Heat stress tolerance responses in developing tomato anthers

Global warming already has and will significantly impact crop productivity and yield in the near future. In order to meet the forecasted requirements of the future agricultural production, a proper assessment of crops environmental stress tolerance needs to be designed and implemented, from the laboratory to field. Genetic variation in the ability of tomatoes to set fruit under high temperature conditions has made selection for heat tolerance possible and multiple opportunities for improvement exist, as tolerance to high temperatures is a multi-genic character involving a complex network of chaperones and other protective proteins acting together to defend the cells from heat injury. Breeding programs involved in the development of heat tolerant cultivars should identify and make use of such tolerance traits already available in collected or wild germplasm. The goal of this thesis was to characterize the response to high temperatures in meiotic tomato anthers with contrasting responses to heat and to identify genes that could be related to thermo-tolerance mechanisms during gamete development. Several molecular tools such as transcriptomic profiling by cDNA-AFLP and microarray analysis, RT-PCR or in situ RNA hybridisation were used to achieve this goal. The second chapter reviews the effects of heat stress on reproductive flower development, candidate tolerance pathways and methods for production of heat tolerant crops. The third chapter provides a general overview of the expression changes occurring in the developing anthers of a sensitive tomato genotype following exposure to a (short and) moderate high temperature stress (MHS). Using a combination of cDNA-AFLP, RT-PCR, and in situ RNA hybridisation, we characterized and verified the general transcriptional response to heat of tomato plants. Our results revealed that approximately 1% of the examined transcript-derived fragments exhibit alterations in expression pattern and the majority of these were down-regulated The putative functions associated with the genes identified by cDNA-AFLP indicated involvement of heat shock, metabolism, antioxidant and developmental processes. Based upon the observed transcriptional changes in response to MHS and on literature sources, we identified a number of candidate transcripts to be involved in heat-tolerance. The spatial expression of several such candidate genes was further examined using in situ RNA hybridisation and this showed that the investigated genes are expressed in the tapetum or/and in developing microspores. Furthermore, the expression of several candidate genes has been quantified by RT-PCR in additional genotypes with different degrees of heat tolerance. The results suggested a correlation between gene expression levels, pollen germination rates and tolerance to heat (Chapter 4). In the fourth chapter we proceeded to profile the response to heat of meiotic anthers in a tolerant and a sensitive tomato genotype and investigated the expression of the identified candidate genes in several pairs of contrasting genotypes. Using microarray analysis (for an extensive overview of the meiotic response to heat) and RT-PCR, we were able to clearly distinguish differential responses of the tolerant genotype. After 2h of moderate heat stress, the heat-tolerant genotype exhibits fewer transcriptional changes than the heat-sensitive genotype. In the heat-tolerant genotype, the majority of changes in gene expression is represented by up-regulation, while in the heat-sensitive genotype there is a general trend to down-regulate gene expression soon after MHS.  Moreover, the heat-tolerant genotype also shows a different level of constitutive gene expression profiles when compared to the heat-sensitive genotype indicating a difference in genetic adaptation with regards to increased temperatures. The putative functions associated with the genes identified by microarray profiling indicate involvement of heat shock, antioxidant, metabolic, and cell development pathways. Based upon the observed differences in response to MHS we selected a number of candidate transcripts involved in heat-tolerance and confirmed their expression pattern in different tomato genotypes with contrasting responses to heat. The results suggested that the candidate genes are involved in the activation of protection mechanisms in the tomato anthers during moderate heat stress and, could therefore contribute to normal growth and development of the male gametophyte and implicitly a successful fruit set under adverse temperatures. In the fifth chapter we tested the hypothesis that heat tolerance is associated with maintenance of organ identity, fertility and lower ABA levels during heat stress (for several tomato genotypes) and analysed the dynamics of ABA accumulation under temperature stress in several tomato genotypes with contrasting responses to heat. Furthermore, pollen germination tests were performed and  additional physiological aspects of anther development for each genotype were analysed  as well. The general trend observed was the accumulation of lower relative levels of ABA at the end of the experimental period compared to the initial stages in more tolerant genotypes and of higher levels in the sensitive genotypes. We concluded from these results that the morphological changes in the floral tissues and the overall changes in ABA levels are correlated with the molecular responses under increased temperature in the genotypes analysed. Whether these correlations are causally related is not clear; therefore more research is needed to resolve these issues. The sixth chapter examines our analysis of the heat stress response in meiotic tomato anthers in a broader scientific context. I discuss the different aspects of our results and present several candidate genes involved in plant thermo-tolerance. In addition, I also discuss the potential involvement of plant growth regulators in plants´ responses to heat stress and suggest various potential follow-up experimental strategies