Induction of C4 Genes Evolved Through Changes in Cis Allowing Integration Into Ancestral C3 Gene Regulatory Networks Pallavi
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bioRxiv preprint doi: https://doi.org/10.1101/2020.07.03.186395; this version posted January 13, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Induction of C4 genes evolved through changes in cis allowing integration into ancestral C3 2 gene regulatory networks 3 4 5 6 7 Pallavi Singh#, Sean R. Stevenson#, Ivan Reyna-Llorens, Gregory Reeves, Tina B. Schreier and 8 Julian M. Hibberd* 9 10 11 Department of Plant Sciences, University of Cambridge, Downing street, Cambridge CB2 3EA, 12 United Kingdom. 13 14 *Correspondence to [email protected] 15 #Both authors contributed equally 16 17 KEY WORDS 18 C4 photosynthesis, evolution, light, de-etiolation, transcriptome, DNaseI-SEQ, cis-elements, light- 19 responsive elements, Gynandropsis gynandra. 20 21 RUNNING TITLE: The transcriptional landscape during induction of C4 photosynthesis. 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.03.186395; this version posted January 13, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 22 ABSTRACT 23 C4 photosynthesis has evolved independently in over sixty lineages and in so doing repurposed 24 existing enzymes to drive a carbon pump that limits the RuBisCO oxygenation reaction. In all 25 cases, gene expression is modified such that C4 proteins accumulate to levels matching those of 26 the photosynthetic apparatus. To better understand this rewiring of gene expression we undertook 27 RNA- and DNaseI-SEQ on de-etiolating seedlings of C4 Gynandropsis gynandra, which is sister to 28 C3 Arabidopsis. Changes in chloroplast ultrastructure and C4 gene expression were coordinated 29 and rapid. C3 photosynthesis and C4 genes showed similar induction patterns, but C4 genes from 30 G. gynandra were more strongly induced than orthologs from Arabidopsis. A gene regulatory 31 network predicted transcription factors operating at the top of the de-etiolation network, including 32 those responding to light, act upstream of C4 genes. Light responsive elements, especially G-, E- 33 and GT-boxes were over-represented in accessible chromatin around C4 genes. Moreover, in vivo 34 binding of many G-, E- and GT-boxes was detected. Overall, the data support a model in which 35 rapid and robust C4 gene expression following light exposure is generated through modifications in 36 cis to allow integration into high-level transcriptional networks including those underpinned by 37 conserved light responsive elements. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.03.186395; this version posted January 13, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 38 INTRODUCTION 39 Photosynthesis fuels most life on Earth and in the majority of land plants Ribulose 1,5- 40 bisphosphate Carboxylase Oxygenase (RuBisCO) catalyses the initial fixation of atmospheric 41 carbon dioxide (CO2) to generate phosphoglyceric acid. However, oxygen (O2) can competitively 42 bind to the RuBisCO active site to form a toxic product 2-phosphoglycolate (Bowes, Ogren, and 43 Hageman 1971). Although 2-phosphoglycolate can be metabolised by photorespiration (Bauwe, 44 Hagemann, and Fernie 2010; Tolbert and Essner 1981) this takes place at the expense of carbon 45 and energy and it is thought that the evolution of carbon concentrating mechanisms allowed rates 46 of photorespiration to be reduced. C4 photosynthesis is one such example and is characterised by 47 compartmentation of photosynthesis, typically between mesophyll and bundle sheath cells (Hatch 48 1987). This compartmentalisation involves cell-preferential gene expression and allows increased 49 concentrations of CO2 to be supplied to RuBisCO sequestered in bundle sheath cells (Furbank 50 2011). Rather than RuBisCO initially fixing carbon, in C4 species fixation is initiated by - 51 phosphoenolpyruvate carboxylase (PEPC) combining HCO3 to form a C4 acid in the mesophyll. 52 Diffusion of C4 acids into bundle sheath cells and subsequent decarboxylation results in elevated 53 partial pressures of CO2 around RuBisCO facilitating efficient carboxylation and reducing the 54 requirement for significant rates of photorespiration. C4 photosynthesis results in higher water and 55 nitrogen use efficiencies compared with the C3 state, particularly in dry and hot climates. C4 crops 56 of major economic importance include maize (Zea mays), sugarcane (Saccharum officinarum), 57 sorghum (Sorghum bicolor), pearl millet (Pennisetum glaucum) and finger millet (Setaria italica) 58 (Sage and Zhu 2011). Although C4 photosynthesis is a complex trait characterized by changes in 59 anatomy, biochemistry and gene expression (Hatch 1987) it has evolved convergently from C3 60 ancestors in more than sixty independent lineages that together account for ~8,100 species (Sage 61 2016). Parsimony would imply that gene networks underpinning this system are derived from those 62 that operate in C3 ancestors. 63 Compared with the ancestral C3 state, expression of genes encoding components of the C4 64 pathway are up-regulated, and restricted more precisely to either mesophyll or bundle sheath cells. 65 Our present understanding of these changes to C4 gene regulation is mostly based on studies 66 designed to understand the regulation of individual C4 genes (Hibberd and Covshoff 2010). For 67 example, a number of cis-regulatory motifs controlling the cell preferential expression of C4 genes 68 have been identified (Brown et al. 2011; Burnell, Suzuki, and Sugiyama 1990; Glackin and Grula 69 1990; Gowik et al. 2004; Kajala et al. 2012; Reyna-Llorens et al. 2018; Williams et al. 2016). Whilst 70 some cis-elements appear to have evolved de novo in C4 genes to pattern their expression 71 (Matsuoka et al. 1994; Nomura et al. 2000, 2005; Stockhaus et al. 1997), others appear to have 72 been recruited from pre-existing elements present in C3 orthologs (Brown et al. 2011; Kajala et al. 73 2012; Reyna-Llorens et al. 2018; Williams et al. 2016) and in these cases, there is evidence that 74 individual cis-elements are shared between multiple C4 genes. In contrast to the analysis of 75 regulators of cell specific expression, there is much less work on mechanisms that underpin the 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.03.186395; this version posted January 13, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 76 up-regulation and response to light of genes important for the C4 pathway compared with the 77 ancestral C3 state. For example, whilst many C4 pathway components and their orthologs in C3 78 species show light-dependant induction (Burgess et al. 2016) the mechanisms driving these 79 patterns are still largely unknown. One possibility is that cis-elements referred to as Light 80 Responsive Elements (LREs) that have been characterized in photosynthesis genes in C3 plants 81 (Acevedo-Hernández, León, and Herrera-Estrella 2005; Argüello-Astorga and Herrera-Estrella 82 1996) are acquired by genes of the core C4 pathway. The response of a seedling to light is the first 83 major step towards photosynthetic maturity, but how C4 genes are integrated into this process is 84 poorly understood. Seedlings exposed to prolonged darkness develop etioplasts in place of 85 chloroplasts (Pribil, Labs, and Leister 2014). Etioplasts lack chlorophyll but contain membranes 86 composed of a paracrystalline lipid–pigment–protein structure known as the prolamellar body 87 (Bahl, Francke, and Monéger 1976). De-etiolation of seedlings therefore marks the initiation of 88 photosynthesis and presents a good model system to study the dynamics and regulatory 89 mechanisms governing photosynthesis in both C3 and C4 species. 90 Here, we used a genome-wide approach to better understand the patterns of transcript 91 abundance and potential regulatory mechanisms responsible for these behaviours underpinning C4 92 photosynthesis. We integrate chromatin accessibility, predicted transcription factor binding sites, as 93 well as binding evidence obtained in vivo and expression data to show that C4 pathway 94 components in Gynandropsis gynandra, the most closely related C4 species to Arabidopsis 95 thaliana, are connected to the top of the de-etiolation regulatory network. Photomorphogenic and 96 chloroplast-related transcription factors were up-regulated upon exposure to light and showed 97 highly conserved dynamics compared with orthologs from C3 Arabidopsis. Further analysis using 98 an analogous dataset from Arabidopsis allowed us to compare the extent to which regulatory 99 mechanisms are shared between the ancestral C3 and derived C4 systems. Firstly, this showed 100 that the cistrome of C4 genes from G. gynandra has diverged from C3 orthologs in Arabidopsis. The 101 C4 cistrome of G. gynandra showed an increased importance for homeodomain and Teosinte 102 branched1/Cincinnata/Proliferating cell factor (TCP)