Proteomics of Heat-Stress and Ethylene-Mediated Thermotolerance Mechanisms in Tomato Pollen Grains

Heat stress is a major cause for yield loss in many crops, including vegetable crops. Even short waves of high temperature, becoming more frequent during recent years, can be detrimental. Pollen development is most heat-sensitive, being the main cause for reduced productivity under heat-stress across a wide range of crops. The molecular mechanisms involved in pollen heat-stress response and thermotolerance are however, not fully understood. Recently, we have demonstrated that ethylene, a gaseous plant hormone, plays a role in tomato (Solanum lycopersicum) pollen thermotolerance. These results were substantiated in the current work showing that increasing ethylene levels by using an ethylene-releasing substance, ethephon, prior to heat-stress exposure, increased pollen quality. A proteomic approach was undertaken, to unravel the mechanisms underlying pollen heat-stress response and ethylene-mediated pollen thermotolerance in developing pollen grains. Proteins were extracted and analyzed by means of a gel LC-MS fractionation protocol, and a total of 1,355 proteins were identified. A dataset of 721 proteins, detected in three biological replicates of at least one of the applied treatments, was used for all analyses. Quantitative analysis was performed based on peptide count. The analysis revealed that heat-stress affected the developmental program of pollen, including protein homeostasis (components of the translational and degradation machinery), carbohydrate, and energy metabolism. Ethephon-pre-treatment shifted the heat-stressed pollen proteome closer to the proteome under non-stressful conditions, namely, by showing higher abundance of proteins involved in protein synthesis, degradation, tricarboxylic acid cycle, and RNA regulation. Furthermore, up-regulation of protective mechanisms against oxidative stress was observed following ethephon-treatment (including higher abundance of glutathione-disulfide reductase, glutaredoxin, and protein disulfide isomerase). Taken together, the findings identified systemic and fundamental components of pollen thermotolerance, and serve as a valuable quantitative protein database for further research

Molecular control of Arabidopsis male germline development by DAZ1 and DAZ2

The male germline of flowering plants is a simple lineage of two cell types: generative (germ) cell and sperm cells. An asymmetric division of the microspore during pollen (male gametophyte) development produces the germ cell, which goes on to divide to form a pair of sperm that later fuse with egg and central cells at double fertilisation. The production of functional sperm cells depends upon cell cycle progression and cell specification, and three regulatory proteins – the MYB transcription factor DUO1, and the C2H2 zinc finger proteins DAZ1 and DAZ2 - acting in a gene network have been identified in Arabidopsis to be essential for both of these processes. Expression of DAZ1/DAZ2 is triggered by DUO1, and DAZ1/DAZ2 show functional redundancy. Understanding the mechanisms by which DAZ1 and DAZ2 control male germline development was a major aim of this thesis. The first objective was to explore sequence diversity among DAZ1/DAZ2 flowering plant homologues. This study established that the number of zinc finger domains differed between species, and mutating the zinc finger domains of DAZ1 had different effects on DAZ1 function. Functional analysis of DAZ1 EAR motifs showed that these sequences are important for in planta activity. DAZ1 and DAZ2 interact with the transcriptional co-repressor TPL, and a second objective was to investigate the spatiotemporal expression pattern of TPL and its family members in pollen, with male germline expression was observed for TPL and TPR2. The final objective was to identify genes under the regulation of DAZ1/DAZ2. While DAZ1 was predicted to be a transcriptional repressor, through transcriptomics it was revealed that a broad suite of genes were positively regulated by DAZ1, overlapping with targets of DUO1. The findings communicated in this thesis provide new insights into the molecular mechanisms controlling male germline development

Proteomics of Heat-Stress and Ethylene-Mediated Thermotolerance Mechanisms in Tomato Pollen Grains

Heat stress is a major cause for yield loss in many crops, including vegetable crops. Even short waves of high temperature, becoming more frequent during recent years, can be detrimental. Pollen development is most heat-sensitive, being the main cause for reduced productivity under heat-stress across a wide range of crops. The molecular mechanisms involved in pollen heat-stress response and thermotolerance are however, not fully understood. Recently, we have demonstrated that ethylene, a gaseous plant hormone, plays a role in tomato (Solanum lycopersicum) pollen thermotolerance. These results were substantiated in the current work showing that increasing ethylene levels by using an ethylene-releasing substance, ethephon, prior to heat-stress exposure, increased pollen quality. A proteomic approach was undertaken, to unravel the mechanisms underlying pollen heat-stress response and ethylene-mediated pollen thermotolerance in developing pollen grains. Proteins were extracted and analyzed by means of a gel LC-MS fractionation protocol, and a total of 1,355 proteins were identified. A dataset of 721 proteins, detected in three biological replicates of at least one of the applied treatments, was used for all analyses. Quantitative analysis was performed based on peptide count. The analysis revealed that heat-stress affected the developmental program of pollen, including protein homeostasis (components of the translational and degradation machinery), carbohydrate, and energy metabolism. Ethephon-pre-treatment shifted the heat-stressed pollen proteome closer to the proteome under non-stressful conditions, namely, by showing higher abundance of proteins involved in protein synthesis, degradation, tricarboxylic acid cycle, and RNA regulation. Furthermore, up-regulation of protective mechanisms against oxidative stress was observed following ethephon-treatment (including higher abundance of glutathione-disulfide reductase, glutaredoxin, and protein disulfide isomerase). Taken together, the findings identified systemic and fundamental components of pollen thermotolerance, and serve as a valuable quantitative protein database for further research.© 2018 Jegadeesan, Chaturvedi, Ghatak, Pressman, Meir, Faigenboim, Rutley, Beery, Harel, Weckwerth and Firo