Functional Study Of A Novel Stay-green Gene,ZjSGR,from Zoysia Japonica

The shorter green period of Zoysia japonica is becoming a prominent barrier preventing its further market promotion.Nevertheless,to date,limited genetic resources are available for zoysiagrass,and the chlorophyll degradation mechanism are far from being well-illustrated.Stay-green genes(SGRs)play crucial roles in chlorophyll degradation and also play multiple roles in plant responses to senescence and other environmental stresses.The objectives of this study were to identify the stay-green protein gene from Zoysia japonica,explore its functional roles and investigate the underlying regulatory mechanism,for purpose of providing references for further genetic breeding.Thus,ZjSGR and the 5' upstream sequence were isolated from Zoysia japonica.Furthermore,the expression characters,subcellular localization,overexpression and complementary tests,promoter activity analysis,chloroplasts ultrastructure observation and RNA-seq analysis were employed in this study.The results are as bellow:1.Using genome walking method,the cDNA and gDNA sequences were obtained and deposited in the NCBI database with accession numbers KP148820 and KP148819,respectively.A 1029 bp upstream fragment of the coding sequence was also isolated.Bioinformatics analysis revealed ZjSGR belonged to the STAY-GREEN superfamily.Multiple darkness and hormone responsive motifs were identified in the promoter sequence.2.The quantitative real-time PCR(qRT-PCR)results showed that the ZjSGR transcript was detected in all the tissues,including roots,stems,and leaves,but with the highest expression level detected in leaves.Furthermore,expression of ZjSGR could be strongly induced by senescence,darkness.ABA or MeJA treatment.3.Agrobacterium-mediated transient transformation of Nicotiana benthamiana results proved ZjSGR was localized in the chloroplasts.What's more,transient overexpression of ZjSGR could accelerate the degradation of chlorophyll in tobacco leaves.4.Using the floral dip method,ZjSGR-overexpressing Arabidopsis thaliana transgenic lines were generated.The results showed that overexpression of ZjSGR decreased chlorophyll content and impaired photosynthesis in Arabidopsis.5.ZjSGR could rescue the stay-green phenotype of nye1-1 under dark inducement suggesting ZjSGR is a functional homolog of AtNYE1 from Arabidopsis.6.Histochemical analysis revealed that strong GUS activity was found in the leaves of ZjSGRpro::GUS-overexpressing Arabidopsis seedlings.In detail,GUS signal was detected in the vein and petiole and trichomes of the leaves.After ABA,MeJA or dark inducement,stronger GUS activity was detected.7.Transmission electron microscopy(TEM)observation revealed that overexpression of ZjSGR could affect the normal development of chloroplasts or weaken its structural stability in both transient transformed tobacco and stable transformed Arabidopsis plants.8.RNA-seq analysis using ZjSGR-overexpressing Arabidopsis showed that overexpression of ZjSGR accelerated the senescence process of transgenic Arabidopsis by regulating hormone signal transduction and the expression of transcription factors.In addition,ZjSGR could also play roles in plant development and responses to environmental stresses other than chlorophyll degradation.Taken together,the results showed ZjSGR could play crucial roles in chlorophyll degradation and also multiple roles in plant development and senescence.This study provides a better understanding of the roles of SGRs,and new insight into the chlorophyll degradation and senescence mechanisms in plants.