1. Introduction Drought stress and climate change Three strategies of plants in response to water stress 3

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1 Contents 1. Introduction Drought stress and climate change Three strategies of plants in response to water stress Three closely related species of Linderniaceae family are experimental model plants to study molecular bases of desiccation tolerance Protection mechanisms in response to dehydration LEA proteins and association with desiccation tolerance Carbohydrate Compatible solutes (proline) Role of ABA in abiotic stress Regulation of stress and ABA-inducible genes ABA-induced genes are bind to ABRE cis-element bzip transcription factors bzip transcription factors belonging to Sl-class bzip bzip transcription factors and dimerization capacity Association of the desiccation tolerance in seeds and the vegetative tissues during evolution Circadian clock system and association with stress Promoter analyses to study mechanisms of desiccation tolerance in C. plantagineum Promoter architecture of LEA like in C. plantagineum, L. brevidens and L. subracemosa Studying promoter function using transient transformation methods Critical factors in developing the Agrobacterium-medmted plant transformation protocol for Linderniaceae species Objectives of the study 22

2 2. Materials and Methods Materials Plant Material Buffers, solutions and media Bacteria E. coli DH10B (Lorrovv and Jessee, 1990) Agrobacterium tumefaciens GV3101/pmP90RK (Koncz and Shell 1986) Plasmid vectors pjet pbtlo-gus pgj pbin Primers (5'-3') Sequences Membranes, enzymes and markers Kits Instruments and other devices Chemicals Software, programs and online tools Methods Growth conditions L. brevidens, L. subracemosa and C. plantagineum Arabidopsis thaliana L. cv Columbia Seed sterilization and cultivation Bacterial growth conditions Bacterial glycerol stock preparation Plasmid purification 35 II

3 DNA plasmid purification in E. coli, mini- prep Plasmid DNA purification of A. tumefaciens cells plasmid DNA purification in large scale, Maxi prep Nucleic acid extraction from the plant materials Extraction of Genomic DNA from C. plantagineum Extraction of total RNA from C. plantagineum Extraction of genomic DNA from A. thaliana Extraction of total RNA from A. thaliana Purification of extracted DNA: Agarose gel electrophoresis Estimation of DNA and RNA RNA blot analyses Staining of membranes Preparation of a 32 P-dCTP hybridisation probe (Feinberg and Vogelstein, 1983) Hybridization procedures Quantitative estimation of extracted protein Cloning of DNA fragments Polymerase chain reaction (PCR) Restriction endonuclease treatments Dephosphorylation Ligation Preparation of competent cells and bacterial transformation methods Preparation of rubidium chloride competent cells for E. coli Preparation of calcium chloride competent cells for E. coli Preparation of electrocompetent cells of A. tumefaciens Bacteria transformation 46 III

4 Transformation of E. coli cells by heat shock method Transformation of A. tumefaciens cells by electroporation method Plant transformation Stable transformation of A. thaliana by floral dip method Transient transformation Agrobacterium-mediated transient transformation, FAST assay Biolistic method Vitality test Screening methods Screening of bacteria colonies Screening of transgenic plants RT-PCR analyses Site-directed mutagenesis Designing of primer and introducing of mutations PCR reaction and digestion of parental DNA Relative water content Plant stress treatments Biological and biochemical methods Proline determination Determination of chlorophyll content Lipid peroxidation assay (MDA assay) GUS expression in plant leaves or seedlings Histochemical GUS assay via tissue staining Fluorometric GUS assay via X-Gluc substrate Results 59 IV

5 3.1 Optimization of Agrobacterium-mediated transient transformation in C. plantagineum, L. brevidens and L. subracemosa Parameters optimized in Agrobacterium-mediated transient transformation of three Linderniaceae species Leaf size Silvvet concentration Bacteria density Duration of co-cultivation Preparation and cloning of the LEA-like 11-24::GUS construct from C. plantagineum, L. brevidens, L. subracemosa in pbin19 binary vector Reliability of the optimized method and transcript analysis Activity of the LEA-like promoter fragments in leaves via Agrobacteriummediated transient transformation in the homologous genetic background Activities of the LEA-like promoter fragments in a heterologous genetic background Expression of the Cp LEA-like promoter fragment Expression of Lb LEA-like promoter fragment Expression of Ls LEA-like promoter fragment Trans-activation of CpbZIPl transcription factor in Cp LEA-like GUS promoter Generation of "Cp LEA-like 11-24::GUS" and "35S::CpbZlPl+Cp LEA-like 11-24: :GUS" constructs Analysis of C. plantagineum leaves transiently transformed with ' 'Cp LEA-like 11-24::GUS" and ' 35S::CpbZlPl+Cp LEA-like 11-24::GUS" constructs Histochemical and fluorometric detection of GUS activity Viability test using Fluorescein diacetate (FDA) Transcript expression analyses of GUS gene 77 V

6 3.2.3 Trans-activation study of Cp LEA-like promoter by CpbZIPl transcription factor Generation of Arabidopsis transgenic plants containing either ' 'Cp LEA-like ] 1-24::GUS" or,"35s::bzip+cp LEA-like G(7S"cassette Screening of the transgenic lines Analysis of Cp LEA-like promoter activity in the presence or absence of CpbZIPl in Arabidopsis GUS activity in 7 day-old seedlings GUS activity in 14 day old seedlings GUS activity in 21 day old seedlings Transcript expression analyses of CpbZIPl and Cp LEA-like coding gene Expression of the CpbZIPl gene in leaves and roots of C. plantagineum exposed to various abiotic stress conditions Gene expression under dehydration condition at different time points Gene expression under ABA treatment at different time points Gene expression profiling of C. plantagineum leaves and roots subjected to elevated sodium chloride concentration Kinetic expression of the CpbZIPl and Cp LEA-like gene in C. plantagineum leaves Sequence similarity of CpbZIPl protein with other plant species Generation and molecular characterisation of transgenic plants ectopically expressing the CpbZIPl gene, (i5s::cpbzipl, S-lines) Screening of the transgenic plants overexpressing the CpbZIPl gene and correlation of CpbZIPl expression with the level of dwarfism Phenotypic analyses of S-lines plants CpbZIPl overexpressing (S lines) plants under salt stress 98 VI

7 3.4.4 CpbZIPI overexpressing (S lines) plants under drought stress Photosynthesis rate in non-stressed plants overexpressing CpbZIPI gene CpbZIPI overexpressing (S-lines) plants under dark stress Effect of nitrogen source on the growth rate of transgenic plants overexpressing CpbZIPI gene Effect of proline in rescuing the growth of transgenic plants Utilization of proline as a nitrogen source in transgenic plants overexpressing the CpbZIPI gene Proline content in transgenic plants overexpressing CpbZIPI Effect of proline on root elongation of transgenic plants overexpressing CpbZIPI Discussion Optimization of a new transient transformation method was essential for studying the promoter function in C. plantagineum Agrobacterium-mediated transient transformation has been successfully optimized in two desiccation tolerant and one desiccation sensitive Linderniaceae members Promoter activities can be analysed in the three species of Linderniaceae by Agrobacterium co-cultivation method Trans-regulatory factors responsible for Cp LEA-like promoter are present in drought tolerant and sensitive species GUS activity was not increased in trans-activation of Cp LEA-like promoter by CpbZIPI protein CpbZIPI transcript expression under abiotic stress CpbZIPI gene from C. plantagineum is slightly induced under drought and ABA treatments in leaves The CpbZIPI gene from C. plantagineum is induced by salt stress in roots Distinct expression pattern of CpbZIPI in response to different abiotic stresses 121 VII

8 4.4 Molecular and functional analyses of the CpbZIPl protein in transgenic plants overexpressing CpbZIPl Dwarf phenotype in transgenic plants Moderate stress tolerance of transgenic plants The involvement of CpbZIPl protein in energy homeostasis/ starvation Seedlings overexpressing CpbZIPl utilize remobilized nitrogen to continue the growth under hypoosmotic conditions More proline accumulated in overexpressing CpbZIPl lines in control condition Kinetic expression analyses of CpbZIPl and Cp LEA-like Expression of Cp LEA-like and CpbZIPl gene depends on the time of the day Summary References 135 VIII