As right as rain: deciphering drought-related metabolic flexibility in the C4-CAM Portulaca

Journal of Experimental Botany, Vol. 73, No. 14 pp. 4615–4619, 2022 https://doi.org/10.1093/jxb/erac179 eXtra Botany © The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Insight As right as rain: deciphering drought-related metabolic flexibility in the C4–CAM Portulaca IvanReyna-Llorens1,*, and SylvainAubry2,*, 1 Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Campus UAB, Bellaterra, Barcelona, Spain 2 Department of Plant and Microbial Biology, University of Zürich, 8008 Zürich, Switzerland * Correspondence: ivan.reyna-llor[email protected] or [email protected] This article comments on: Ferrari RC, Kawabata AB, Ferreira SS, Hartwell J, Freschi L. 2022. A matter of time: regulatory events behind the synchronization of C4 and crassulacean acid metabolism gene expression in Portulaca oleracea. Journal of Experimental Botany 73, 4867–4885. Optimization of carbon and water usage in plants is a widespread strategy to survive in hot and dry environments. Based on the ancestral C3 photosynthesis, two major carbon -concentrating mechanisms (CCMs) evolved, allowing spatial (C4) or temporal (Crassulacean acid metabolism, CAM) segregation of carbon-fixing activities. While C4 and CAM have generally been considered mutually exclusive, they share most of their biochemical machinery. An exception to the rule is found among the Caryophyllaceae, in the Portulaca genus. Described as an ‘inducible’ CAM, the C4 species Portulaca oleracea accumulates malate transiently under drought stress and in a reversible manner. Concentrating mostly at the transcriptome level, Ferrari et al. try deciphering the complex interplay between C4 and CAM under various drought conditions. A better understanding of how the two carbon-fixing mechanisms are coordinated could shed light on key regulatory mechanisms necessary to improve C4 crops under changing environments. Most of the enzymatic machinery that is required to run C4 and CAM cycles has derived from ancestral C3 species, where these enzymes mostly played anaplerotic roles (Silvera et al., 2010; Aubry et al., 2011). Both C4 and CAM are impressive examples of convergent evolution, with at least 66 and 40 independent origins, respectively (Silvera et al., 2010; Sage et al., 2012). Typically, in C4, a subset of proteins is limited to bundle sheath or mesophyll cells, allowing concentrating of CO2 around the central carboxylase Rubisco, thus reducing the penalties of photorespiration (Fig. 1). While in species running CAM, carbon fixation by a phosphoenolpyruvate carboxylase (PEPC) is transposed into the dark phase, connected to malate accumulation, its efflux from the vacuole and subsequent decarboxylation take place during the day. Interestingly, both CAM and C4 not only enable more efficient carbon fixation, but also generally improve water use efficiency. The inverse pattern of stomatal opening in CAM species is primarily aimed at limiting water loss during the light phase, while C4 leaves operate at lower stomatal conductance compared with C3 (Aubry et al., 2016; Males and Griffiths, 2017). It is noteworthy that within both CAM and C4 species, a whole range of non-canonical adaptations evolved, moving away from ‘prototypical’ models, for example CAM species still fixing carbon on a 24h basis or various C3–C4 intermediates (Owen and Griffiths, 2013; Schlüter and Weber, 2020). Consideration of this flexibility is important when thinking in terms of engineering C4 in a C3, CAM in a C3, or, our focus here, CAM in a C4 (Box 1). Several anatomical, physiological, and biochemical barriers have led to the assumption that CAM and C4 photosynthesis were incompatible (Sage, 2002). In particular, concomitant mesophyll (C4-)carboxylation and (CAM-)decarboxylation may result in futile cycles. Meanwhile, at least three genera primarily using C4 photosynthesis (namely Portulaca, Spinifex, and Trianthema) present temporary acidification and reversed stomatal behaviour under drought stress, signatures of CAM (Gilman et al., 2022). The Portulaca family belongs to the Caryophyllales, where eight of the 23 families encompass C4 and C3–C4 species (Voznesenskaya et al., 2010). The peculiar metabolic flexibility of Portulaca oleracea, a facultative CAM species using an NAD-malic enzyme (ME)-type C4 metabolism, is a good model to study interplays between various transcriptional, translational, and metabolic regulations. This paper is available online free of all access charges (see https://academic.oup.com/jxb/pages/openaccess for further details) Downloaded from https://academic.oup.com/jxb/article/73/14/4615/6660818 by Consejo Superior de Investigaciones Cientificas (CSIC) user on 17 January 2023 4616 | Interestingly, despite strong anatomical constraints, at the biochemical level CAM inception appears to be more flexible (and reversible) than the C4 pathway: while there is no such thing as a facultative/inducible C4 pathway, there are some examples of inducible CAM in both C3 and C4 backgrounds (Wai et al., 2019; Ferrari et al., 2022; Gilman et al., 2022). Engineering CAM into naturally non-CAM crops may improve water use efficiency and stress resilience (Schiller and Bräutigam, 2021). CAM and the circadian clock In their contribution, Ferrari and colleagues evaluate the extent to which the underlying circadian clock regulation might modulate the shift to temporal gene expression under CAM induction by drought stress. Generally, studies on obligate CAM (Mesembryanthemum crystallinum, Kalanchoe fedtschenkoi, and K. laxiflora) show that circadian clock elements are mostly unaffected by drought, in terms of both phasing and amplitude. Nevertheless, diurnal variations appear to be a necessary requirement to ensure proper CAM-related metabolic fluxes (Boxall et al., 2020). In recent years, our understanding of the complexity of gene circuits responsible for the core clock oscillation improved dramatically (Millar, 2016). Ferrari and colleagues report that none of these genes was significantly affected by drought. Two levels of regulation are possibly coordinating CO2 uptake in CAM: the circadian oscillator control and the metabolite control (Dodd et al., 2002). While C4 photosynthesis is essentially based on cell differentiation, it is also important to consider the circadian clock not necessarily ‘ticking’ at the same pace in all cells and tissues (Greenwood and Locke, 2020), and therefore not regulating all genes involved in carbon metabolism in the same way in all cells. To unravel the complex metabolic interplay underlying C4 to CAM transition in stressed P. oleracea, more studies on cell-specific variations of the circadian clock under stress as well as the conservation of gene regulatory networks (specifically cis-elements of clock target genes) that are under control of the core clock genes appear very relevant. Central role of diurnal PPCK in the C4–CAM transition Portulaca oleracea is a bona fide C4 species with the capacity of switching from C4 to CAM in response to drought stress (Voznesenskaya et al., 2010). By taking advantage of the facultative nature of this species, Ferrari and colleagues assessed the contribution of both the clock and drought in the activation of the transcriptional programme for CAM induction. In P. oleracea, transcriptional induction and repression of both CAM and C4 genes were mainly affected by drought conditions. On the other hand, while the circadian clock is fundamental for CAM activation, its disruption only affected the expression of PPCK-E1 among the genes involved in CAM. Phosphoenolpyruvate carboxylase kinase (PPCK) is responsible for activating the CAM carboxylase PEPC that controls the initial fixation of CO2 during the night (Hartwell et al., 1999). The influence of both clock and drought on PPCK could be essential for CAM induction and control, acting as a fine-tuning switch that limits PEPC activity to the dark period once CAM has been established. Understanding how clock and drought signals converge in the regulation of PPCK is essential for engineering CAM in both C3 and C4 species (Box 2). Hormonal cues involved in the C4 to CAM transition At the transcriptional level, the C4–CAM system in P. oleracea seems to be triggered by the water status of the plant. Ferrari Box 1. Bringing microgenomics into CAM research The complexity of the metabolic interplay and diversity in C4–CAM species ran into several limitations that may be alleviated by recent advances in molecular biology and genomics. In the C4 field, where the cell-specific component might have been more obvious, research efforts have been undertaken to unravel evolutionary, biochemical, and metabolic complexity (Schlüter and Weber, 2020). While phylogenomics approaches have provided compelling results towards the origins of the CAM syndrome and its plasticity, only few recent works address CAM using microgenomics (Abraham et al., 2016). Such an approach will be made easier by the sequencing of genomes from CAM species and may allow monitoring of steady-state transcript abundance, and cell-specific transcriptional and translational regulatory processes in individual cell types that would, in turn, result in a better understanding of gene regulatory networks underlying C3/C4–CAM interplay. For example, whether and how water storage cells present in many succulent species (e.g. P. oleracea) are influencing the C4 cycle, or the extent to which diurnal malate pools in the mesophyll are influencing the rhythmicity of stomatal apertures remain open questions that may only be addressed using cell-specific approaches. A more precise description of the C4–CAM switch that reflects the actual plasticity of the CAM syndrome is required: not all plants could be switched into CAM, not all species switch in a similar time frame (Dodd et al., 2002), and, finally, not all cells may adapt their metabolism to the same extent. Downloaded from https://academic.oup.com/jxb/article/73/14/4615/6660818 by Consejo Superior de Investigaciones Cientificas (CSIC) user on 17 January 2023 | 4617 Box 2. C4–CAM: a physiology still to be explored The C4–CAM switch appears to be a possible way to complement C4-centric carbon concentration, particularly for some of the crops that may encounter increasingly arid conditions in the near future. From an engineering perspective, an inducible CAM system would possibly allow plants to tolerate more extreme environments (where C4 plants underperform) while still relying on C4 metabolism under optimal conditions. However, in order to achieve this ambitious goal, several gaps in our understanding of the C4–CAM interplay need to be addressed. A first point might be to try to understand the actual limits of C4 hydraulics towards drought stress and the extent to which succulence may affect metabolic regulation and C4 pathways. Secondly, the major change in malate homeostasis would require temporal and spatial changes in gene expression (namely alterations of PEPC, MDH, and NAD-ME) that would necessarily impact the whole tissue (Fig. 1). Down-regulation in C4 gene expression upon CAM induction might require rewiring of higher order gene regulatory networks such as the circadian and other light signalling networks. Typically, a proper PPCK activation in time and during drought will be crucial to kick-start carboxylation into the CAM route. A better understanding of these processes is a prerequisite for any attempt considering CAM as a valuable asset for improving crop resilience. chlp CO2 CO2 HCO3CA PEPC PPDK-RP RBCS OAA Asp Asp OAA Mal mMDH CO2 NAD-ME CBBcycle Pyr Pyr PEP Ala Ala Mesophyll Guard Cells Guard Cells Bundle Sheath Glu 2-KG AspATAspAT Glu 2-KG AlaAT ATP NADH NAD C4 C4-CAM PPDK CO2 HCO3CA PEPC (CAM) RBCS OAA mMDH CO2 NAD-ME CBBcycle Pyr Mesophyll Bundle Sheath AspAT AspAT Night Day cMDH ? Mal Malic acid PPC2 Day CO2 ? Mal OAA ? ? Pyr Ala Ala Glu 2-KG AlaAT PPDK-RP PEP ATP ADP+Pi ADP+Pi PPDK PEPCC4 PPCK C4 C4-CAM Drought Watering mito mito chlp chlp chlp vac vac Fig. 1. Working model illustrating the C4–CAM switch in the NAD-ME-type C4 Portulaca oleracea. A transversal section of a P. oleracea leaf is illustrated in the middle, guard cells in grey, mesophyll cells in yellow, and bundle sheath cells in orange. Enzymes highlighted in red represent those C4 enzymes that need to be down-regulated or repressed for the activation of the CAM cycle. At the same time, enzymes in blue denote those enzymes that would have to be activated or modified during CAM induction. Abbreviations: Chlp, chloroplasts are in green; mito, mitochondria in red; vac, vacuole; CA, carbonic anhydrases; PEPC, phosphoenolpyruvate carboxylase; AspAT, aspartate amino acid transferase; AlaAT, alanine amino acid transferase; NAD-ME, NAD-dependent malic enzyme; PPDK, pyruvate.orthophosphate dikinase; PPDK-RP, PPDK regulatory protein; RBCS, Rubisco; mMDH, mitochondrial malic dehydrogenase; cMDH, chloroplastic malic dehydrogenase; PPCK, PEPC kinase; Mal, malate; Ala, alanine; Pyr, pyruvate; Glu, glutarate; 2-KG, 2-ketoglutarate; OAA, oxaloacetate. Downloaded from https://academic.oup.com/jxb/article/73/14/4615/6660818 by Consejo Superior de Investigaciones Cientificas (CSIC) user on 17 January 2023 4618 | and colleagues further explored this connection by assessing the activities of abscisic acid (ABA) and cytokinins (CKs). ABA is known to mediate abiotic stress responses in plants and has been associated with CAM expression in other species such as agave or pineapple (Chen et al., 2020). The role of CKs in CAM is less clear, yet some evidence suggests its involvement in CAM induction in the C3+CAM M. crystallinum (Wakamatsu et al., 2021). Indeed, endogenous levels of ABA in P. oleracea increased in correlation with a reduction in osmotic potential and intracellular acidification proper of CAM metabolism. The correlation between ABA metabolism and CAM induction was also observed at the transcriptional level, where ABA biosynthesis genes and several components of the ABA signalling pathway were up-regulated under drought conditions and down-regulated during re-watering. CK genes, on the other hand, showed a more delayed induction during re-watering, suggesting a potential role at later stages of C4–CAM induction. In fact, addition of exogenous CK reverted the suppression of C4 genes in drought-stressed plants while exogenous ABA triggered transcription of the CAM PPC1E1c gene. Despite this, neither ABA nor CK treatments induced a change in intracellular ∆H+, implying that ABA is not sufficient to trigger CAM in P. oleracea. The exact role of these two phytohormones for the CAM switch remains unclear. To find potential links between ABA and CK signalling networks and the C4–CAM induction in P. oleracea, Ferrari and colleagues defined a group of transcription factor candidates based on a gene co-expression network built from previously published transcriptomics data (Ferrari et al., 2020). Indeed, a group of nine transcription factor candidates responded to drought conditions. Similarly, their expression was influenced by the exogenous addition of either ABA or CK, which is consistent with the patterns observed for both CAM and C4 PEPC genes. Based on these results, the authors proposed a transcriptional regulatory network for C4–CAM induction in P. oleracea. This work paves the way for further characterization of the transcription factors as well as the development of P. oleracea as a model system to address CAM induction in the C4 context, similar to M. crystallinum for C3+CAM induction (Wakamatsu et al., 2021). Perspectives Ferrari and colleagues take advantage of whole-transcriptome analysis to try to decipher CAM dynamics, its interplay with the C4 pathway, and drivers of its induction upon drought stress. While this approach is interesting, a comprehensive view over the C4 to CAM transition is still missing. For example, few transcripts actually do match peak enzyme activities, and variation in transcript abundance may not entirely correlate with protein abundance and activity, nor with the actual metabolite signatures (Abraham et al., 2016). Therefore, it would be interesting also to collect proteomics data and test if protein abundance matches the enzyme activities better than transcripts, or if the enzyme regulation relies on further post-translational mechanisms. Recent genome sequencing of Portulaca amabilis, a diploid facultative CAM using NADP-type C4, identified a specific PEPC orthologue for each carboxylation as well as evidence for cross-linking nocturnal acid production into the C4 cycle (Gilman et al., 2022). The extent to which P. amabilis and P. oleracea coordinate their carbon fluxes in a comparable way remains to be determined. Data presented here open a whole new perspective over the way the C4–CAM switch could have been recruited to bring some fitness improvements in arid conditions for already efficient CCMs. Exploring further the genomic space of Portulaca will allow identification of regulatory elements both in cis and in trans, and help in deciphering the regulatory network underlying this complex metabolic interplay. As for any other biochemical pathways (e.g. C4 acid decarboxylations, Furbank, 2011), metabolism ‘subtypes’ are useful intellectual constructs to try making order out of chaos, but often do not match reality. Above all, these categorizations should not limit us in the quest to understand the fantastic plasticity of plant metabolism. Acknowledgements This paper is dedicated to the memory of our colleague Chris John. Funding This work was supported by grant CEX2019-000902-S funded by MCIN/AEI/10.13039/501100011033, by the CERCA Programme/ Generalitat de Catalunya, and by the Swiss National Science Foundation (#31003A_172977). Keywords: C4, CAM, photosynthesis, Portulaca oleracea. References Abraham PE, Yin H, Borland AM, et al. 2016. Transcript, protein and metabolite temporal dynamics in the CAM plant Agave. Nature Plants 2, 16178. Aubry S, Aresheva O, Reyna-Llorens I, Smith-Unna RD, Hibberd JM, Genty B. 2016. A specific transcriptome signature for guard cells from the C4 plant Gynandropsis gynandra. Plant Physiology 170, 1345–1357. Aubry S, Brown NJ, Hibberd JM. 2011. The role of proteins in C3 plants prior to their recruitment into the C4 pathway. Journal of Experimental Botany 62, 3049–3059. Boxall SF, Kadu N, Dever LV, Kneřová J, Waller JL, Gould PJD, Hartwell J. 2020. 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