Research On The BrWRKY8 Regulation Mechanism On The Pak Choi Leaf Senescence Mediated By Exogenous Brassinolides

Pak choi(Brassica rapa subsp.chinensis),as one of the most consumed vegetables on Chinese dinner tables with high nutritional value,rich in nutrients such as ascorbic acid,glucosinolates,and sulforaphane.However,due to constraints such as storage and shelf life,harvested pak choi is prone to quality deterioration,such as yellowing and decay.The aging process induced by unfavorable post-harvest conditions has a particularly severe impact on the quality deterioration of pak choi.This paper focuses on the significant demand for "high-quality,healthy,and nutritious" products in the market and addresses key issues such as the unclear biological mechanisms of post-harvested deterioration and the lack of regulatory technology for pak choi.Based on the personalized bottleneck problem of pak choi color after harvested,the paper takes the delay of pak choi green leaf senescence by plant hormone brassinolide as the starting point,explores the regulatory mechanism of the senescence association gene Br WRKY8 by brassinolide,and reveals a new coupling mechanism of " pak choi color-preservation functional agent-leaf senescence association genes".The paper innovatively proposes a new theory for preservation and freshness of pak choi and conducts the following research:(1)The experiment was designed to investigate the effect of the hormone 2,4-epibrassinolide(EBR)on the aging process of pak choi.The optimal concentration of EBR that delays pak choi leaf senescence was selected through an equal gradient concentration treatment method.Undering 1.5 μM EBR treatment,the degree of leaf yellowing was the lightest,with the color difference values of L* and h* were significantly different from the control,and the total chlorophyll degradation level was relatively lower,with significant reductions in the degradation rates of chlorophyll a,chlorophyll b,and their derivatives.At the same time,exogenous EBR reduced the activity of chlorophyll metabolism-related enzymes,downregulated the transcription levels of aging association and chlorophyll degradation genes.In addition,some endogenous hormone contents and their synthesis metabolic pathways were also affected to varying degrees,among which the contents of jasmonic acid(JA)and abscisic acid(ABA)decreased most significantly.The results indicate that EBR treatment can delay pak choi leaf senescence to a certain extent.(2)Using the pak choi aging-related transcriptome database and RT-q PCR analysis,agingrelated transcription factors of pak choi were selected.Through KEGG(Kyoto Encyclopedia of Genes and Genomes)and GO(Gene Ontology)analysis,the WRKY transcription factor family was found to play an important role in pak choi leaf senescence,and after systematic and strict screening,six key senescence-related transcription factors(Br WRKY8,Br WRKY44,Br WRKY48,Br WRKY51,Br WRKY61,and Br WRKY75)were obtained,among which Br WRKY8 is regulated by EBR treatment and highly responsive to pak choi senescence,and its biological functions were further explored.(3)The full-length transcript of Br WRKY8 was cloned from pak choi c DNA,and its localization in the nucleus was determined through transient expression in tobacco.Spatial and temporal expression analysis revealed that Br WRKY8 was highly expressed in senescent leaves,suggesting a positive correlation with leaf senescence.To clarify the biological function of Br WRKY8,the full-length sequence was inserted into an overexpression vector containing the35 S promoter and transformed into Arabidopsis.Homozygous overexpression lines OE-19 and OE-23 were obtained through continuous self-crossing.Experimental results showed that overexpression of OE-19 and OE-23 promoted leaf senescence in Arabidopsis and reduced chlorophyll content,while increasing the expression of endogenous senescence genes,indicating that Br WRKY8 accelerated the process of leaf senescence in Arabidopsis.In addition,to reveal the downstream regulatory network of Br WRKY8,it was found through a dual-luciferase reporter gene detection system and yeast one-hybrid assay that Br WRKY8 specifically binds to W-box elements in the promoters of downstream Br SAG21 and Br SGR2,promoting their expression.In summary,exogenous EBR has an inhibitory effect on post-harvested leaf senescence and yellowing in pak choi,which is related to the decreasing in chlorophyll metabolism enzyme activity,upregulation of SAGs expression,and changes in endogenous hormone levels.Through transcriptomics and RT-q PCR analysis,it was found that the differential changes in pak choiqing WRKY transcription factors were related to the aging process and EBR treatment.By analyzing the regulatory network of the aging-promoting factor Br WRKY8,it was found that Br WRKY8 could directly activate SAGs expression and promote leaf senescence.This study revealed the molecular mechanism of brassinosteroid regulation of pak choi aging and yellowing at the molecular level.The research results provide a scientific basis and technical support for plant hormone regulation of pak choiqing post-harvest quality and storage resistance,which is of great significance for ensuring the quality and safety of agricultural products and reducing post-harvest deterioration and decay of fresh agricultural products.