Cloning Of Senescence-regulating Genes And Genetic Manipulation Of Leaf Senescence In Plants

Leaf senescence is the terminal phase of leaf development that involves massive programmed cell death and nutrient remobilization. The senescence process is highly coordinated and tightly regulated by genetic program, involves differential expression of thousands of senescence associated genes (SAGs). It has great impact on crop production by altering photosynthesis duration, and by affecting the nutrient remobilization efficiency. This research work is designed to decipher the underlying molecular and genetic mechanism of leaf senescence in the model plant Arabidopsis and agronomic crop tobacco. The main aim of the research work is to identify Leucine rich repeat receptor like kinase (LRR RLK) genes involved in regulation of leaf senescence and to device strategies for senescence induction systems in an agricultural setting. Two experimental projects have been designed to accomplish the desired goals;1) Screening of Arabidopsis T-DNA insertion accessions for identification of leucine-rich repeat receptor-like kinase (LRR-RLK) genes regulating leaf senescence. Receptors like kinases play important roles in plant development and defense. LRR-RLKs represent the largest subfamily of putative RLKs in plants. However, few members in this subfamily have been identified for their role in leaf senescence. In this study, reverse genetic approach was employed to identify homozygous Arabidopsis accessions for T-DNA insertion. PCR based genotyping was performed and 26 mutants were confirmed for homozygous T-DNA insertion. Arabidopsis lines with T-DNA insertions were phenotypically screened for leaf senescence. Six novels LRR-RLK genes were identified where loss of function displayed altered leaf senescence phenotypes. Based on phenotypic performance, two genes were selected for characterization and functional analysis. These genes were renamed as Senescence-associated Receptor like Kinase i.e., SRKI (At4g08850) and SRK2 (At2g37050). Knock out mutants of SRKI displayed delay leaf senescence. For gain of function analysis, SRKI was isolated from cDNA and cloned in over-expression vector pCHF3. Loss of function mutation also significantly delayed leaf senescence in SRK2. While no obvious alteration in the overall developmental process was observed, such as the timing of leaf emergence and growth. Mutants of srk-2 retained high levels of chlorophyll contents as compare to wild type (Col-0). Higher values of Fv/Fm ratios were noted in leaves of srk-2 than the wild type. For gain of function analysis, SRK2 was isolated from cDNA and cloned in over-expression vector pCHF3. Arabidopsis thaliana (Col-o) was transformed and TO seeds were collected. RNA was extracted at four different leaf senescence stages (young, fully expanded, early and late senescence) from plants of Col-0 background and srkl and srk2 mutants for expression analysis. The role of these SRKI and SRK2 in controlling leaf senesce will further be confirmed by gain of function and complementary tests. These findings will significantly contribute in understanding the genetical regulatory mechanisms of leaf senescence.2) Inducible gene expression system for leaf senescence regulation in tobacco. Inducible systems offer researchers the possibility to deregulate gene expression levels at particular stages of plant development and in particular tissues of interest. AtNAP is a transcription factor gene and a universal regulator of leaf senescence. Its sequence homologues exist in various plant species. In this study, NAP (from Arabidopsis/tobacco cDNA) genes were isolated and cloned in ethanol inducible construct. The cloning constructs consists of a small shuttle vector containing the AlcA promoter followed by a MCS and terminator (pBJ36_AlcA) and a binary vector containing the 35S promoter driving the AlcR gene (pMLBART_AlcR). Agrobacterium mediated transformation of tobacco (Nicotiana tabacum cv Hongda) was conducted with the inducible gene expression constructs harboring NAP genes. We demonstrated that direct application of ethanol or exposure of whole plants to ethanol vapour are effective means of leaf senescence induction in tobacco.Tissue culture plantlet (TO) treated with ethanol vapor induced senescence symptoms after 10 days. Leaf senescence induction was achieved with 03% ethanol as a foliar spray in controlled conditions. Treatment of a single leaf resulted in induction of senescence in the treated leaf only. Detached leaves from transgenic adult plants resulted in whole leaf senescence after 02 weeks of treatment. Homozygous transgenic lines will be generated for field studies. In this study, we described a novel strategy for manipulating leaf senescence in tobacco through the utilization of alc system and NAP based technolgy.Leaf senescence has great significance and intensively investigated in the model plant A. thaliana. Here, we focused on some basic investigation to identify LRR RLKs genes and their function in leaf senescence. In addition, basic findings from Arabidopsis have been translated into practical application for manipulating leaf senescence in agronomic crop of tobacco. We conclude that our findings will enhance the basic understanding of regulatory mechanisms of leaf senescence and development of tecnology targeting the leaf senescence trait will broaden its utility for crop improvement.