Synergistic binding of bHLH transcription factors to the promoter of the maize NADP-ME gene used in C₄ photosynthesis is based on an ancient code found in the ancestral C₃ state

Abstract

C4 photosynthesis has evolved repeatedly from the ancestral C3 state to generate a carbon concentrating mechanism that increases photosynthetic efficiency. This specialised form of photosynthesis is particularly common in the PACMAD clade of grasses, and is used by many of the world’s most productive crops. The C4 cycle is accomplished through cell-type specific accumulation of enzymes but cis-elements and transcription factors controlling C4 photosynthesis remain largely unknown. Using the NADP-Malic Enzyme (NADP-ME) gene as a model we aimed to better understand molecular mechanisms associated with the evolution of C4 photosynthesis. Two basic Helix-Loop-Helix (bHLH) transcription factors, ZmbHLH128 and ZmbHLH129, were shown to bind the C4NADP-ME promoter from maize. These proteins form heterodimers and ZmbHLH129 impairs trans-activation by ZmbHLH128. Electrophoretic mobility shift assays indicate that a pair of cis-elements separated by a seven base pair spacer synergistically bind either ZmbHLH128 or ZmbHLH129. This pair of cis-elements is found in both C3 and C4 species of the PACMAD clade. Our analysis is consistent with this cis-element pair originating from a single motif present in the ancestral C3 state. We conclude that C4 photosynthesis has co-opted an ancient C3 regulatory code built on G-box recognition by bHLH to regulate the NADP-ME gene. More broadly, our findings also contribute to the understanding of gene regulatory networks controlling C4 photosynthesis.

Genetic diversity of C4 photosynthesis pathway genes in Sorghum bicolor (L.)

“…The absence of large sequence variation at the gene level is also consistent with previous evolutionary studies suggesting that relatively minor changes to pre-existing regulatory networks and the use of pre-existing cis-elements were often sufficient to recruit genes into the C 4 pathway [43][44][45]. The C 4 isoform of the NADP-ME gene found in maize and sorghum is one such gene that has been found to be activated for C 4 photosynthesis via subtle changes to its promoter, while the rest of the gene is highly conserved [32]. This is consistent with the low diversity in this gene family observed in our study.…”

“…The recent assembly of whole-genome sequences for a wide range of wild and cultivated sorghum species [23][24][25], provides an excellent opportunity to explore genetic diversity of genes related to the C 4 photosynthetic pathway. Several high-throughput comparative transcriptomics and evolutionary studies using C 3 and C 4 phylogenetically related species and cell-specific gene expression have elucidated the key genes and regulatory networks that underpin the C 4 photosynthetic pathway [4,[26][27][28][29][30][31][32][33][34]. In the present study, we explored the genetic variation in genes that have previously been identified as core C 4 genes, mined their allelic diversity and investigated signatures of selection during domestication in sorghum.…”

Comparative transcriptomics method to infer gene coexpression networks and its applications to maize and rice leaf transcriptomes

“…We find nine TFs that may regulate BS-preferred C 4 enzyme genes, including four TFs (ZmGATA12, ZmbHLH43, ZmERF, and ZmNAC ) for ZmNADP-ME (NADP-MALIC ENZYME, Zm00001d000316), four TFs (ZmMYB48, ZmMYB88, ZmMYB56, and ZmbHLH118) for ZmPCK (PHOSPHOENOLPYRUVATE CARBOXYKINASE, Zm00001d028471), and one TF (ZmMYBr17) for ZmRBCS2 ( Table 1). The four TFs we identified for ZmNADP-ME are different from the two TFs, ZmbHLH128 (Zm00001d054038) and ZmbHLH129 (Zm00001d014995), identified by Borba et al (21), which showed no strong preferential expression in BS or M cells (20). For M-preferred C 4 enzyme genes, we find two TFs (ZmABI33 and ZmRAV) that may up-regulate ZmCA (CARBONIC ANHYDRASE, Zm00001d044099) and that ZmRAV may also up-regulate ZmPEPC (PHOSPHOENOLPYRUVATE CARBOXYLASE, Zm00001d046170) ( Table 1).…”

Genome-Wide Transcription Factor Binding in Leaves from C3 and C4 Grasses

“…Some of these conserved and cell-specific motifs have been previously described to have a relevant role in photosynthesis. For instance, in whole leaves of maize and S. italica, we found significant enrichment of the bHLH129 motif (Supplemental Data Set 11) that has been proposed to act as a negative regulator of NADP-ME (Borba et al, 2018).…”