2 Convergent GRN evolution at the spatial-temporal level: Carbon Concentrating Mechanisms
Dry habitats are often also sunny, placing further specific restraints on plant physiology and specifically photosynthesis: high light intensity and high temperature. As plants acclimate to drought by restricting their water loss through closing their stomata, they also restrict CO2 uptake, reducing the available CO2 for photosynthesis. High light becomes damaging if the energy obtained from light absorption is not used by the photosynthetic electron transport chain due to the absence of CO2. Simultaneously, high temperature reduces the solubility of gases so that RuBisCO activity shifts away from carboxylation towards more oxygenation, increasing photorespiration and leading to wasted energy (Edwards, 2019). To adapt to these constraints, carbon concentrating mechanisms (CCMs) have evolved in numerous lineages to enable efficient photosynthesis in dry, hot, and high light environments while improving water use efficiency.
CCMs work by separating the initial carbon fixing away from the photosynthetic carbon fixing (Calvin cycle) either temporally across the diurnal cycle, or spatially across different cell types or compartments. Both the temporal separation, as seen in Crassulacean Acid Metabolism (CAM), and spatial separation, as seen in C4photosynthesis, have evolved independently in over 60 lineages (Edwards & Ogburn, 2012). The evolutionary paths to both CAM and C4 photosynthesis have been recently reviewed and discussed in great detail (Bräutigam, Schlüter, Eisenhut, & Gowik, 2017; Chen, Xin, Wai, Liu, & Ming, 2020; Edwards, 2019; Heyduk, Moreno-Villena, Gilman, Christin, & Edwards, 2019; Niklaus & Kelly, 2019; Schlüter & Weber, 2020; Sedelnikova, Hughes, & Langdale, 2018) and in short, both require two main aspects: 1) an anatomical adaptation and 2) co-option of the carbon concentrating metabolic pathway to the correct spatiotemporal location. The main anatomical adaptation for CAM is enlarged storage vacuole to store the malate synthesized during the night and enable the day-night CCM (Luttge, 1987). In many C4 plants, the spatial separation is across two cell types, mesophyll and bundle sheath, and to achieve this, C4 leaves adapt with so-called Kranz anatomy with enlarged bundle sheath cells with increased plastid numbers and increased vein density (Haberlandt, 1904). The regulation of Kranz anatomy is proving to be a complex process (Sedelnikova et al., 2018), and although it has readily evolved convergently in some plant clades, it is starting to appear that not all plant clades are pre-conditioned for the C4 photosynthesis to evolve (Edwards & Ogburn, 2012). Conversely to complex leaf anatomy and its regulation, all the enzymes required for both the CAM and C4 metabolic pathways, such as phosphoenolpyruvate (PEP) carboxylase and malate dehydrogenase, are present in all plants serving other functions. To co-opt these enzymes for CCMs, the expression and regulatory patterns have evolved to be spatially and temporally specific (Brown et al., 2011; Burgess et al., 2016; Christin et al., 2013; Dunning, Moreno-Villena, et al., 2019; Gowik et al., 2004; Heyduk, Ray, et al., 2019; Kajala et al., 2012; Ming et al., 2015; Rondeau et al., 2005; Schulze et al., 2013; Williams et al., 2016; Yang et al., 2017). Whole genome sequencing has enabled a level of understanding of how these CCMs evolved in plants. The first C4 (Sorghum bicolor ) (Paterson et al., 2009) and CAM (Phalaenopsis equestris ) (Cai et al., 2015) genomes provided insights about redirection of genes involved in C3 photosynthesis and expansion of ancient and recent gene families. Transcriptomic approaches are also offering new evidence about convergent evolution of genes and regulatory pathways underlying these CCMs. For example, the genome and temporal transcriptome sequencing of the CAM species Kalanchoë fedtschenkoi revealed that the independent emergences of CAM from C3 have been based on rewiring of diel gene expression patterns along with protein sequence mutations (Yang et al., 2017). Furthermore, the pineapple genome (Ananas comosus (L.) Merr.), another CAM species, indicated that the transition from C3 to CAM was based on regulatory neofunctionalization of pre-existing genes and regulation of circadian clock components through evolution of novelcis -regulatory elements (Ming et al., 2015).
To resolve how gene expression patterns and GRNs have evolved in convergent C4 lineages, comparative leaf transcriptomics have been utilized, including comparisons of C3, C4 and intermediate C3-C4 leaves, developmental gradients, specific cell types and environmental cues (Aubry, Kelly, Kümpers, Smith-Unna, & Hibberd, 2014; Bräutigam et al., 2011; Burgess et al., 2016; Dunning et al., 2017; Gowik et al., 2004; Li et al., 2010). From these comparisons shared routes to C4 are starting to emerge. Transcriptomic comparison across the monocot-dicot divide revealed deep evolutionary conservation of C4 leaf development pathways and that certain homologous cell type-specific regulators were co-opted during the independent evolutions of C4 photosynthesis (Aubry et al., 2014). Not only is it possible to co-opt the same orthologs as another species, but it is also possible to co-opt the very gene from another species. Transcriptomics of closely related Alloteropsis grasses revealed that recurrent evolution of C4 among the group was enabled by co-opting genes across species boundaries, specifically by introgressions of the C4 components (Dunning et al., 2017). Subsequently, it was discovered that grasses have utilized lateral gene transfer also to speed up C4 evolution (Dunning, Olofsson, et al., 2019; Phansopa, Dunning, Reid, & Christin, 2020). Furthermore, the understanding of C4 enzymes’ regulatory networks in ancestral C3 state was elucidated also by a transcriptomics approach: comparison of how light and chloroplasts regulate C4 enzymes in closely related C3 and C4 plants. This linked the C4 enzymes into a pre-existing C3regulatory network, explaining the readiness of C4 to evolve at the molecular level (Burgess et al., 2016). Regardless of these examples of GRN readiness for C4 evolution, or “genetic potentiation”, the C4 lineages are not evenly distributed across the angiosperms (Edwards, Osborne, Strömberg, Smith, & Consortium, 2010). This can be due either to the different lineages facing different evolutionary pressures, or differences in either genetic or anatomic potentiation (Heyduk, Moreno-Villena, et al., 2019), and remains a question to be addressed in the future.
Counter-intuitively, C4 and CAM can exist in the same leaf. A recent transcriptomic approach was taken to dissect the behaviour of both C4 and drought-induced CAM in the same plant, Portulaca oleracea , offering insight on how the regulatory networks of shared enzymes might be able to coexist while responding to different environmental and temporal cues (Ferrari et al., 2020). With more C4 and CAM genomes and transcriptomes becoming recently available (Phytozome v.12.1, Goodstein et al. (2012)), the door is open for better understanding of convergent evolution of CCMs and other parallel drought adaptations in these species.