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

波动光（Fluctuating light, FL）通常与干旱胁迫（drought stress, DS）相伴发生；这种复合胁迫（FL-DS）会影响水稻在波动光与干旱胁迫响应过程中的内在水分利用效率（intrinsic water use efficiency, iWUE）模式。然而，水稻在FL-DS复合胁迫下调控内在水分利用效率的分子机制仍未被阐明。
本研究对200份水稻种质资源在20天FL-DS复合胁迫下的内在水分利用效率动力学特征进行了定量分析。通过全基因组关联分析（genome-wide association studies, GWAS），我们在光敏色素互作因子（phytochrome-interacting factor, OsPIF4）基因中鉴定到一处与新性状iWUEFL显著关联的基因变异。该基因变异在高iWUEFL亚群中呈高表达水平。
我们将OsPIF4基因中包含9个单核苷酸多态性（single nucleotide polymorphisms, SNPs）的区域划分为两种单倍型；通过原生质体瞬时转化实验发现，启动子区域v3 SNP的突变可显著降低启动子活性，且相较于其他SNPs的突变，其对波动光的敏感性最强。
与野生型（Wild Type, WT）相比，v3 SNP突变体（PIF4v3m）和OsPIF4过表达材料在FL-DS胁迫下的iWUEFL分别降低至少20%和升高25%。基于转录组分析，我们鉴定到在PIF4v3m与野生型之间表达差异最显著的两个基因：OsSAL1和OsNHX1。与野生型相比，PIF4v3m材料的腺苷3',5'-二磷酸（adenosine 3',5'-diphosphate, PAP）水平降低85%，而OsSAL1基因的表达量升高73%。
此外，本研究结果揭示，OsPIF4可通过结合OsSAL1和OsNHX1的G-box基序，分别发挥转录抑制OsSAL1与转录激活OsNHX1的双重调控作用。在FL-DS胁迫下，该调控机制可使iWUEFL相较于野生型分别提升16%（sal1突变体）和10%（OsNHX1过表达材料）。水稻OsPIF4的单倍型分布与其种质来源地的光照条件显著相关。本研究阐明了OsPIF4通过调控叶绿体逆行信号通路与液泡离子交换过程，在FL-DS胁迫下提升水稻iWUEFL中的关键作用。
整体实验设计：本提交数据包含对经FL-DS胁迫处理的OsPIF4过表达水稻叶片进行染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）的样本数据。此类实验的核心目标是探究与气孔运动关键调控因子SAL1相结合的结合位点及基因组区域。