Molecular Dissection Of Arabidopsis Light Signaling Protein FHY3 In Regulation Of Leaf Senescence

Senescence,the final stage of leaf development,is tightly regulated by numerous internal cues such as age,development,and metabolites and external environmental signals such as light,temperature,and stress.In annual plants such as Arabidopsis thaliana and many crops,leaf senescence indicates that the whole plant will soon die and allows the remobilization of nutrients into growing organs,such as young leaves,storage organs or developing seeds.Therefore,leaf senescence is critical for plant fitness and crop productivity.In general,senescence occurs in an age-dependent manner and initiates from the tip of the mature leaf.However,the initiation and process of leaf senescence are affected by variable environmental light conditions.The process of leaf senescence is promoted by a low ratio of red to far-red(R:FR)light(shade),FR light,or extended darkness and is repressed by a high ratio of R:FR light or R light.However,the precise regulatory mechanisms by which plants assess external light signals and their internal cues to initiate and control the process of leaf senescence remain largely unknown.At the molecular level,several well-studied SENESCENCE-ASSOCIATED GENES(SAGs),such as SAG12,SAG13,SAG29,SAG101,and SAG113,are strongly expressed in senescing leaves.In addition,multiple types of leaf senescence-related transcription factor genes,such as NAC and WRKY family members,are highly expressed in senescing leaves.These transcription factors control leaf senescence by regulating the transcription of different targets,including a large set of SAGs.The WRKY family is one of the two biggest transcription factor families specific in plants and multiple WRKY transcription factors have been identified as key players in the regulation of leaf senescence.Arabidopsis WRKY28,a member of WRKY transcription factor family,plays an important role in salicylic acid synthesis,plant defense response and germ-line specification,while the role of WRKY28 in leaf senescence has not been reported.Arabidopsis FAR-RED ELONGATED HYPOCOTYL3(FHY3)and its closest homolog FAR-RED IMPAIRED RESPONSE1(FAR1)are transposase-derived transcription factors that play key roles in the phytochrome A(phyA)-mediated FR light signaling pathway.Recent studies have implicated FHY3 and FAR1 in multiple cellular processes involved in growth and development.However,the physiological and molecular roles of FHY3 in leaf senescence remain unknown.In this study,we discovered that the light-signaling protein FHY3 negatively regulates age-induced and light-mediated leaf senescence in Arabidopsis and investigated the molecular mechanism of this pathway.The main results are as follows:(1)The fhy3-4 mutant exhibited early senescence compared to wild-type No-0 plants at various developmental stages(3,5,and 7 weeks old).To further explore whether the early leaf senescence of the fhy3 mutants is caused by the functional disruption of FHY3,we observed the phenotypes of FHY3pro:FHY3-YFP fhy3-4(FHY3-YFP)transgenic plants and FHY3pro:FHY3-GR fhy3-4(FHY3-GR)transgenic plants.As expected,the expression of FHY3-YFP or FHY3-GR in the fhy3-4 mutant rescued its early leaf senescence phenotype to a large extent.These results suggest that FHY3 negatively regulates age-dependent leaf senescence.(2)FHY3 negatively regulates light-and hormone-mediated leaf senescence.Phenotypic analysis showed that,compared to No-0 plants,the fhy3-4,far1-2,and fhy3-4 far1-2 mutants senesced earlier under both high and low R:FR light conditions.While,compared to the wildtype plants,the far1-2 and fhy3-4 far1-2 mutants underwent senescence earlier under high R:FR light vs.low R:FR light conditions,suggesting that FHY3 and FAR1 play important roles in light-regulated leaf senescence,especially in inhibiting senescence mediated by high R:FR light.The phenotype analysis of detached leaves treated with various hormone proved that FHY3 participates in Ethylene-,JA-and SA-induced precocious senescence.(3)FHY3 directly inhibits the expression of WRKY28 during leaf senescence.Analysis of the promoter sequence of WRKY28 revealed two typical FHY3 and FAR1 binding site(FBS;CACGCGC)cis-elements.Yeast one-hybrid assays and ChIP-qPCR assays verified the specific binding of FHY3 and FAR1 to the FBS cis-elements of the promoter of WRKY28.Transient expression assays in Arabidopsis protoplasts and qRT-PCR assay showed that the expression of WRKY28 were negatively regulated by FHY3 during the aging process.(4)WRKY28 positively regulates leaf senescence and FHY3 inhibits senescence by repressing WRKY28.We contructed the WRKY28-ox lines and found that over-expression of WRKY28 promote leaf senescence,indicating that WRKY28 is a positive regulator of leaf senescence.Moreover,we constructed wrky28 and fhy3-4 wrky28 lines using CRISPR-Cas9 system.The wrky28 mutant showed considerably delayed leaf senescence and the disruption of WRKY28 in fhy3-4 wrky28 restored the early senescence phenotype of fhy3-4 plants,indicating that FHY3 represses leaf senescence through controlling the transcript levels of WRKY28.(5)The early leaf senescence phenotype of fhy3-4 was largely restored by crossing with sid2 or S3H-ox,in which the SA contents and leaf senescence rate were altered,suggesting that FHY3 and WRKY28 regulate leaf senescence by controlling the SA contents.Moreover,the phenotype analysis of detached leaves treated with SA suggested that over-expression of WRKY28 led to a higher level of SA-induced leaf senescence compared to No-0 leaves,and the WRKY28 mutation largely rescued the precocious leaf senescence of fhy3-4 plants treated with SA.These results suggest that FHY3 represses WRKY28 expression to control SA biosynthesis,accumulation,and responses,subsequently repressing leaf senescence.(6)Phenotypic analysis showed that both FHY3 and WRKY28 were effective in mediating high R:FR or low R:FR light-regulated senescence and the FHY3-WRKY28 transcriptional regulatory module might play a predominant role in preventing leaf senescence under high R:FR light conditions.(7)qRT-PCR results showed that the expression of FHY3 and WRKY28 gradually increased throughout leaf development.To further assess the regulation of FHY3 at the posttranscriptional level,we observed LUC protein accumulation in FHY3pro:FHY3-LUC fhy3-4 transgenic plant.The results showed that FHY3 expression is induced by aging signals at both the transcriptional and post-transcriptional level.qRT-PCR analysis and phenotypic analysis revealed that the FHY3-WRKY28 transcriptional module is present at the seedling stage and plays a more important role in preventing leaf senescence under high R:FR vs.low R:FR light conditions.This study reveals the physiological and molecular functions of FHY3 and WRKY28 in leaf senescence and provides insight into the regulatory mechanism by which plants integrate dynamic environmental light signals and internal cues to initiate and control leaf senescence.