Comparative transcriptome profiling of tomato (Solanum lycopersicum L.) pollen grains during maturation and in response to acquired thermotolerance conditions

Global temperature increase poses a serious challenge for agricultural production worldwide, affecting yield in many crops including vegetable crops. While most crop plants can survive temperature increases during their vegetative growth periods, the reproduction phase is highly heat-stress (HS)-sensitive. Impaired pollen development and functioning under HS is implicated as the major cause for yield reduction. To better understand HS effect on pollen and identify pollen thermotolerance mechanisms, we established conditions that enable developing pollen grains to acquire thermotolerance (ATT conditions), using tomato as a model system. High-throughput sequencing at cDNA level was performed by Massive Analysis of 3’cDNA using Illumina HiSeq 2000 technology, generating a total of 6430 and 4660 transcripts differentially expressed (p ≤ 1e-05) during pollen development/maturation and following response of developing pollen to ATT, respectively. Gene Onthology functional analysis showed that transcripts related to maintenance of protein homeostasis (translation, proteolysis, protein folding) were enriched during pollen maturation and following the ATT treatment in our study, highlighting these processes as central for enabling pollen maturation and maintenance of pollen functioning under HS. The transcriptomic data was compared to available pollen proteomic data based on the same experimental setup and an overlap of 47% was detected between differentially expressed proteins and transcripts following ATT conditions, highlighting genes/proteins involved in protein folding, oxidation-reduction and translation, and validating transcriptomic results. Involvement of mitochondria and endoplasmic reticulum in pollen heat acclimation, and activation of several HSPs including sHSPs and HSP101, for protecting pollen cellular components including the translational machinery, are indicated. The results of this study can serve as a valuable resource of genes for future research on improving pollen thermotolerance. Establishment of the capacity of developing tomato pollen grains to acquire thermotolerance (ATT) using greenhouse-grown plants. Then, use of the established ATT conditions to test the effect on expression profiles of developing/maturing and mature pollen grains. Developing pollen grains are represented by pooling three developmental stages (3, 2 and 1 days before flower opening).

全球气温升高对全球农业生产构成严峻挑战，会降低包括蔬菜作物在内的多种农作物的产量。尽管多数农作物在营养生长阶段可耐受温度升高，但生殖阶段对热胁迫（heat-stress, HS）极为敏感。热胁迫下花粉发育与功能受损被认为是产量下降的主要诱因。为深入解析热胁迫对花粉的影响并鉴定花粉耐热性机制，本研究以番茄为模式系统，建立了可使发育中花粉粒获得耐热性的培养条件（以下简称ATT条件）。本研究采用Illumina HiSeq 2000平台开展3'cDNA大规模分析（Massive Analysis of 3’cDNA）以进行转录组高通量测序，分别在花粉发育/成熟阶段以及发育中花粉响应ATT条件后，鉴定出6430和4660个差异表达转录本（p值≤1×10^-5）。基因本体论（Gene Ontology, GO）功能富集分析显示，在花粉成熟阶段及ATT处理后，与蛋白质稳态维持（翻译、蛋白水解、蛋白质折叠）相关的转录本显著富集，表明这些过程是花粉在热胁迫下完成成熟并维持功能的核心环节。本研究将转录组数据与基于相同实验体系获取的已公开花粉蛋白质组数据进行比对，发现在ATT条件下差异表达的蛋白与转录本之间存在47%的重叠比例，其中富集了参与蛋白质折叠、氧化还原及翻译过程的基因/蛋白，从而验证了转录组测序结果的可靠性。研究表明，线粒体与内质网参与了花粉热驯化过程，同时多种热休克蛋白（Heat Shock Protein, HSP）包括小热休克蛋白（small Heat Shock Protein, sHSP）和HSP101被激活，以保护花粉细胞组分（包括翻译机器）。本研究结果可为未来开展提升花粉耐热性的相关研究提供宝贵的基因资源。本研究通过温室栽培植株，建立了发育中番茄花粉粒获得耐热性的体系（ATT）。随后利用所建立的ATT条件，探究其对发育/成熟花粉及成熟花粉表达谱的调控效应。本研究中发育中花粉粒以开花前3天、2天和1天的三个发育阶段的混合样本作为代表。