter, por
interesarse siempre por nuestro trabajo y apoyarlo, incluso compartiendo material
científico y dejándonos usar su laboratorio, donde utilizamos aparatos cuyo ruido
continuo no es precisamente agradable.
Gracias a Paquita, por aguantar con tan buen humor ese ruido y por su
inestimable ayuda con mis plantas, la bibliografía, las fotocopias, etc.
Gracias a Laura, porque siempre estaba dispuesta a compartir sus
conocimientos con los demás, y me ayudó mucho cuando quisimos que en nuestro
laboratorio se hiciera, además de Bioquímica, Biología Molecular.
Gracias a Sergio, compañero de penas y alegrías, siempre pendiente de mi
trabajo y animándome, lo que me he reído con él, y lo que he sufrido cuando me
martirizaba con el reggaeton.
Gracias a Iván, porque aunque también ha llegado hace poco, se ha acoplado
perfectamente a nosotros y se ha convertido en un miembro indispensable de
nuestra “familia”.
Gracias a Laci, uno de los mejores húngaros que hay, y eso que los hay muy
buenos. Gracias por tratarme tan bien en Budapest, donde me siento como en mi
casa y donde siempre quiero volver, por su ayuda con mi trabajo allí y aquí, por su
buen humor aunque las cosas se tuerzan en el laboratorio, y por su amistad.
Gracias miles a los compañeros de pasillo con los que tantísimas risas he
compartido, son los mejores: José Ángel, Juande, Antonio, Álvaro, Sol, Amada
(que no era del pasillo, pero como si lo fuera), Eloy..., cómo echo de menos a los
que están fuera. Gracias a Mari Carmen, José Carlos y Ana Vílchez por ser como
son. Gracias a María, Raúl y Antonio por ayudarme a recuperar archivos
importantes (vitales, debería decir). Gracias a todos los demás compañeros
“precarios” de la Estación Experimental del Zaidín, sois muchos para enumeraros,
me gustaría que todos os sintierais incluidos en este agradecimiento. Gracias a
Andrés Belver por prestarnos material de laboratorio y ayudarnos a encontrar el
material de centrífuga misteriosamente desaparecido. Gracias a Alberto Bago por
dejarme usar su microscopio con cámara de fotos que ayudó a que mi primera
publicación científica viera la luz, y a la ya doctora Adriana Marulanda por
enseñarme a usarlo, y por ser sufrida y cariñosa compañera en el largo camino del
doctorado. Gracias a Mariam y a Ana Chueca, y a Julio López Gorgé. Gracias a
Antonio Jesús Castro por darme un buen consejo sobre proteómica. Mis geles de
proteínas en el rango básico no existirían de no ser por él. Gracias a Tino Krell por
su ayuda en la parte de esta tesis dedicada a proteómica. Gracias a todos los
empleados de mantenimiento y del departamento de informática, y sobre todo, a
Antonio Melgar, por estar pendiente de las cámaras de cultivo, día y noche, fines
de semana, festivos, vacaciones…, gracias por todas esas veces que le he hecho
venir y él lo ha hecho sin rechistar.
Gracias a todos mis compañeros de fatigas del CIB, que hacían que a una le
entraran ganas de levantarse por las mañanas y enfrentarse al día a día cuando el
estado de ánimo no es el mejor. Gracias al “dostol” Alfonso, Margot, Miriam,
Gema, Antonio, Mercedes, Carolina y Pablo. Gracias por tantos y tan buenos
momentos.
Gracias a Pili, la mejor compañera de piso del mundo. Gracias por estar
siempre dispuesta a hablar y a escuchar, por ser como es. Siempre nos quedará el
Athenas… (aunque cada vez nos cobren un precio diferente).
Gracias a mi otra compañera de piso en Madrid, Esther, porque aunque no
pueda decir que es la mejor compañera de piso (entre otras cosas porque apenas
aparecía por allí), sí que puedo afirmar que cuando se le conoce se le quiere
incondicionalmente.
Gracias a Merce Romero, por ser mi guía en el mundo del intercambio de
gases. Su entusiasmo es contagioso, da gusto trabajar con ella. Gracias también a
Enrique Moriones, Jesús Navas y Rafael Fernández, así como a todos los que nos
juntábamos en el cuarto de becarios y alrededor de la mesa del desayuno en La
Mayora, en especial a Elena, mi compañera de tapas.
Gracias también a los becarios de Cayo: “Isas”, Luis y Clara Todos forman
un grupo excelente, con el que da gusto colaborar.
I would like to say thanks to Zuzana Benedikty for making easier my first
stay abroad in such a place like Nové Hrady (probably, Zuzana cannot understand
why I say “such a place”). Also, I should say thanks to Julie Soukupová and
Ladislav Nedbal, because they are very good hosts, always taking care about us
when we go to the Czech Republic, and helping us with FluorCam in Granada. In
this point, I have to thank also Martin Trtilek, our favourite fluorometer designer.
I would like to say thanks to Dominique Van Der Straeten and Laury Chaerle
for receiving me in their lab and for being so helpful with me.
I would like to say thanks to Eva Sarvari, Magdolna Droppa, Karoli Bokak
and Zoltan Zsigeti for making me feel like home in Budapest, for showing me very
interesting demonstrations, Balaton Lake, the excellent Hungarian food, and, over
everything, for being so very good people.
Gracias a toda mi familia y amigos, por todo ese apoyo que me dan y que
nunca me puede faltar. A la familia de Pepe, por preguntar siempre “eso que tú
haces, ¿para qué sirve?”. Especiales gracias a Antonio, Raquel, Karmele,
Guillermo y Miguel, porque siempre confiaron en mi. Especiales gracias también a
Paco, por interesarse siempre en lo que yo hacía, desde que estudiaba en la
universidad.
Hay cuatro personas para los que la palabra GRACIAS (así, con mayúsculas)
se me queda pequeña. Es imposible expresar con palabras mi enorme gratitud, pero
lo intentaré. A mis padres, Manuel y Carmen, GRACIAS por estar siempre ahí, por
empeñarse en que yo tuviera una educación universitaria, por querer siempre lo
mejor para mi. Me han dado todo lo mejor que unos padres puedan dar a sus hijos.
Lucho a diario por estar a la altura de sus expectativas y por no defraudarlos jamás,
pues se merecen todo lo mejor, y sin embargo, se conforman conmigo. A mi
hermana, Mari Carmen, GRACIAS por cuidar siempre de su hermana pequeña, por
no olvidar jamás un cumpleaños y por ser la mejor hermana que se puede desear y
de la que me gustaría poder disfrutar más. A mi pareja, Pepe, GRACIAS por dar
alegría a mi vida como sólo él sabe hacerlo, por todo lo que significa para mi,
pasado, presente, y sobre todo, futuro. Os quiero mucho a los cuatro, GRACIAS
por devolverme tanto amor.
A mis padres
A mi hermana
A Pe
ÍNDICE
ABREVIATURAS.................................................................................................. 19
RESUMEN ............................................................................................................. 23
SUMMARY............................................................................................................ 25
A. INTRODUCCIÓN ............................................................................................. 27
A.1. El estrés en las plantas. La interacción planta-virus como
modelo de estrés biótico en el cloroplasto ................................................. 27
A.2. Técnicas biofísicas de imagen aplicadas al estudio de la
detección precoz del estrés vegetal ............................................................ 34
A.2.1. Termografía ........................................................................ 35
A.2.1.1. La transpiración: el origen del calor
desprendido por las plantas .............................................. 35
A.2.1.2. Termografía, una técnica de imagen................... 37
A.2.1.3. Aplicaciones de la termografía a la
biología vegetal ................................................................ 39
A.2.1.3.1. Selección de mutantes......................... 39
A.2.1.3.2. Cultivo de vegetales en el espacio ...... 39
A.2.1.3.3. Transporte hídrico en plantas.............. 40
A.2.1.3.4. Tolerancia a la congelación ................ 41
A.2.1.3.5. Calidad de los frutos ........................... 41
A.2.1.3.6. Alteraciones ocasionadas por
patógenos ............................................................. 41
A.2.1.3.7. Muerte celular ..................................... 43
A.2.1.3.8. Teledetección ...................................... 44
A.2.2. Fluorescencia verde-azul .................................................... 46
A.2.2.1. Origen de la fluorescencia verde-azul ................ 48
A.2.2.2. Cocientes de fluorescencia ................................. 50
A.2.2.3. Fluorescencia inducida por luz UV de imagen ... 51
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
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Índice
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
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Abreviaturas
ABREVIATURAS
1D: monodimensional
2D: bidimensional
2D-DIGE: electroforesis bidimensional empleando fluoróforos (2-D difference gel
electrophoresis)
ΦPSII: rendimiento cuántico del fotosistema II
AB: abaxial
ABA: ácido abscísico
AD: adaxial
AS: asintomática
BGF: fluorescencia verde-azul (blue-green fluorescence)
BN-PAGE: geles de poliacrilamida nativos azules (blue native gels)
CCD: charge-coupled device
CF1: porción catalítica de la ATP sintasa
Chl: clorofila
Chl a: clorofila a
Chl b: clorofila b
Chl-F: fluorescencia emitida por la clorofila
Chl-FI: fluorescencia emitida por la clorofila de imagen
CMV: virus del mosaico del pepino (cucumber mosaic virus)
CP: proteína de cubierta del virus (coat protein)
Cyt. f: citocromo f
dpi: días post-inoculación
F440: fluorescencia azul
F520: fluorescencia verde
F690: fluorescencia roja
F740: fluorescencia en el rojo lejano
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
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Abreviaturas
21
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
22
Resumen – Summary
RESUMEN
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
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Resumen – Summary
SUMMARY
In previous works our research group has shown that the infection of
Nicotiana benthamiana plants with the pepper mild mottle virus (PMMoV) induces
a decrease of the levels of some chloroplastidic proteins and their encoding
mRNAs. Thermoluminescence measurements showed the presence of oxidative
stress in infected plants, disturbances both on the PSII donor side and assimilatory
potential in the host plant, as well as the induction of a PSI cyclic electron transport
to attend the increasing ATP demand due to the viral infection. The chlorophyll
fluorescence imaging appeared also as an useful tool to follow plant virus
infection, as well as to analyse the protective mechanisms developed by the
chloroplast against biotic stress.
In the present Ph.D. work, we have used three imaging techniques to study
the impact of PMMoV in Nicotiana benthamiana. (i) Thermographic imaging
allowed the visualization of changes in the transpiration pattern of the leaves by
measuring their temperature. We could demonstrate that asymptomatic leaves from
infected plants suffered a progressive temperature increase, starting in the tissues
surrounded the main veins and expanding later to the rest of the leaf blade. This
effect preceded the virus entrance on the leaf. Temperature increment on
symptomatic leaves was homogeneous and occurred after symptom appearance. In
both cases, the temperature increase correlated with a transpiration decrease, due to
the viral induced-stomatal closure. Simultaneous measurements of red chlorophyll
fluorescence imaging showed alterations on the fluorescence pattern of the
asymptomatic leaves correlated with the viral entrance and spreading on these
leaves. (ii) Imaging UV-induced fluorescence showed an increment in the blue-
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
green fluorescence, especially in the abaxial surface of the leaves from infected
plants. HPLC analysis of leaf extract has shown that chlorogenic acid is the main
emitter of this kind of fluorescence. We have also shown that the fluorescence
ratios F440/F690 and F440/F740 were suitable for following the infection with
PMMoV in both adaxial and abaxial surfaces of the leaves. (iii) Red chlorophyll
fluorescence imaging showed that NPQ is a good indicator of the viral infection in
the absent of symptoms. We could detect the infection in the asymptomatic leaves
previously to viral immunolocalization by means of advanced statistical
approaches using data obtained from the fluorescence kinetics.
The last part of this work continues the analysis of the chloroplast proteome
of Nicotiana benthamiana as well as of the changes induced by the infection with
PMMoV. Using bidimensional electrophoresis in the pH range 6-11, we have
increased the number of proteins identified in this proteome and showed some of
them that were up-(PsbQ) or down-regulated (PSI proteins, RuBisCO large
subunit, ribosomal protein) by the viral infection.
26
A. Introducción
A. INTRODUCCIÓN
El término estrés fue definido por primera vez en los años 30 por Hans Selye
(1936). Desde entonces, el concepto se ha ido modificando y mejorando hasta
elaborarse definiciones más específicas de estrés para las plantas. El estrés vegetal
se definiría como la alteración del estado fisiológico provocada por factores, de
naturaleza abiótica y biótica, que tienden a alterar el equilibrio (Gaspar et al. 2002),
o bien como cualquier condición desfavorable para el metabolismo, crecimiento y
desarrollo de los vegetales que, en la mayor parte de los casos, pueden recuperarse
cuando desaparecen los agentes estresantes (Lichtenthaler 1998). Sin embargo,
cuando se exceden los límites de tolerancia de la planta y se sobrepasa la capacidad
adaptativa de la misma, el resultado puede ser el daño permanente o incluso la
muerte (Larcher 1987). La respuesta de la planta al estrés consta de cuatro fases:
respuesta o reacción de alarma, resistencia, fase final o de agotamiento cuando se
sobrecarga la respuesta adaptativa de la planta y finalmente, la fase de regeneración
parcial o total de las funciones fisiológicas cuando el factor causante del estrés
desaparece (Lichtenthaler 1998). El concepto de estrés se ha extendido aún más
diferenciando entre eu-estrés y dis-estrés. El primero es un estrés suave que activa
el metabolismo celular y estimula la actividad fisiológica de la planta, siendo un
elemento positivo y una fuerza impulsora del crecimiento vegetal. En cambio, el
segundo es cualquier condición desfavorable, bien de alta intensidad o de larga
duración, que afecta negativamente a la planta, sobrecargando los mecanismos que
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
28
A. Introducción
29
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
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A. Introducción
demostrando que incluso los niveles de la proteína PsbO pueden descender debido
a la infección por TMV (virus del mosaico del tabaco), cuya helicasa muestra gran
afinidad por esta proteína del OEC (Abbink et al. 2002). Nuestro grupo también ha
demostrado que el estrés abiótico es capaz de producir cambios en la acumulación
de las proteínas de este complejo (Barón et al. 1995a).
Además de la bajada en la eficiencia fotosintética, en la mayoría de
interacciones planta-patógeno compatibles, también tiene lugar un incremento en
respiración, cambios en la partición del fotosintato y alteraciones en la
acumulación de almidón (Lucas et al. 1993, Shalitin y Wolf 2000, Hull 2002,
Maule et al. 2002).
El estudio de la acción de los virus vegetales más allá de la fase luminosa de
la fotosíntesis ha demostrado que en las plantas infectadas se producen graves
alteraciones en el metabolismo de carbohidratos (acumulación de almidón y
azúcares solubles en hoja), y en su transporte a través del floema, que podrían
afectar la expresión génica y rutas metabólicas del huésped, y llevar a una síntesis
deficiente de complejos, enzimas y pigmentos fotosintéticos en la planta infectada
(Scholes et al. 1994, Wright et al. 1995b, Havelda y Maule 2000, Herbers et al.
2000). Los azúcares, directa o indirectamente, provocan la retroinhibición de la
fotosíntesis (Krapp y Stitt 1995), la regulación de la expresión génica (Herbers et
al. 2000) e incluso la represión transcripcional de genes fotosintéticos (Sheen et al
1990). La iluminación de tejidos con alto contenido en hidratos de carbono puede
llevar a una sobre-reducción de la cadena de transporte electrónico y a la
desorganización del aparato fotosintético a través de efectos selectivos sobre la
expresión génica de proteínas de membrana y estromáticas (Maxwell et al. 1995).
Algunos autores proponen que las señales redox procedentes del cloroplasto y el
contenido de azúcares son reguladores activos de la expresión de genes nucleares
codificadores de proteínas cloroplastídicas (Oswald et al. 2001), mientras que para
otros es todavía una incógnita si el control de la expresión génica en las plantas
infectadas se realiza por señales redox o por los niveles de carbohidratos (Osmond
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
et al. 1998, Lohaus et al. 2000). Herbers et al. (2000) muestran, en fases tempranas
de la infección con potyvirus, una regulación transcripcional de genes cab (que
codifican proteínas de unión a clorofila a y b) como la proteína del complejo mayor
de antena del PSII (LHCII), y el gen rbcS (subunidad menor de la ribulosa 1,5-
bifosfato carboxilasa/oxigenasa) de la RuBisCO. Para estos autores, los azúcares
también podrían actuar como amplificadores de las respuestas de defensa durante
la interacción planta-patógeno.
Además, se ha visto que la infección viral puede afectar procesos como los
de apertura estomática (Chaerle et al. 1999, Chaerle et al. 2004) o difusión en el
mesófilo, que controlan la asimilación de CO2 (Sampol et al. 2003). También, la
infección viral puede afectar al metabolismo secundario vegetal, propiciando la
acumulación de compuestos de naturaleza fenólica (Nicholson y Hammerschmidt
1992, Dixon y Paiva, 1995, Kofalvi y Nassuth 1995, Dixon et al. 2002) que
absorben luz ultravioleta y/o fluorescen en el verde o el azul (Buschmann y
Lichtenthaler 1998, Cerovic 1999, Buschmann et al. 2000, Balogun y Teraoka
2004). Estudios de nuestro grupo con hojas de Nicotiana benthamiana infectadas
con PMMoV e iluminadas con luz ultravioleta confirman estos datos (Sajnani
2005).
Actualmente, al aparato fotosintético se le asigna una función dual, fijando
por un lado la energía solar y por otro, actuando como sensor ambiental mediante
la generación de señales redox que pueden actuar en conexión con las de otros
orgánulos celulares, regulando la expresión de los genes fotosintéticos
cloroplastídicos y nucleares en respuesta a los distintos factores de estrés
(Pfannschmidt 2003). El cloroplasto está implicado en el equilibrio entre procesos
fotoprotectores y procesos fotoinhibitorios. Los primeros tratan de disipar la
energía de excitación excesiva que no puede utilizarse en fotosíntesis, mientras que
los segundos tienen lugar cuando se excede la capacidad de fotoprotección y
antioxidante de la planta y se inicia la fotooxidación de los complejos
fotosintéticos, fundamentalmente el PSII, por una superproducción de especies
32
A. Introducción
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
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A. Introducción
utilidad como detectores precoces del estrés vegetal, haciendo especial hincapié en
las técnicas de imagen empleadas en la elaboración de la presente Tesis Doctoral.
A.2.1. Termografía
A menudo pensamos en el concepto de temperatura como la medición del
calor desprendido por el cuerpo de los animales; de hecho, solemos clasificarlos
como de “sangre fría” o “sangre caliente”. Sin embargo, nos es más difícil
relacionar la temperatura con el mundo vegetal, aún cuando es posible medir la
temperatura de la superficie de las hojas mediante la detección de la radiación
infrarroja que todos los objetos, a temperatura ambiente, emiten con una longitud
de onda de 8-14 µm (Chaerle y Van Der Straeten 2000, 2001). La termografía es la
técnica que posibilita la medida de la radiación infrarroja y se ha aplicado
extensamente en medicina (para un breve resumen de muchas de sus aplicaciones
que escapan a los objetivos de esta tesis, ver Chaerle 2000) y es ampliamente
utilizada por los ejércitos para detectar, mediante el calor que emiten, objetos en
situaciones de escasa o nula visibilidad (www.militaryinfrared.com).
El calor desprendido por el metabolismo vegetal tiene un papel muy
secundario en la temperatura de los vegetales, con una notable excepción: las flores
de las plantas “termogénicas”, la mayoría pertenecientes a la familia Araceae, que
pueden controlar su temperatura mediante el control de la respiración (Chaerle
2000, Chaerle y Van Der Straeten 2000, 2001). Los cambios en la temperatura de
las hojas en plantas no termogénicas resultan, principalmente, de alteraciones en la
transpiración y dependen, por tanto, de la conductancia estomática (Fuchs y Tanner
1966, Jones et al. 1998).
35
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
H 2O ESTOMAS
CERRADOS
-
-
T hoja
-
CO2
O2
CO2
-
O2 -
H2O -
ESTOMAS
ABIERTOS T hoja
Fig. 1: Relación entre apertura estomática, transpiración y temperatura de las hojas. T: temperatura.
36
A. Introducción
37
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
Fig. 2: A la izquierda, brazo del robot para la obtención de imágenes termales, de reflectancia y de
Chl-FI, perteneciente al grupo de la Dra. Dominique van der Straeten, de la Universidad de Gante. A
la derecha, detalle de las cámaras armadas en el brazo del robot: de fluorescencia, de termografía y de
reflectancia (de izquierda a derecha, respectivamente).
38
A. Introducción
39
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
T (ºC)
1.0
Gravedad (g)
2.0
0.01
4 8 12 16 20
Tiempo (s) de caída libre
Fig. 4: Relación entre temperatura de las hojas y gravedad (tomado de Kitaya et al. 2003).
40
A. Introducción
41
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
A B
C
4h
26 h
3 días
13 h 39 h
5 días
73 h 8 días
Térmica Vídeo Reflectancia Térmica Térmica Vídeo
Fig. 5: A) Las lesiones necróticas producidas por TMV en hojas de tabaco Xanthi XX y B) las
espontáneas en tabaco transgénico bO, se detectan presintomáticamente gracias a la termografía de
imagen (tomado de Chaerle 2000). C) El descenso de temperatura ocasionado por P. cubensis en
pepino también se detecta presintomáticamente gracias a esta técnica (tomado de Lindenthal et al.
2005).
42
A. Introducción
43
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
44
A. Introducción
sido especialmente secos, y las demandas de agua, crecientes. Por tanto, el uso
racional y responsable de los recursos hídricos y la adopción de un régimen de
riego eficiente es de vital importancia. La termografía de imagen ha demostrado su
eficacia en este terreno, ya que la temperatura de los cultivos es un poderoso
indicador del estado hídrico de las plantas y una herramienta potente para
proporcionar información sobre el uso del agua en agricultura, permitiendo
desarrollar regímenes de riego apropiados (Cohen et al. 2005, Leigh et al. 2006).
Jones (2004b) revisa los diversos métodos disponibles para establecer regímenes de
riego, y subraya la importancia de la termografía entre todos ellos. El reciente
desarrollo de cámaras térmicas portátiles ha extendido enormemente las
oportunidades de análisis de las propiedades térmicas de cultivos y ha expandido la
información disponible relativa a las condiciones de crecimiento de las plantas.
Esta técnica permite el análisis semiautomático de grandes áreas de cultivo con una
mayor reproducibilidad de la que podría conseguirse con la porometría. (Jones et
al. 2002). Recientemente, Leigh et al. (2006) han desarrollado un método para
medir fidedignamente la temperatura de los cultivos bajo luz solar directa.
SAR
SAR
Fig. 6: Teledetección: estimación de la temperatura de los cultivos de algodón empleando una cámara
térmica situada a 5 m de distancia del cultivo. SAR: superficie artificial de referencia (tomado de
Cohen et al. 2005).
45
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
46
A. Introducción
Fig: 7: Espectro de emisión de fluorescencia inducido por luz UV en una hoja verde de maíz. Los
máximos de emisión están indicados (tomado de Lichtenthaler y Miehé 1997).
47
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
48
A. Introducción
B Epidermis
A paredes celulares
Parénquima en
empalizada
cloro-
fila a
paredes
celulares
Parénquima lagunar
Epidermis
Mesófilo
Fig. 8: Asimetría de una hoja bifacial de dicotiledónea como Nerium oleander (adelfa). A)
micrografía de epifluorescencia (200x) de la emisión en el visible de una sección transversal de una
hoja de adelfa. Adaxial, ad; abaxial, ab. Se ha indicado la posición de la epidermis AD y AB, que
fluoresce en el azul; así como la situación del parénquima en empalizada y el lagunar, que lo hace en
el rojo (tomado de Mantha et al. 2001). B) Espectros de emisión de la epidermis AD y de las células
del mesófilo bajo la misma. La intensidad de la BGF de las células del mesófilo es menor que la de la
epidermis libre de pigmentos fotosintéticos, ya que BGF es reabsorbida por las Chls (tomado de
Buschmann et al. 2000).
Dados los distintos orígenes de BGF y Chl-F y que cada una de ellas puede
variar independientemente en respuesta a factores ambientales o fisiológicos,
además de influirse mutuamente, a veces es necesario emplear cocientes de
fluorescencia en lugar de valores de fluorescencia absolutos. A continuación,
49
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
detallaremos los cocientes más empleados para describir las respuestas de las
plantas al estrés.
50
A. Introducción
51
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
Resolución espacial
Distribución de la señal a lo largo del área
Buena resolución espectral foliar
Instrumentos de bajo coste Localización de señales
- No representativo Patrón de señales
- Sólo un píxel por medida Buena confianza estadística
52
A. Introducción
53
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
(Stober y Lichtenthaler 1993b, Agati et al. 2002, Mantha et al. 2001, Hideg et al.
2002).
A escala macroscópica, la efectividad de la MCFI ha sido probada en
numerosas ocasiones (Heisel et al. 1996, Lang et al. 1996, Lichtenthaler et al.
1996, Lichtenthaler y Miehé 1997, Langsdorf et al. 2000, Krizek et al. 2001,
Lichtenthaler et al. 2005, Lenk y Buschmann 2006).
Otra ventaja que ofrece la MCFI es la posibilidad de su aplicación para la
detección de estreses en campo, incluso mediante teledetección.
54
A. Introducción
55
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
al. 2000, Apostol et al. 2003, Corp et al. 2003, Mercure et al. 2004, Lichtenthaler et
al. 2005). Este tipo de estrés generalmente ocasiona incrementos en F440/F690 y
F440/F740 (Fig. 10), mucho antes de que el déficit de nitrógeno cause una pérdida
del contenido de Chls (por tanto, los índices señalados son detectores del estrés
más tempranos que el cociente F690/F740). Se sabe que la carencia de este
nutriente provoca la acumulación de fenoles en la epidermis de las hojas, que
absorben la radiación y dejan pasar menos cantidad de luz al mesófilo. Gracias a la
fluorescencia inducida por UV se ha podido realizar un mapa de la disponibilidad
de nitrógeno en un campo de maíz (Apostol 2006). La fluorescencia inducida por
luz UV también ha sido de gran utilidad a la hora de detectar deficiencias de hierro
(Morales et al. 1994), toxicidad por exceso de cinc (Schuerger et al. 2003) o déficit
de potasio (Chappelle et al. 1984).
-N +N -N +N
Figura 10: Imágenes de los cocientes F440/690 y F690/F740 en hojas de remolacha crecidas con
(+N) o sin (-N) aporte de nitrógeno (tomado de Lichtenthtaler et al. 2005).
56
A. Introducción
F690/F735
lento proceso de desecación
(240 min) en las hojas verdes
del musgo Tortula rurales. El
cero en el eje X representa el
estado seco antes de la
rehidratación del musgo
(tomado de Takács et al.
Tiempo (min) 2000).
57
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
A.2.2.4.7. Hojas variegadas. Las partes claras de las hojas variegadas emiten
gran cantidad de BGF en comparación con las partes verdes; si trazáramos un
transepto a lo largo de una hoja variegada, veríamos que F440 y F690 muestran un
contraste negativo (Lichtenthaler et al. 2005) (Fig. 12). Además, las zonas claras
poseen trazas de Chl no funcional (Lichtenthaler et al. 1996), por lo que los
cocientes F440/F690 y F440/F740 son mucho mayores que en las partes verdes de
las mismas (Buschmann y Lichtenthaler 1998).
58
A. Introducción
59
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
60
A. Introducción
61
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
H + H + Ciclo de
Benson-Calvin
2 H2O cadena
e-
P680 transportadora de P700 NADPH
O2 electrones
Fig. 13: Transformación de la luz absorbida por las antenas de ambos fotosistemas en tres
distintos tipos de energía: fotosintética (procesos fotoquímicos), fluorescencia y calor (procesos no
fotoquímicos).
CONDICIONES CONDICIONES DE
FISIOLÓGICAS ESTRÉS
Chl* a Chl* a
fotosíntesis calor
hν
ν calor hν
ν fluorescencia
fluorescencia fotosíntesis
Chl a Chl a
Fig. 14: La clorofila excitada por la luz (Chl*) vuelve a su estad basal mediante tres procesos en
equilibrio. Bajo condiciones fisiológicas, los procesos fotoquímicos son los predominantes. Bajo
situaciones de estrés, los procesos no fotoquímicos y la fluorescencia aumentan en detrimento de la
fotosíntesis.
62
A. Introducción
63
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
conocido como máximo rendimiento cuántico del PSII o FV/FM, que nos indica la
eficiencia fotosintética máxima de nuestro sistema. Normalmente, una planta sana
de cualquier especie tiene un índice FV/FM cercano a 0,8 (Björkman y Demming
1987), pero cuando se enfrenta a situaciones de estrés, este valor suele disminuir.
Continuando con la cinética de inducción de fluorescencia de Kautsky, tras el
inicial aumento de fluorescencia, se registra un descenso de la misma, debido a que
QA va cediendo electrones a la cadena transportadora y los centros de reacción se
van “abriendo”. Este descenso en la emisión de fluorescencia hasta un estado
estacionario de equilibrio (FS) se conoce como quenching, y tiene dos orígenes: el
fotoquímico (qP, ocasionado por el desencadenamiento de la fotosíntesis) y el no
fotoquímico (qN).
A B
FM
quenching
Fluorescencia
F0 FS
Tiempo
Luz saturante
Fig. 15: A) Esquema de la cadena de transporte de electrones en el PSII, donde QA es uno de los
aceptores de electrones (tomado de www.uqtr.ca/labcarpentier/eng/research.htm). B) Cinética de
inducción de la fluorescencia: el efecto Kautsky.
64
A. Introducción
FM
qN
Fluorescencia
F’M
FV
qP
F
F0 Luz actínica
F’0
Tiempo
Pulsos saturantes
65
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
FM' − Ft ∆F
Φ PSII = = '
FM' FM
FM − FM' ∆F
NPQ = '
= 'M
FM FM
66
A. Introducción
67
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
NPQ = qT + qI + qE
68
A. Introducción
69
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
70
A. Introducción
A De-epoxidación
B
Directo
I II H PsbS H
Protonación
∆ pH
Zea
H H H H
PsbS H PsbS H
III IV
PsbS
Indirecto
Violaxantina Zeaxantina
Fig. 17: A) Modelo del cambio conformacional del LHCII (rectángulos verdes) para el NPQ. La
proximidad entre los rectángulos (LHCII) representa la extensión del cambio conformacional que
causa la disipación de la energía y que controla la eficiencia del quenching. El grosor de las flechas
indica la cantidad de energía disipada en cada estado. Los estados de quenching van del I-IV. I
representa el estado adaptado a oscuridad, mientras que IV es el estado de qE predominante, que se
alcanza después de varios minutos de exposición a la luz. El estado III es el estado de transición de qE
que se alcanzaría inmediatamente después de encender la luz y antes de que la de-epoxidación tenga
lugar. El estado II es un estado de “memoria”, que se encontraría a los pocos minutos de apagar la luz
tras haber expuesto la planta previamente a la luz, y se correspondería con qI. B) Posibles
mecanismos de actuación de PsbS. El quenching sería directo, si PsbS protonada y unida a
zeaxantina actuara disipando el exceso de energía en forma de calor; en cambio, el quenching
indirecto tendría lugar si la proteína PsbS protonada indujera el quenching de antena catalizando los
cambios conformaciones entre los estados I y III, y entre II y IV (reproducido a partir de Horton et al.
2005).
71
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
72
A. Introducción
Unidad de detección
Unidades de excitación
Unidad de
control
Fig. 18: Modelo abierto del FluorCam (PSI, www.psi.cz) con sus distintos componentes.
73
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
74
A. Introducción
75
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
distinguibles gracias a las técnicas de Chl-FI. Las zonas que muestran una baja tasa
fotosintética coinciden con aquellas que mostraban un mayor NPQ (Repka 2002,
Berger et al. 2004).
Estos resultados contrastan con los de Aldea et al. (2006), quienes detectaron
un descenso de actividad fotosintética incluso en áreas alejadas del punto de
inoculación de Phyllosticta en varias coníferas como consecuencia del daño físico
infligido por el hongo en los RC del PSII (Fig. 19B).
dpi 0 8 11 14
A +
ΦPSII
_
NPQ
+
B ΦPSII
Fig. 19: Heterogeneidad espacio- temporal en la fotosíntesis de plantas infectadas por hongos. A)
Evolución de la infección por Albugo candida (punto de inoculación indicado por una flecha) en
hojas de Arabidopsis thaliana (tomado de Chou et al. 2000). B) Infección por Phyllostactica en hojas
de Cercis canadensis (tomado de Aldea et al. 2006).
76
A. Introducción
Control P. syringae
FV/FM
ΦPSII
qN
Fig. 20: La infección con Pseudomonas syringae en Arabidopsis thaliana produce un descenso de los
parámetros de quenching indicados en la imagen (tomado de Bonfig et al. 2006).
77
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
78
A. Introducción
linurón suministrado en su solución nutritiva, sufrieron más daños que aquellas que
crecieron con linurón más la mencionada bacteria, lo que fue rápidamente
descubierto mediante Chl-FI, mientras que la termografía de imagen detectó el
efecto de forma mucho más tardía (Chaerle et al. 2003).
Fig. 21: Emisión de fluorescencia de una hoja de tabaco infectada con TMV a diferentes días post-
inoculación (indicados en cada panel). El patrón de emisión es heterogéneo en el espacio y en el
tiempo (tomado de Balachandran et al. 2004).
79
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
80
A. Introducción
rodeados de una delgada línea de bajo NPQ (Fig. 22). Este patrón espacio-temporal
es muy característico de este tobamovirus en N. benthamiana, pudiéndose
correlacionar la zona de alto NPQ con el patrón de distribución del virus en las
hojas asintomáticas.
Control
PMMoV-I
dpi 7 14 17 21 24 28
Fig. 22: La infección de N. benthamiana por PMMoV-I da lugar a un patrón de NPQ espacio-
temporal heterogéneo en hojas asintomáticas (tomado de Pérez-Bueno 2003).
avispa
hongo
Fig. 23: El daño ocasionado por insectos y por hongos causa un descenso de la actividad fotosintética
más allá del área afectada y provoca efectos antagonistas en la temperatura (ºC) de las hojas (tomado
de Aldea et al. 2006).
81
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
82
A. Introducción
menor, por lo que esta técnica también puede emplearse para determinar el grado
de madurez y vida media de los mismos (Ciscato 2000, Nedbal et al. 2000b).
La Chl-FI pudo poner de manifiesto presintomáticamente la infección de
limones por Penicillium digitatum (Fig. 24) (Nedbal et al. 2000b). Más
recientemente, el daño ocasionado por toxinas de Trichothecium roseum o por
podredumbre negra en manzanas también pudo detectarse mediante Chl-FI (Ariana
et al. 2006, Žabka et al. 2006).
Butz et al. (2005) han realizado una revisión sobre los métodos desarrollados
para el control no destructivo de la calidad de los frutos, concluyendo que la
combinación de diferentes técnicas, entre las que se encuentras las de imagen, es la
mejor opción para conseguir este propósito.
F0
FV
FM
F0/FM
Tiempo 0 h 48 h 66 h 84 h
Fig. 24: Detección presintomática de la infección por Penicillium digitatum en limones (tomado de
Nedbal et al. 2000b).
83
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
84
Fig. 25: Esquema del sistema experimental y de las técnicas más empleadas en proteómica.
Prefraccionamiento
Prefraccionamiento ESI MALDI
dela
de lamuestra
muestra Separación
Separación Separaciónde
Separación de
(recomendado)
(recomendado) proteica
proteica Digestióntríptica
Digestión tríptica Ionización
Ionización iones
iones
Electroforesis: Cromatografía:
MS:
•1D •Monodimensional
•Trampa iónica MS/MS:
•SDS-PAGE •Bidimensional
•Cuadrupolo (Q) •TOF/TOF
•BN-PAGE •Multidimensional
•TOF •QTOF
•2D
•FTICR
•IEF/SDS-PAGE
•2D-DIGE Detección:
Detección:
•BN/SDS-PAGE dela
•Medidade
•Medida lamasa
masa
Identificación de la muestra dela
•Medidade
•Medida laabundancia
abundancia
•Huella peptídica (PMF) •ICAT
•ICAT
•Secuenciación de novo •ITRAQ
•ITRAQ
A. Introducción
Interacciones proteína-proteína (SELDI-TOF MS) •Mutantes
(Interactómica) •Estreses
•Estados de desarrollo
85
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
86
A. Introducción
87
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
mental. Este problema ha sido subsanado por la técnica 2D-DIGE (2D difference
gel electrophoresis, Ünlü et al. 1997, Tonge et al. 2001, Alban et al. 2003), que
permite el análisis de varias muestras en un solo gel gracias al empleo de
fluoróforos (Görg et al. 2004); así se reducen las variaciones metodológicas en las
posiciones y abundancia de las proteínas. Este método permite economizar tiempo
y esfuerzo, ya que conlleva el uso de un menor número de geles. Las aplicaciones
de la 2D-DIGE incluyen el estudio de la expresión diferencial bajo varias
88
A. Introducción
Fig. 27: Esquema de la ionización de péptidos mediante MALDI. El láser provoca la desorción e
ionización de la muestra (tomado de www.cnic.es/proteomica).
89
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
Cátodo
Ánodo
Fig. 28: Representación esquemática del proceso de ESI (tomado de Jonson 2001).
90
A. Introducción
91
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
92
A. Introducción
confrontar los espectros generados por MS y MS/MS: NCBI (National Center for
Biotechnology Infomation, http://www.ncbi.nlm.nih.gov/), EBI (European
Bioinformatics Institute, http://www.ebi.ac.uk/Databases/proeomic.html), Swiss-
Prot y TrEMBL (http://www.expasy.org/sprot). Bases de datos específicas para
plantas son: TAIR (The Arabidopsis Information Resource,
http://www.arabidopsis.org, Huala et al. 2001), TIGR (genome rice annotation,
http://www.tigr.org/tdb/e2k1/osa1/index.shtml, Goff et al. 2002, Yu et al. 2002),
una base de datos específica para plantas arbóreas, con Populus trichocarpa como
planta modelo (http://genome.jgi-psf.org/Poptr1/Poptr1.home.html), PPdb (Plastid
Proteome database, http://ppdb.tc.cornell.edu/, Sahnoun et al. 2000), Plastid
Protein DB (PL_prot, http://www.pb.ipw.bio.ethz.ch/), PROTICdb
(http://moulon.inra.fr/~bioinfo/PROTICdb, Ferry-Dumazet et al. 2005), AMPDB
(Arabidopsis Mitochondrial Protein Database, http://www.ampdb.bcs.uwa.edu.au/,
Heazlewood y Millar 2005), http://www.gramene.org/ y http://www.plantgdb.org/.
La modificación en la expresión proteica provocada por el estrés en las
plantas puede ser cuantificada gracias a métodos como el ICAT o el ITRAQ, que
añaden distintas modificaciones químicas a los péptidos provenientes de muestras
diferentes que pueden ser detectadas mediante MS/MS (Gygi et al. 1999, 2000,
Dunklye et al. 2004, Newton et al 2004, Whitelegge 2004, Jones et al. 2006a,
Lilley y Dupree 2006)
Dado que la secuencia de muchas de las proteínas maduras de los
organismos eucariotas no coincide con la secuencia aparente deducida a partir del
DNA, la proteómica abre paso a la llamada post-traductómica, ya que permite la
deducción de sitios de splicing, PTMs y proteolisis de péptidos señal (Aebersold y
Mann 2003, Hirano et al. 2004, Seo y Lee 2004, Kwon et al. 2006, Whitelegge et a.
2006). La proteómica también abre las puertas a la llamada interactómica (Hirano
et al. 2004), que estudia las interacciones proteína-proteína o proteína-ligando. Una
de las técnicas que permite dicho estudio es el empleo de chips de proteínas
(SELDI-TOF MS, Issaq et al. 2002).
93
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
94
A. Introducción
95
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
96
A. Introducción
97
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
98
B. Objetivos / Objectives
B. OBJETIVOS
99
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
OBJECTIVES
100
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1. Unit Plant Hormone Signalling and Bioimaging, Ghent University, K. L. Ledeganckstraat 35, B-
9000 Gent (Belgium).
2. Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del
Zaidín. C/ Profesor Albareda, nº 1. C.P. 18008. Granada (Spain).
3. Department of Plant Breeding, Estación Experimental La Mayora. 29750 Algarrobo-Costa, Málaga
(Spain).
4. These authors have equally contributed to this work and are placed in alphabetical order
*For correspondence,
mbaron@eez.csic.es; fax: +34958129600; dominique.vanderstraeten@ugent.be; fax : +3292645333
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
Keywords: Biotic stress - Infrared gas analysis - Nicotiana benthamiana – Pepper mild mottle virus –
Stomatal conductance – Thermography.
Introduction
Thermal and chlorophyll-fluorescence imaging (Chl-FI) have proved to be
valuable non-destructive tools to study the spatial and temporal heterogeneity of
leaf transpiration and photosynthesis under biotic stress. Thermography visualises
leaf surface temperature and thus reveals the pattern of transpiration over a leaf.
This technique is well-suited to monitor pest and pathogen infections, which often
lead to changes in plant water status (Chaerle and Van Der Straeten 2000, 2001).
Presymptomatic increases in temperature were visualised at tobacco mosaic virus
(TMV) infection sites in resistant tobacco during the hypersensitive reaction (HR)
(Chaerle et al. 1999). A bacterial-toxin induced HR was also successfully revealed
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by thermography (Boccara et al. 2001). Still at the lab scale, downy mildew
infection in cucumber was visualised at an early stage by thermal imaging
(Lindenthal et al. 2005, Oerke et al. 2006). By remote airborne thermal imaging,
Schmitz et al. (2004) could distinguish low and high nematode infestation in sugar
beet fields, due to the effect of infestation on plant water relations.
Each representative of the wide range of plant-pathogen interactions likely
affects different plant physiological processes to a varying extent. Using several
imaging techniques in parallel increases the chance of highlighting these
pleiotropic effects. Using thermography, Chaerle et al. (2004) observed opposite
effects on leaf temperature in a necrotrophic fungal infection versus a viral-induced
HR. Chl-FI carried out in parallel on the same plants revealed an increase in
chlorophyll fluorescence (Chl-F) in both infection types, albeit with different
kinetics. Chl-FI was also used to reveal disease signatures, which could allow
diagnosis in the absence of symptoms in the visible spectrum (Chaerle and Van
Der Straeten 2001, Nedbal and Whitmarsh 2004). Chl-FI of fungi-infected oat and
Arabidopsis plants (Rolfe and Scholes 1995, Scholes and Rolfe 1996, Chou et al.
2000), as well of fungi and bacterial-infected tomato (Berger et al. 2004),
correlated changes in Chl-F emission with alterations of carbohydrate metabolism
and photosynthetic gene expression. In addition, changes in source-sink
relationships during fungal infection were highlighted by Chl-FI (Esfeld et al.
1995, Weiss et al. 1998). The same technique visualised the inhibition of
photosynthetic electron transport during infection of tobacco with Phytophthora
nicotianae (Scharte et al. 2005). Studies on the early responses in Chl-F emission
to viral infection demonstrated photoinhibitory damage in the infected plants
(Balachandran and Osmond 1994, Balachandran et al. 1994, Balachandran et al.
1997, Osmond et al. 1998, Lohaus et al. 2000).
In the plant-pathogen system Nicotiana benthamiana infected with either the
Italian or the Spanish strain of pepper mild mottle virus (PMMoV-I, PMMoV-S),
the oxygen evolving complex (OEC) of the photosynthetic electron transport chain
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was identified as the main target of this tobamovirus (Barón et al. 1995, Rahoutei
et al. 1998, 1999, 2000, Pérez-Bueno et al. 2004). In the present work we have
simultaneously monitored changes in photosynthesis and transpiration in N.
benthamiana leaves infected with PMMoV-S or PMMoV-I by Chl-FI and
thermography, respectively. To support thermal image data, stomatal conductance
(gs) measurements were carried out with an infrared gas analyser (IRGA); in
addition, stomatal imprints were used to investigate both stomatal density and
aperture. Immunolocalization of viral spreading was followed by tissue printing to
reveal a possible correlation with the fluorescence and thermographic patterns of
infected plant leaves.
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Imaging
Imaging was carried out in a custom-made walk-in chamber with built-in
gantry robot monitoring a 2×1m area. Twenty-four plants representing the three
different treatments were imaged within one experiment. Each treatment group was
split in 2 for respectively AS and S leaf imaging. Illumination and temperature was
set equal to the conditions in which the plants were grown before imaging: A
FLIR/Agema (FLIR Systems, West Malling, Kent, UK) THV900LW camera (pixel
resolution 272×136) was used to capture the thermal images of the PMMoV
infection process. Thermographic measurements on AS and S leaves were
reproducible for the three independent experiments. The fluorescence and colour
images had a size of 300×300 pixels. The Chl-FI system consists of a miniature
black and white CCD camera with <0.1 Lux sensitivity, fitted with a cut-off high
pass red filter (B+W 092) to detect light above 650 nm. The camera is surrounded
by a ring of six small halogen lamps, shielded by a blue cut-off low-pass filter
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
(Schott BG-39) to provide excitation light below 650 nm, with an excitation
maximum at 500 nm (Chaerle et al. 2004). Fluorescence images were grabbed after
respectively 1 second of 250 µmol m-2 s-1 PAR (FL) and immediately after an
additional second of 1000 µmol m-2 s-1 PAR (FH). Colour reflectance images were
captured under chamber illumination.
The ImageJ software package (rsb.info.nih.gov/ij, Thévenaz et al. 1998) was
used to register (i.e. adjust for the difference in field of view and magnification
between the three imaging sensors) and process the captured images. The 8-bit
fluorescence images were normalized for optimal visualization, using the same
scaling factor based on the maximum pixel intensity over all selected images across
all time points. This allows a relative comparison of fluorescence intensity for the
AS leaves of control, PMMoV-I, and PMMoV-S infected plants per panel, as well
as between the different time points.
Leaf temperature data were obtained with AGEMA Research software. Leaf
temperature was measured by drawing a region of interest (roi) corresponding to
the leaf outline. Temperature profiles across the leaves were obtained by drawing a
line perpendicular to the main vein, covering 60 (80) pixels for small (large)
leaves. The main vein was located in the middle of the line (approx. 30 or 40 pixels
to the leaf edge).
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considered for data calculation. AS leaves were measured at 4, 7, 10, 14, 17, 19,
22, 26 and 28 dpi, whereas S leaves were measured at 7, 10, 19, 24 and 28 dpi.
Stomatal imprints
Leaf impressions were obtained from both surfaces of the leaves at 16 dpi
according to Romero-Aranda et al. (2001), on the same leaves used for IRGA
measurements. Imprints were examined by a light microscope (Leica DMRB,
Leica Microsystems, Switzerland) at 200 x magnification, and stomata were
counted on forty fields selected at both sides of the mid-rib. To determine stomatal
aperture of adaxial leaf surface, impressions were analysed by a light microscope
(Nikon Eclipse E200, Nikon Inc. Instruments, Groups USA), and photographed by
a Nikon Coolpix 4500. Thirty stomata (fifteen at every side of the mid-rib) were
measured.
Statistics
Statistical analysis was carried out with SigmaPlot 8.0 software. Student t-
test was used to compare control with infected plants, and PMMoV-I infected with
PMMoV-S infected plants. Significance level was set to 0.005. Graphs show mean
values and their corresponding standard errors. Significant differences between
treatments (according to Student t-tests) are indicated on bar-graphs with different
small letters.
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
Tissue prints
Localization of PMMoV coat protein (CP) in both AS and S leaves was
performed by tissue print analysis using specific antiserum against viral CP
(Alonso et al. 1989). Non-specific binding was reduced by preabsorbing the
immune antiserum with acetone powder obtained from stems and leaves of young
healthy N. benthamiana plants, as described by Ruiz del Pino et al. (2003). Whole
leaves from healthy and infected plants were imprinted on polyvinylidene fluoride
(PVDF) sheets and assayed as described by Maliga et al. (1995). Membranes were
first incubated with the PMMoV-CP specific antiserum at the appropriate dilutions
and subsequently with goat anti-rabbit alkaline phosphatase-IgG (Sigma Aldrich).
Bound antibodies were detected using nitro-blue tetrazolium/5-bromo-4-chloro-3-
indolyl phosphate (NBT/BCIP, Roche).
Results
Visual symptoms
During the imaging experiments, plants infected with PMMoV-S at the 6th or
7th leaf stage developed new curly young leaves (symptomatic [S] leaves) at 5 dpi,
while in plants infected with PMMoV-I these symptoms appeared between 5 and 7
dpi. Growth was obviously inhibited in plants infected with either one of the virus
strains, stunting was evident at 14 dpi. Plants infected with PMMoV-S were totally
senescent at 22 dpi, whereas PMMoV-I infected plants started to recover from the
symptoms (as evaluated by emergence of new unaffected leaves) at 21 dpi.
Recovery occurred irrespective of the time of appearance of the first symptoms.
Asymptomatic (AS) leaves were totally developed at the infection time and did not
show any symptoms along the infection process. In the batch of plants on which
gas exchange was measured, infection was delayed by 2 days: plants infected with
PMMoV-S and PMMoV–I showed symptoms at 7 and 8-9 dpi, respectively.
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Fig. 1: Colour reflectance (R), thermal (T) and Chl-F (FH, under high excitation light; FL, under low
excitation light) imaging of AS leaves of N. benthamiana, at 4 dpi (A), 7 dpi (B), 10 dpi (C), 14 dpi
(D), 17 dpi (E), 22 dpi (F), 26 dpi (G) and 28 dpi (H). Image width is 60 mm for all panels.
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
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plants. The latter presented a higher temperature associated with the vicinity of the
veins from 7 dpi on (Fig. 2, right panel). Subsequently, the region of higher
temperature expanded to the surrounding tissues, finally affecting the whole leaf.
Fig. 2: Infection-induced local increases in leaf temperature in AS leaf tissues adjacent to major
veins. Temperature profiles are shown along a 60 to 80 pixel line drawn in the middle of an AS leaf,
and centred on the midrib (vein type I; for N. benthamiana vein classification, see Roberts et al.
1997). The thermal images shown were captured at 12 dpi. Every vertical dotted line represents the
position of veins type I and II. Temperature difference between horizontal solid lines is 0.1ºC. The T-
values indicate the lowest pixel value for every temperature trace. From bottom to top: 4, 7, 10, 12,
14, 17 and 22 dpi.
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
At 17 dpi, temperature differences among the line profile nearly levelled out. The
overall temperature of the leaf was nearly a degree higher in PMMoV-S infected
plants as compared to the corresponding control plants. At this time point, the AS
leaf of the PMMoV-I infected plant still showed a marked contrast between the
main vein and the adjacent tissue. At 22 dpi AS leaves from both PMMoV-S and
PMMoV-I infected plants, exhibited a higher average temperature than those of
control plants.
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Asymptomatic Symptomatic
24
A aaa aaa aaa abb abc abb aab aa aa B
23
T (ºC)
22
21
20
4 7 10 14 17 19 22 26 28 4 7 10 14 17 19 21 24 28
t (days post-inoculation)
Asymptomatic Symptomatic
0.9 aaa aaa abb ab ab
C D
gs (mol H2O·m-2·s-1)
0.8
0.7 aaa aaa aaa aaa aaa aabb aab aa aa
Control
0.6 PMMoV-I
0.5 PMMoV-S
0.4
0.3
0.2
0.1
0.0
5 7 10 14 17 19 21 26 28 7 10 19 24 28
t (days post-inoculation)
Fig. 3: Virus-induced temperature changes in N. benthamiana leaves (A and B). Data are mean values
from four different plants, error bars represent standard error. Virus-induced stomatal conductance
changes in N. benthamiana leaves (C and D). Data are mean values from at least six different plants,
error bars represent standard error. Letters show the significant differences, according to statistical
analysis (p<0.005). No significant differences were found in panel A.
equal to the control value, which is in agreement with the complete recovery of
these plants (Fig. 3B, 4D).
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
Fig. 4: Colour reflectance (R), thermal (T) and Chl-F (FH, under high excitation light; FL, under low
excitation light) imaging of S leaves of N. benthamiana, at 7 dpi (A), 14 dpi (B), 21 dpi (C) and 28
dpi (D). At each time-point newly-emerged leaves are shown. PMMoV-S infected plants did not
produce new leaves after 14 dpi and died at 22 dpi. Image width represents 60 mm for all panels.
compared with the controls. In contrast, S leaves from both infected plants
displayed a decrease in stomatal conductance compared with the control, which
was significant at 19 dpi (Fig. 3D). Indicative of the recovery phase, gs values of S
leaves from PMMoV-I infected plants increased at both 24 and 28 dpi, compared
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with gs values at 19 dpi, but remained significantly different from control gs values.
However, for these S leaves, the ratio of gs values from control plants over gs
values from PMMoV-I infected plants decreased markedly from 9.68 (19 dpi) over
2.27 (24 dpi) to 1.77 (28 dpi).
Asymptomatic Symptomatic
Adaxial Abaxial Adaxial Abaxial
50 120
Stomatal density
100
(stomata/mm2)
40
80
30
60
20 Fig. 5: Stomatal density and
40
aperture measurements after
10 20 imprinting. Both stomatal density
0
4 0
4 and aperture of AS (left) and S
a b c (right) leaves of Nicotiana
Stomatal aperture
a b b
3 3 benthamiana are shown at 16 dpi.
Data are mean values, error bars
(µ m)
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
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Chlorophyll content
To investigate a possible correlation between changes in leaf temperature or
in Chl-F with those in Chl content, this parameter was also measured. In AS leaves
from PMMoV-I and PMMoV-S infected plants Chl content was not significantly
different from control, and moreover remained higher at later time points as
compared to control (Fig. 6A).
In S leaves, Chl content was the highest in infected plants at 7 dpi (Fig. 6B).
Chl content decreased significantly at 14 and 21 dpi for PMMoV-S infected plants,
and for PMMoV-I only at 21 dpi. PMMoV-S infection thus showed a more severe
response as expected. At 28 dpi, Chl content for the PMMoV-I infected plants
reached control values, corresponding to total recovery.
Asymptomatic Symptomatic
16 aabb aabb abc aa
14
A Control
B
Chl content (a.u.)
12 PMMoV-I
PMMoV-S
10
8 a a a a a a a a a aabb aabb a a a a a
6
4
2
0
3 7 10 14 17 21 28 7 14 21 28
t (days post-inoculation)
Fig. 6: Chl content of AS (A) and S (B) N. benthamiana leaves. Data are mean values from six
different plants, error bars represent standard error. Letters show the significant differences between
infected and control plants, according to statistical analysis (p<0.005).
Viral immunolocalization
To establish a possible correlation between both thermal and fluorescence
patterns in infected plant leaves, and PMMoV localisation, tissue prints were
carried out using an antibody against the viral coat protein. In AS leaves of plants
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
infected with PMMoV-S, virus was detected at 14 dpi, mainly in the leaf base
along the major veins (Fig. 7A). At 21 dpi the virus was present in most of the
main veins. In plants infected with PMMoV-I, the virus localisation pattern was
similar, but delayed in time; the first detection occurred at 17 dpi, and the virus had
spread through the whole main vein system at 24-28 dpi. Non-specific binding
could be seen at 10 dpi in both AS leaf and in its corresponding control (Fig. 7A).
Both viral infections were detectable on S leaves at every dpi assayed, and even in
the recovery phase of plants infected with PMMoV-I (Fig. 7B).
Fig. 7: Tissue prints carried out using a specific antibody against the viral CP at different post-
infection times (dpi) from AS (A) and S leaves (B).
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The same asymptomatic leaf was measured from 4 to 28 dpi. Newly emerging symptomatic leaves
were measured when 2 days old, from 4 until 28 dpi.
Discussion
Early detection by thermography of a viral infection was previously reported
(Chaerle et al. 1999, 2004). This response was however specific for the local
hypersensitive response (HR) induced at the TMV infection site in resistant
tobacco plants, since parallel imaging of a TMV-infected leaf of a susceptible
tobacco plant did not reveal changes in temperature (within 7 dpi, the timeframe of
the thermal effect of the HR; Chaerle et al. 1999). To our knowledge, the present
work on PMMoV infection in N. benthamiana is the first to demonstrate
thermographic visualisation of a systemic viral infection in asymptomatic leaves.
Importantly, thermography revealed symptoms prior to detection of the virus by
tissue print or Northern blot (Pérez-Bueno 2003) (Table 1).
Asymptomatic leaves from plants infected with either one of the PMMoV
viral strains displayed an expanding temperature increase initiating adjacent to the
major veins, and starting at respectively 7 (PMMoV-S ) and 10 dpi (PMMoV-I)
(Table 1). This difference correlates with the lower virulence of PMMoV-I.
Control leaves only display a higher leaf vein temperature, due to the lack of
stomata in the epidermis covering leaf veins; transpiration is thus low, resulting in
less cooling (Chaerle et al. 2003). The tissue next to the major veins has a similar
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
stomatal density to the tissue further towards the leaf border (data not shown),
hence transpiration and leaf temperature are equal under control conditions.
A linear correlation between Chl content and temperature of AS leaves could
not be established (Fig. 6). Thus, factors other than premature leaf aging (of which
a decrease in Chl content is indicative) were responsible for the observed early
temperature increase in virus-infected plants. In control AS leaves, a late, gradual
increase in leaf temperature (Fig. 1; on average starting at 21-23 dpi) paralleled a
decrease in Chl content (Fig. 6). This correlates with a reduction in stomatal
conductance (see Fig. 3C), which is related to leaf age and thus to aging of the
stomatal apparatus (Wardle and Short 1983, Willmer et al. 1988).
The observed temperature increase is most likely due to stomatal closure and
the consequent reduction of transpiration. Temperature is known to vary with leaf
transpiration and therefore is a function of stomatal conductance (Fuchs and
Tanner 1966, Jones et al 1998). A number of reports show that transpiration, as
determined by either gas exchange or porometry measurements, is reduced in
several plant-viral interactions (Keller et al. 1989, Linsey and Gudauskas 1975,
Sridhar et al. 1994, Clover et al. 2001, Ryšlavá et al. 2003, Zhou et al. 2004,
Rowland et al. 2005). However, these methods lack the spatial information
provided by thermography, which has enough spatial resolution to monitor the
variability of stomatal conductance across the leaf surface (Jones, 1999; Jones et al.
2002).
Both average stomatal aperture and number of stomata per unit leaf surface
influence transpiration (Jones 1998). Studies on different host-virus systems show
either a reduction of leaf stomatal conductance (Levy and Marco 1991, Sampol et
al. 2003, Bertamini et al. 2004, Zhou et al. 2004, Guo et al. 2005a, b) or no effect
(Alleyne et al. 1989). To our knowledge no information is available showing an
effect of viral infection on stomatal density. Upon PMMoV-infection of Nicotiana
benthamiana plants, stomatal density of abaxial and adaxial leaf surfaces did not
change.
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
Acknowledgements
M.P. was recipient of a MEC fellowship, which included financial support
for a short-term research stay at Ghent University. L.C. is a post-doctoral fellow of
the Research Foundation - Flanders.
This research was supported by grants from the Spanish Ministry of Science
and Education (MEC, grants BIO2001-1937-C02-02 and BIO2004-04968-C02-02,
to M.B.) and by a grant (G.0015.01) from the Research Foundation - Flanders to
D.V.D.S. The authors are very grateful to Drs. Isabel García Luque and Maite
Serra (Centro Investigaciones Biológicas, CSIC, Madrid) for providing all
PMMoV solutions and antibodies against the viral coat protein.
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1. Department of Biochemistry, Cell and Molecular Biology of Plants. Estación Experimental del
Zaidín, Spanish Council for Scientific Research (CSIC). C/ Profesor Albareda, nº 1. C.P. 18008.
Granada (Spain).
2. Department of Plant Physiology and Molecular Plant Biology, Eötvös Loránd University, Pázmány
Péter sétány 1/c, 1117 Budapest, Hungary.
3. Department of Plant Nutrition, Aula Dei Experimental Station, Spanish Council for Scientific
Research (CSIC), Apdo. 202, E-50080 Zaragoza, Spain.
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Introduction
In the last decade, ultraviolet (UV)-excited fluorescence signals have
emerged as a sensitive and specific tool to evaluate the physiological state of plants
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
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C. Resultados
(2007) monitored the hypersensitive response (HR) to tobacco mosaic virus (TMV)
infection by a BGF increase linked to scopoletin accumulation.
In the present work we follow, a systemic viral infection by an MCF imaging
setup in Nicotiana benthamiana plants infected with either the Spanish or the
Italian strain of Pepper Mild Mottle Virus (PMMoV-S, PMMoV-I; the origin of
these strains was previously reported in Alonso et al. 1989 and Wetter et al. 1984,
respectively). In previous studies we have showed that the oxygen-evolving
complex (OEC) of photosystem II (PSII) is the main target of this tobamovirus in
the chloroplast (Barón et al. 1995; Rahoutei et al. 1998, 1999, 2000; Pérez-Bueno
et al. 2004). Kinetic Chl-FI studies showed a correlation between NPQ (non-
photochemical quenching) and the PMMoV spreading in leaves without showing
any symptoms (Pérez-Bueno et al. 2006). Imaging thermography could detect the
infection earlier than the former technique in the form of an increase in leaf
temperature caused by stomatal closure (Chaerle et al. 2006). In order to give a
more complete picture of the host plant response to PMMoV infection, information
is lacking about secondary metabolites implicated in signalling processes and
response to pathogens in N. benthamiana. Hence, we have monitored changes in
both BGF and Chl-F in N. benthamiana leaves infected with either PMMoV-S or
PMMoV-I by a compact flash-lamp MCF setup. Fluorescence values (F440, F520,
F690 and F740) and fluorescence ratios (F440/F520, F440/F690, F440/F740 and
F690/F740), as well as their corresponding images, of whole leaves from control
and infected plants were monitored along the infection. In an attempt to identify the
compounds involved in the BGF emission, imaging was complemented by high
performance liquid chromatography (HPLC) analysis of leaf extracts. To correlate
changes in fluorescence parameters with the virus entrance in the symptomatic
young leaves, inmunolocalization of viral coat protein (CP) by tissue printing was
carried out.
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Fluorescence Imaging
Fluorescence imaging measurements were carried out in control and virus
infected plants at different post-infection times: 3, 5, 7, 10, 12, 14, 17 (for plant
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infected with both viral strains), 21, 24 and 28 dpi (for controls and plants infected
with PMMoV-I).
Fluorescence excitation and detection was essentially the same as described
earlier (Lenk and Buschmann 2006).
Fluorescence emission images of the abaxial (AB) and adaxial (AD) surfaces
of leaves were carried out with a compact flash-lamp fluorescence imaging system
(FL-FIS, Buschmann and Lichtenthaler 1998; Lichtenthaler and Babani 2000).
Excitation light was provided by a pulsed xenon lamp with a UV-transmitting filter
(transmission maximum at 355 nm). Fluorescence images were acquired with a
CCD-camera gated synchronously with the flash lamp. Leaves were placed at 390
mm from the camera, obtaining images with 60 mm width. The multispectral
fluorescence imaging for the blue (F440), green (F520), red (F690) and far-red
(F740) fluorescence was acquired sequentially for the identical field of view, each
with the appropriate filter that were built into a filter wheel in front of the CCD-
camera. The acquisition of the fluorescence emission images required the
accumulation of 800 images.
Images were processed by specific software (Camille 1.05 software,
Photonetics, Kehl, Germany). The raw image was corrected for non-uniform
excitation with the pixels of an image showing uniform fluorescence (blue
fluorescence image of a white paper). Black and white images of both measured
fluorescence intensity and fluorescence ratios (F440/F520, F440/F690, F440/F740
and F690/F740, acquired by a division of fluorescence intensities pixel by pixel)
were set at the appropriate intensity and either false colour or black and white scale
were applied by ImageJ software package (rsb.info.nih.gov/ij).
Numerical data were obtained by Camille 1.05 software, in a region of
interest corresponding to the leaf outline. Fluorescence profiles across the leaves
(carried out by ImageJ, http://rsb.info.nih.gov/ij) were obtained by drawing a line
perpendicular to the main vein, covering 266 pixels of AS leaves. The main vein
was located in the middle of the line (133 pixels to the leaf edge).
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Fluorophore determination
Phenolic compounds were extracted in warm (at 70 ºC) from 20 mm2 leaf
disks with methanol for 30 min. The extract solution was brought to a volume of 5
or 10 ml (depending on type of sample) with methanol, and then frozen at –80 ºC
until analysis.
Leaf extracts were analyzed by an HPLC method described previously (Morales et
al. 2005). This method revealed the presence of one (majority) phenolic acid,
identified as chlorogenic acid by comparison of its retention time and UV
absorption spectrum with that of chlorogenic acid (pure standard) in methanol.
Prior to analysis, the sample extracts were thawed and filtered through a 0.45 µm
polyvinylidene fluoride (PVDF) filter. High-performance liquid chromatography
was performed with a HPLC system (Waters Alliance 2795 HPLC system, Waters
Corp., Mildford MA, USA) equipped with on-line degasser, autosampler module (4
ºC) and column oven (40 ºC). A diode array detector (Waters 2996 UV-visible)
was used for analyte detection. The column used was an analytical HPLC column
(Waters Symmetry C18, 250 mm x 4.6 mm i.d., dp = 5 µm) with a 20-mm guard
column of the same material. The mobile phase consisted of a gradient system of
solution A, MilliQ water-acetic acid (97.5:2.5; v/v) and solution B, HPLC gradient
grade methanol-acetic acid (97.5:2.5; v/v) at a flow rate of 1.0 ml min-1. The
gradient program was as follows: linear gradient from solution A-B (70:30) to
solution A-B (51:49) in 15 min, isocratic at solution A-B (51:49) for 10 min,
followed linear gradient to solution A-B (70:30) in 5 min, finally isocratic at
solution A-B (70:30) for 5 min. Injection volume was 15 µl. Automated
instrumentation was realized with an HPLC data system (HyStar, Bruker Daltonik
GmbH, Bremen, Germany). The data were analyzed by a data processing software
package (HyStar PostProcessing, Bruker Daltonik GmbH, Bremen, Germany).
Quantification was based on peak area measurements at 325 nm. Standard
solutions were prepared with chlorogenic acid, and were examined by HPLC in
order to determine its retention time (4.1 min). A multipoint calibration was
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C. Resultados
Statistics
Statistical analysis was carried out with specific software (SigmaPlot 8.0
software, Systat Software Inc., San Jose, California, USA). Student t-test was used
to compare control with infected plants, and PMMoV-I infected with PMMoV-S
infected plants. Graphs show mean values (obtained from at least 4 samples per
leaf type) and their corresponding standard errors. Tables 1 and 2 indicate
significant differences between treatments, according to Student’s t-tests and the
three significance levels tested: p<0.05 (*), p<0.01 (**) and p<0.001 (***).
Tissue prints
Localization of PMMoV CP in S leaves was performed by tissue print
analysis using specific antiserum against viral CP (Alonso et al. 1989). As
described by Ruíz del Pino et al. (2003), non-specific binding was reduced by
preabsorbing the immune antiserum with acetone powder obtained from stems and
leaves of young healthy N. benthamiana plants. Whole S leaves from infected and
their respective equivalent leaves in control plants were imprinted on PVDF sheets
and assayed as described by Maliga et al. (1995). Membranes were first incubated
with the PMMoV-CP specific antiserum at the appropriate dilutions and
subsequently with goat anti-rabbit alkaline phosphatase-IgG (Sigma-Aldrich, St.
Louis, USA). Bound antibodies were detected using nitro-blue tetrazolium/5-
bromo-4-chloro-3-indolyl phosphate (NBT/BCIP, Roche, Basel, Switzerland).
Results
Here we present the images corresponding to fluorescence emission at the
different wavelengths analysed and to ratios between them. In addition, values of
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
these parameters averaged in the whole leaf surface are showed. In order to avoid
the tedious enumeration of dpi in which significant differences according to the
statistic (see Material and Methods) are found between PMMoV infected plants
and controls, this information is displayed in tables 1 and 2.
F440 (x10 )
F440 (x10 )
3
3
PMMoV-S
1.2 2.0
1.0 1.5
0.8 1.0
0.6 0.5
1.8
0.4 2.5
0.0
1.6
2.0
1.4
F520 (x103)
F520 (x103)
1.2 1.5
1.0 1.0
0.8
0.5
0.6
0.4 80
0.0
80
60
F690 (x10 )
F690 (x10 )
3
3
60
40
40
20 20
0
80 0
60
60
F740 (x10 )
F740 (x103)
3
40
40
20
20
0
3 5 7 10 12 14 17 21 24 28 3 5 7 10 12 14 17 21 24 28 3 5 7 10 12 14 17 21 24 28 3 5 7 10 12 14 17 21 24 28
Fig. 1. Time-course of blue (F440), green (F520), red (F690) and far red (F740) fluorescence
emission of asymptomatic (left) and symptomatic (right) leaves of Nicotiana benthamiana control and
PMMoV-infected plants. Averages of the whole leaf at every dpi assayed are displayed, error bars
indicate standard error. AB and AD surfaces of the leaves are shown. Fluorescence intensity is given
in arbitrary units.
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C. Resultados
Table 1. Summary of the statistical analysis of the intensity of the fluorescence emitted in the blue,
green, red and far-red region by intact leaves of control and PMMoV infected N. benthamiana plants.
Table displays days post-inoculation where significant differences were found at p<0.05 (*), p<0.01
(**) and p<0.001 (***). Asymptomatic leaves: old leaves which remain symptomless during all the
infection process; F440, blue fluorescence; F520, green fluorescence; F690, red fluorescence; F740,
far red fluorescence; PMMoV-I and –S, Italian and Spanish strains of the Pepper Mild Mottle Virus;
n.s. no significant differences were found at any dpi assayed; symptomatic leaves, young leaves
displaying curling from the first week of the infection time.
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C. Resultados
Fig. 2. Images of blue (F440), green (F520), red (F690) and far red (F740) fluorescence emission of
AS (a) and S (b) leaves of Nicotiana benthamiana control and PMMoV-infected plants. AB and AD
surfaces of the leaves are shown. Images obtained before (5 dpi) and after (17 dpi) fluorescence
changes occurred are displayed. The applied colour scale is shown for every panel. Different
asymptomatic (old) and symptomatic (young) leaves sizes are due to leaf age.
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
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C. Resultados
case of PMMoV-I infection and during the recovery phase, F440 values of these
leaves decreased reaching control values.
Both surfaces of control young leaves displayed a very homogeneous F440
and F520 pattern during the period analysed (Fig. 2b). S leaf of PMMoV-infected
plants did no differ from controls at 5 dpi (increased values registered in the AD
surface were not significant); however, a BGF increment in the whole leaf (both
surfaces) was evident at 17 dpi, the vein areas showing the highest values, more
apparent in the AB surface of the S leaves and in PMMoV-S infected plants, as in
the case of AS leaf patterns of infected plants.
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
Chl-F. Control plants emitted the strongest F690 (Fig. 1), as in AS leaves.
The AB side of S leaves from PMMoV-S infected plants exhibited the first
significant changes (5-10 and 17 dpi; Table 1). The AD surface only showed
significant differences as early as 7 dpi and from 12 to 17 dpi. PMMoV-I infected
plants showed decreased F690 values in the AD surface at 7-21 dpi, and 28 dpi.
The AB surface displayed lower F690 emission than control plants at 10 and 17-21
dpi. Only this leaf surface showed the effects of recovery phase.
F740 emission followed a very similar kinetics to F690 (check Table 1 for
significant time points).
In contrast to the defined red Chl fluorescence pattern from AS leaves of
infected plants, no clear pattern emerged from F690 images of S leaves (Fig. 2b).
The only remarkable fact is the lower fluorescence intensity of S leaves from
infected plants at 5 and 17 dpi in both surfaces of the leaves, which is significant at
5 dpi in the case of AB side of S leaves of PMMoV-S infected plants, and at 17 dpi
in all cases. Also imaging of F740 was comparable to that of F690 (Fig. 2b).
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C. Resultados
increase of this ratio was evident in the AB side of plants infected with both viral
strains, exhibiting the vein areas the highest values.
F440/F690. As Figure 4 shows, AS leaves of infected plants displayed
higher values of F440/F690 than control leaves early in the time-course, being
more evident in the AD side of the leaves. The earliest change was registered in the
AD side of the AS leaves of PMMoV-I infected plants (Table 2). These results
were confirmed by imaging (Fig. 5a). Even being an increase of F440/F690 clear at
17 dpi in the AB surface of the AS leaves of infected plants, the AD ones
evidenced the highest values of the ratio at this post-infection time. It was possible
to observe a decreasing gradient in this parameter from the vein (showing the
highest values of the ratio) to the mesophyll. Control AS leaves did not show clear
changes along the experimental time.
F440/F740. The kinetics of this parameter was very similar to that of
F440/F690 (Fig. 4). F440/F740 detected significant differences later than
F440/F690 in both virus infections (Table 2). However, recovery phase of
PMMoV-I infected plants could be detected at 28 dpi in both surfaces of the AS
leaf, if we consider that any significant difference between the values of the ratio
between control and infected plants are evident at this post-infection time (Table
2).
Images of F440/F740 displayed a very similar situation to F440/F690, albeit
with higher values than the former (Fig. 5a).
F690/F740. In general, F690/F740 values, inversely correlated with the Chl
content in leaf, were higher in the AB surface than in the AD one. In both leaf
surfaces and for the 3 samples analysed, F690/F740 slightly diminished until 10-12
dpi, from this point the ratio raised, being more evident this phenomenon on the
AD side.
AS leaves of PMMoV-S infected plants displayed the highest ratio and
consequently the lowest Chl content at 17 dpi, only significant in the AB leaf
surface (Table 2, Fig. 5). However, the AB surface of AS leaves from PMMoV-I
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
infected plants did not differ significantly along the experiment, when compared
with the controls. In this kind of leaves, the AD leaf surface displayed a higher Chl
content than controls at various time-points (12, 14, 28 dpi, Table 2).
F440 / F520
F440 / F520
1.0 1.5
0.8
1.0
0.6 Control
0.4 PMMoV-I 0.5
0.2 PMMoV-S
0.08
0.0 0.16
0.0
0.07 0.14
0.06 0.12
F440 / F690
F440 / F690
0.05 0.10
0.04 0.08
0.03 0.06
0.02 0.04
0.01 0.02
0.06
0.00 0.18
0.00
0.16
0.05
0.14
F440 / F740
F440 / F740
0.04 0.12
0.10
0.03
0.08
0.02 0.06
0.04
0.01
0.02
0.00
1.8 0.00
1.8
1.6 1.6
F690 / F740
1.0 1.0
0.8 0.8
0.6 0.6
3 5 7 10 12 14 17 21 24 28 3 5 7 10 12 14 17 21 24 28 3 5 7 10 12 14 17 21 24 28 3 5 7 10 12 14 17 21 24 28
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C. Resultados
Table 2. Summary of the statistical analysis of the different fluorescence ratios of control and
PMMoV infected N. benthamiana plants. Table displays days post-inoculation where significant
differences were found at p<0.05 (*), p<0.01 (**) and p<0.001 (***). Asymptomatic leaves: old
leaves which remain symptomless during all the infection process; F440, blue fluorescence; F520,
green fluorescence; F690, red fluorescence; F740, far red fluorescence; PMMoV-I and –S, Italian and
Spanish strains of the Pepper Mild Mottle Virus; n.s. no significant differences were found at any dpi
assayed; symptomatic leaves, young leaves displaying curling from the first week of the infection
time.
PMMoV Asymptomatic leaf Symptomatic leaf
Strain AB surface AD surface AB surface AD surface
-I 14-28** 17**, 28* n.s. n.s.
F440/F520
-S 12-14*, 17** 17** n.s. 17*
7*, 10**, 12-14***, 5*, 7**, 10***, 7*, 10-14***, 17-
-I 10-17***, 21-24*
17-21**, 24* 12-17**, 21*, 28* 21**, 28**
F440/F690
7-10*, 12***, 14**,
-S 12-14*, 17** n.s. 7*, 10**, 12-17*
17*
*** ** ** ***
10-12 , 14-21 , 7 , 10 , 12- 7*, 10-14***, 17-
-I 10-17***, 21-24*
24* 14**, 17-21* 21**, 28*
F440/F740
7-10*, 12-14***,
-S 14*, 17** 14* 7*, 10**, 12-17*
17**
* *
-I n.s. 12-14 , 28 10-12* 12*, 14**, 28*
F690/F740
-S 17* n.s. 10-12*, 14-17** 7*, 17*
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
Fig. 5. Images of F440/F520, F440/F690, F440/F740 and F690/F740 fluorescence ratios of AS (a)
and S (b) leaves of Nicotiana benthamiana control and PMMoV-infected plants. AB and AD surfaces
of the leaves are shown. Images obtained before (5 dpi) and after (17 dpi) fluorescence changes
occurred are displayed. The applied colour scale is shown for every panel. Different asymptomatic
(old) and symptomatic (young) leaves sizes are due to leaf age.
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C. Resultados
Table 2). Recovery phase of the symptoms on PMMoV-I infected plants was
registered as a decrease in this ratios, more evident in the AB surface where no
significant differences with control values could be detected at the end of the
infection process.
At 5 dpi, both surfaces of control S leaves showed a very uniform F440/F690
and F440/F740 pattern, with slightly increased values of these ratios on the main
veins (Fig. 5b). At 17 dpi, this situation continued without great changes. PMMoV
infected plants showed similar F440/F690 and F440/F740 values at 5 dpi compared
to the controls. Increased values registered in the AD surface were not significant
(Table 2). Nevertheless, an increment of the ratios was clear in S leaves of infected
plants at 17 dpi. The whole leaf exhibited increased F440/F690 values, emitting the
vascular tissues the highest amount of fluorescence. This vein-associated pattern,
very similar to that found in AS leaves of infected plants, was more apparent in the
AB surface of the S leaves.
F690/F740. AD surface of the 3 treatments displayed lower values of
F690/F740 than the AB one (Fig. 4). Changes in the ratios related to a decrease in
Chl content of S leaves from PMMoV-S-infected plants were only visible at 17 dpi
in the AD surface (Table 2), whereas AB one displayed less Chl content from 10 to
17 dpi (Fig. 4). On the other hand, the two surfaces of the S leaf of PMMoV-I
infected plants exhibited a different behaviour depending on the considered leaf
surface (Fig. 5b, Table 2.)
Fluorophores determination
HPLC analyses of methanolic extracts from S leaves of Nicotiana
benthamiana plants revealed the presence of chlorogenic acid (Fig. 6), whose
concentration increase during the viral infection. Significant differences with
respect to the controls appeared from 7 to 21 dpi for PMMoV-S plants, whereas
PMMoV-I infected plants displayed differences only at 14 dpi. The delay is in
agreement with the lower virulence of this strain; at the beginning of the recovery
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
phase (21 dpi), no significant differences could be detected between control and
PMMoV-I- infected plants.
0.20
Control ***
Chlorogenic acid (mg·mm-2) (x10-2)
PMMoV-I
PMMoV-S
0.15
0.00
7 14 21 28
t (days post-inoculation)
Discussion
Albeit in the last years UV-excited MCF imaging has emerged as a sensitive
and specific tool for presymptomatic and non-destructive monitoring of changes in
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C. Resultados
the physiological state of plants, the application of this technique has not gained
ground in the investigation of pathogen attacks, especially viral infections.
Nevertheless, Chl-FI has been broadly employed to study such interactions (van
Kooten et al. 1990; Balachandran and Osmond 1994; Balachandran et al. 1994;
Técsi et al. 1994; Osmond et al. 1998; Lohaus et al. 2000; Chaerle et al. 2004,
2006; Pérez-Bueno et al. 2006). Information about BGF imaging and the
corresponding accumulation of secondary metabolites in virus-infected plants is
only available for the HR induced by TMV (Chaerle et al. 2007); the present
contribution is the first in elucidating such changes during a systemic infection.
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
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C. Resultados
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
phloem (Roberts et al. 1997; Cheng et al. 2000; Oparka and Santa Cruz 2000).
Tissue prints taken from 7-28 dpi of the young leaves showed a quick viral
accumulation in the whole leaf during the early infection steps (Chaerle et al 2006).
In the present work, we could detect the PMMoV-S entrance in the leaf at 4 dpi
and later (5-6 dpi) for the less virulent strain (PMMoV-I), the first visual symptoms
appearing one day later. This confirms the rapid viral movement to S leaves in
contrast to AS leaves (Chaerle et al. 2006), and could explain the absence of a
dynamic imaging Chl-F pattern in S leaves (Fig. 2b). However, averaged whole
leaf Chl-F values could distinguish infected leaves from controls, but never in a
presymptomatic way (Fig. 1, Table 1). These changes in Chl-F emission preceded
the decrease of the Chl content (Chaerle et al. 2006), as F690/F740 ratio showed
(Fig. 4, Table 2).
PMMoV detection in the S leaf (Table 1) either preceded alterations in BGF
emission or the two processes took place concomitantly (F520 of AB surface of S
leaves of PMMoV-S infected plants). As in the case of AS leaves, and due to the
same reasons described above, the changes in fluorescence emission on the AB
surface of S leaves were more significant. The accumulation of phenolic
compounds in S leaves induced by PMMoV, contrary to AS ones, did not precede
virus detection. The decrease of BGF values in the S leaves of infected plants
between 21-28 dpi, compared with previous points of measurement, was indicative
of the symptom recovery phase affecting the young leaves in PMMoV-I infected
plants.
To identify the major compounds responsible for the emission of BGF in N.
benthamiana plants, HPLC analyses of leaves were carried out. Wolfbeis et al.
(1985) reviewed a series of natural compounds known to be present in leaves, that
emit BGF upon UV excitation. The two main classes of BGF emitters are HCAs
and nicotinamide and flavine nucleotides (Lang et al. 1991; Morales et al. 1994;
Cerovic et al. 1999). In the methanolic extracts of S leaves from control and
PMMoV-infected N. benthamiana plants, among the HCAs, chlorogenic acid was
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C. Resultados
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
Acknowledgements
This research and MP´s contract was supported by grants from the Spanish
to M.B.A.) The authors are very grateful to Drs. Isabel García Luque and Maite
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C. Resultados
PMMoV solutions and antibodies against the viral coat protein, and to Ruth
Sagardoy for her assistance with the HPLC analyses which were supported by an
19187/05/I-EC – to F.M.. The travel associated with this work was supported by
(HH2004-0032 in Spain).
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Pineda, M.1, Soukupová, J.2, Matouš, K.2, Barón, M1,*. and Nedbal, L2,*.
1. Department of Biochemistry, Molecular and Cell Biology of Plants. Estación Experimental del
Zaidín, Consejo Superior de Investigaciones Científicas. C/ Profesor Albareda, nº 1. C.P. 18008.
Granada, Spain
2. Institute of Systems Biology and Ecology, Academy of Sciences of the Czech Republic, Zámek
136, Nové Hrady 37333, Czech Republic / Institute of Physical Biology, University of S. Bohemia,
Zámek 136, Nové Hrady 37333, Czech Republic.
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infection earlier than the standard Chl-F parameters (4 and 6 dpi: PMMoV-S and –
I, respectively).
Introduction
Chlorophyll fluorescence imaging (Chl-FI) is an useful tool to study the
spatial and temporal heterogeneity of leaf photosynthesis under biotic stress
(Nedbal and Whitmarsh 2004), allowing the diagnosis and quantification of
different stress responses on the basis of their characteristic disease-specific
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signatures (Chaerle et al. 2004, 2007). Chl-FI, combined with other imaging
techniques, allowed distinguishing between infections with different pathogens
(Chaerle et al. 2004). Changes in red fluorescence emission (Chl-F) could be
related with physiological alterations on infected plants (Scholes and Rolfe 1996,
Chou et al. 2000, Berger et al. 2004, 2007, Bonfig et al. 2006, Swarbrick et al.
2006). Photoinhibitory damage in infected plants (Balachandran and Osmond
1994, Balachandran et al. 1994) has been also detected by the measurement of
changes on fluorescence parameters.
Recent Chl–FI instruments allow application of complex measuring
protocols with different dark adaptation-times of the plant and periods of constant
actinic light, both supplemented by saturating bright flashes. Thus, different types
of light protocols have been used to reveal differences between healthy and
stressed plants: obtaining fluorescence values after just one saturating flash
(Chaerle et al. 2004, 2006), Kautsky kinetics (Kautsky and Hirsch 1931), harmonic
oscillations (Nedbal and Brezina 2002, Nedbal et al. 2003, Matouš et al. 2007),
quenching analysis with different number of saturating flashes (Pérez-Bueno et al.
2006) and combined with various actinic light intensities (Berger et al. 2007,
Matouš et al. 2007).
During the quenching analysis, huge information is provided because of the
great number of fluorescence parameters obtained associated to the photosynthetic
function. Some coefficients are related with photochemical processes, as FV/FM and
ΦPSII, which indicate the maximum and effective quantum yield of photosystem
(PSII) (Genty et al. 1989); or with the non photochemical processes, the most
straightforward is NPQ (non photochemical quenching, Bilger and Björkman
1990). A coefficient giving information about the vitality of the plants (RFD,
Lichtenthaler and Rinderle 1988) has also been broadly used, and can be calculated
from Kautsky kinetics and quenching analysis. Lists of Chl-F parameters related to
different physiological functions have been reviewed by several authors (e.g.,
Schreiber et al. 1986, Roháček and Barták 1999, Maxwell and Johnson 2000).
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Images of them obtained in every kinetic point could provide a good/early stress
indicator to follow pathogenesis (Pérez-Bueno et al. 2006).
However, standard Chl-F parameters not always reveal the maximal
differences between healthy and infected tissues along the infection, when complex
spatial and temporal changes are occurring. In this context, Soukupová et al. (2003)
proposed a simple experimental algorithm to assess the impact of the treatment by
fungal phytotoxins in canola and white mustard. More recently, Berger et al.
(2007) and Matouš et al. (2007) described combinatorial imaging methods to map
bacterial infection in Arabidopsis. This method searches small sets of images,
obtained during Chl-F transient, giving the highest contrast between the stressed
and control leaf tissue and can be used to image the pathogen action during the
early infection phase.
The experimental host-pathogen system studied was Nicotiana benthamiana
plants infected with the Italian and Spanish strains of the pepper mild mottle virus
(PMMoV-I and –S, respectively). In previous studies (Barón et al. 1995, Rahoutei
et al. 1998, 1999, 2000, Pérez-Bueno et al. 2004), we have analysed the effect of
both PMMoV-I and –S infection upon photosynthesis in these plants, and propose
the oxygen-evolving complex (OEC) as main target of tobamovirus on the electron
transport chain. Chl-FI studies of Nicotiana benthamiana plants infected with
PMMoV-I revealed that NPQ was a good reporter of the infection process as early
as 17 days post-inoculation (dpi). In leaves, which remain asymptomatic (AS)
during the infection process, NPQ changes were parallel to the virus spreading in
these leaves (Pérez-Bueno et al. 2006). Chaerle et al. (2006) studied thermal and
fluorescence responses in Nicotiana benthamiana plants infected with either
PMMoV-S or –I. They found that infection with PMMoV-S induced earlier
alterations than PMMoV-I (14 dpi vs. 17 dpi) in the fluorescence pattern of AS
leaves from infected plants. However, as the first reporter of viral infection was
observed the temperature increment registered near the main veins (7 dpi in the
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case of PMMoV-S infection, 10 dpi in the case of PMMoV-I one), which expanded
to the rest of the leaf tissues in successive days.
The aim of the present study was to identify differences between the
infection processes induced by distinct strains of the same virus (PMMoV), using
conventional Chl-F parameters that are related to well-established photosynthetic
processes or pathways. In addition, we investigated PMMoV infection in Nicotiana
benthamiana plants using novel protocols and statistical approaches that were
earlier shown to reveal very early phases of infection with pathogenic bacteria
(Berger et al. 2007, Matouš et al. 2007).
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
where the blue color represents the lowest value and the red color represents the
highest one.
Combinatorial imaging
For selection of the relevant images during tobamoviral infection on
Nicotiana benthamiana, we used the method of Sequential Forward Floating
Search (Pudil et al. 1994). As a separability criterion we used error rate of k-NN
classifier (k=3), which is a method classifying unknown objects (in our case Chl-F
kinetics) on the base of their Euclidian distance to the training samples.
Here, we use measurements of Chl-F transient under protocol 2 as a dataset
for the combinatorial imaging. Measurements (4 repetitions) carried out in AS
leaves of every plant group –control, PMMoV-I and PMMoV-S-infected- were
analyzed. The Chl-F transients of control leaves were used as the first (control)
class and those of plants infected with PMMoV-S as the second (infected) class.
PMMoV-S infected plants were chosen because of the higher virulence of this
virus strain (Rahoutei et al. 2000). The classifier was trained to find the differences
between controls and PMMoV-S infected leaves at 16 dpi, the middle phase of the
infection, and to resolve these two classes. When the set of the 3 most contrasting
features (images) with the lowest error rate was found, it was used to evaluate the
difference among control and PMMoV-S as well as PMMoV-I infected plants
during the whole infection.
LDA (Linear Discriminant Analysis) algorithm (Fukunaga 1990) was used to
find the linear combination of the 3 most contrasting images into one. This
resultant image was shown in false colour scale. The method is described in detail
by Matouš et al. (2007).
IRGA measurements
Photosynthesis rate (A) was determined on a 100 mm2 leaf area at the leaf
apex by IRGA (infrared gas analyzer) in an open system at ambient partial
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Results
Study of standard Chl-F parameters during quenching analysis (protocol 1)
We have analysed different conventional Chl-F parameters after
measurements using protocol 1, in order to find those that can discriminate the
infection with two similar strains of the PMMoV in the early infection steps and in
absence of symptoms. NPQ and ΦPSII were found to be the best reporters of
PMMoV infection in N. benthamiana plants among the standard parameters.
Figure 1b displays dynamics of NPQ20 distribution over the leaves during the
first 28 dpi. NPQ20 was the kinetic point showing the biggest differences between
control and PMMoV-infected plants along the whole infection. Prior to 17 dpi, we
could not find any significant response in NPQ20 pattern, except in the case of AS
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
leaf from PMMoV-S infected plant, which displayed certain higher NPQ20 beside
the petiole at 14 dpi, depending on the sample assayed.
Considering the signal over the whole leaf, NPQ was higher in the infected
plants compared to control at 17 dpi (Fig.1a) and the relationship became inverse
between 19 and 28 dpi (Fig. 1b).
Fig. 1: Images of classical Chl-F parameters obtained by measurements using protocol 1 in AS leaves
from control and both PMMoV-I and PMMoV-S infected plants. (a) NPQ kinetic at 17 dpi. (b) NPQ20
pattern at different dpi (displayed at the bottom of the figure). (c) ΦPSII pattern at different dpi. The
2
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surrounding the main veins than in interveinal tissues. The ΦPSII2 pattern was more
homogeneous over the whole leaf in case of PMMoV-I and control plants. This
parameter allows a good differentiation between the infections with either viral
strain.
Fig. 2: Analysis of data obtained under fluorescence measuring protocol 1 or by means of IRGA in
AS leaves from control and infected plants. (a) Net photosynthesis rates (A) measured at different dpi
by the IRGA; (b) Linear correlation between A and ΦPSII . Correlation factor (r) were 0.927
300
(control), 0.956 (PMMoV-I infected plants) and 0.859 (PMMoV-S infected plants).
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
from 14 dpi onwards. A significant decrease of this rate compared with the control
values was observed in infected plants at 17 dpi (PMMoV-S) and 19 dpi (PMMoV-
I). This delay agrees with the less virulence of the last viral strain. In addition, we
have made a correlation analysis between ΦPSII300 (PSII quantum yield at the steady
state, i.e. at 300 s of the induction kinetic) averaged in the whole leaf and the rate
of net photosynthesis (Fig. 2b). A positive correlation was established in the case
of control and infected plants, with r = 0.927 (control plants), r = 0.956 (PMMoV-
I-infected plants) and r = 0.859 (PMMoV-S-infected plants).
Fig. 3: Images obtained under measuring protocol 2 at different dpi (indicated at the bottom of the
figure). NPQ at 5 s after switching off the actinic light at low light (a) and high light (b).
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Fig. 4: Chl fluorescence transients of AS leaves measured with protocol 2 at different dpi. A brief
picture of the protocol is at the top of the figure, and the distinct peaks (FM, FP, FM(1)’, FM(2)’, FM(1)’’
and FM(2)’’) are depicted in the 9 dpi transient Chl-F kinetic.
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
light). Fig. 3 shows the NPQ images of the AS leaves obtained at the first
saturating pulse in the dark under both LL and HL, where the contrast between
control and infected leaves was the highest.
Since the analysis of standard Chl-F parameters was not successful in
revealing differences between controls and infected plants prior to 16 - 17 dpi, we
evaluated difference between the Chl-F transients of control and infected plants
under measuring protocol 2 (Fig. 4). The integrative Chl-F signal over the AS
leaves of infected plants started to differ from control at 13 dpi under LL; and
became more evident at 16 dpi under both light intensities. The differences
between the Chl-F transients of control and infected plants were observed during
the Chl-F decline from the local transient maximum level (FP).
Fig. 5: Kautsky kinetics registered under protocol 3 at low (LL, a) and high light (HL, b) in AS leaves
from control and infected plants (left panels). Differences (%) between control and infected plants
(right panels) at different dpi.
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
Fig. 6: Images of F5/F0 parameter under high light (HL) at different dpi captured using measuring
protocol 3.
Fig. 7: Feature selection analysis of AS leaves measured by protocol 2. The colour scale indicates the
intensity of the signal.
We found the biggest difference during the transient decrease from FP, in the
images [I(t)] taken at t1 = 5 s, t2 = 21 s, and t3 = 736 s (i.e., at 5 and 21 s of LL, and
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Discussion
Chl-FI has been broadly applied to study biotic stress on plants (Nedbal and
Whitmarsh 2004), since it makes possible to follow the physiological alterations
that stress factors induce in leaves from infected plants (Lichtenthaler and Miehé
1997). This technique was successful to follow the effect of folivory damage
(Zangerl et al. 2002, Aldea et al. 2006, Tang et al. 2006), as well as fungal (Scholes
and Rolfe 1996, Chou et al. 2000, Swarbrick et al. 2006), bacterial (Berger et al.
2004, 2007, Bonfig et al. 2006) and viral infections (Balachandran and Osmond
1994, Balachandran et al. 1994, Osmond et al. 1998, Lohaus et al. 2000, Chaerle et
al. 2004). Our previous works with PMMoV-infected plants showed that images of
both Chl-F under one saturating flash (Chaerle et al. 2006) and NPQ (Pérez-Bueno
et al. 2006) could reflect the viral distribution on leaves which remain
asymptomatic during the viral infection. Some authors (Osmond et al. 1998,
Lohaus et al. 2000) have proposed that NPQ images reproduce the state of
symptom development
In this study, conventional and combinatorial Chl-F imaging of leaf was used
to detect the early phase of infection and to differentiate the infection with two
viral strains of PMMoV on Nicotiana benthamiana plants. .
According to Pérez-Bueno et al. (2006), in the present study PMMoV-I
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
infection induced in the AS leaf from the host plant a complex and characteristic
NPQ pattern from 16-17 dpi onwards (Fig. 1a, 1b, 3). This pattern is better defined
in PMMoV-S infected plants. NPQ pattern at 17 dpi corresponds with the viral
location in these leaves at the same infection points, tested by tissue print in our
previous work (Chaerle et al. 2006, Pérez-Bueno et al. 2006). Changes on NPQ
pattern could not be attributed to a viral-induced decrease of the leaf Chl content;
only at the last infection steps Chl amount decreased even in the case of control
plants (Rahoutei et al 2000, Chaerle et al. 2006). At this time, senescence processes
are predominant in the old leaves, since non-photochemical as well as
photochemical processes are totally down-regulated, as the lowest NPQ and ΦPSII
values reflect (Fig. 1). According to our results and those by other authors (Chou et
al. 2000, Berger et al. 2004), changes in NPQ values in the regions colonized by
the pathogen are a relevant phenomenon in plant pathogenesis. NPQ increase may
have different origins. It is well established that virus induce disturbances in the
donor site of PSII due to a decrease in the amount of the extrinsic protein levels
(Rahoutei et al. 2000, Pérez-Bueno et al. 2004) that may lead to the appearance of
dissipative PSII centres (van Wijk et al. 1993, Critchley and Russell 1994). Viruses
also induce disturbances in the Benson-Calvin cycle that can increase the
intrathylakoidal pH gradient, which modulates the NPQ process (Ruban and
Horton 1995). Other process regulating the intrathylakoidal pH gradient is the
cyclic electron transport around PSI (photosystem I) (Horton et al. 2005), which is
stimulated during viral infection (Sajnani et al. 2007). The higher NPQ values
during the viral infection could be a host defence response delaying photoinhibition
until the final infection steps (Balachandran et al. 1997), as revealed as a late (19-
21 dpi onwards for PMMoV-S and –I, respectively) decrease in FV/FM (images not
shown; point measurements by Rahoutei et al. 2000).
Analysis of the ΦPSII kinetic at different infection points revealed that, only at
2 s after switching on the actinic light (ΦPSII2), AS leaves from PMMoV-S-infected
plants displayed a differential pattern from control plants from 17 to 21 dpi,
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undetectable in the case of PMMoV-I infection (Fig. 1b). This allows the
differentiation between the infections with every viral strain. The ΦPSII2 pattern, as
the NPQ one, also correlates with the viral presence (Chaerle et al. 2006, Pérez-
Bueno et al. 2006) in the infected leaf.
Changes on both standard Chl-F parameters correlated with a decrease on the
net photosynthesis rate from 17-19 dpi (PMMoV-S and PMMoV-I, respectively) in
infected plants (Fig. 2a). Chl-F imaging demonstrates to be more powerful than
IRGA points measurements to detect the PMMoV-I infection, showing earlier
changes (17 dpi) in the spatial pattern of the fluorescence parameters.
The standard Chl-F parameters measured under protocol 1 and 2 detected the
PMMoV-infection as early as 16 – 17 dpi (Fig. 1,3). However if the combinatorial
imaging was applied (Matouš et al. 2007), the first significant changes between
control and infected plants were visible at 4 and 6 dpi (for PMMoV-S and –I,
respectively; Fig. 7). This is in accordance with previous results, demonstrating
that such method could identify the infection process earlier than classical Chl-F
parameters (Berger et al. 2007, Matouš et al. 2007).
When we analysed the Chl-F transients under two levels of actinic
irradiances (Kautsky effect, measuring protocol 3), the first differences between
PMMoV-infected and control plants were displayed at 9 dpi during the Chl-F
increase and decline from the FP (Fig. 5). This result is in well accordance with the
data obtained from statistical analysis of Chl transient under light protocol 2, which
indicated Chl-F images at 5 s of the kinetics as one of the most contrasting. These
changes were reflected in the F5/F0 images of the leaves (Fig. 6), displaying a
heterogeneous Chl-F pattern in infected plants. Regions emitting less Chl-F yield
corresponded with zones of higher NPQ (Fig 1a, 1b) and viral presence (Chaerle et
al. 2006, Pérez Bueno et al. 2006). In the later post-infection days, there was an
increment of kinetic time points displaying differences between infected and
control plants (Fig. 5), that could be associated to the last infection steps connected
with the senescence process. Changes on the parameter F5/F0 may reflect PMMoV-
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
induced alterations on the Benson-Calvin cycle, since this time point is very near to
the kinetic peak corresponding to the activation of the cycle (Ireland et al. 1984);
the fact that the differences between AS leaves from infected plants and control are
emphasized by HL is an additional evidence for it.
Images obtained by the combinatorial imaging and those of F5/F0 identified
the viral infection before the virus is detected in AS leaves by tissue printing
(Chaerle et al. 2006, Pérez-Bueno et al. 2006).
Summarizing, Chl-F was a very useful tool to follow the PMMoV infection
on AS leaves of N. benthamiana plants. The technique provided specific signatures
(Chaerle et al. 2004, 2007) of the pathogenesis induced by two different strains of
the same virus, allowing us to distinguish between PMMoV-I and PMMoV-S
infection. Classical Chl-F parameters gave us information about the physiological
processes taking place in the AS leaves (decrease in photosynthesis and increase in
the energy dissipated as heat), correlated with the viral movement on these leaves.
Analysis of Chl-F transients by advanced statistical approach and F5/F0 let us to
detect the infection earlier than classical parameters. The use of complementary
imaging biophysical techniques, as thermography or multicolour fluorescence
induced by ultraviolet light, would provide a signature of the complete PMMoV
infection process, allowing us to elaborate stress-catalogues that might be used for
diagnostic and quantification of the stress responses (Chaerle et al. 2007).
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1: Department of Biochemistry, Molecular and Cellular Biology, Estación Experimental del Zaidín.
CSIC, Granada, Spain.
2: Corresponding author: mbaron@eez.csic.es
*: These authors have contributed equally to this work and are placed in alphabetical order.
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Introduction
Proteomics is nowadays an irreplaceable tool to study the response of living
organisms to their environment. However, less attention has been applied to plant
proteome, although some revisions about this topic were published in last years
(Rossignol 2001, Bagginsky and Gruissem 2004, Cánovas et al. 2004, Newton et
al. 2004, Rose et al. 2004, van Wijk 2004, Rossignol et al. 2006). In order to
improve the understanding of plant proteome expression, a number of studies are
focused in organelles subproteomes, as those of chloroplast and mitochondria
responsible of the plant energy metabolism. 2-DE (two dimensional gel
electrophoresis), consisting in isoelectrofocusing (IEF) followed by SDS-PAGE
electrophoresis (IEF/SDS-PAGE), was one of the first techniques applied to study
the chloroplast proteome. This approach has demonstrated to be successful for the
analysis of both luminal and peripheral thylakoid proteins from Pisum sativum
(Peltier et al. 2000) and Arabidopsis (Kieselbach et al. 2000, Peltier et al. 2002,
Schubert et al. 2002). 2D-BN/SDS-PAGE (Blue-native polyacrylamide gel
electrophoresis followed by SDS-PAGE), commonly used to characterize
chloroplast protein complexes in plants and algae (Schägger et al. 1994), has also
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
OEC is one of the main targets of the viral infection in the chloroplast (Rahoutei et
al. 2000). The proteomic analysis of the OEC from the infected plants showed that
the PsbO and PsbP proteins correspond to protein families in Nicotiana
benthamiana, and the content of the PsbP polypeptides decreased dramatically with
respect to PsbO during the progress of the infection. Interestingly, there was a
differential decrease of the different PsbP proteins, indicative of a distinct
regulation of their expression during pathogenesis (Pérez-Bueno et al. 2004).
In the present work we go further insight in the analysis of the chloroplast
proteome of Nicotiana benthamiana by 2-DE and mass spectrometry (MS)
followed by database searching. In addition, changes in this chloroplast proteome
induced by the infection with PMMoV-S were studied after 14 days post-
inoculation (dpi). Viral infection induced the down-regulation of several
photosynthetic proteins, while expression of other chloroplast proteins was
enhanced by the virus.
N. benthamiana is one of the most common experimental hosts and it is used
in many studies about compatible interaction plant-virus, because of its ability to
be infected by many different viral families. Progress in the knowledge of the N.
benthamiana chloroplast proteome would be of high interest in the field of the
impact of biotic stress on photosynthesis.
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Sample isolation
Chloroplast-enriched preparations from virus-infected plants and their
corresponding controls were isolated according to Reche et al. (1997) at 14 dpi.
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
used for alkaline pH range gels, pI (isoelectrical point) standards (2-D SDS-PAGE
Standards, BioRad) were added to them after the quantification.
SDS-PAGE
Focused IEF strips were incubated at room temperature for 20 min with two
changes of equilibration buffer Tris-HCl 50 mM pH 8.8 containing 6 M urea, 2%
(w/v) SDS, 0.02% (w/v) bromophenol blue, 30% glycerol, and 0.5% (w/v) DTT.
Strips were then layered on the top of a SDS 12% polyacrylamide gel and sealed
with melted 0.8% agarose in running buffer. 2 gels were run simultaneously, one
for each treatment, in order to reduce variations. Running conditions were 17 mA
for 30 min and 48 mA for 5-6 h. After electrophoresis, gels were silver stained (pH
6-11 map; Yan et al. 2000) or Sypro Ruby stained (pH 4-7 map; Bio-Rad,
Steinberg et al. 1996). To ensure the reproducibility of the results, triplicate 2D
gels were made from 3 independent chloroplast preparations, each from a different
batch of plants. Figures show the most representative gels.
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Results
Chloroplastidic proteins from both control and PMMoV-S infected Nicotiana
benthamiana plants were separated by 2-DE and the gels were analysed to study
the changes induced by the viral infection. To improve the gel resolution and avoid
troubles due to the difficult focusing of proteins with alkaline pI, we made separate
2D maps for the low (4-7) and high (6-11) pH range.
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
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203
Table 1 (continued). Proteins identified by MALDI/TOF-TOF from the 2D gels showed in Figs. 1 and 3 and marked by the same number. Each
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
Fig. 2. Changes in the expression levels of different chloroplastidic proteins (pH 4-7 range) detected
by 2-DE. Left panels correspond to control plants and right panels to PMMoV-S infected plants. All
the proteins (excepting # 55, the coat protein of PMMoV-S) present lower expression levels in
infected plants. Numbers correspond with those of Table 1.
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
Fig. 3. Silver-stained 2D maps of chloroplast preparations from control (A) and PMMoV-S infected
(B) Nicotiana benthamiana plants. Proteins were separated in the first dimension in a linear (pH 6-11)
IPG gel strips and in the second dimension in 12% polyacrylamide vertical gels. Identified spots by
means of MALDI/TOF-TOF are listed in Table 1. Black and gray numbers in (A) represent proteins
suffering no changes or down-regulated, respectively, during the PMMoV-S infection. Numbers
enclosed in circles in (B) display up-regulated proteins in infected plants. Black boxes frame human
keratins. Dotted arrows in (A) represent pI standards, named by letters and listed in Table 2.
210
Table 2. pI standards identified by MALDI/TOF-TOF from gels displayed in Fig. 3. Each polypeptide is annotated with the homology found,
including protein name, species and the corresponding protein accession number. Theoretical and experimental pIs and Mr are also provided.
Homologies include the % coverage reached, the number of matched and unmatched peptides, and the MASCOT score details. Scores>76 are
significant (p<0.05).
C. Resultados
g Albumin Bos taurus gi|162648 71 5.8 10.0 8.9 145 11 4 21
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Discussion
2-DE has been applied successfully to the analysis of the chloroplast
proteome, and especially, to the peripheral and luminal one in plants as
Arabidopsis and Pisum sativum (Kieselbach et al. 2000, Peltier et al. 2000, van
Wijk 2001, Whitelegge 2003). Few authors have investigated changes in the
chloroplast proteome under abiotic or biotic stress factors, Andaluz et al. (2006)
analysed the effect of Fe-deficiency in Beta vulgaris, Cui et al. (2005) that of cold
stress in Oryza sativa and Jones et al. (2006) evaluated changes in the chloroplast
proteome of Arabidopsis plants infected with Pseudomonas syringae pv. tomato.
We have also carried out a proteomic analysis of the OEC from N. benthamiana
infected with PMMoV (Pérez-Bueno et al. 2004); the OEC proteins were the most
abundant one in the chloroplast proteome and we had previously described them as
target of the infection with this tobamovirus (Rahoutei et al. 1998). In the present
work we go further insight in the analysis of the chloroplast proteome of N.
benthamiana by 2-DE and MS followed by database searching.
More than 200 spots could be visualised in the 2D gels from chloroplast
preparations isolated from control plants: 150 spots in the pH range 4-7, most of
them in the 20-43 kDa Mr region (Fig. 1A, B), and 53 spots in the pH range 6-11
(Fig. 3A, B). From the analysis by MALDI-TOF/TOF MS, 71 proteins were
identified (35,5%); in addition, several LHCII proteins were visible in the gels
(Fig.1A) and identified by Western Blot (data not shown).
It was very remarkable the high resolution grade reached in the case of PsbO
and PsbP proteins, extrinsic proteins of the OEC (Fig. 1A). Our previous works
only let us identify 4 isoforms of every protein family (Rahoutei et al. 1998, Pérez-
Bueno et al. 2004), both encoded by a multigene family with at least 4 isogenes
(Pérez-Bueno 2003). In turn, we have identified in the present work 6 spots
corresponding to PsbO and 10 belonging to PsbP (Table 1). Multigene families
coding for different isoforms of PsbO have been described in Pisum sativum
(Wales et al. 1989), Licopersicum esculentum (Görlach et al. 1993) and
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Arabidopsis thaliana (Kieselbach 2000, Peltier et al. 2002, Schubert et al. 2002).
Gene families for PsbP were found in Nicotiana tabacum (Hua et al. 1991a and b,
Hua et al. 1993, Takahashi et al. 1991), Arabidopsis thaliana (Peltier et al. 2002)
and Sinapis alba (Merkle et al. 1990). It has been demonstrated in Nicotiana
tabacum that the 4 PsbP isoforms are functionally equivalents in vivo, but it is
required the expression of the 4 isogenes for optimal PSII activity (Ishihara et al.
2005). We have also demonstrated that the expression level of the distinct PsbP
isoforms is differentially affected by the PMMoV infection in Nicotiana
benthamiana (Pérez-Bueno et al. 2004). Analysis of the 2 PsbO isogenes from A.
thaliana revealed that they were not equivalent; the down-regulation of the
expression from every isogene induced that of other PSII proteins and a reduced
activity of this photosynthetic complex (Murakami et al. 2005).
The higher number of PsbP and PsbO spots found in this work compared
with our previous one (Pérez-Bueno et al. 2004), could be explained because in the
earlier work we have used self-made disc gels for the IEF; in the present one we
carried out the first dimension by ready made IPG Strip gels. This technique allows
higher resolution, more reproducible patterns and better focusing of the basic
proteins (Corbett et al. 1994; Gorg et al. 2000). From the 6 and 10 spots detected
for the PsbO and PsbP proteins, respectively, some of them may be the result of
post-transcriptional modifications, because we have found only 4 isogenes
codifying every protein family (Pérez-Bueno 2003). Further analysis of these spots
carried out by electrospray ionization-MS will clarify this point.
The reported decrease due to the viral infection on the levels of PsbP
isoforms (Pérez-Bueno et al. 2004) was not evident in the present acidic 2D maps.
Being the OEC proteins the most prominent ones in this pH range, the PsbO and
Psb P signals were saturated (Fig. 2), because we have loaded a higher amount of
protein in these gels in order to identify minor proteins in the N. benthamiana
chloroplast proteome.
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In the alkaline 2D map (pH 6-11) we detected two spots for the PsbQ protein
(pI 9.4 and 9.5; Fig. 3A, B and Table 1), in accordance with the 2 psbQ isogenes
that we have reported for N. benthamiana (Pérez-Bueno 2003). Two PsbQ
isoforms were identified by 2-DE on chloroplast preparations (Kieselbach et al.
2000, Schubert et al. 2002). However, Peltier et al. (2000) could only detect one
spot in 2D gels from Pisum sativum chloroplasts. The 2D gels in the alkaline pH
range displayed increased levels of the PsbQ proteins in chloroplast from PMMoV-
S infected plants (Fig. 3B). Ifuku et al. (2005a and b) showed that under standard
growth conditions, PsbP protein, but not PsbQ, is indispensable for the normal PSII
function in vivo. In contrast, Yi et al. (2006) found that under stressful low light
conditions, the PsbQ suppression led to a loss of different PSII components,
decrease in PSII quantum yield and disturbances on electron transfer from QA- to
QB. Other authors (Ifuku and Sato 2002, Ifuku et al. 2005a) conclude that PsbQ is
required for optimum PSII function under conditions in which PsbP is impaired.
This could explain why PsbQ are accumulated in PMMoV-S plants, whereas PsbP
expression is diminished.
The different subunits of the CF1 are together with the OEC proteins the
most abundant polypeptides in the acidic pH range (Fig. 1A, B). These data are in
accordance with the 2D-maps from the thylakoid proteome of Arabidopsis thaliana
investigated by Friso et al. (2004). We have found in our gels all subunits of the
CF1 fragment from the ATP synthase. Due to the analogy with Friso et al. (2004)
gels, we propose that proteins with the same Mr close to CF1 α and CF1 β (Fig. 1A)
would be protein trains of these subunits.
We have also identified several high Mr chaperons (Fig. 1, Table 1): the 60
kDa chaperonin collaborating in RuBisCo assembly (Buchanan et al. 2000) and
Hsp70 involved in PSII stress protection (Schroda et al. 2001). The FtsH-like
related to PSII repair (Nixon et al. 2005, Cheregi et al. 2005) and turnover
(Zaltsman et al. 2005a and b) was also present in the 2D gels. Two of the identified
spots in our 2D maps (hypothetical protein and the thylakoid lumenal 16.5 kDa
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proteins) have unknown function; the hypothetical protein amount decreased in the
PMMoV-S infected plants.
Comparative analysis of the chloroplastidic proteome from control and
PMMoV-S infected plants revealed that the expression levels of some proteins
implicated in Benson-Calvin cycle, electron transport chain and nitrogen
metabolism decreased during the PMMoV-S infection (Fig. 2).
The proteins involved in the Benson-Calvin cycle affected by the PMMoV
infection are: RbcL, RbcS, Rca, SFBA, SBPase, and PRK. The decrease on the
amounts of both RuBisCO subunits and Rca is in agreement with the diminished
transcript levels of these proteins during pathogenesis (Pérez-Bueno, 2003). The
degradation of the RcL subunit under different stress conditions (UV radiation,
ozone, osmotic, etc.) is for some authors a consequence of oxidative modifications
on this enzyme (Houtz et al. 2003 and references therein). Oxidative stress is
usually induced in infected plants (Foyer and Harbinson 1994, Vranová et al.
2002). The Rca has a dual function, activating the RuBisCO complex, and as a
chaperonin, protecting protein synthesis under stress conditions (Sánchez de
Jiménez et al. 1995, Rokka et al. 2001). SFBA is codified by two genes in
Nicotiana with differential expression levels under salt stress (Yamada et al. 2000).
A similar situation seems to occur in our PMMoV-infected plants. We have
identified three proteins spots corresponding to SFBA, being number 52 the most
affected by the PMMoV-S infection. Probably, the exceeding spot is due to post-
translational modifications. To evaluate the impact of the diminished levels of the
SBPase -one of the key regulatory enzymes of the Benson-Calvin cycle- and PRK
in infected plants, we take as model transgenic plant showing lower expression
levels of both enzymes. Decrease on SBPase amounts induced limitations on CO2
fixation and leaf carbohydrates content (Harrison et al. 1998); diminished PRK
expression could be compensated by the higher activity of the remaining PRK in
the plant (Banks et al. 1999). In general it is well documented that the decline on
the Benson-Calvin enzymes levels induced adverse effects on CO2 fixation,
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carbohydrates accumulation and plant growth (Haake et al. 1998, Raines 2003).
We have also documented (unpublished data, chapter C.3 in this work) a decrease
of the net photosynthesis rate during the infection with PMMoV.
The plastid GS, a key protein in the nitrogen metabolism, is also diminished
in PMMoV-S infected plants. Senescent plants present also decreased protein and
mRNA levels of this enzyme, whereas the cytosolic one is induced (Masclaux-
Daubresse et al. 2005, Teixeira et al. 2005). PMMoV-S infection on Nicotiana
benthamiana shows similarities with senescence processes. In clover (Trifolium
repens L.) senescence induced diminished levels of PsbO, both RuBisCO subunits
and Rca, as well as GS (Wilson et al. 2002). This down-regulation pattern is
similar to that found in symptomless leaves of PMMoV-infected Nicotiana
benthamiana plants for the transcripts of these proteins (Pérez-Bueno 2003), and
confirm that viral infection accelerates senescence processes.
In the 2D maps of the alkaline pH range from Nicotiana benthamiana
chloroplasts appeared some PSI proteins, as the PsaD and PsaE (Fig. 3A, B, Table
1). These highly hydrophilic proteins, together with PsaC, that could be
distinguished in the low pH range gels (Fig. 1A, Table 1), are located on the PSI
stromal side being involved in the ferredoxin-docking (Sétif et al. 2002). PsaD and
PsaE are codified by nuclear genes, existing two isogenes for both of them in
Arabidopsis thaliana (Naver et al. 1999, Ihnatowicz et al. 2004) and Nicotiana spp.
(Obokata et al. 1993, 1994, Yamamoto et al. 1993). In the case of Nicotiana
sylvestris, the two psaE isogenes codify for 4 different polypeptides; however, we
could detect only 2 PsaE spots in N. benthamiana. In N. sylvestris PsaD is also a
protein family with two isoforms (PsaD1 and PsaD2), functionally redundant,
encoded by 2 isogenes. However, we could detect up 8 PsaD spots, whose
expression levels diminished due to the viral infection. Because of the high
homology between PsaD1 and PsaD2 proteins (96/95% similarity/identity,
Ihnatowicz et al. 2004), they could not be distinguished by the MALDI-TOF/TOF
analysis. The psaD genes are differentially expressed during leaf development in
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
Acknowledgements
This research and M.P. contract was supported by grants from the Spanish
Ministry of Science and Education (BIO2004-04968-C02-02, to M.B.) and FEDER
Funds. The authors are very grateful to Drs. Isabel García Luque and Maite Serra
(Centro Investigaciones Biológicas, CSIC, Madrid) for providing all PMMoV
solutions and antibodies against the viral coat protein. In addition, they thank to
J.A. López, E.Camafeita and E. Calvo from the Proteomic Unit of the National
Centre for Cardiovascular Research (Madrid, Spain) for the skilful assistance with
the MALDI/TOF-TOF analysis and protein identification.
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D. Discusión
D. DISCUSIÓN
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
PMMoV-I PMMoV-S
dpi AS S AS S
Infección detectada por Presecia del virus en la
4 “selección de base
características”
F690 cara AB F520 cara AD Curvamiento
F440/F690 cara AD Hoja completa con virus
5
F520 cara AB
F690 cara AB
Infección detectada por Curvamiento
6 “selección de Hoja completa con virus
características”
F690 cara AD Curvamiento Temperatura en la Ácido clorogénico
F740 ambas caras F690 cara AD zona de venas F440 ambas caras
F440/F690 cara AB F740 cara AD mRNA: psaK, rca, F520 cara AD
F440/F740 cara AD F440/F690 cara AD gox F690 cara AD
mRNA CP detectado mRNA CP detectado Desplazamiento leve F740 ambas caras
mRNA: psaK, gox mRNA: lhcb1, rca, banda AG hacia F440/F690 ambas
rbcS, psaK, gox, rbcL y mayores temperaturas caras
psbA Aparición banda HTL2 F440/F740 ambas
Proporción AG/B caras
7 Desplazamiento banda F690/F740 cara AD
AG hacia mayores Chl
temperaturas mRNA CP detectado
mRNA: lhcb1, rca,
rbcS, psaK, gox, rbcL y
psbA
proporción AG/B
Desplazamiento banda
AG hacia mayores
temperaturas
Alteración patrón F5/F0 Alteración patrón F5/F0
9
en régimen de HL en régimen de HL
Temperatura en la F440 ambas caras F440 cara AB F690/F740 cara AB
zona de venas F520 cara AB F520 cara AB
F440/F740 cara AB F690 cara AB
10 F740 cara AB
F440/F690 cara AB
F440/F740 ambas caras
F690/F740 cara AB
F440 cara AB F690/F740 cara AD F440/F520 cara AB
12 F520 cara AB F440/F690 cara AB
F690/F740 cara AD
226
D. Discusión
PMMoV-I PMMoV-S
dpi AS S AS S
Enanismo planta Temperatura en hoja Enanismo planta Temperatura en hoja
completa completa completa completa
Chl Ácido clorogénico Patrón Chl-F alterado Chl
F440/F520 cara AB F520 cara AD Chl mRNA: psbO, psbP,
CP no detectada en mRNA: psbO, psbP, F440/F740 ambas psbQ
geles 2D psbQ caras Banda C
proteínas (mezcla hoja Banda C Presecia del virus en la Banda HTL2
ASIN +SIN): 18-20, 29, base
30, 32, 34-37, 42-50 CP detectada en geles
mRNA: lhcb1, rbcS, 2D
rca, psbO, psbP, psbQ proteínas: 7-15, 18-
Desplazamiento bandas 20, 28-30, 34-37, 42-54,
AG y B hacia mayores 57-67, 69
14 temperaturas o aparecen proteínas
Proporción AG/B (mezcla hoja ASIN
Banda C +SIN):
16, 21, 32, 65, 66, 70,
71
mRNA CP detectado
mRNA: lhcb1, rbcS,
psbO, psbP, psbQ
Desplazamiento
significativo bandas AG
y B hacia mayores
temperaturas
proporción AG/B
banda C
16 apertura estomática apertura estomática apertura estomática apertura estomática
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D. Discusión
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
campo, hace aún más valiosos nuestros datos obtenidos durante la infección por
PMMoV en N. benthamiana.
Los cambios en BGF suceden de forma más acentuada en la cara AB de las
hojas (Fig. 1-5, sección C.2), así que en general son recomendables las medidas en
el envés de la hoja para la detección del estrés biótico. La diferencia en la emisión
de fluorescencia de la cara AD y AB incluso en una hoja sana está relacionada con
la diferente anatomía y contenido de Chl de ambas caras de la hoja. Las hojas de
plantas dicotiledóneas como N. benthamiana son bifaciales (Fig. 8A), lo que
supone que la luz de medida puede penetrar mejor en la cara AB debido al menor
empaquetamiento del parénquima lagunar y lograr una mayor eficiencia de
excitación (A.2.2.1, Sajnani 2005). Asimismo, el contenido de Chl de la cara AD
es mayor (Cerovic et al. 2002 y referencias incluidas), como lo demuestran
nuestros datos del cociente F690/F740 (Fig. 4 y 5, sección C.2), que es
inversamente proporcional al contenido de Chl de las hojas (Lichtenthaler et al
1986, Stober y Lichtenthaler 1992, Babani y Lichtenthaler 1996, Gitelson et al.
1998, 1999, Buschmann et al. 2000); la mayor cantidad de Chl de la cara AD
reabsorbe la BGF emitida por la propia hoja con mayor eficiencia que en la cara
AB (Buschmann y Lichtenthaler 1998, Cerovic et al. 1999).
Aunque el incremento en BGF inducido por la infección viral afecta a toda la
hoja AS, las imágenes obtenidas en plantas infectadas muestran un característico
patrón espacial a 17 dpi, registrando las venas principales de la hoja los mayores
niveles de BGF (Fig. 2, 3, sección C.2). El incremento de BGF en el resto de la
hoja es mayor en el caso de F520 que en el de F440, probablemente por una
acumulación de compuestos que fluorescen en el verde bajo estreses que actúan a
largo plazo (Buschmann y Lichtenthaler 1998) y que hasta el momento no hemos
identificado debido a la presencia mayoritaria de ácido clorogénico, como veremos
más adelante; además F520 es reabsorbida por otros pigmentos fotosintéticos con
menor eficacia que F440 (Lichtenthaler et al. 1996). Las hojas S presentan también
un patrón similar de BGF aunque peor definido, con altos valores de esta emisión
230
D. Discusión
231
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
232
D. Discusión
233
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
sección C.1, anexo 1); sin embargo, el grado de apertura de los estomas a 16 dpi
era menor en las plantas infectadas. Por tanto, el cierre estomático estaría
provocando el incremento de temperatura, lo que fue confirmado mediante las
mediciones realizadas con el IRGA (Fig. 3C, 3D, sección C.1). Otros estudios
empleando diferentes sistemas virus-planta huésped ya habían demostrado una
reducción de la conductancia estomática del vegetal (Levy y Marco 1991, Sampol
et al. 2003, Bertamini et al. 2004, Zhou et al. 2004, Guo et al. 2005a y b). Sin
embargo, el IRGA detectó el cierre estomático más tarde que la termografía de
imagen, a partir de 19 dpi en hojas S y en la hoja AS de las plantas infectadas con
PMMoV-S, o incluso no lo detecta, como es el caso de la hoja AS de las plantas
infectadas con PMMoV-I (Fig. 3C, 3D, sección C.1). La aparente falta de
sensibilidad del IRGA podría deberse a que, durante la medida, la pinza se situó en
el extremo apical de la hoja, donde el incremento de temperatura tiene lugar más
tarde que en las zonas centrales (Fig. 1, sección C.1). Ya que este aparato no puede
revelar patrones espaciales de transpiración, la termografía de imagen resulta más
resolutiva que otras técnicas equivalentes, y podemos afirmar con Jones (1999) que
posee suficiente resolución espacial como para proporcionar información sobre la
variabilidad de la conductancia estomática a lo largo de la superficie de una hoja.
Para tratar de correlacionar el movimiento del virus en las hojas AS con el
efecto térmico, se realizaron impresiones de hojas donde la proteína de la cápside
viral fue detectada con un anticuerpo específico. Los resultados mostraron que el
virus comienza a penetrar en las hojas AS con posterioridad al aumento de
temperatura (14 y 17 dpi, en plantas infectadas con PMMoV-S e –I,
respectivamente; Fig. 7, Tabla 1, sección C.1), de acuerdo con resultados previos
de Northern Blot (Pérez-Bueno 2003).
Respecto al agente directo causante del cierre estomático durante la infección
viral, está bien documentada la acumulación de varias sustancias que afectan la
apertura estomática durante las interacciones planta-patógeno: ácido salicílico
(Malamy et al. 1990, Van Der Straeten et al. 1995, Chaerle et al.1999), óxido
234
D. Discusión
nítrico (Del Río et al. 2004, Mur et al. 2005), las hormonas vegetales ABA y
auxina (Whenham y Fraser 1981, Whenham et al. 1986, Grabov y Blatt 1998) y
hasta compuestos fenólicos (Bhatia et al. 1986, Nicholson y Hammerschmidt 1992,
Kofalvi y Nassuth 1995, Chaerle et al. 2001, Balogun y Teraoka 2004). Al ácido
clorogénico se le atribuye la propiedad de inhibir la apertura estomática (Plumbe y
Willmer 1986) y la de cerrar los estomas (Einhellig y Kuan 1971). Sin embargo, el
aumento de BGF en las hojas AS y S no correlaciona temporalmente con el
aumento de temperatura de las mismas (Tabla 1, C.1 y C.2), ni el patrón de BGF
(Fig. 2 y 3, sección C.2) con el efecto expansivo del aumento de temperatura en la
hoja AS (Fig. 1 y 2, sección C.1). Por tanto, la subida de temperatura no parece ser
consecuencia directa de la acumulación de fenoles, si bien éstos, una vez presentes
en las hojas, podrían contribuir al descenso de la conductancia estomática. La
sustancia causante del cierre estomático y/o de la acumulación de ácido
clorogénico está aún por determinar.
Más tardíos que los cambios en el patrón termal de las hojas de plantas
infectadas son los que afectan a la emisión de Chl-F, que aparecen a 9 dpi en la
hoja AS. Se detectan registrando la curva de Kautsky con el FluorCam a bajas (LL,
50 µmol m-2 s-1) y altas (HL, 200 µmol m-2 s-1) intensidades de luz actínica. Tras
aplicar estudios estadísticos a las cinéticas de inducción de Chl-F (Fig. 5, C.3), se
encontró un parámetro que evidenciaba diferencias entre las hojas AS de las
plantas infectadas con ambas cepas virales y sus respectivos controles en medidas
realizadas a HL, mucho antes que con los protocolos tradicionales (17 dpi; Pérez-
Bueno et al. 2006, Fig. 1, sección C.3, Tabla 2). Este parámetro es la emisión de
Chl-F a los 5 s de la cinética de inducción de fluorescencia (es decir, tras el
encendido de la luz actínica), con respecto a la fluorescencia basal o F0 (F5/F0). Las
imágenes del mismo evidencian un patrón asociado a venas característico de
plantas infectadas que no se vislumbra en las hojas correspondientes de las plantas
control (Fig. 6, sección C.3). El cambio en la emisión de Chl-F a los 5 s de la
cinética con respecto a los controles, ocurre en torno al pico de emisión de
235
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
236
D. Discusión
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
Fig. 29: La cadena de transporte electrónico fotosintético formada por los cuatro macrocomplejos
proteicos embebidos en la membrana tilacoidal del cloroplasto: PSII, Cyt b6/f y la ATP sintasa.
Las proteínas del PSI localizadas en los geles de rango básico, PsaD y PsaE
(8 y 2 polipéptidos, respectivamente), son altamente hidrofóbicas y localizadas en
el lado estromático de este fotosistema junto con PsaC, que aparecía en los geles
del rango ácido. Todas ellas forman el complejo encargado del anclaje de la
238
D. Discusión
239
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
240
D. Discusión
241
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
termografía, las imágenes han sido mucho más resolutivas a la hora de realizar el
seguimiento de la infección viral en la hoja AS.
Con anterioridad a 17 dpi, no hay cambios destacables en las imágenes de los
diferentes parámetros analizados: F0, FM, FV/FM, Rfd, ΦPSII, ΦII, NPQ, qCN, qTQ.
A 17 dpi, el parámetro que maximizó las diferencias entre plantas control e
infectadas y mostró un complejo patrón espacio-temporal en estas últimas fue el
NPQ, en especial NPQ20 (Fig. 1, sección C.3), de acuerdo con los resultados
previos de nuestro grupo durante la infección por PMMoV-I empleando otro
sistema de Chl-FI diferente (Pérez-Bueno et al. 2006). En el presente trabajo, los
experimentos con el FluorCam mostraron también un patrón heterogéneo de NPQ
en las hojas AS de plantas infectadas con PMMoV-S. En las etapas finales de la
infección, las hojas AS de las plantas infectadas muestran los valores más bajos de
NPQ, demostrando que los procesos de senescencia son predominantes y que los
procesos no fotoquímicos están totalmente regulados a la baja. Diferentes trabajos
señalan al NPQ como uno de los mejores indicadores de estrés en plantas
infectadas, revelando una compleja heterogeneidad metabólica en la zona de
infección (Chou et al. 2000, Repka 2002, Berger et al. 2004, Swarbrick et al.
2006). Autores como Osmond et al. (1998) y Lohaus et al. (2000) encuentran que
NPQ reflejaba el desarrollo estado de de los síntomas provocados por un
geminivirus restringido a floema en plantas de Abutilon striatum Dicks. La ventaja
de nuestro trabajo con respecto a los anteriormente citados, es que amplía las
posibilidades de uso del NPQ al detectar la infección en zonas asintomáticas de la
planta (Pérez-Bueno et al. 2006), situación similar a la encontrada en nuestros
trabajos de colaboración con el grupo del Dr. C. Ramos en plantas de judía
infectadas con distintas cepas de Pseudomonas (comunicación personal).
El incremento del NPQ durante la infección viral es una respuesta defensiva
de la planta infectada que podría retrasar el proceso de fotoinhibición hasta las
fases finales del proceso infectivo (Wright et al. 1995a, b, Balachandran et al.
1997). De hecho, la fotoinhibición sólo es evidente en las plantas infectadas con
242
D. Discusión
243
Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
244
D. Discusión
valores próximos a los de las plantas control (Tabla 2). En este sentido, registramos
una disminución de la temperatura de las hojas S (Fig. 3B y 4, sección C.1) y una
tendencia al alza de la conductancia estomática (Fig. 3D, sección C.1). Muchos de
los parámetros calculados con el equipo de MCFI tienen una clara tendencia a
alcanzar los valores de las plantas control, tanto en hoja S como AS (Fig. 1, 4,
Tabla 1, 2, sección C.2), lo que sorprende, ya que la fase de recuperación sólo
afecta a las hojas jóvenes de la planta huésped. El descenso de BGF en la hoja S se
corresponde con una caída en los niveles de ácido clorogénico, como cabía esperar
(Fig. 1, 6, sección C.2). Los niveles de Chl se recuperan completamente a 28 dpi
(Fig. 6, sección C.1), así como los de la mayor parte de transcritos de proteínas
cloroplastídicas analizadas (lchb1, rca, rbcS, psaK, gox, rbcL, psbA, psbO, psbP, y
psbQ; Pérez Bueno et al. 2003).
La fase de recuperación es un fenómeno del que se conoce muy poco. Es
posible que la recuperación de la enfermedad sea consecuencia del silenciamiento
de RNA mediado por virus, por el cual se produce de forma específica la
destrucción enzimática del RNA viral (Hiriart et al. 2002, Mlotshwa et al. 2002), y
por la activación coordinada de mecanismos de defensa generales de la planta
durante los procesos de floración y/o senescencia (Somssich y Hahlbrook 1998,
Malek y Dietrich 1999, Ji y Ding 2001, Obregón et al. 2001). Sin embargo, durante
la fase de recuperación aún es posible detectar el virus mediante Northern Blot
(Pérez-Bueno 2003) o impresiones de hojas (Fig. 7B, sección C.1), y por el
momento se desconoce si estos focos de acumulación están formados por células
que fueron invadidas antes del proceso de recuperación o simplemente éste no es
completamente eficaz.
Llegados a este punto, resumiremos el trabajo presentado, considerando
también los trabajos previos de nuestro laboratorio y aquellos realizados en
colaboración con el grupo de la Dra. I García Luque (CIB. CSIC. Madrid) en el
sistema huésped-patógeno Nicotiana benthamiana-PMMoV. En esta Tesis se han
caracterizado, mediante varios sistemas de captación de imágenes, diversas
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D. Discusión
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Técnicas de imagen y proteómicas aplicadas al seguimiento de la interacción virus-planta huésped
podrían ser buenos indicadores del movimiento viral. El NPQ se postularía como
un mecanismo de defensa del cloroplasto para evitar la fotoinhibición. El ΦPSII se
ha revelado como un parámetro diferenciador de la infección por PMMoV-S,
puesto que muestra un patrón característico que no presentan las hojas AS de las
plantas control o infectadas con PMMoV-I. Las alteraciones que tienen lugar en la
cinética de emisión de Chl-F de hojas de plantas infectadas aparecen en un punto
cercano a FP, lo que es indicativo de perturbaciones en el ciclo de Benson-Calvin.
Esta hipótesis parece confirmarse con nuestros resultados previos y los expuestos
en esta Tesis, que muestran descensos en los niveles de transcritos y de proteínas
implicadas en esta ruta metabólica.
Considerando los resultados de todas las técnicas utilizadas en esta Tesis
Doctoral, es evidente una vez más la enorme complejidad de la interacción
compatible planta-virus, que resulta dependiente de otros factores ambientales y
del estado de desarrollo del vegetal en el momento de la infección. La respuesta de
la planta a la infección viral, que es un factor de estrés biótico, tiene patrones
comunes a la que presenta frente a factores adversos de origen abiótico y, en
determinados momentos, se asemeja a los procesos de senescencia.
La activación del metabolismo secundario, el equilibrio entre procesos
fotoinhibitorios y fotoprotectores así como entre fotoquímica y fase asimilatoria de
la fotosíntesis, la inhibición de procesos fotoquímicos y activación de rutas
electrónicas alternativas, el descenso en los niveles de proteínas claves en las dos
fases del proceso fotosintético y la inhibición progresiva de genes nucleares y
cloroplastídicos que las codifican, es clave para la respuesta de la planta huésped al
patógeno.
Los resultados de este trabajo abren nuevas líneas de investigación que se
abordarán próximamente. Por un lado, el estudio del proteoma cloroplastídico de
N. benthamiana utilizando técnicas proteómicas sin geles permitirá aumentar el
número de proteínas identificadas y caracterizar las modificaciones post-
traduccionales que pueden sufrir, así como sus isoformas. El empleo de la técnica
248
D. Discusión
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250
E. Conclusiones / Concluding remarks
E. CONCLUSIONES
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252
E. Conclusiones / Concluding remarks
E. CONCLUDING REMARKS
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302
G. Anexos
Control
PMMoV-I
PMMoV-S
dpi 7 14 17 19 21 24 28
0 1
303