C4 photosynthesis has arisen from the ancestral C3 state in over sixty lineages of angiosperms. It is widely accepted that an early step in C4 evolution is associated with the appearance of so-called C2 photosynthesis caused by loss of glycine decarboxylase activity from mesophyll cells followed by activation in the bundle sheath. Although changes in cis to a distal enhancer upstream of the P-subunit of GLYCINE DECARBOXYLASE (GLDP) from C2 Moricandia enable loss of expression from mesophyll cells, the mechanism then allowing GLDP expression in the bundle sheath is not known. Here we identify a MYC-MYB transcription factor module previously associated with the control of glucosinolate biosynthesis as the basis of this foundational event in the evolution of C2 photosynthesis. Specifically, we find that in the C3 state this MYC-MYB module already patterns GLDP expression to bundle sheath cells. As a consequence, when GLDP expression is lost from the mesophyll, the MYC-MYB dependent expression in the bundle sheath is revealed. Evolution of C2 photosynthesis is thus associated with a MYC-MYB based transcriptional network already present in the C3 state. This work identifies a molecular genetic mechanism underlying the bundle sheath accumulation of glycine decarboxylase required for C2 photosynthesis and thus a fundamental step in the evolution of C4 photosynthesis.

Chloroplast biogenesis is dependent on master regulators from the GOLDEN2-LIKE (GLK) family of transcription factors. However, glk mutants contain residual chlorophyll, indicating that other proteins must be involved. Here, we identify MYB-related transcription factors as regulators of chloroplast biogenesis in the liverwort Marchantia polymorpha and angiosperm Arabidopsis thaliana. In both species, double-mutant alleles in MYB-related genes show very limited chloroplast development, and photosynthesis gene expression is perturbed to a greater extent than in GLK mutants. Genes encoding enzymes of chlorophyll biosynthesis are controlled by MYB-related and GLK proteins, whereas those allowing CO2 fixation, photorespiration, and photosystem assembly and repair require MYB-related proteins. Regulation between the MYB-related and GLK transcription factors appears more extensive in A. thaliana than in M. polymorpha. Thus, MYB-related and GLK genes have overlapping as well as distinct targets. We conclude that MYB-related and GLK transcription factors orchestrate chloroplast development in land plants.

Chloroplast biogenesis is dependent on master regulators from the GOLDEN2-LIKE (GLK) family of transcription factors, but glk mutants contain residual chlorophyll and therefore other proteins must also be involved. Here we identify MYB-related transcription factors as regulators of chloroplast biogenesis in the liverwort Marchantia polymorpha and angiosperm Arabidopsis thaliana . In both species, double mutant alleles in MYB-Related genes show very limited chloroplast development, and photosynthesis gene expression is perturbed to a greater extent than in mutants of GLK. In M. polymorpha MYB-related genes act upstream of GLK, while in A. thaliana this relationship has been rewired. In both species, genes encoding enzymes of chlorophyll biosynthesis are controlled by MYB-related and GLK proteins whilst those allowing CO2 fixation, photorespiration and photosystem assembly and repair require the MYB-related proteins. Thus, MYB-related and GLK genes have overlapping as well as distinct targets. We conclude that together MYB-related and GLK transcription factors orchestrate chloroplast development in land plants.### Competing Interest StatementThe authors have declared no competing interest.

Leaves of shade-avoiding plants such as Arabidopsis (Arabidopsis thaliana) change their growth pattern and position in response to low red to far-red ratios (LRFRs) encountered in dense plant communities. Under LRFR, transcription factors of the phytochrome-interacting factor (PIF) family are derepressed. PIFs induce auxin production, which is required for promoting leaf hyponasty, thereby favoring access to unfiltered sunlight. Abscisic acid (ABA) has also been implicated in the control of leaf hyponasty, with gene expression patterns suggesting that LRFR regulates the ABA response. Here, we show that LRFR leads to a rapid increase in ABA levels in leaves. Changes in ABA levels depend on PIFs, which regulate the expression of genes encoding isoforms of the enzyme catalyzing a rate-limiting step in ABA biosynthesis. Interestingly, ABA biosynthesis and signaling mutants have more erect leaves than wild-type Arabidopsis under white light but respond less to LRFR. Consistent with this, ABA application decreases leaf angle under white light; however, this response is inhibited under LRFR. Tissue-specific interference with ABA signaling indicates that an ABA response is required in different cell types for LRFR-induced hyponasty. Collectively, our data indicate that LRFR triggers rapid PIF-mediated ABA production. ABA plays a different role in controlling hyponasty under white light than under LRFR. Moreover, ABA exerts its activity in multiple cell types to control leaf position.

ABSTRACT C 4 photosynthesis has arisen from the ancestral C 3 state in over sixty lineages of angio-sperms. It is widely accepted that an early step in C 4 evolution is restriction of glycine decarboxylase activity to bundle sheath cells to generate the so-called C 2 pathway. In C 2 Moricandia species, changes to the cis -regulatory region controlling expression of the P-subunit of GLYCINE DECARBOXYLASE ( GLDP ) in mesophyll cells enables this trait, but the mechanism underpinning GLDP expression in the bundle sheath is not known. We identify a MYC-MYB transcription factor module previously associated with the control of glucosinolate bio-synthesis as the basis of GLDP expression in bundle sheath cells. In C 3 Arabidopsis thaliana this module drives GLDP expression in bundle sheath cells along with as yet unidentified factors driving expression in mesophyll cells. In the C 2 species Moricandia arvensis, GLDP expression is lost from mesophyll cells and the MYC-MYB dependent expression in the bundle sheath is revealed. Evolution of C 2 photosynthesis is thus associated with a MYC-MYB based transcriptional network already present in the C 3 state. This work identifies a molecular genetic mechanism underlying the bundle sheath accumulation of glycine decarboxylase required for C 2 photosynthesis and thus a foundational step in the evolution of C 4 photosynthesis.

C4 photosynthesis has evolved by repurposing enzymes found in C3 plants. Compared with the ancestral C3 state, accumulation of C4 cycle proteins is enhanced. We used de-etiolation of C4Gynandropsis gynandra and C3Arabidopsis thaliana to understand this process. C4 gene expression and chloroplast biogenesis in G. gynandra were tightly coordinated. Although C3 and C4 photosynthesis genes showed similar induction patterns, in G. gynandra, C4 genes were more strongly induced than orthologs from A. thaliana. In vivo binding of TGA and homeodomain as well as light-responsive elements such as G- and I-box motifs were associated with the rapid increase in transcripts of C4 genes. Deletion analysis confirmed that regions containing G- and I-boxes were necessary for high expression. The data support a model in which accumulation of transcripts derived from C4 photosynthesis genes in C4 leaves is enhanced because modifications in cis allowed integration into ancestral transcriptional networks.

Compared with the ancestral C 3 state, C 4 photosynthesis enables higher rates of photosynthesis as well as improved water and nitrogen use efficiencies. In both C 3 and C 4 plants rates of photosynthesis increase with light intensity and so are maximal around midday. We report that in the absence of light or temperature fluctuations, photosynthesis in maize peaks in the middle of the subjective photoperiod. To investigate molecular processes associated with these changes, we undertook RNA-sequencing of maize mesophyll and bundle sheath strands over a 24-hour time-course. Cell-preferential expression of C 4 cycle genes was strongest between six and ten hours after dawn when rates of photosynthesis were highest. For the bundle sheath, DNA motif enrichment and gene co-expression analyses suggested members of the DOF and MADS-domain transcription factor families mediate diurnal fluctuations in C 4 gene expression, and trans -activation assays in planta confirmed their ability to activate promoter fragments from bundle sheath expressed genes. The work thus identifies transcriptional regulators as well as peaks in cell-specific C 4 gene expression coincident with maximum rates of photosynthesis in the maize leaf at midday.

AbstractChloroplast biogenesis is dependent on master regulators from the GOLDEN2-LIKE (GLK) family of transcription factors, butglkmutants contain residual chlorophyll and therefore other proteins must also be involved. Here we identify MYB-related transcription factors as regulators of chloroplast biogenesis in the liverwortMarchantia polymorphaand angiospermArabidopsis thaliana. In both species, double mutant alleles in MYB-Related genes show very limited chloroplast development, and photosynthesis gene expression is perturbed to a greater extent than in mutants of GLK. InM. polymorphaMYB-related genes act upstream of GLK, while inA. thalianathis relationship has been rewired. In both species, genes encoding enzymes of chlorophyll biosynthesis are controlled by MYB-related and GLK proteins whilst those allowing CO2fixation, photorespiration and photosystem assembly and repair require the MYB-related proteins. Thus,MYB-relatedand GLK genes have overlapping as well as distinct targets. We conclude that together MYB-related and GLK transcription factors orchestrate chloroplast development in land plants.

Compared with the ancestral C3 state, C4 photosynthesis occurs at higher rates with improved water and nitrogen use efficiencies. In both C3 and C4 plants, rates of photosynthesis increase with light intensity and are maximal around midday. We determined that in the absence of light or temperature fluctuations, photosynthesis in maize (Zea mays) peaks in the middle of the subjective photoperiod. To investigate the molecular processes associated with these temporal changes, we performed RNA sequencing of maize mesophyll and bundle sheath strands over a 24-h time course. Preferential expression of C4 cycle genes in these cell types was strongest between 6 and 10 h after dawn when rates of photosynthesis were highest. For the bundle sheath, DNA motif enrichment and gene coexpression analyses suggested members of the DNA binding with one finger (DOF) and MADS (MINICHROMOSOME MAINTENANCE FACTOR 1/AGAMOUS/DEFICIENS/Serum Response Factor)-domain transcription factor families mediate diurnal fluctuations in C4 gene expression, while trans-activation assays in planta confirmed their ability to activate promoter fragments from bundle sheath expressed genes. The work thus identifies transcriptional regulators and peaks in cell-specific C4 gene expression coincident with maximum rates of photosynthesis in the maize leaf at midday.

In C4 plants, the enzymatic machinery underpinning photosynthesis can vary, with, for example, three distinct C4 acid decarboxylases being used to release CO2 in the vicinity of RuBisCO. For decades, these decarboxylases have been used to classify C4 species into three biochemical sub-types. However, more recently the notion that C4 species mix and match C4 acid decarboxylases has increased in popularity and, as a consequence, the validity of specific biochemical sub-types has been questioned. Using five species from the grass tribe Paniceae, we show that, while in some species transcripts encoding multiple C4 acid decarboxylases accumulate, in others, transcript abundance and enzyme activity is almost entirely from one decarboxylase. In addition, the development of a bundle sheath isolation procedure for a close C3 species in the Paniceae enables the preliminary exploration of C4 sub-type evolution.

C 4 photosynthesis evolved repeatedly from the ancestral C 3 state, improving photosynthetic efficiency by ~50%. In most C 4 lineages, photosynthesis is compartmented between mesophyll and bundle sheath cells, but how gene expression is restricted to these cell types is poorly understood. Using the C 3 model Arabidopsis thaliana , we identified cis -elements and transcription factors driving expression in bundle sheath strands. Upstream of the bundle sheath preferentially expressed MYB76 gene, we identified a region necessary and sufficient for expression containing two cis -elements associated with the MYC and MYB families of transcription factors. MYB76 expression is reduced in mutant alleles for these transcription factors. Moreover, downregulated genes shared by both mutants are preferentially expressed in the bundle sheath. Our findings are broadly relevant for understanding the spatial patterning of gene expression, provide specific insights into mechanisms associated with the evolution of C 4 photosynthesis and identify a short tuneable sequence for manipulating gene expression in the bundle sheath.

Temperature is a key environmental variable influencing plant growth and survival. Protection against high temperature stress in eukaryotes is coordinated by heat shock factors (HSFs), transcription factors that activate the expression of protective chaperones such as HEAT SHOCK PROTEIN 70 (HSP70); however, the pathway by which temperature is sensed and integrated with other environmental signals into adaptive responses is not well understood. Plants are exposed to considerable diurnal variation in temperature, and we have found that there is diurnal variation in thermotolerance in Arabidopsis thaliana, with maximal thermotolerance coinciding with higher HSP70 expression during the day. In a forward genetic screen, we identified a key role for the chloroplast in controlling this response, suggesting that light-induced chloroplast signaling plays a key role. Consistent with this, we are able to globally activate binding of HSFA1a to its targets by altering redox status in planta independently of a heat shock.

Daphne Ezer, Samuel J.K. Shepherd, Anna Brestovitsky, Patrick Dickinson, Sandra Cortijo, Varodom Charoensawan, Mathew S. Box, Surojit Biswas, Katja E. Jaeger, and Philip A. Wigge Sainsbury Laboratory, University of Cambridge, Cambridge CB2 1LR, United Kingdom Department of Biochemistry, Faculty of Science, and Integrative Computational BioScience Center, Mahidol University, Bangkok 10400, Thailand Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, United Kingdom

Plants have significantly more transcription factor (TF) families than animals and fungi, and plant TF families tend to contain more genes; these expansions are linked to adaptation to environmental stressors. Many TF family members bind to similar or identical sequence motifs, such as G-boxes (CACGTG), so it is difficult to predict regulatory relationships. We determined that the flanking sequences near G-boxes help determine in vitro specificity but that this is insufficient to predict the transcription pattern of genes near G-boxes. Therefore, we constructed a gene regulatory network that identifies the set of bZIPs and bHLHs that are most predictive of the expression of genes downstream of perfect G-boxes. This network accurately predicts transcriptional patterns and reconstructs known regulatory subnetworks. Finally, we present Ara-BOX-cis (araboxcis.org), a Web site that provides interactive visualizations of the G-box regulatory network, a useful resource for generating predictions for gene regulatory relations.