Walking the C₄pathway: past, present, and future

Journal of Experimental Botany, Vol. 68, No. 2 pp. e1–e10, 2017 doi:10.1093/jxb/erx006 DARWIN REVIEW Walking the C4 pathway: past, present, and future Robert T. Furbank1,2,* 1 ARC Centre of Excellence for Translational Photosynthesis, The Australian National University, Research School of Biology, 134 Linnaeus Way, Acton ACT 2601, Australia 2 CSIRO Agriculture, Clunies Ross St, Acton ACT 2601, Australia Received 7 March 2016; Accepted 4 April 2016 Editor: Donald Ort, University of Illinois This article is was originally published in JXB volume 67 issue 14, pages 4057-4066. Please cite as: Robert T. Furbank; Walking the C 4 pathway: past, present, and future. J Exp Bot 2016; 67 (14): 4057-4066. doi: 10.1093/jxb/erw161 Abstract The year 2016 marks 50 years since the publication of the seminal paper by Hatch and Slack describing the biochemical pathway we now know as C4 photosynthesis. This review provides insight into the initial discovery of this pathway, the clues which led Hatch and Slack and others to these definitive experiments, some of the intrigue which surrounds the international activities which led up to the discovery, and personal insights into the future of this research field. While the biochemical understanding of the basic pathways came quickly, the role of the bundle sheath intermediate CO2 pool was not understood for a number of years, and the nature of C4 as a biochemical CO2 pump then linked the unique Kranz anatomy of C4 plants to their biochemical specialization. Decades of “grind and find biochemistry” and leaf physiology fleshed out the regulation of the pathway and the differences in physiological response to the environment between C3 and C4 plants. The more recent advent of plant transformation then high-throughput RNA and DNA sequencing and synthetic biology has allowed us both to carry out biochemical experiments and test hypotheses in planta and to better understand the evolution-driven molecular and genetic changes which occurred in the genomes of plants in the transition from C3 to C4. Now we are using this knowledge in attempts to engineer C4 rice and improve the C4 engine itself for enhanced food security and to provide novel biofuel feedstocks. The next 50 years of photosynthesis will no doubt be challenging, stimulating, and a drawcard for the best young minds in plant biology. Key words: Bundle sheath, C4 decarboxylation, C4 photosynthesis, Kranz anatomy, PEP carboxylase, Rubisco. Clues to the existence of C4 plants Many historical articles have been written about the discovery of C4 photosynthesis, including a number by Hal Hatch and Roger Slack themselves (Hatch, 1992a, 1997; Hatch and Slack, 1998). Table 1 attempts to present a timeline on which are mapped important observations made before elucidation of the biochemical pathway in 1966, and key discoveries thereafter which have shaped our understanding of the mechanism and future work in C4 photosynthesis research. As pointed out many times in the literature (e.g. Hatch, 1987; Sage, 2012), the evolution of the C4 pathway required the combination of complex anatomical and biochemical specialization. The C4 photosynthetic mechanism requires a spatial separation of the biochemical ‘CO2 pump’ from the site of Rubisco and, while this spatial separation can occur within a single cell (Voznesenskay et al., 2001), it has most commonly evolved as ‘Kranz anatomy‘. In Kranz C4 species, phophoenolpyruvate © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com Downloaded from https://academic.oup.com/jxb/article-abstract/68/2/e1/2932224 by guest on 03 June 2020 * Correspondence: Robert.furbank@anu.edu.au e2 | Furbank Table 1. A timeline of key observations leading to the discovery of the major elements of the C4 pathway and mechanism Date of observation Nature of observation Reference 1904 (original observation 1884) 1927 1944 1954 Kranz anatomy High water use efficiency Biochemical specialization of bundle sheath and mesophyll Preliminary labelling evidence for unusual labelling patterns in sugarcane Dimorphic chloroplasts Demonstration of radiolabelling into malate and aspartate in maize leaves Low CO2 compensation point and high rates of photosynthesis of C4 leaves First published report of labelling patterns in sugarcane by Hawaiian group Publication of pulse–chase radiolabelling of sugarcane leaves and proposal of the C4 pathway Extraction of Rubisco from C4 leaves and cellular location of PEPC and Rubisco Haberlandt (1904) Shantz and Piemeisel (1927) Rhoades and Carvalho (1944) Kortchak et al. (unreferenced; Hawaiian Sugar Planters Association annual report) Hodge et al. (1955) Karpilov (1960) 1955 1960 1962/63 1965 1969 / 1970 1970/1971 1971–1976 1976 Evidence for an intermediate CO2 pool in the bundle sheath and the co-operative function of mesophyll and bundle sheath Elucidation of the three C4 acid decarboxylation pathways Comprehensive discussion of diffusion of metabolites between mesophyll and bundle sheath cells Fig. 1. A simplified biochemical scheme of NADP-ME-type C4 photosynthesis as known by the early 1970s. Enzymes are numbered as follows: 1, carbonic anhydrase; 2, PEP carboxylase; 3, malate dehydrogenase; 4, NADP-malic enzyme; 5, pyruvate orthophosphate dikinase. Pathways and cell types are cross-referenced to a laser confocal fluorescence micrograph of a transverse section of maize leaf. False colouring indicates differences in the emission spectrum at 685 nm and 745 nm due to the dimorphic chloroplasts where little PSII is present in the bundle sheath (purple, while C3 levels of Chl a and PSII are present in the mesophyll (pink). Thylakoids in the mesophyll show strong granal stacking, while those in the bundle sheath are agranal, typical of many NADP-ME types (indicated in the scheme). For a full explanation, see Furbank et al. (2009). (PEP) carboxylase (PEPC) and associated components of the C4 pump are located in the mesophyll cells, adjacent to the atmosphere in the leaf intercellular spaces, while Rubisco and the photosynthetic carbon reduction cycle, plus the enzymes required Kortschak et al. (1965) Hatch and Slack (1966) Bjorkman and Gauhl (1969); Slack et al. (1969); Edwards et al. (1970); Berry et al. (1970) Berry et al. (1970); Hatch (1971) Edwards et al. (1971); Hatch and Kagawa (1976) Hatch and Osmond (1976) to decarboxylate C4 acids, are located in the bundle sheath cells (see Fig. 1). While the significance at the time was unknown, the existence of the Kranz anatomical specialization in grasses was reported >60 years before the biochemical pathway was elucidated (Haberlandt, 1904). Observations concerning bundle sheath cell-specific starch accumulation (Rhodes and Carvalho, 1944) and the existence of structurally dimorphic chloroplasts in the bundle sheath and mesophyll cells (Hodge et al., 1955) further suggested that there was biochemical specialization accompanying these interesting anatomical features. During the historical period where observations of specialized anatomy were building for C4 grasses, physiological measurements indicating that these plants were ‘special’ were also being reported. As early as the 1920s it was reported that grasses we now know to be C4 exhibited whole-plant water use efficiencies (as g dry biomass accumulated per g water lost) double that obtained for C3 cereals (Shantz and Piemeisel, 1927). Forty years later, leaf-level characterization of photosynthetic gas exchange revealed that these plants had very low CO2 compensation points (Moss, 1962), high rates of photosynthesis in air at high light and high temperature (Hesketh, 1963; El Sharkaway and Hesketh, 1964), and very high growth rates (Loomis and Williams, 1963). These observations all set the scene for our understanding of the significance of the C4 pathway in affording C4 plants unique advantages over their C3 counterparts in appropriate environmental conditions. Elucidation of the C4 biochemical pathway While important discoveries are often firmly based in serendipity (Hatch wrote a short retrospective of his career and role in the C4 discovery entitled ‘I can‘t believe my luck’; Hatch, 1992b), the history of the discovery of the C4 pathway Downloaded from https://academic.oup.com/jxb/article-abstract/68/2/e1/2932224 by guest on 03 June 2020 1966 Moss (1962); Hesketh (1963) C4 photosynthesis review | e3 labelled first in illuminated sugarcane leaves, and that 3-phosphoglycerate (3-PGA) and sugars followed, pre-dated Hatch and Slack (1966) by a number of years (see Hatch and Slack, 1998). However, the Australian and Hawaiian groups were unaware of the Russian work until the late 1960s, and interpretation of the labelling was again confounded by lack of certainty in identifying the labelled compounds and possible artefacts in quenching the leaves after labelling (see Hatch and Slack, 1998). It appears that another young Australian plant scientist, Barry Osmond, also had similar evidence for labelling of malate as the first product of photosynthesis in the genus Atriplex. Apparently senior colleagues in the USA cast doubt on the results, due to the presence of C3 species in the genus, uncertainty about the appropriateness of the nonsteady-state labelling method used, and of course concern as to whether it was ‘politic’ to air somewhat controversial views on the mechanism of photosynthesis. However, Osmond eventually gained the confidence to publish these results in 1967 (Osmond, 1967). Apparently, over a beer at an Australian Biochemical Society conference in Hobart in 1965, Hatch and Slack decided to explore Korstchak’s results in earnest, and designed the labelling experiments which led to their definitive paper. The defining feature of the approach of Hatch and Slack, as mentioned above, was the ‘pulse–chase’ technique. By ensuring the leaves of the plant to be labelled were performing steady-state photosynthesis, then applying a short pulse of radiolabelled CO2, flushing with air, and rapidly quenching leaves over a timecourse of the chase, Hatch and Slack were able to determine that carbon 4 of malate and aspartate was labelled initially and that this radiolabel was passed to carbon 1 of 3-PGA. This pulse–chase approach finally ruled out the possibility that radiolabel was finding its way into photosynthetic products through isotopic exchange or anapleurotic pathways. The transmission of carbon from C4 acids to 3-PGA is now the yardstick by which plants are identified definitively ‘C4’ and not just incorporating label into malate via PEPC for other metabolic or osmotic processes (as is the case for many reproductive structures sometimes mooted to carry out C4). In the following 2 years, similar labelling patterns to that seen in sugarcane were reported for a range of C4 grasses and dicots (Hatch et al., 1967; Osmond, 1967; Johnson and Hatch, 1968). It is of note that Hatch and Slack did not propose an intermediate CO2 pool in their biochemical model and wrongly assumed that a trans-carboxylation reaction transferred fixed carbon from malate to 3-PGA. This view was supported by observations that ‘fraction I protein’, now known as Rubisco, was apparently not present at high activities in C4 leaves (e.g. Slack and Hatch, 1967). We now know that Rubisco is difficult to extract from the lignified and suberized bundle sheath cells of C4 leaves without extensive grinding and that Rubisco is the major carboxylating enzyme for the photosynthetic carbon reduction cycle of C4 plants (see below). Delineation of the enzymes involved in C4 photosynthesis proceeded over the next 5 years, with pyruvate orthophosphate dikinase (PPdK), the mesophyll chloroplast-localized enzyme regenerating PEP from pyruvate Fig. 1), proving to be one of the most recalcitrant to identify and purify (reviewed in Hatch and Slack, 1998). Downloaded from https://academic.oup.com/jxb/article-abstract/68/2/e1/2932224 by guest on 03 June 2020 reads almost like a Television script. The historical transcript is littered with reports of data pre-dating the published discovery by a decade which went unpublished when researchers were told their work was artefactual by high-profile scientists; papers published in foreign language journals which went unnoticed; pivotal experiments penned on the back of a beer mat at a hotel; all making entrancing stories for Hatch’s young cohort of researchers training in his laboratory over the years. Hopefully the timeline of Table 1 and the text below convey the flurry of research activity in this field in the 1960s and 1970s, and the sense of amazement I felt on hearing the story. The technique which laid the foundations for the C4 biochemical mechanism was of course radioisotope labelling. Calvin and colleagues used this technique in the 1950s, in combination with newly developed analytical techniques such as paper chromatography, to elucidate the C3 photosynthetic pathway, employing 14C labelling mostly in algae (the history of this is reviewed comprehensively in Bassham, 2003). Initially limited by the availability of radiolabel, by the 1960s techniques for labelling higher plant leaves and analysing the 14 C-labelled products were better developed, including, but less commonly, the use of ‘pulse–chase’ labelling. Here a short pulse of 14CO2 was applied and the fate of that carbon determined after ‘chasing’ by a period of illumination of the labelled leaf in unlabelled air. It was this ‘pulse–chase’ technique which resulted in the comprehensive elucidation of the C4 pathway in 1966 (Hatch and Slack, 1966). While the unique morphology and physiological attributes of C4 plants had not initially been linked to any biochemical attributes, as early as the 1950s there was evidence that some grasses, such as sugarcane, exhibited ‘odd’ labelling patterns when subjected to 14CO2. Snippets of data appeared in annual reports of the Hawaiian Sugar Planters Association Experimental Station by the researchers H.P. Kortschak, C.E. Hart, and G.O. Burr. These experiments received little or no exposure in international journals; however, Hatch and Slack were working in the David North laboratories, the research arm of Colonial Sugar Refiners at the time of their discovery, and had strong links with the Hawaiian laboratories. Most of the early labelling experiments in Hawaii were plagued by technical difficulties in correctly identifying the labelled compounds and interpretation of labelling results. This was because labelling time-courses rather than pulse–chase were used, and there were suspicions that labelling in malate and other C4 compounds was due to artefacts of quenching leaves or the many other pathways which could distribute radiolabel into intermediates of the photosynthetic pathway and beyond. It is rumoured that Kortschak and colleagues visited the laboratories of Calvin and shared their labelling data, only to be told that the label in malate was probably artefactual. The dangers of artefactual labelling are detailed in Bassham’s review of the discovery of the C3 photosynthetic biochemistry (Bassham, 2003). It was not until 1965 (Kortschak et al., 1965) that any of the Hawaiian work was published in a readily accessible journal. In the early 1960s, a young Russian researcher, Yuri Karpilov, also found labelling patterns similar to those of the Hawaiian group and published the results in Russian in a rather obscure journal (Karpilov, 1960). However, there is no doubt that the discovery that malate and aspartate were e4 | Furbank Anatomy, biochemistry, and an intermediate CO2 pool in C4 photosynthesis Three variations on a biochemical theme? The isolated bundle sheath strands discussed above played no small part in providing evidence for three variations on the pathway of C4 photosynthesis, delineated by the identity of the enzyme used to decarboxylate C4 acids in the bundle Fig. 2. Fluorescence micrographs of bundle sheath strands isolated from the NAD-ME monocot Panicum miliaceum by blending in isotonic buffer. (A) A low magnification epifluorescence micrograph of a single bundle sheath strand detecting autofluorescence (excitation 450–490 nm, emission >515 nm). Each single isolated strand is generally 200–250 μm in length. (B) A higher magnification laser confocal micrograph (×40) where the strands have been incubated in 6-carboxyfluorescein, a green fluorescent dye which can only pass into cells symplastically (through plasmodesmata), and the image has been generated from a single optical section which transects the cells in the cylindrical strand shown in (A), overlaying the green fluorescence channel with red chlorophyll fluorescence [excitation 488 nm plus 543 nm, emission 500–535 nm (green) and 600–740 nm (red)]. The green fluorescence indicates that the dye is moving into the cytoplasm of cells through plasmodesmatal pores. Scale bar=20 μm. Downloaded from https://academic.oup.com/jxb/article-abstract/68/2/e1/2932224 by guest on 03 June 2020 The realization that the C4 pathway is, in fact, a CO2concentrating mechanism and the result of the superimposition of biochemical specialization on Kranz anatomy and a C3 cycle appears not to be attributable to any single published work, and the flurry of activity in the USA, Canada, and Australia following the publication of the 14C labelling data in 1966 meant that a great deal of pivotal work must have been occurring simultaneously around the globe. Hatch credits a conference in Canberra in 1970 (the proceedings of which were edited by Hatch et al., 1971) for stimulating discussion and synthesizing ideas on C4 photosynthesis, and several seminal papers from the conference proceedings, to which a number of well-known international researchers in photosynthesis contributed, support this view. Whether stimulated by this meeting or not, workers in Canada and the USA were all quick to assemble the jigsaw puzzle of the physiology, anatomy, and the biochemistry of C4 photosynthesis. There were a number of independent reports of the cell-specific localization of enzymes from C4 leaves, notably PEPC and Rubisco in the mesophyll and bundle sheath cells, respectively (Bjorkman and Gauhl, 1969; Edwards et al., 1970), and NADP-malic enzyme (NADP-ME) in the bundle sheath cells (Berry et al., 1970) plus the intracellular localization of enzymes in the C4 pathway (Slack et al., 1969) which stimulated the proposal for an intermediate CO2 pool and later the CO2-concentrating function of C4 photosynthesis. Berry et al. (1970) showed that a pool of labelled CO2 accumulated in the bundle sheath at high specific activity in C4 plants, thus demonstrating, possibly for the first time, the role of an intermediate CO2 pool in C4 photosynthesis. Interestingly, both Slack et al. (1969) and Berry et al. (1970) also proposed the importance of abundant plasmodesmata in the exchange of metabolites between the two photosynthetic cell types of C4 leaves. While it was some years later that the significance of elevated CO2 in the bundle sheath cells in reducing photorespiration was explored further (see Hatch and Osmond, 1976), the foundations were laid by these early, elegant studies including those made in the laboratories of Tregunna and Downton linking tropical grass anatomy, physiology, and photorespiration to the newly discovered C4 pathway (reviewed in Berry, 2012). Circumstantial evidence for reduced photorespiration in C4 plants (reviewed in Berry, 2012) and the link between C4 photosynthesis in grasses, the CO2 compensation point, oxygen inhibition of photosynthesis, and superior performance of C4 plants was made between 1968 and 1969 (Downton and Tregunna, 1968; Black et al., 1969). As an aside, it is worth noting the key role that separation of bundle sheath and mesophyll cells of relatively high purity from C4 leaves played in the piecing together of the C4 pathway described above and subsequently in examining bundle sheath cell processes in general. While not claiming that they had isolated bundle sheath ‘strands’, Bjorkman and Gauhl (1969) showed that the differential grinding technique could produce preparations highly enriched in ‘fraction 1 protein’ or ‘carboxydismutase’ activity and low in PEPC activity from C4 leaves, debunking the myth that C4 plants did not fix CO2 via Rubisco and also postulating an intermediate CO2 pool. Edwards et al. (1970) reported that leaves of the C4 grass Digitaria sanguinalis could be blended in isotonic buffer to produce relatively pure mesophyll and bundle sheath cell preparations, ideal for examining cell-specific localization of C4 enzymes. The ability to separate mesophyll from bundle sheath in C4 leaves is presumably due to the thickened layer between the cell types providing a shear point during blending and the relative robustness of the cell walls of each cell type. Hatch and Kagawa (1976) later showed that bundle sheath ‘strands’ (vascular strands covered in bundle sheath cells) could be isolated from a number of species representative of the three variants of the C4 pathway. These strands (Fig. 2) were remarkable in that they remained fully photosynthetically functional and permeable to a range of metabolites. Isolated bundle sheath strands have subsequently been shown to have intact and functional plasmodesmatal pores penetrating their cell walls (Weiner et al., 1988), and have proven a valuable workhorse for biochemical evaluation of C4 photosynthesis, sucrose biosynthesis, and transport in a range of species (e.g. Lunn et al., 1997). C4 photosynthesis review | e5 30% of flux through the decarboxylation reaction (reviewed in von Caemmerer and Furbank, 2016). These pathways have very different energy requirements in bundle sheath and mesophyll cell types and implications for quantum efficiency (see Furbank et al., 1990; Y. Wang et al., 2014; von Caemmerer and Furbank, 2016). Which is the ‘best’ or most efficient way of carrying out C4 photosynthesis is unknown, and a better understanding of this seems pivotal for future crop engineering strategies. With ease of access by plant biochemists to revolutionary techniques in transcriptional analysis, gene sequencing, and other ‘omics’ platforms, a combination of traditional biochemistry and gene technology will undoubtedly provide new insights into how flexible these pathways are in response to the environment and the efficiency of C4 photosynthesis. Transgenic C4 plants The flurry of incisive C4 biochemistry and physiology in the two decades following the discovery of the C4 pathway yielded a huge body of information on these important and fascinating plants. However, a number of seemingly intractable problems have plagued progress in understanding many of the intricate aspects of the C4 pathway. In regard to enzyme Fig. 3. A scheme of the three C4 decarboxylation types as we now believe they operate, including the option of aspartate and malate being the transported C4 acid in the NADP-ME type in (A), NAD-ME type in (B), and PCK type in (C) (adapted after Furbank, 2011. Evolution of the C4 photosynthetic mechanism: are there really three C4 acid decarboxylation types? Journal of Experimental Botany 62 (9): 3103–3108. Copyright Society of Experimental Biology). Metabolite abbreviations: PEP, phosphoenolpyruvate; OAA, oxaloacetate, Asp, aspartate; Ala, alanine; Pyr, pyruvate; Mal, malate. Chloroplasts and thylakoids are coloured green, mitochondria blue, and the decarboxylation reactions are coloured red. Enzymes in the pathways are numbered as follows: 1, phosphoenolpyruvate (PEP) carboxylase; 2, NADPH-malate dehydrogenase; 3, NADP-malic enzyme; 4, pyruvate orthophosphate dikinase; 5, Rubisco; 6, aspartate aminotransferase; 7, alanine aminotransferase; 8, NAD-malate dehydrogenase; 9, NAD-malic enzyme; 10, phosphoenolpyruvate (PEP) carboxykinase. Due to the complexity, the combination of NADP-ME types utilizing malate and aspartate and both NADP-malic enzyme and PEP carboxykinase as decarboxylation enzymes is not shown. Downloaded from https://academic.oup.com/jxb/article-abstract/68/2/e1/2932224 by guest on 03 June 2020 sheath (Edwards et al., 1971; Hatch et al.,1975; Hatch and Kagawa, 1976). These biochemical variants are shown in Fig. 3. One can only imagine the complexity of the work involved in building these complicated pathway maps from a combination of 14C labelling, organelle and cell separations, and enzyme assays. It is not surprising that a degree of simplification was made in assigning these biochemical types, and suggestions were made quite early on that some dicots were of ‘mixed’ type. It is now becoming apparent that even in many ‘classical’ C4 NADP-ME species such as maize and sugarcane, both NADP-ME and PEP carboxykinase (PEPCK) are present in the bundle sheath and may potentially contribute to C4 acid decarboxylation. Even the separation of C4 plants into aspartate or malate ‘formers’ is blurred, with some NADP-ME types capable of utilizing both metabolites as the translocated C4 acid (Meister et al., 1996; Furbank, 2011; Brautigam et al., 2014; Y. Wang et al., 2014). Quantitative measurements of flux through the various pathway options are currently lacking, however, and evidence from enzyme assays and immunoblots indicate that the PEPCK pathway in the classical NADP-ME types may account for as little as 10–15% of flux (Koteyeva et al., 2015). It has also been known for >25 years that PEPCK types are most commonly if not exclusively using NAD-ME for up to e6 | Furbank A revolution in genomics and nextgeneration sequencing In the past decade, there has been a transformational advance in our capacity to sequence genomes rapidly and cheaply and to carry out sequence-based RNA expression analysis (Egan et al., 2012). While there has barely been a field of biology left untouched by these new technologies, there has been a major impact on C4 photosynthesis research, both in the study of evolution of C4 plants and in gene discovery. Sequence-based molecular phylogenies have provided new insight into the evolutionary relationships of C3 and C4 plants and provided a number of surprises in terms of the relationships between clades in the grasses (Grass Phylogeny Working Group II, 2012). Combining these new phylogenies with whole-genome sequencing and RNA expression analysis though RNAsequencing (RNA-seq, e.g. via the 1KP initiative, www.onekp. com/) has enabled the identification of a suite of genes under selection during the evolution of C4 photosynthesis and related transcription factors which may be responsible for evolution of C4 molecular specialization (Aubry et al., 2014). This is achieved by comparing sequence information from closely related C3 and C4 species and C3–C4 intermediates. The evolution of C4 photosynthetic traits has recently been reviewed comprehensively (Brautigam and Gowik, 2016). A particularly powerful platform for gene discovery, driven in part by the desire to engineer C4 photosynthesis into C3 plants (von Caemmerer and Furbank, 2012), has been the use of RNA-seq to examine gene expression patterns along a leaf developmental gradient in C4 monocots (Li et al., 2010). This has now been achieved to compare rice with the C4 plants Setaria, Sorghum, and maize (Ding et al., 2015), with additional information from C4 dicots such as Gynandropsis and Cleome (Kulahoglu et al., 2014; Williams et al., 2016). In addition, many data sets are enriched by separation of transcript pools from mesophyll and bundle sheath cells, providing information on the likely importance of a particular transcript in regulating expression of C4 genes and allowing clustering analysis of expression of known C4 transcripts with expression of genes of unknown or dubious function (Aubry et al., 2014; Williams et al., 2016). Combining these transcriptional data sets with other ‘omics’ measurements on the same tissues and careful microscopy for leaf cellular and subcellular morphology (Li et al., 2010) provides a powerful research tool for testing hypotheses on regulation of C4 leaf development and searching for new genes important in defining the C4 paradigm. Such data sets have also provided insight into the biochemical complexities of potential crossover between the three C4 decarboxylation types (Bräutigam et al., 2014) and the plethora of membrane transporters required for metabolites to transverse the plastids of C4 plants (reviewed in Weber and von Caemmerer, 2010). An example of the utility of such transcriptional approaches in teasing apart the gene regulation required for C4 plants to evolve has been work carried out on the regulatory gene network involved in conferring Kranz anatomy in C4 leaves. For example, of the gross morphological differences between a C3 and C4 monocot leaf, of note is the difference in vein spacing, with the former having 6–9 mesophyll cells between vascular bundles and the latter rarely having more than two. These leaf developmental patterns are determined early in meristematic tissue during leaf development and, until recently, candidate genes determining C4 leaf anatomy have been elusive (see Slewinski, 2013). An opportunity to use RNA-seq to examine this issue is provided by making comparisons of genes expressed in maize husk development, tissues with more C3like vein spacing, with development of true leaves with C4 vein Downloaded from https://academic.oup.com/jxb/article-abstract/68/2/e1/2932224 by guest on 03 June 2020 regulation, many of the C4 enzymes are polymeric and prone to losing regulatory function during isolation (e.g. PEPC, PPdK, and NADP-ME; see Ashton et al., 1990]. Probing regulation of photosynthetic flux by measurements of metabolite levels is complicated by compartmentation between cell types as well as organelles. Even interpretation of standard physiological measures such as gas exchange and chlorophyll fluorescence are complicated by the two-cell photosynthetic system. By the early 1990s, the growth of the C4 photosynthetic community had slowed considerably, compared with the heydays of the previous two decades. Many researchers attributed this to the lack of a good, easily transformable model genetic system for C4 plants. While researchers in C3 photosynthesis were rapidly transforming and analysing tobacco with gene suppression and overexpression constructs (Sonnewald et al., 1991; Hudson et al., 1992), gene promoter–reporter fusions (Willmitzer, 1988), and mining Arabidopsis t-DNA insertion collections (Krysan et al 1999), C4 researchers were reliant on maize genetic resources generated by transposon tagging which were not as well developed and slower to obtain results from (Settles et al., 2007, and references therein). While maize and sugarcane genetic transformation were possible, they were difficult to achieve and feasible only in industrial laboratories. The development of a genetic transformation system for Flaveria bidentis, an NADP-ME dicot (Chitty et al., 1994), enabled a range of experiments on cell-specific gene regulation (Stockhaus et al.,1997; Engelmann et al., 2008) and a comprehensive analysis of the limitations to photosynthetic flux afforded by each of the enzymes of the C4 pathway (Furbank et al., 1997; von Caemmerer and Furbank, 2016, and references therein). The system was used to validate C4 photosynthetic models and examine enzyme regulation (see von Caemmerer and Furbank, 2016). However, a dicot annual from the Asteraceae, while of great utility in basic research, was not particularly relevant to the commercial monocot C4s such as maize, sorghum, sugarcane, Miscanthus, and switchgrass. The C4 biofuels community was a key driver for the development of the NADP-ME grass Setaria viridis (green foxtail millet) as a C4 model grass (Brutnell et al., 2010). This grass has all the hallmarks of an ideal model system including Agrobacterium-mediated genetic transformation at reasonable efficiency. While still in its infancy, this system offers the promise of manipulating photosynthetic proteins, cell wall components, and transporters in a plant closely related to important commercial crops. C4 photosynthesis review | e7 Unresolved questions about the C4 mechanism As a research field becomes ‘mature’, there is a perception by the non-specialist that ‘we must have it all figured out by now’! The C4 pathway is one of the most complicated biochemical pathways likely to be placed in front of a university undergraduate, and attempts at C4 engineering have only served to reveal additional uncertainties about how the pathway operates, how it is regulated at the level of gene expression and post-translationally, and some of the physical properties of the anatomical specialization of Kranz C4 leaves. One area of uncertainty and dispute has been the concentration of CO2 achieved by the CO2-concentrating mechanism in bundle sheath cells of C4 plants. We now know that the role of the biochemical CO2 pump in the mesophyll cells of C4 leaves is to concentrate CO2 at the site of Rubisco, potentially to levels of inorganic carbon 10-fold higher than atmospheric equilibrium (see Furbank and Hatch, 1987; Hatch et al., 1988 von Caemmerer and Furbank, 2003). The advantages of C4 photosynthesis in reducing photorespiration and allowing Rubisco to operate close to its Vmax, however, are dependent on growth environment and come at an energetic cost. The generic statement that the C4 mechanism concentrates CO2 to levels up to ‘10-fold atmospheric levels’ is not precise enough to allow modelling of such efficiencies and is based on radiolabelling accompanied by some rather complex assumptions concerning the equilibrium of CO2 and bicarbonate in the bundle sheath compartment (see Furbank and Hatch, 1986). Subsequent estimates with other technologies varied widely, and the important parameter of ‘leakiness’ (the proportion of carbon fixed by PEPC which leaks back to the mesophyll intercellular spaces) is dependent upon the diffusion gradient of CO2 and hence the pool size in the bundle sheath (Farquhar, 1983; Furbank et al., 1990; von Caemmerer and Furbank, 2003). Since the energy consumption of the mesophyll CO2 pump and the efficiency of the pump in reducing photorespiratory flux are largely dependent on these parameters, a quantitative understanding of these processes would seem paramount. If the diffusion properties of the mesophyll–bundle sheath interface were known, from the ratio of the velocity of PEPC and the net rate of CO2 fixation by Rubisco, the calculation would be mathematically trivial. Unfortunately, the leakage of CO2 from the bundle sheath can only be indirectly calculated and, most recently, this is done using carbon isotope discrimination and modelling (reviewed in von Caemmerer et al., 2014). Questions pertinent to C4 engineering in C3 plants such as the necessity or otherwise of having a suberized lamella in bundle sheath cell walls as a CO2 diffusion barrier, whether cellular positioning of organelles is important, and what is the appropriate PEPC/ Rubisco ratio for optimal performance are all unanswered. To add to this quandary on CO2 diffusion, it has now been shown that, as is the case if C3 leaves, mesophyll conductance to CO2 may be important in limiting flux to PEPC in C4 leaves (von Caemmerer et al., 2014), and this can only be estimated by a combination of carbon and oxygen isotope analysis carried out during measurements of CO2 fixation and modelling. While the text above concentrates on the biochemistry and physiology of C4 photosynthesis, our understanding of gene regulation and molecular biology of the C4 mechanism is also rather incomplete. As discussed above, the developmental regulation of C4 leaf anatomy is only now being elucidated, and the elements controlling transcriptional regulation of C4 genes are finally being examined. The need to regulate the expression in C4 leaves tightly so that PEPC, PPdK, and malate dehydrogenase (MDH) are exclusively (or predominantly) in the mesophyll cells, and Rubisco, the PCR cycle, and enzymes of C4 acid decarboxylation in the bundle sheath cells has been appreciated for 40 or more years and has spawned many research projects, particularly with the advent of plant genetic transformation (reviewed in Hibberd and Covshoff, 2010). Surprisingly, few cis-elements have been isolated from C4 promoters which confer cell specificity (the MEM1 element in the PEPC promoter being one of the exceptions; reviewed in Hibberd and Covshoff, 2010). Recent work using transcriptomics approaches has implicated a novel cis-element in conferring mesophyll specificity to carbonic anhydrase which is also present in the 5'-untranslated region of the PPdK gene (Williams et al., 2016). Bundle sheath-specific promoters are rare, and no cis-elements have been isolated which confer high expression exclusively to this compartment. Interestingly, many C4 gene promoters function in a cell-specific or preferential manner when tested in C3 plants, suggesting that the trans factors necessary for cell specificity are present in these plants. However, the identity of these trans factors remains elusive, although next-generation sequencing tools being applied to the problem are beginning to bear fruit, producing some candidate genes (Aubrey et al., 2014). A renaissance in C4 photosynthesis research? A timeline for citations of papers in which the term ‘C4 photosynthesis’ is in the title or abstract from the early 1970s Downloaded from https://academic.oup.com/jxb/article-abstract/68/2/e1/2932224 by guest on 03 June 2020 spacing (Wang et al., 2013). Comparison of C4 leaf primordial RNA pools with RNA from husk primordia of similar developmental age (Wang et al., 2013) combined with analysis of maize mutants with disruption of the Scarecrow gene (see Slewinski 2013) have now resulted in at least a partial model of how Kranz anatomy develops. How easily this network can be installed in a C3 plant and whether the bundle sheath cells and chloroplasts of C3 grasses are appropriately equipped to accept C4 biochemistry are currently unanswered questions. As such large transcriptional and genomic sequence data sets have become available online, in silico mining has become a common practice for the new generation of young researchers interested in testing hypotheses on gene function and designing gene constructs for transgenic engineering. The power of these data sets is massive, and the current limitation to their rapid adoption in C4 photosynthesis research seems to be higher level bioinformatics training required to mine, filter, and interpret such data appropriately. e8 | Furbank advocacy of Dr John Sheehy, a consortium of researchers tasked with installing a C4 pathway into rice was formed (Sheehy et al., 2007; von Caemmerer et al., 2012). In 2008 the Bill and Melinda Gates foundation funded these efforts as an ‘Apollo Project’ of equivalent aspiration as it was to put a man on the moon, committed to a 20 year timeline. The project has now entered Phase 3, with six previous years of investment and now four more years of research commitment by 16 laboratories in 11 countries. This financial boost to C4 research no doubt continued to build people and citations, and certainly the high profile of C4 photosynthesis research was assured by the advocacy of such a charitable donor. C4 engineering has now become a major effort in the plant science community alongside many other international efforts to engineer improved photosynthetic performance in our cereal grain crops (Furbank et al., 2015). What is the future of C4 photosynthesis research? As always, digging into the history of the research field as we have above can provide insight into the future. In the late 1960s, only isolated pockets of researchers were working on aspects of C4 photosynthesis. While this cohort grew over the next decade with the excitement of the discovery of a new photosynthetic pathway, progress was hampered by the large distances between researchers (particularly for the Australian groups), the lag time for publication of results, and in particular difficulties of communication (no internet or E-mail). This is evidenced by the early ignorance of C4 researchers in Australia, the USA, and Russia that they all actually had essentially the same data sets but had interpreted them differently! Although Hal Hatch maintains that isolation improved his and Roger Slack’s focus and deductive power in those days, online journals, ease of conference travel, massive online data sets, and E-mail have all had a major impact on the way we carry out research. How the cohort of young researchers in C4 photosynthesis has grown over the last decade is nothing short of phenomenal. In this context it is worth recounting a conversation had with the Bill and Melinda Gates Foundation co-ordinator at our first C4 rice consortium meeting of ~20 researchers in 2008. The question was asked ‘you all seem to know each other well; how is that so?’ The answer given was met with a look of disbelief, that we basically encapsulated a large proportion of the field of active C4 researchers and many of us had known each other for 20–30 years, or indeed trained the younger folk present. It was pointed out to us that the age profile was worryingly high (except for some notable exceptions), but, as the consortium and global interest in engineering the C4 pathway has grown, the future of the field now seems assured. The young age profile of the scientific meeting in 2016 in Canberra, celebrating 50 years since the pivotal description of the C4 pathway, is a testament to the vibrant, exciting future of C4 photosynthesis research. Hopefully there is a new generation of plant biologists willing to challenge dogma and push the boundaries of our knowledge of C4 photosynthesis with every new tool available. (For readers interested in how C4 photosynthesis researchers are connected, Supplementary Fig. 2 and the associated URL provide a network diagram of C4 photosynthesis Downloaded from https://academic.oup.com/jxb/article-abstract/68/2/e1/2932224 by guest on 03 June 2020 to the present day (earlier records from the 1960s appear to be unreliable in the citations database) can be found in Supplementary Fig. 1 at JXB online. The approach used in the analysis was to count the number of citations for papers published in each year of publication. This should capture elements of both the volume of papers published and the interest in citing earlier work both from within the C4 photosynthesis community and in related fields. While the number of plant science researchers and the journals in which research can be published has burgeoned since the discovery of C4 50 years ago, and the accessibility of the internet and availability of digitized journal articles would have contributed to bursts of citations, it is interesting to note the pattern of these citations and postulate what has caused these patterns. The flurry of activity following the discovery of C4 biochemistry is largely omitted from this analysis, but would barely rate as a blip on this timeline, apart from a few seminal papers, due to the small community of C4 researchers but undoubtedly biased by the small number of dedicated journals and the smaller total number of researchers in plant biology. It was not until papers of the late 1990s that citations have received quite a large boost. This may partly be due to the burgeoning interest at this time in gene discovery, gene regulation, and molecular biology of C4s, and the availability of genetic resources in the maize community. Delving into the publications of this period reveals a broad range of topics, but the regular appearance of papers entitled ‘Cloning and characterization of expression of the enzyme … involved in C4 photosynthesis’ is obvious. Current interest in evolutionary models for C4, C3, and C2 photosynthetic relationships which began in the 1990s probably also play a part, and the first papers in molecular evolution of C4 photosynthesis appear in the literature based on the PEPC gene sequence at that time. The opportunity to use promoter–reporter fusions in stable transformation to study C4 gene regulation and the large number of publications examining control of C4 photosynthetic flux in transgenic Flaveria may have contributed to these citations. While citations continued to climb in the following decade, another ‘flush’ of citations began in the mid 2000s, and this precipitous rise has continued unabated since that time. While purely a personal perspective, it cannot be coincidental that three major events occurred in plant biology around this time. First, the interest in plant-based biofuels began in earnest, with major investments in lignocellulosic processes (culminating in the British petroleum investment in the Energy Biosciences Institute at Berkley in the USA in 2007) spurring even more interest in Miscanthus and switchgrass, both C4s. Secondly, it was becoming apparent that the world was approaching another food crisis, with a plateau in annual cereal yield gains and a burgeoning population putting major pressure on food supplies and prices. In rice (and now in wheat), breeders were reporting that traditional breeding targets for yield potential in C3 cereals such as grain number and harvest index had been exhausted and that biomass and photosynthetic performance were the new breeding targets (Sheehy et al., 2007). Following two pivotal meetings at the International Rice Research Institute and the inexhaustible C4 photosynthesis review | e9 research papers and authors showing their relationships, highlighting speakers at the C4 Photosynthesis Conference in 2016; created by Adam Carroll, ANU.) Supplementary data Supplementary data are available at JXB online. Figure S1. Total citations of C4 photosynthesis-linked papers published in each year. Figure S2. C4 photosynthesis publication network. The interactive version is available at http://www.metabolomeexpress.org/c4pubnet.php. I would like to acknowledge the funding support of the Australian Research Council. 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