ABF2, ABF3, and ABF4 Promote ABA-Mediated Chlorophyll Degradation and Leaf Senescence by Transcriptional Activation of Chlorophyll Catabolic Genes and Senescence-Associated Genes in Arabidopsis

Chlorophyll （Chl） degradation is an integral process of leaf senescence, and NYE1/SGR1 has been demonstrated as a key regulator of Chl catabolism in diverse plant species. In this study, using yeast one-hybrid screening, we identified three abscisic acid （ABA）-responsive element （ABRE）-binding transcription factors, ABF2 （AREB1）, ABF3, and ABF4 （AREB2）, as the putative binding proteins of the NYE1 promoter. Through the transactivation analysis, electrophoretic mobility shift assay, and chromatin immunoprecipitation, we demonstrated that ABF2, ABF3, and ABF4 directly bound to and activated the NYE1 promoter in vitro and in vivo. ABA is a positive regulator of leaf senescence, and exogenously applied ABA can accelerate Chl degradation. The triple mutant of the ABFs, abf2abf3abf4, as well as two ABA-insensitive mutants, abil-1 and snrk2.2/2.3/2.6, exhibited stay-green phenotypes after ABA treatment, along with decreased induction of NYE1 and NYE2 expression. In contrast, overexpression of ABF4 accelerated Chl degradation upon ABA treatment. Interestingly, ABF2/3/4 could also activate the expression of two Chl catabolic enzyme genes, PAO and NYCl, by directly binding to their promoters. In addition, abf2abf3abf4 exhibited a functional stay-green phenotype, and senescence-associated genes （SAGs）, such as SAG29 （SWEET15）, might be directly regulated by the ABFs. Taken together, our results suggest that ABF2, ABF3, and ABF4 likely act as key regulators in mediating ABA-triggered Chl degradation and leaf senescence in general in Arabidopsis.

Age-Triggered and Dark-Induced Leaf Senescence Require the bHLH Transcription Factors PIF3, 4, and 5

Leaf senescence can be triggered and promoted by a large number of developmental and environmental fac- tors. Numerous lines of evidence have suggested an involvement of phytochromes in the regulation of leaf senescence, but the related signaling pathways and physiological mechanisms are poorly understood. In this study, we initially identi- fied phytochrome-interacting factors （PIFs） 3, 4, and 5 as putative mediators of leaf senescence. Mutations of the PIF genes resulted in a significantly enhanced leaf longevity in age-triggered and dark-induced senescence, whereas overexpressions of these genes accelerated age-triggered and dark-induced senescence in Arabidopsis. Consistently, loss-of-function of PIF4 attenuated dark-induced transcriptional changes associated with chloroplast deterioration and reactive oxygen species （ROS） generation. ChlP-PCR and DualoLuciferase assays demonstrated that PIF4 can activate chlorophyll degradation regulatory gene NYE1 and repress chloroplast activity maintainer gene GLK2 by binding to their promoter regions. Finally, dark-induced ethylene biosynthesis and ethylene-induced senescence were both dampened in pif4, suggesting the involvement of PIF4 in both ethylene biosynthesis and signaling pathway. Our study provides evidence that PIF3, 4, and 5 are novel positive senes- cence mediators and gains an insight into the mechanism of light signaling involved in the regulation of leaf senescence.

The NPR1-WRKY46-WRKY6 signaling cascade mediates probenazole/salicylic acid-elicited leaf senescence in Arabidopsis thaliana

Endogenous salicylic acid(SA) regulates leaf senescence, but the underlying mechanism remains largely unexplored. The exogenous application of SA to living plants is not efficient for inducing leaf senescence. By taking advantage of probenazole(PBZ)-inducedbiosynthesisof endogenous SA, we previously established a chemical inducible leaf senescence system that depends on SA biosynthesis and its core signaling receptor NPR1 in Arabidopsis thaliana. Here,using this system, we identified WRKY46 and WRKY6 as key components of the transcriptional machinery downstream of NPR1 signaling. Upon PBZ treatment, the wrky46 mutant exhibited significantly delayed leaf senescence. We demonstrate that NPR1 is essential for PBZ/SA-induced WRKY46 activation, whereas WRKY46 in turn enhances NPR1 expression. WRKY46 interacts with NPR1 in the nucleus, binding to the W-box of the WRKY6 promoter to induce its expression in response to SA signaling. Dysfunction of WRKY6 abolished PBZ-induced leaf senescence, while overexpression of WRKY6 was sufficient to accelerate leaf senescence even under normal growth conditions, suggesting that WRKY6 may serve as an integration node of multiple leaf senescence signaling pathways. Taken together,these findings reveal that the NPR1-WRKY46-WRKY6 signaling cascade plays a critical role in PBZ/SA-mediated leaf senescence in Arabidopsis.

REF6 promotes lateral root formation through de-repression of PIN1/3/7 genes

The H3K27 methyltransferase CLF inhibits lateral root(LR) formation through depositing the repressive H3K27me3 mark to the chromatin of PIN1, a key polar auxin transporter gene. Here, we show that the H3K27me3 demethylase REF6 promotes lateral root primordium initiation and LR emergence. REF6 directly binds to the chromatin of PIN1/3/7. Dysfunction in REF6 results in increased levels of H3K27me3 on PIN1/3/7 and suppressed expression of PIN genes. Genetic analysis of the clf ref6 double mutant revealed an antagonistic action between CLF and REF6, in terms of LR formation.Our findings indicate that H3K27 methylation and demethylation activities are likely coordinated to ensure proper LR organogenesis.

Reverse Genetic Identification of CRN1 and its Distinctive Role in Chlorophyll Degradation in Arabidopsis

Recent identification of NYE1/SGR1 brought up a new era for the exploration of the regulatory mechanism of Chlorophyll （Chl） degradation. Cluster analysis of senescence associated genes with putative chloroplast targeting sequences revealed several genes sharing a similar expression pattern with NYE1. Further characterization of available T-DNA insertion lines led to the discovery of a novel stay-green gene CRN1 （Co-regulated with NYE1）. Chl breakdown was significantly restrained in crnl-1 under diversified senescence scenarios, which is comparable with that in acdl-20, but much more severe than that in nyel- 1. Notably, various Chl binding proteins, especially trimeric LHCP II, were markedly retained in crnl-1 four days after dark-treatment, possibly due to a lesion in disassociation of protein-pigment complex. Nevertheless, the photochemical efficiency of PSII in crnl-1 declined, even more rapidly, two days after dark-treatment, compared to those in Col-0 and nyel-1. Our results suggest that CRN1 plays a crucial role in Chl degradation, and that loss of its function produces various side-effects, including those on the breakdown of Ch-protein complex and the maintenance of the residual photosynthetic capability during leaf senescence.