Molecular investigation of signalling components in sugar sensing and defence in Arabidopsis thaliana

Abstract

As sessile organisms, plants are continually exposed to signals from environmentally adverse conditions and internal developmental cues. Gathering this information, processing it, and generating a proper physiological response is determinant in plant growth and fitness. Synchronized responses are mediated by phytohormones through the whole plant, as well as small peptide hormones over shorter distances via cell-cell communication. Phytohormones are able to diffuse through the plasma membrane and are perceived by their corresponding intracellular receptors, while peptide hormones are not permeable and interact with receptors localized on the plasma membrane to trigger intracellular downstream transduction cascades. In addition, nutrients and especially sugars are also known to be strong signalling molecules engaged in regulation of many aspects of plant developmental and physiological processes. The work presented in this thesis has been divided into three parts: 1) Identification and expression analysis of the INFLORESCENCE DEFICIENT IN ABSCISSION (IDA)/IDA-LIKE (IDL) and PAMP-INDUCED SECRETED PEPTIDE (PIP)/PIP-LIKE (PIPL) gene families in Arabidopsis, 2) Functional characterization of three PIP peptides and their role in regulation of plant immunity, and 3) Functional analysis of an LRR-RLK and its role in sensing of extracellular sugars status and regulation of growth in Arabidopsis. In part one, a survey of the Arabidopsis genome for genes encoding peptides with similarity to IDA and IDA-LIKE1-5 identified 14 new genes, including three new IDA-LIKE members and eleven genes constituting a new family termed PAMPINDUCED SECRETED PEPTIDEs (PIP)/PIP-LIKE (PIPL). All encoded peptides harbor an N-terminal signal peptide for the secretory pathway and a C-terminal conserved SGPS motif that is believed to function as part of final active peptide. Transcriptional analysis suggests that the new identified genes can be divided into two groups; genes that are involved in growth and development and stress inducible genes. Exogenous application of C-terminal region of PIP-LIKE 3 followed by microarray analysis revealed differential expression of genes involved in defence responses and cell wall modification. In the second part, we focused on three members of PIP/PIPL family: PIP1, PIP2 and PIP3. In silico analysis showed a high degree of co-regulation between these genes. Expression of PIP genes was induced strongly by flg22 and Botrytis cinerea treatments, indicating a possible role in regulation of plant immunity. Transcriptional profiling of seedlings treated with synthetic peptides encompassing the conserved C-terminal region of PIP1 and PIP2 showed that many genes involved in JA and SA biosynthesis and signalling as well as redox homeostasis related genes were differentially regulated. However, a set of starvation condition marker genes such DINs and bZIP11 were also among the regulated genes. Phenotype assays using pathogens with different life styles (Pst DC3000 and B. cinerea) showed that transgenic plants over-expressing PIP3 are more susceptible to both pathogens compared to wt and pip3 plants. Gene expression analysis upon B. cinerea infection showed that genes involved in JA and SA biosynthesis and signalling in PIP3 over-expression background are up-regulated compared to wt and pip3 plants. PIP1 and PIP3 over-expression plants also showed altered phenotype when grown on high concentrations of sucrose, indicating a possible role in nutrient sensing and partitioning. Based on our data, we conclude that PIP3 plays a role in growth and defence trade-off, probably through modulation of nutrient partitioning towards growth and attenuation of immunity. In the last part, we have identified and functionally characterized an LRR-RLK, hereafter termed as Sugar Responsive RLK1 (SRR1) with a potential role as extracellular sugar sensor. Phenotype assays of insertion mutant lines showed that plants lacking this gene suffer from growth retardation when grown on sugar-free medium. Transcriptome analyses of plants in presence and absence of exogenous sugar in the growth medium revealed a massive transcriptional reprogramming of srr1 plants in the sugar-free medium compared to wt. Functional categorization of differentially regulated genes revealed that anabolic biological processes were repressed while catabolic pathways were induced upon starvation in the srr1 background. This global switch seems to be essential when plants enter starvation condition in order to sustain a functional metabolism. A comparative analysis of SRR1 regulated pathways to SnRK1 and TOR-kinase mediated signalling pathways exhibited a striking similarity, suggesting a role for SRR1 in sensing of the main cellular metabolite status. SRR1 overexpression plants showed increased tolerance to high concentrations of sucrose, further supporting this hypothesis. pSRR1:GUS studies and qRT-PCR analysis revealed that SRR1 is induced by exogenous sugars, with strongest effect of sucrose. Measurements of major sugars showed that starch production is impaired in srr1 plants. Based on our observations, we conclude that SRR1 is involved in sensing of extracellular sugars and connects sugar availability to growth and development, probably through regulation of main metabolite sensory machinery.