Genetic characterization of natural variation regulating thermal responses in plant development

Temperature affects several aspects of plant growth and development. The predicted rises in global temperature is expected to have an impact on worldwide crop productivity. Plants alter their physiological and developmental strategies in order to survive day to day and seasonal fluctuation in their growth temperature. In order to predict the impact of temperature on plants and to develop varieties that can cope with varied temperatures, we need to have a better knowledge of temperature response in plants at the molecular level. It is currently unclear as to how plants perceive and respond to varying temperatures. In this thesis, I employed model plant Arabidopsis thaliana as a tool to identify new factors involved in this process in plants. In this thesis, I have screened for natural variation in Arabidopsis accessions in temperature-response, followed by gene identification and characterization. First, Cvi-0, collected from Cape Verde Island, was identified to be insensitive to higher temperature. Using Quantitative Trait Loci (QTL) mapping with recombinant inbred lines derived from a cross between Cvi-0 and the reference strain Col-0 (CviColRILs), I showed that a QTL tightly linked to the blue light receptor CRYTOCHROMOME 2 (CRY2) contributes to natural variation in hypocotyl elongation and flowering response to temperature. The role for the CviCRY2 allele in response to temperature was supported by quantitative knockdown experiment with artificial microRNAs (amiRNAs) in Cvi-0. In addition, transgenic complementation experiments with CviCRY2 allele in the Ler-0, Col-0 and cry2 mutant backgrounds suggest that the role of CRY2 in regulating temperature response is dependent on the genetic background indicating the presence of modifiers for this response. Second, I discovered that Sij4 strain, collected from central Asia, is insensitive to temperature-induced hypocotyl elongation, and displays a temperature-dependent growth defect in their first leaves (thus named “abnormal first leaves (afl)”). Both traits show high genetic correlation (afl) (rG=0.88) indicating common genetic basis. Using Sij4ColF2 and Sij4LerF2 populations, I fine mapped the AFL locus to a 6 kb fragment, which includes a previously uncharacterized gene At2g31580. I demonstrate At2g31580 is AFL through transgenic complementation and artificial microRNA mediated knock-down experiments. I show that AFL regulates cell cycle at the G2/M transition through a combination of flow cytometry, transcriptome analysis and by using the cell cycle marker CYCB1. CyclinB1,1 (CYCB1,1), a key gene in the regulation of cell cycle, was not mis-expressed on transcriptional level, but a strong pCYCB1,1-CYCB1,1-GFP signal accumulated in mutant cells suggested that inhibition of CYCB1,1 degrading during G2/M phase transition. This was associated with increased DNA content suggestive of endoreduplication. Furthermore, I have shown that plants compromised for AFL function are more prone to DNA damage, suggesting a role for AFL in DNA repair. In summary, my studies on natural genetic variation in Arabidopsis identify a new factor AFL in regulating cell elongation and cell proliferation in response to higher temperature. In this thesis I review temperature response in plants and then report novel functions for two genes, CRY2 and AFL, in higher temperature response. In the first chapter I review our understanding of temperature response in plants and the associated mechanisms. I also provide an introduction to natural variation in Arabidopsis. In the second chapter I describe the results from screen I have carried out to find natural variants with altered thermal response and then go on describe the genetic basis of the temperature insensitivity phenotype in the Cvi-0 strain of Arabidopsis thaliana. In Chapter 3, I describe the genetic and molecular basis of temperature insensitivity in Sij-4, another strain I picked up from the screen. The natural afl mutant allele in Sij4 can be used as system to address fundamental questions of AFL beyond temperature response such as cell cycle regulation in plants. My finding on CRY2 opens up avenues for studying temperature and light interactions. The implications of this study as well as future areas for research are also discusses