Functional Characterization Of STAY-GREEN Gene In Perennial Ryegrass

Leaf yellowing is the common hallmark of leaf senescence.Stress-induced chlorophyll(Chl)and protein degradation promoted premature of leaf senescence.The promoted premature of leaf senescence not only decreased the turf quality,such as shorted the green period of canopy,but also reduced forage quality.Therefore,blocking the Chl degradation by RNA interference Chl degradation genes in perennial ryegrass(Lolium perenne L.),which can be used as turf and forage,technically could delay leaf yellowing and increase its forage quality.Analysis of the regulation mechanism of Chl degradation during leaf senescence is essential for molecular breeding process of perennial ryegrass.In this study,we cloned a SATY-GREEN gene(LpSGR)from perennial ryegrass,which functions as a master regulator in Chl degradation pathway.We also determined its function and mechanism on the hormonal-regulation of Chl degradation.The STAY-GREEN gene,LpSGR,was cloned according to the known SGR genes from rice(Oryza sativaL.)and Arabidopsis(Arabidopsis thaliana)using RACE-PCR method.Its NCBI GeneBank accession number is AQM55942.The coding sequence(CDS)of LpSGR is consisted by 864 bp of nucleotides and coding a protein contains 287 amino acids.LpSGR protein has a typical SGR motif.The genomestructure of LpSGR contains two introns and three exons,which is the same as SGR genes in brachypodium,rice,sorghum,and maize,while deferent from SGR genes in poplar(Populus spp.),soybean(Glycine max L.),Arabidopsis,and medicago(medicago truncatula),which have three introns and four exons.The CDS aliment results indicated that LpSGR shared 82%,81%,80%,78%,and 67%nucleotide sequence similarity to that of BdSGR,OsSGR,ZmSGR,SbSGR,and AtSGR respectively.Overexpression of LpSGR promoted the Chl degradation in wild tobacco(Nicotiana benthamiana)and Arabidopsis.Both LpSGR and AtSGR rescued the stay green phenotypes of leaves and seedpods of Arabidopsis sgr mutant.LpSGR was localized in chloroplast,and it was also physically interactive with other Chl degradation proteins,such as LpNOL,LpNYC1,and LpPPH,in chloroplast.These results indicated that,the same as AtSGR,LpSGR coding a Magnesium-Dechelatase,which not only catalyzes the degradation of Chl a to pheophytin a,but also regulates Chl breakdown pathway through physicially interactive with orther Chl-catabolism enzymes(CCEs).The transcription of LpSGR was positively regulated by the senescence signaling,which was manifested by its relative expression level was almost 1000 folder higher in senescent leaf than that in expanding leaf.Transcription factors in ABA and ethylene signaling pathway,such as LpABF3,LpABI5,ORE1,and EIN3,can enhanced the activity of LpSGR's promoter by directly binding to its promoter,and further increased the transcription of LpSGR.Transcription factors in CK signaling pathway,such as LpARRl,LpARR10,and LpARR12,can't directly recognize the promoter of LpSGR,but they can physically interactive with ABA signaling pathway transcription factors,such as LpABF3 and LpABI5.Therefore,CK may inhibited the expression of LpSGR by indirectly regulated its promoter's activity through the interaction with LpABF3 and LpABI5.Melatonin treatment delayed dark-and heat-induced leaf yellowing by directly or in directly regulated the expression of LpSGR.RNA interference of LpSGR blocked the Chl degradation pathway in perennial ryegrass.Under dark treatment,Chl contents in transgenic lines were significantly higher than that in wild type.SGR protein can combined with the light harvesting complex(LHC)proteins;therefore,RNA interference of LpSGR can delayed the degradation of LHC proteins.Compared to wild type,the crud protein content,in vitro dry matter digestibility(IVTDMD),and total fat content were significantly higher in transgenic lines.The Magnesium(Mg)content,acid detergent fiber(ADF),neutral detergent fiber(NDF),and crude ash(ASH)content were significant lower in transgenic lines than that in wild type.These results indicated that LpSGR not only regulates Chl degradation,but also affects the forage quality in perennial ryegrass during leaf senescence.The transcriptomic changes in RNAi:LpSGR transgenic lines and wild type indicatethat RNA interference of LpSGR altered the transcription of genes which involved in plant hormone signaling pathways.The changed plant hormone signaling pathways may delay leaf senescencein transgenic lines.The transcription of ABA,ethylene,and JA signaling genes were significantly higher in senescent leaves than mature leaves in wild type perennial ryegrass.B-ARRs and A-ARRs are two types of transcription factors in CK signaling pathway were also up-regulated during leaf senescence.However,the transcription of ABA,ethylene,and JA signaling genes were significantly down-regulated in transgenic lines during leaf senescence.These results were consisted with the phenotypic and physiological changes of transgenic lines and wild type during leaf senescence.RNA interference of LpSGR also altered the expression of photosynthesis,protein synthesis,lipid metabolism,and saturated or/and unsaturated fatty acid metabolism related genes.These changes may lead to the increase of crud protein content and forage quality in RNAi:LpSGR transgenic lines.This study provided afundamental knowledge on manipulating STAY-GREEN genes for molecular breeding of perennial ryegrass.