Research Article Phaseolus Vulgaris L.) : Genetic Control, Differential
Research Article Phaseolus Vulgaris L.) : Genetic Control, Differential
Research Article Phaseolus Vulgaris L.) : Genetic Control, Differential
Research Article
Superoxide-Dismutase Deficient Mutants in Common
Beans (Phaseolus vulgaris L.): Genetic Control, Differential
Expressions of Isozymes, and Sensitivity to Arsenic
Copyright © 2013 D. Talukdar and T. Talukdar. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Two common bean (Phaseolus vulgaris L.) mutants, sodPv 1 and sodPv 2, exhibiting foliar superoxide dismutase (SOD) activity of
only 25% and 40% of their mother control (MC) cv. VL 63 were isolated in EMS-mutagenized (0.15%, 8 h) M2 progeny. Native-
PAGE analysis revealed occurrence of Mn SOD, Fe SOD, Cu/Zn SOD I and Cu/Zn SOD II isozymes in MC, while Fe SOD, and
Mn SOD were not formed in sodPv 1 and sodPv 2 leaves, respectively. In-gel activity of individual isozymes differed significantly
among the parents. SOD deficiency is inherited as recessive mutations, controlled by two different nonallelic loci. Gene expressions
using qRT PCR confirmed higher expressions of Cu/Zn SOD transcripts in both mutants and the absence of Fe SOD in sodPv 1
and Mn SOD in sodPv 2. In 50 𝜇M arsenic, Cu/Zn SODs genes were further upregulated but other isoforms downregulated in the
two mutants, maintaining SOD activity in its control level. In an F2 double mutants of sodPv 1 × sodPv 2, no Fe SOD, and Mn SOD
expressions were detectable, while both Cu/Zn SODs are down-regulated and arsenic-induced leaf necrosis appeared. In contrast
to both mutants, ROS-imaging study revealed overaccumulation of both superoxides and H2 O2 in leaves of double mutant.
1. Introduction quite stable, due in large part to copper and zinc binding and
oxidation of an intramolecular disulfide [3]. Copper serves as
Superoxide radicals are ubiquitously generated in many the catalyst for superoxide disproportionation, whereas zinc
biological oxidations within all compartments of the cell. and the disulfide participate in proper protein folding [3].
The toxicity of superoxide radicals has been attributed to Arsenic (As) is a ubiquitous toxic metalloid without
their interaction with other cellular constituents, in particular known biological functions in higher plants [4, 5]. In recent
with hydrogen peroxide [1]. Superoxide dismutases (SODs; times, the impact of irrigation with high As contaminated
EC 1.15.1.1) are a family of metalloenzymes that catalyze the groundwater on soil and crops has drawn huge attention due
disproportionation of superoxide radicals into H2 O2 and O2 to transfer of As to the food chain via the groundwater-soil-
and constitute the first line of defense against the toxicity of plant system [6–8]. As can induce oxidative stress through
superoxide radicals [1, 2]. Based on the metal cofactor used generation of reactive oxygen species (ROS) [4, 5], and
by the enzyme, SODs are classified into three groups as iron moderate accumulation of ROS significantly affects nuclear
SOD (Fe SOD), manganese SOD (Mn SOD), and copper- gene expression [9]. ROS sensors could be activated to
zinc SODs (Cu/Zn SOD) [3]. SOD isozymes are located in induce signaling cascades that ultimately impinge on gene
different cellular compartments [3]. Fe SODs are located in expression. Alternatively, components of signaling pathways
the chloroplast, Mn SODs in the mitochondrion and the could be directly oxidized by ROS. Finally, ROS might change
peroxisome, and Cu/Zn SODs in the chloroplast, the cytosol, gene expression by targeting and modifying the activity of
and possibly the extracellular space. Cu/Zn SOD is normally transcription factors [9].
2 BioMed Research International
Table 1: Oligonucleotides used for qRT-PCR analysis of target gene expressions of superoxide dismutase (SOD) isozymes in Phaseolus vulgaris
L. mother cv. VL 63 and two of its SOD-deficient mutant lines. F1—forward primers and R1—reverse primers.
Jr. and Fridovich [28]. In this method, 1 ml of solution with DNaseI (Chromous Biotech, Bangalore, India) at 37∘ C
containing 50 mM K-phosphate buffer (pH 7.8), 9.9 mM L- for 30 min. The quality of total RNA samples was determined
methionine, 57 𝜇M NBT, and 0.025% triton-X-100 was added spectrophotometrically (Systonic, Kolkata, India) and by 1%
into small glass tubes, followed by 20 𝜇L of enzyme extract. agarose gel electrophoresis with 500 bp DNA ladder. First-
Reaction was started by adding 10 𝜇L of riboflavin solution strand cDNA was synthesized from DNase-treated RNA
(0.044 mg mL−1 ), and the absorbance of solution was mea- with oligo-dT primer and with MmuLV reverse transcriptase
sured at 560 nm. SOD activity was expressed as U (unit) mg−1 enzyme kit (Chromous Biotech, Bangalore, India) following
protein. One unit of SOD was equal to that amount which manufacturer’s instructions. Quantitative RT PCR of first
causes a 50% decrease of SOD-inhibited NBT reduction. SOD stand cDNA was run on ABI Step-One Real Time PCR
isozymes were individualized by native PAGE on 10% acry- machine. Amplification was done in a total reaction volume
lamide gels and were localized by a photochemical method of 50 𝜇L, containing template (first strand cDNA) 2.0 𝜇L,
[28]. Activity-staining gels were incubated for 30 min in forward primer 2.0 𝜇L, reverse primer 2.0 𝜇L, 2 X PCR SYBR
50 mM K-phosphate buffer at pH 7.5 containing 2 mM KCN green ready mixture (Fast Q-PCR Master Mix, Chromous
or 5 mM H2 O2 or 5 mM NaN3 . Cu/Zn-SODs are inhibited by Biotech, India, Cat no. QCR 05/QCR 06), 25.0 𝜇L, and
KCN and H2 O2 ; Fe SODs are inactivated by H2 O2 and NaN3 DEPC water 19.0 uL. The SOD primers for four isozymes
but resistant to KCN and Mn SODs are resistant to all three (Cu/Zn-SODs I and II, Fe SOD, and Mn SOD) of PvSOD
inhibitors. Quantification of the bands was performed using a (Table 1) were constructed by Primer Express software with
Gel Doc system (Bio-Rad Laboratories, Chennai, TN, India) the search of available GenBank/legume databases [30]. The
coupled with a high sensitive CCD camera. Band intensity qRT-PCR cycling stages consisted of initial denaturation at
was expressed as relative transmittance units. Based on the 94∘ C (3 min), followed by 35 cycles of 94∘ C (5 s), 62∘ C (10 s),
observed variations, isozyme bands were assigned to putative 72∘ C (10 s), and a final extension stage at 72∘ C (2 min).
loci following the earlier adopted principles [29]. Only clearly A melting curve analysis was performed after every PCR
visible bands were scored in the present study. reaction to confirm the accuracy of each amplified product.
Samples for qRT PCR were run in five biological replicates
2.4. Genetic Control and Allelism Test of SOD-Deficient Muta- and two technical replicates. DEPC water for the replacement
tions. Inheritance of mutations controlling SOD deficiency of template was used as negative control. RT-PCR reaction
was traced in segregating populations of F2 generation mixtures were loaded onto 2% agarose gels in TAE buffer. A
derived from MC × mutants. For allelism test, intercrosses 100 bp DNA ladder was run on every gel. The mRNA levels
were made between mutants. Chi-square test was employed were normalized against a common bean ubiquitin as the
to test the goodness of fit between observed and expected housekeeping gene and the relative (to control) expression of
values for all crosses. Zymograms of SOD isoforms of leaf F1 target genes was calculated as 2−ΔΔCt ; Ct is cycle threshold,
and F2 plants were analysed and compared with parents. following Livak and Schmittgen [31].
2.5. RNA Isolation and Relative Gene Expression through 2.6. Detection and Imaging of Superoxide and H2 O2 Radi-
Quantitative RT PCR. Gene expression levels of different cals by Confocal Laser Scanning Microscopy. Detection and
SOD isozymes of MC, MuC, F2 -segregating progenies, and imaging of superoxide radicals in leaf sections was carried
As-treated plants of Phaseolus vulgaris (PvSOD) were ana- out using the fluorescence probe dihydroethidium (DHE),
lyzed by quantitative reverse transcription polymerase chain following the earlier method [32]. Bean leaf segments of
reaction (qRT-PCR) technique. Total RNA was isolated from approximately 30 mm2 were incubated for 1 h at 25∘ C, in
the young leaves of 14d-old Phaseolus leaves (control and darkness, with 10 𝜇M DHE prepared in 5 mM Tris-HCl buffer
treated in separate sets of experiment) using the RNA iso- at pH7.4, and samples were washed twice with the same buffer
lation kit (Chromous Biotech, Bangalore, India) and treated for 12 min each. After washing, leaf sections were embedded
4 BioMed Research International
70 A 1 2 3 4 5 6 7 8 9 10
a
60
Relative transmittance (%)
b
50 B b
Mn SOD
A
40 A a
A c a Fe SOD
30 c
b Cu/Zn SOD I
b Cu/Zn SOD II
20 d C
c
c D B
10 Figure 3: Inhibition (KCN, H2 O2 , and NaN3 ) study and visualiza-
tion of SOD isoforms in native PAGE of leaf extracts of Phaseolus
0 vulgaris cv. VL 63 (mother variety) and two SOD-deficient mutant
MC SodPv 1 SodPv 2 Treated Treated Treated
mother sodPv 1 sodPv 2 lines, sodPv 1, and sodPv 2; lane 1: mother control (no inhibitor),
lane 2: mother variety (KCN), lane 3: sodPv 1 (KCN), lane 4: sodPv 2
Mn SOD Cu/Zn SOD I (KCN), lane 5: mother variety (H2 O2 ), lane 6: sodPv 1 (H2 O2 ), lane
Fe SOD Cu/Zn SOD II 7: sodPv 2 (H2 O2 ), lane 8: mother variety (NaN3 ), lane 9: sodPv 1,
and lane 10: sodPv 2. Note that complete absence of band at lane 7;
Figure 2: Activity of individual isozymes of superoxide dismutase Mn SOD is absent in sodPv 2 mutant, and Fe SOD and Cu/Zn SODs
(SOD) in mother control (MC), two mutants, sodPv 1 and sodPv are inhibited by H2 O2 treatment.
2, and 50 𝜇M arsenic- (As-) treated mother variety VL-63 and two
mutant lines in Phaseolus vulgaris L. Band intensities were expressed
as relative transmittance (T) units. The results are means ± SE of 1 2 3 4 5 6 7 8 9 10
at least three replicates, and same letters (four different types to
represent four isozymes) above error bars denote nonsignificant
(𝑃 > 0.05) differences among means by Duncan’s multiple range
test. Mn SOD
Fe SOD
Cu/Zn SOD I
Cu/Zn SOD II
1 2 3 4 5 6 7
Mn SOD
Fe SOD
Cu/Zn SOD I
Cu/Zn SOD II
Figure 5: (A) Inhibition study of SOD isozymes produced by double mutant obtained from crosses between sodPv 1 and sodPv 2 mutants of
Phaseolus vulgaris L.; lane 1: mother control (no inhibitor), lanes 2 & 3: double mutants (no inhibitor), lane 4: double mutant (KCN), lane 5:
double mutant (H2 O2 ), and lanes 6 & 7: double mutants (NaN3 ). Note that both Cu/Zn SODs I and II were inhibited by KCN and H2 O2 in
lanes 4 and 5, while visualized as, and normal faint bands by NaN3 at lanes 6 & 7, (B) appearance of necrotic spots ( → ) on pod wall of double
mutant.
zymogram pattern of respective parents and was controlled Dramatic changes in gene expression pattern of SOD
by interactions between two different nonallelic loci. The isozymes were observed in leaves of mother variety exposed
recessive nature of null mutations in SOD isozyme loci was to 50 𝜇M As. In comparison with unstressed condition (MC),
also reported in sunflower, maize, soybean, and other plants relative mRNA levels of Cu/Zn SODs, Mn SOD, and Fe SOD
[37–39]. Obviously, in the presence of dominant alleles of were increased by 2-fold, 1.5-fold, and 3-fold, respectively,
both loci (SOD Pv1-SODPV2-), SOD activity and zymograms in treated mother (Figures 6 and 7). The Results suggested
of isozymes were normal in the present MC plants. Absence upregulation of Cu/Zn SOD, Mn SOD, and Fe SOD genes in
of dominant alleles in the loci (either in the form of sodPv response to As treatment of mother variety. Compared to MC,
1-SODPv 2- or SODPv 1-sodPv 2-) led to origin of two total SOD-specific activity was markedly increased in As-
SOD-deficient mutants, differing distinctly in total SOD treated mother variety. Certainly, enhanced activity of Cu/Zn
activity level and isozyme banding pattern. In the last class SODs and Fe SOD isoforms mainly contributed to this rise
of progeny plants, double mutant recessive to both loci which can be attributed to As-induced transcriptional upreg-
(sodPv 1 and sodPv 2) resulted in extremely deficient SOD ulation of both Cu/Zn SOD and Fe SOD genes. However, the
level accompanied with phenotypic anomalies. declining activity of Mn SOD isoform, as manifested in native
gel, was not in harmony with enhanced level of its mRNA
transcript, indicating possible regulation of this isozyme at
3.3. Gene Expression of SOD Isozymes under Un-Stressed posttranscriptional level.
and As-Treated Conditions. The expression patterns of the In As-treated sodPv 1 and sodPv 2 mutants, mRNA tran-
Cu/ZnSODs I and II, Mn SOD, and Fe SOD genes in leaves of scripts of Cu/Zn SODs as amplified in qRT-PCR (Figure 6)
Phaseolus seedlings and two mutant lines under unstressed, were increased by about 2-2.2-fold over their corresponding
and As-treated conditions were investigated by qRT-PCR levels in untreated control (MuC) and >3.0-fold in relation
(Figure 2). Under unstressed condition, the mRNA tran- to MC (Figure 7). By contrast, reduced level of mRNA
scripts of Mn SOD, Fe SOD, Cu/Zn SODs I, and Cu/znSOD transcripts was observed for Mn SOD in sodPv 1 and for Fe
II isoforms were amplified by PCR in MC (Table 1, Figure 6). SOD in sodPv 2 mutant exposed to As treatment (Figures
The mRNA transcripts of Cu/Zn SODs were also detectable 6 and 7). The results indicated As-induced upregulation of
in both sodPv 1 and sodPv 2 mutants, but there were no Cu/Zn SODs genes in both mutants but concomitant down-
detectable transcripts of Fe SOD in sodPv 1 mutant and no Mn regulation of Mn SOD in sodPv 1 and Fe SOD in sodPv 2
SOD in case of sodPv 2 mutants (Figure 6). Besides Cu/Zn mutant. Overexpression of mRNA genes controlling Cu/Zn
SODs I and II, genes encoding Mn SOD transcripts were SODs led to higher activity of this isozyme in zymogram,
expressed in sodPv 1 while those of Fe SOD were detected in which was manifested as increasing band intensity and
sodPv 2 mutant leaves (Figure 6). Relative expression pattern thickness in native PAGE. Likewise, declining activity of Mn
(compared to respective gene of MC) indicated that genes SOD in sodPv 1 and Fe SOD in sodPv 2 mutant exposed to
encoding Mn SOD as well as Cu/Zn SODs I and II transcripts 50 𝜇M As treatment was presumably due to down-regulation
in sodPv 1 exhibited significantly (𝑃 < 0.05) higher level of their respective genes governing mRNA transcripts. The
than those of MC values (Figure 7). Higher transcript levels of results indicated differential regulation of SOD isozymes
Cu/Zn SODs and Fe SOD compared to MC were also deduced in both mutants, subjected to As treatment, presumably,
in sodPv 2 mutant leaves (Figure 7). Between the two mutants, balancing SOD activity in MuC levels (25% in sodPv 1
gene expression of Cu/Zn SODs transcripts was markedly and 45% in sodPv 2 of MC). Differential regulations of
higher in sodPv 2 mutant (Figures 6 and 7). SOD isoforms were also reported in cadmium-treated pea,
BioMed Research International 7
1 2 3 4 5 6 4.5
4 ∗
DM-treated
MC
SodPv 1
SodPv 2
Treated
Treated
sodPv 1
Treated
sodPv 2
DM-untreated
mother
SodPv 1
transgenic alfalfa, salinity-induced lentils, and in many other 3.4. Gene Expression of F2 -Segregating Progeny Plants under
plants experiencing stresses [32, 40–42]. Since Mn SOD Unstressed and As-Treated Conditions. Compared to their
and Fe SOD are proved to be mitochondrial and plastidic respective parent, there were no significant alterations in
isoforms, respectively, their down-regulation in the present mRNA levels and morphology of F2 -segregating progeny
mutants of common beans could have adverse effect on plants either under untreated or As-treated conditions (data
plant growth, as observed in Nicotiana seedlings [43] and not shown) except in the double mutant. Compared to sodPv
in transgenic Arabidopsis [19]. Interestingly, no As-induced 1 and sodPv 2 single gene mutant phenotypes, relative expres-
oxidative damage and consequent retardation of plant growth sion of mRNA genes encoding Cu/Zn SODs I and II was
was visible in any part of the present mutants, despite down- significantly low in the double mutant plants under untreated
regulation of MnSOD in sodPv 1 and Fe SOD in sodPv condition (Figures 7 and 8(a)). Furthermore, no mRNA
2 mutant under As exposure. Obviously, upregulations of transcripts of Mn SOD and Fe SOD were detected. The
Cu/Zn SOD I and II mRNA gene counterbalanced down- results indicated origin of double knockout mutant for SOD
regulation of other SOD isoforms in the mutant plants, and deficiency in segregating mutant progeny of Phaseolus. Upon
presumably, are holding the key in response of both mutants exposure of the mutant to 50 𝜇M As for 7 days in hydroponics,
to high As exposure. Compared to chloroplast, the amount Cu/Zn SODs transcript level is reduced further (Figures 7
of ROS produced by mitochondria is rather minor [44] and and 8), suggesting As-induced down-regulation of the gene.
chloroplasts bear a particular risk of oxygen toxicity because Declining level of mRNA transcripts and complete absence
8 BioMed Research International
Figure 9: Representative imaging of superoxide radicals and H2 O2 productions in Phaseolus vulgaris L. leaves by CLSM. Images are developed
from several optical sections collected by confocal microscopy showing the autofluorescence (blue; excitation at 633 nm, emission at 680 nm)
and fluorescence due to DHE and DCF-DA. A–E, (I) superoxide-dependent DHE fluorescence (red) in leaf cross sections from mother control
(A) and arsenic (As)-treated (B–K) leaves; (B) As-treated sodPv 1 mutant, (C) As-treated mother plants, (D) for the negative control, leaves
were incubated with 1 mm TMP, a superoxide scavenger, (E) magnified view of leaf mesophyll with red fluorescence and autofluorescence, (I)
red fluorescence in leaf section of double mutant, F–H, J, and (K) H2 O2 -dependent DCF-DA fluorescence (green) in leaf cross sections; (F)
MC leaves, (G) As-treated mother plants, (H) As-treated sodPv 2 mutant, (J) green fluorescence in leaf tissues along with epidermal hair of
double mutant, (K) as a negative control, and leaves were incubated with 1 mm ascorbate (ASC), which acts as a H2 O2 scavenger. The results
are representative of at least three independent experiments. Ep: Epidermis; Hr: hair; Ms: mesophyll cells; Scl: sclerenchyma; Xyl: xylem
vessels. Bars = 50 𝜇m.
10 BioMed Research International
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