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Pollen thermotolerance and crop fertility

Final Report Summary - SPOT-ITN (Pollen thermotolerance and crop fertility)

The SPOT-ITN was organized in eight work packages. Five work packages (1-5) concerned the research program with the objective to identify BIOMARKER for BREEDING of tomato with higher pollen thermotolerance. The research aims have been achieved as described below. Work package six was tailored toward ESR/ER training, which is fully achieved as all secondments and training elements are completed. Work package seven concerned the management, which is completed with delivery of this report and the final accounting. Work package eight targeted the outreach. This work package concerned scientific presentations in form of lectures and publications, the latter will be completed after fully exploration of all obtained results. In addition, information obtained have been delivered to the public in form of press releases in all media and in form of public lectures (fully completed). Finally, all information obtained will be delivered to the community by deposition in public database and in the database created in work package 5. Moreover, a special issue in Plant Reproduction will be published in 2016, where results are summarized in form of Reviews and Protocols. In the following the short summary of the achievement is provided, most of which are already published (see list of publications).
The aim to identify plants with pollen thermotolerance in natural collections and to produce PTT lines by forward genetics is achieved. A tomato TILLING population (cv Red setter) was screened for mutation discovery for genes involved in the heat shock response leading to the discovery of lines mutated in the Heat shock binding protein Hsbp. Several heat stress experiments were performed to confirm the relation of the mutants to heat response reactions. Mutant lines were backcrossed in order to eliminate unwanted additional mutations. The phenotype of mutant lines does not differ from the wild type Red setter, and thus, the identified mutants provide an optimal base for further investigations and breeding strategies. Secondly, plasmids containing promoter sequences specific for gene expression in anthers at specific developmental periods were produced and tested. Chimeric genes were introduced by stable transformation in tomato, and the effects of increased gene expression were validated by measuring pollen vitality at high temperatures. Here, increased levels of HsfA2 and HsfB3a transcripts lead to 20-30 % more viable pollen under heat stress. We deliver new plant lines for breeding and new tools for further investigations of pollen fertility in different plant lines. At third, many available tomato lines have been analyzed for pollen thermotolerance, for example the acquired thermotolerance conditions for tomato cv. 3017 were established.
We significantly contributed to the description of the molecular base of protein homeostasis and the identification of chaperone and hormone networks involved in manifestation of protein stability in pollen. This was based on global transcriptome, metabolome and proteome profiling of pollen at different developmental stages, after HS and of PTT lines will be determined. At first we assigned functional categories to the genes of the tomato genome by orthology definition, which was important for all subsequent studies. Secondly, we defined overlapping and contrasting reactions in different tissues by quantitative MACE based transcriptomics for tomato leaves and pollen (cv. Red Setter and Money Maker) with and without heat stress application. Moreover, the transcriptome profile of different pollen stages (cv. Red Setter) or of pollen of other cultivars (e.g. cv. 3017), tomato mutants (e.g. HsfA2) and other combinatory treatments (e.g. with heat stress and hormones) have been defined. Thirdly, we established the protein profile of pollen at various development stages like microsporocyte, tetrad, microspore, polarized microspore and mature pollen; as well as of different compartments of pollen like soluble and membrane inserted protein pools. Further, we have performed the proteomics of heat treated pollen samples from the tomato cultivar Red Setter, considering developmental stages like tetrad, microspore and mature pollen and have identified putative heat treatment responsive protein candidates in tomato pollen using targeted mass accuracy precursor alignment (tMAPA). Fourthly, metabolites in pollen tissue have been analyzed to get insight into the metabolic changes occurring during pollen development and in response to different heat stress regimes, in both heat-sensitive and heat-tolerant tomato genotypes.
By the combination of all four large scale experiments we were able to describe the involvement of ethylene in pollen thermotolerance based on ethylene pre-treatment experiments and the use of tomato ethylene signaling mutants in heat-stress experiments. To this end, we identified ethylene biosynthesis and signaling components relevant for the heat stress response. In the same line, we detected that protein transport is globally important for thermotolerance, but an individual component particularly regulating this process in response to heat stress in pollen was not identified. In contrast, the large scale approach allowed the functional dissection of the heat shock transcription factor family, which is a central base of thermotolerance regulation. We identified HsfA2 regulated genes in wild type and mutant tomato plants by which a central hypothesis for BIOMARKER could be described, namely HsfA2, HsfA7 and Hsbp. All of these results are major findings for future investigations and incorporation of targets for screening strategies.
Moreover, we have shown that heat affects specific cell types and developmental stages in the male flower organs of tomato and is at least in part the result of a loss of male organ identity upon heat. However, we did not find any evidence for long-term intra- and transgenerationally transmitted acquired tolerance. This finding is of importance as we conclude that transgenerational epigenetic modifications do not play a major role in manifestation of acquired thermotolerance. In turn, the analysis of the heat stress-responsive epigenome and small RNA-ome of developing pollen revealed that developmental processes in pollen are accompanied by the overabundant occurrence of a novel class of small RNAs that is potentially able to maintain the epigenetic memory of the cell nucleus across meiosis. It further showed that stress-responsive cytosine methylation is fundamentally different from developmentally regulated methylation and concerns different genomic loci. The identification of small RNAs is of importance as they could serve as BIOMARKER, but the relation of their abundance to thermotolerance needs to be confirmed. The observed heat stress related cytosine methylation profile is a major finding and poses the question, which could not be answered in the course of SPOT-ITN, whether such alteration can be manifested in seedlings and transferred to later developmental stages.
The ultimate goal was a complete profiling of pollen for one cultivar grown under various conditions. After a trial experiment growing Red Setter at Volcani Centre Israel under optimal and mild chronic heat-stress conditions, the large scale experiment was transferred to Metapontum Agrobios / Italy. The phenotype of entire plants and the pollen quality has been explored on the base of 200 plants at control and stress conditions in two seasons. The transcriptome, proteome and metabolome for three pollen stages (tetrads, post-meiotic and mature) under control and stress conditions have been established and deposited in the SPOT-ITN database. The database includes accessory data and tools to allow the proper investigation of datasets for comparative functional analyses. At stage the platform is privately accessible at the moment to all Spot-ITN members at the public release is scheduled after publication of the final analysis and network description, which is anticipated to be completed by the end of 2016.
Summarizing: we identified Tilling and native tomato lines with contrasting pollen thermotolerance as well as at least three factors that can serve as BIOMARKER. Further, we introduced small RNAs and DNA methylation as a second layer at which BIOMARKER can exist. Finally, we provide a complete set of information for different pollen stages for one cultivar, and the network based on these results will be released shortly.