Investigation Of Molecular Mechanism Of GmSARK And AtSARK-Mediated Leaf Senescence

Leaf senescence constitutes the final stage of leaf development and is afine-tuned process regulated by interplays of multiple signaling pathways. Like otherdevelopmental processes, the onset and process of leaf senescence are influenced byspecific developmental signals.Our lab has previously shown that a soybean LRR-receptor-like protein kinase(LRR-RLK) GmSARK, positively regulate soybean leaf senescence. Biochemicalstudy revealed that GmSARK is a dual-specificity protein kinase, which couldautophosphorylate on both serine/threonine and tyrosine residues in vitro. RNAinterference (RNAi)-mediated knockdown of GmSARK expression in soybeandramatically delayed senescence, while the CaMV35S-GmSARK transgenic soybean,tomato, and Arabidopsis all exhibited a stepped-up progression of leaf senescence,leading to premature death. In the GmSARK-RNAi transgenic soybean, the flowerpetals never opened, and the stigma had a curved shape, so that the plants could notpollinate and were lethal. Since no stable transgenic soybean lines could bemaintained, I took advantage of a glucocorticoid-inducible (GVG system, the induceris dexamethasone, DEX) to ectopically express GmSARK in the model plantArabidopsis. I then analyzed GmSARK-mediated leaf senescence extensively, with afocus on how several phytohormones are involved in this process.It was found that exogenous inducible overexpression of GmSARK not only ledto growth-inhibition and precocious leaf senescence, but also produced short andcurvy roots in the GVG-GmSARK transgenic seedlings. Consistent with the soybeandata, overexpression of GmSARK in the adult transgenic Arabidopsis accelerated leafsenescence, and produced abnormal floral organs, resembling those observed inmutants with constitutive ethylene-response. Additionally, quantitative RT-PCRshowed that overexpression of GmSARK induced the expression of criticalsenescence-related transcription factors, including AtNAP, NAC1, NAC2, andWRKY6.During leaf senescence, the most prominent change in cell structure is the breakdown of the chloroplast, which has been used as a biomarker for leaf senescence.Ultrastructural morphology analyses indeed revealed a reduction in thylakoids andaccumulation of huge starch grains in GmSARK-overexpressing chloroplasts.Moreover, the GUS reporter system was used to determine the features of theGmSARK promoter in Arabidopsis. The GUS assay was taken in the GmSARK-GUStransgenic Arabidopsis in the background of three individual mutants, which havesevere defects in the structure and function of chloroplasts. The results suggested apositive feedback loop in which the expression of GmSARK was reinforced by thedisintegration of the structure and function of chloroplasts resulting from itsexpression, to further promote the senescence process during leaf senescence.Gene chip analyses showed that the expression of phytohormone-related geneschanged greatly during GmSARK-induced senescence. More than half of the changedphytohormone-related genes are auxin-and ethylene-related genes. I had focused onthe roles of and relationships among cytokinin, auxin and ethylene inGmSARK-mediated senescence in the following studies.Exogenous application of cytokinin or auxin/ethylene significantly inhibited orenhanced the activities of the GmSARK promoter in GmSARK-GUS transgenicArabidopsis seedlings, respectively. Overexpression of GmSARK also reduced orenhanced the expression level of synthesis and response genes of cytokinin or auxinand ethylene, respectively. In addition, GmSARK-overexpressing seedlings werehypersensitive to exogenous auxin. I further used a synthetic auxin-induciblepromoter, DR5, to detect auxin accumulation and distribution in the transgenicseedlings. It seemed that GmSARK overexpression affected not only in vivo levels ofauxin but also the distribution of auxin in the roots of the GVG-GmSARK transgenicArabidopsis seedlings.Although exogenous application of cytokinin could strongly reduce the activityof GmSARK promoter, it could only partially restore GmSARK-induced earlysenescence. I found that exogenous application of PCIB (p-chlorophenoxyisobutyricacid, an auxin antagonist) and AVG (aminoethoxyvinylglycine, an inhibitor ofethylene biosynthesis) could effectively inhibit GmSARK-induced precocioussenescence. Interestingly, the delay in senescence produced by PCIB was effectively inhibited by exogenous ACC, while exogenous IAA had no effect on the ability ofAVG to restore the precocious leaf senescence induced by GmSARK. Consistently,mutations of either AUX1or EIN2completely reversed the GmSARK-inducedsenescence, and their flower morphology was also indistinguishable from that of themock plants. All these results suggested that auxin and ethylene are directly involvedin the positive regulation of leaf senescence, and that the enhancement of auxin onethylene biosynthesis and response could be the critical step in GmSARK-mediatedleaf senescence. I therefore proposed a model of the leaf senescence process in whichGmSARK and GmSARK-like genes participate. It was hypothesized that the balanceamong cytokinin, auxin and ethylene plays important and synergistic roles in theinitiation and process of senescence.I have identified a functional homolog of GmSARK in Arabidopsis, and named itAtSARK. Biochemical analyses proved that AtSARK is a novel dual-specificityLRR-RLK, which localizes to the plasma membrane. Quantitative RT-PCR revealedthat the expression level of AtSARK was dramatically up-regulated in either thenatural or GmSARK-induced senescence. Leaf senescence in sark-1, a T-DNAinsertional mutant of AtSARK, was significantly delayed, while inAtSARK-overexpressing transgenic Arabidopsis plants it was strongly induced.Meanwhile, the AtSARK-overexpressing transgenic Arabidopsis plants madeabnormal floral organs, resembling those observed in mutants with constitutiveethylene-response, similar to the GmSARK-overexpressing plants. Therefore weinferred that the SARKs (e.g. GmSARK and AtSARK)-mediated pathway may be awidespread mechanism in regulating leaf senescence in higher plants.Finally, I have identified a gene encoding a novel protein phosphatase, namedSSPP (Senescence Suppressed Protein Phosphatase). Quantitative RT-PCR showedthat the expression level of SSPP was dramatically down-regulated in either thenatural or SARKs-induced senescence, suggesting that SSPP may play a negative rolein leaf senescence. Constitutive overexpression of SSPP showed asenescence-delayed phenotype, it also exhibited enhanced tolerance to drought andwas supersensitive to high temperature. It is interesting that the constitutiveoverexpression of SSPP could effectively restore AtSARK-induced senescence. In conclusion, I have investigated the molecular mechanisms of GmSARK andAtSARK involved in the regulation of leaf senescence. These results makecontributions to elucidate the molecular regulatory mechanisms of the signalingpathways involved in leaf senescence, and provide new insights into the molecularbasis of leaf development.