Compartmentation of photosynthesis gene expression in C4 maize depends on time of day

Abstract

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 co-expression 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 as well as peaks in cell-specific C4 gene expression coincident with maximum rates of photosynthesis in the maize leaf at midday.

Investigating thecis-Regulatory Basis of C3and C4Photosynthesis in Grasses at Single-Cell Resolution

“…We investigated the variable enzymes that give each C 4 subtype its unique properties by focusing on two species (S. bicolor and Z. mays) from the NADP-ME subtype (Figure 3A). As expected, chromatin accessibility bias was observed for enzymes previously reported as having cell-type-specific expression patterns, similarly to the core C 4 enzyme set (23,29). Reassuringly, one of the most biased enzymes identified was NADP-ME, the key enzyme of the redox step in NADP-ME subtypes.…”

Single nuclei sequencing reveals C4photosynthesis is based on rewiring of ancestral cell identity networks

“…Previous studies have identified a small number of cis - and trans -regulatory mechanisms that control the specific expression of C 4 genes in mesophyll and bundle sheath cells (Marshall et al, 1997; Gowik et al, 2004; Akyildiz et l., 2007; Brown et al, 2011; Williams et al, 2016; Reyna-Llorens et al, 2018; Borba et al, 2023). The expression patterns driven by these cis -regulatory sequences in C 3 and C 4 species suggest that rewiring of ancestral gene regulatory mechanisms underlies the evolution of cell type specific gene expression required for C 4 photosynthesis.…”

“…For example, DOF transcription factor binding sites were enriched in chromatin regions that were specifically accessible in bundle sheath cells of both rice and sorghum. Supporting this, transient expression assays in C4 maize have shown that DOF transcription factors are able to activate the expression of NADP-ME and PEPCK, two genes that are specifically expressed in the bundle sheath (Borba et al, 2023). Thus, in contrast to the low level of conservation of transcriptional identities detected for the bundle sheath between C3 rice and C4 sorghum, it appears that the cisregulatory landscape regulating cell-type specific expression is conserved between the two species.…”

“…In eukaryotes, the regulation of gene expression in distinct cell types and in response to endogenous and environmental signals is encoded by cis -regulatory elements, the binding sites of transcription factors (Marand et al, 2017). In C 4 leaves mechanical and cell digestion approaches have provided insight into the patterning of gene expression in mesophyll and bundle sheath strands (Markelz et al, 2003; Covshoff et al, 2013; John et al, 2014; Burgess et al, 2019; Borba et al, 2023) and a small number of cis -regulatory mechanisms controlling cell specific expression of C 4 genes have been identified (Gowik et al, 2004; Brown et al, 2011; Williams et al, 2016; Reyna-Llorens et al, 2018; Burgess et al, 2019). However, in almost all studies of C 4 leaves, only bundle sheath strands have been examined, which contain phloem and xylem cells as well as the bundle sheath (Burgess et al, 2019, Borba et al, 2023).…”

“…In C 4 leaves mechanical and cell digestion approaches have provided insight into the patterning of gene expression in mesophyll and bundle sheath strands (Markelz et al, 2003; Covshoff et al, 2013; John et al, 2014; Burgess et al, 2019; Borba et al, 2023) and a small number of cis -regulatory mechanisms controlling cell specific expression of C 4 genes have been identified (Gowik et al, 2004; Brown et al, 2011; Williams et al, 2016; Reyna-Llorens et al, 2018; Burgess et al, 2019). However, in almost all studies of C 4 leaves, only bundle sheath strands have been examined, which contain phloem and xylem cells as well as the bundle sheath (Burgess et al, 2019, Borba et al, 2023). Separation of bundle sheath cells from C 3 leaves has been even more challenging (Aubry et al, 2014) and so molecular mechanisms allowing the rewiring of gene expression during the evolution from C 3 to C 4 photosynthesis are poorly understood.…”