Biochemical, physiological and molecular responses of Ricinus communis seeds and seedlings to different temperatures: a multi-omics approach

<strong>Biochemical, physiological and molecular responses of <em>Ricinus communis</em> seeds and seedlings to different temperatures: a multi-omics approach</strong> <strong>by Paulo Roberto Ribeiro de Jesus</strong> The main objective of this thesis was to provide a detailed analysis of physiological, biochemical and molecular-genetic responses of <em>Ricinus communis </em>to temperature during seed germination and seedling establishment. In Chapter 2, I describe the assessment of 17 candidate reference genes across a diverse set of samples, including several tissues, various developmental stages and environmental conditions, encompassing seed germination and seedling growth in <em>R. communis</em>. These genes were tested by RT-qPCR and ranked according to the stability of their expression using two different approaches: GeNorm and NormFinder. Both GeNorm and Normfinder indicated that <em>ACT</em>, <em>POB</em> and <em>PP2AA1</em> represent the optimal combination for normalization of gene expression data in inter-tissue studies. I also describe the optimal combination of reference genes for a subset of samples from root, endosperm and cotyledonary tissues. The selection of reference genes was validated by normalizing the expression levels of three target genes involved in energy metabolism with the identified optimal reference genes. This approach allowed me to identify stably expressed genes, and, thus, reference genes for use in RT-qPCR studies in seeds and seedlings of <em>R. communis</em>. In Chapter 3, a thermo-sensitive window is identified during seed germination in which high temperatures compromise subsequent seedling development. I assessed the biochemical and molecular requirements of <em>R. communis</em> germination for successful seedling establishment at varying temperatures. For that, I performed metabolite profiling (GC-TOF-MS) and measured transcript levels of key genes involved in several energy-generating pathways such as storage oil mobilization, β-oxidation of fatty acids and gluconeogenesis of seeds germinated at three different temperatures. Transient overexpression of genes encoding for malate synthase (<em>MLS</em>) and glycerol kinase (<em>GK</em>) resulted in higher starch levels in <em>N. benthamiana </em>leaves, which highlights the likely importance of these genes in energy-generating pathways for seedling establishment. Additionally, I showed that γ-aminobutyric acid (GABA), which is a stress-responsive metabolite, accumulated in response to the water content of the seeds during the initial phase of imbibition. In Chapter 4 I undertook a genomics approach using microarray analysis to determine transcriptome changes in three distinct developmental stages during seed germination at 20, 25 and 35ºC that could explain the thermo-sensitive window that is described in Chapter 3. Most of the differences in the <em>R. communis</em> transcriptome occurred between 6 hours of imbibition and the commencement of germination, i.e. radicle protrusion. This coincides with the thermo-sensitive window identified during seed germination in which high temperatures compromise seedling development. The transcriptome data was used to identify heat-stress responsive genes that might be involved in thermotolerance of <em>R. communis</em> during germination. Temperature had a major effect on genes involved in energy generating pathways, such as the Calvin-Benson-Bassham cycle, gluconeogenesis, and starch- and triacylglycerol degradation. Transcripts coding for ATP binding proteins, DNA binding proteins, RNA binding proteins, DNA-directed RNA polymerases I, II, and III, heat shock factor proteins, multiprotein-bridging factor proteins, and zinc finger proteins were also affected by temperature suggesting the whole transcriptional regulatory machinery was altered in response to temperature. Among the downregulated transcripts under high temperature, only three were shared by all three stages: an oxidation-related zinc finger 2, an F-box and wd40 domain protein, and a DNA binding protein/MYB-like transcription factor. Among the upregulated transcripts, nine were shared by all three stages: a BET1P/SFT1P-like protein, 14BB, a low-molecular-weight cysteine-rich protein LCR78, a WD-repeat protein, a GAST1 protein, an adenylate kinase 1/P-loop containing nucleoside triphosphate hydrolases superfamily protein, and four conserved hypothetical proteins. These genes constitute good candidates for further characterization of temperature-responsive genes in <em>R. communis</em>. In Chapter 5, I studied the genetic variation in the effect of temperature on growth of young <em>R. communis</em> seedlings and measured primary and secondary metabolites in roots and cotyledons of three <em>R. communis</em> genotypes, varying in stress tolerance. Seedling biomass was strongly affected by the temperature, with the lowest total biomass observed at 20ºC. The response in terms of biomass production for the genotype MPA11 was clearly different from the other two studied genotypes: genotype MPA11 produced heavier seedlings at all temperatures but the root biomass of this genotype decreased with increasing temperature, reaching the lowest value at 35ºC. In contrast, root biomass of genotypes MPB01 and IAC80 was not affected by temperature, suggesting that the roots of these genotypes are less sensitive to changes in temperature. A shift in carbon-nitrogen metabolism towards the accumulation of nitrogen-containing compounds seems to be the main biochemical response to support growth at higher temperatures. Carbohydrate content was reduced in response to increasing temperature in both roots and cotyledons, whereas amino acids accumulated to higher levels. The results in this chapter show that a specific balance between amino acids, carbohydrates and organic acids in the cotyledons and roots of genotype MPA11 seems to be an important trait for faster and more efficient growth of this genotype at higher temperatures. In Chapter 6, I decided to focus on the differential ability of genotypes MPA11 and IAC80 to sustain root biomass production at higher temperatures. Biomass allocation was assessed by measuring dry weight of roots, stems, and cotyledons of seedlings grown at three different temperatures. Seedlings grown at 25ºC and 35ºC showed greater biomass than seedlings grown at 20ºC. Cotyledon and stem dry weight increased for both genotypes with increasing temperature, whereas root biomass allocation showed a genotype-dependent behaviour. Genotype MPA11 showed a continuous increase in root dry weight with increasing temperature, while genotype IAC80 was not able to sustain further root growth at higher temperatures. Metabolite and gene expression profiles of genotype MPA11 demonstrated an increase in the levels of osmoprotectant molecules, such as galactinol and transcripts of genes encoding antioxidant enzymes and heat shock proteins, to a higher extent than in genotype IAC80. In Chapter 7 I raised the question whether carbohydrate accumulation in <em>R. communis</em> leaves, roots, and seeds, grown at low temperatures, as compared to higher temperatures, results from up-regulation of biosynthetic pathways, from down-regulation of catabolic pathways, or both. To answer this question, transcript levels were measured of genes encoding enzymes involved in starch biosynthesis, starch catabolism, and gluconeogenesis in leaves, roots, and seeds grown at 20ºC and 35ºC. Transcript levels of genes involved in starch catabolism were higher in leaves grown at 20ºC than at 35ºC, but up-regulation of genes involved in starch biosynthesis seems to compensate for this and, therefore, is the likely explanation for higher levels of starch in leaves grown at 20ºC. Higher levels of soluble carbohydrates in leaves grown at 20ºC may have been caused by a coordinated up-regulation of starch catabolism and gluconeogenesis pathways. In roots, starch catabolism and gluconeogenesis seem to be enhanced at elevated temperatures. Higher levels of starch in seeds germinated at low temperatures is associated with higher transcript levels of genes involved in starch biosynthesis. Similarly, higher transcript levels of <em>RcPEPCK</em> and <em>RcFBPase</em> are most likely causal for fructose and glucose accumulation in seeds germinated at 20ºC. This thesis provides important insights in the understanding of the plasticity of <em>R. communis</em> in response to temperature. The knowledge obtained may apply to other species as well. Additionally, based on the transcriptomics data, we selected several candidate genes that are potentially involved in, or required for, proper seed germination and seedling establishment under different temperatures, such as a number of transcription factors, a zinc finger protein, heat-shock proteins, malate synthase and glycerol kinase. Overexpressing Arabidopsis lines transformed with these <em>R. communis</em> genes, as well as Arabidopsis T-DNA lines, in which Arabidopsis homologs of these genes are knocked-out, are being generated for further phenotypical analysis. These overexpression and T-DNA lines should help us to understand the molecular requirements for vigorous seedling growth of <em>R. communis</em> under different environmental conditions. This work was performed at the Laboratory of Plant Physiology, Wageningen University. This research was financially supported by the Brazilian Government through the National Counsel of Technological and Scientific Development (CNPq grant number 200745/2011-5).