PIF4 promotes stomatal movement during fluctuating light coincidant with drought stress via the SAL1-PAP signaling pathway (ChIP-Seq)

Fluctuating light (FL) usually occur concomitantly with drought stress (DS); this combined (FL-DS) condition influences intrinsic water use efficiency (iWUE) pattern during FL and DS response. However, the molecular mechanisms governing iWUE during FL-DS combined events in rice remain unexplored. In this study, we quantified iWUE kinetics in 200 rice accessions exposed to a 20-day FL-DS. Using genome-wide association studies (GWAS), we identified a significant gene variation in the phytochrome-interacting factor (OsPIF4) gene which significantly associated with a novel trait, iWUEFL. This gene variation was highly expressed in the high iWUEFL subgroup. Two haplotypes containing nine SNPs of OsPIF4 were classified, where the mutation of v3 SNP at its promoter shows substantially reduced activities of promoter and greatest sensitivity to FL among mutation of other SNPs via protoplast transient transformation approach. Mutation at v3 SNP (PIF4v3m) and overexpression of OsPIF4 resulted in at least 20% reduction and 25% increase in iWUEFL under FL-DS, compared it to WT, respectively. Two genes (OsSAL1 and OsNHX1) were identified with the most significant differences in expression between PIF4v3m and WT based on transcriptome analysis. PIF4v3m displayed an 85% reduction in adenosine 3', 5'-diphosphate (PAP) levels and a 73% increase in OsSAL1 gene expression compared to WT. Moreover, our results revealed that OsPIF4 has a dual role in repressing OsSAL1 transcriptionally and activating OsNHX1 by binding to their G-box motifs. This regulatory mechanism increased by 16% (sal1) and 10% (OsNHX1-OE) in iWUEFL compared to WT under FL-DS. OsPIF4 haplotypic distribution is strongly associated with the local light regime where accessions originated. This study highlights the key role of OsPIF4 in enhancing iWUEFL during FL-DS by regulating chloroplast retrograde signaling pathways and vacuolar ion exchanges. Overall design: The submission includes samples from chromatin immunoprecipitation sequencing (ChIP-seq) experiments conducted on rice leaves of PIF4 overexpression line exposed to FL-DS. The primary objective of these experiments is to investigate the binding sites and genomic regions associated with SAL1, a key regulator in stomatal movement.