Study On The Mechanism Of Seedlessness Of Grape Induced By GA₃

Grapevine (Vitis vinifera L.) is an economically important fruit crop cultivatedworldwide for the production of table grapes, wine, juice, and dry raisins. Seedlessness of thefruit is an especially desirable trait for consumers. In table grape, seedlessness is one of themost appreciated quality traits. Seedlessness in grapes is caused by either parthenocarpy orstenospermocarpy. The phytohormone gibberellic acid (GA) is widely used in the table grapeindustry to induce seedlessness in seeded varieties. However, there is a paucity of informationconcerning the mechanisms by which GAs induce seedlessness in grapes. In this study, In anattempt to improve our understanding of the mechanisms by which GAs induce seedlessnessin grape, we conducted an in depth characterization of two seeded grape cultivars (‘Kyoho’and ‘Red Globe’), along with a seedless cultivar (‘Thompson Seedless’), following treatmentwith100mg L-1GA3before full bloom. Firstly, morphological effects on grape ovules or seedsdifferent development phase, physiological indexes analysis, anatomic observation of embryodevelopment, and analysis of the transcript levels of related genes were conducted; secondly,an RNA-Seq transcriptome analysis of grape flowers under GA3treatment was carried out; inaddition, domains, phylogeny and evolution, gene structures, gene synteny and expressionpatterns in various grape tissues or organs of grape rboh genes were investigated. The majorfindings were as following:1. The effects of GA3on the grape berries and seeds. The frequency of seedless berrieswas98.6%and85.2%in GA3-treated‘Kyoho’ and ‘Red Globe’ cultivar, respectively. In thecase of ‘Kyoho’ and ‘Red Globe’ cultivar, at the early stage of fruit development, berry weight,transverse diameter and longitudinal diameter from GA3-treated plants were significantlyhigher than those of untreated plants, while seed weight in GA3-treated plants was similar tothat of untreated seeds. From15DAF onwards, seeds from treated‘Kyoho’ plants were foundto weigh significantly less than those from untreated plants; seeds from GA3-treated ‘RedGlobe’ plants were found to be significantly smaller than those harvested from untreatedplants from9DAF onwards. The weight, transverse diameter and longitudinal diameter ofberries from ‘Thompson Seedless’ plants treated with GA3were always higher than those ofuntreated plants. Seeds from GA3-treated ‘Thompson Seedless’ plants weighed significantly more than seeds from untreated plants at3DAF, were similar in weight to untreated controlsat9DAF, and weighed significantly less than untreated controls from15to45DAF. From51DAF onwards, GA3-treated ‘Thompson Seedless’ plants produced seeds that were slightly, butnot significantly, lower in weight than untreated control seeds from the same cultivar.Following GA3application, some ovules had undergone fertilization normally; subsequently,abnormal zygotes and endosperm nuclei became apparent, embryo sacs degenerated; finally,the growth of GA3-treated seeds was completely halted.2. GA3application had an effect on redox homeostasis in grape flowers/berries. At theearlier period following GA3application, H2O2content and MDA content in GA3-treatedsamples were significantly increased, while the antioxidant enzyme (SOD1, SOD2, SOD3,CAT1, CAT2and POD) genes tended to be down-regulated in GA3-treated samples，theircorresponding enzyme activities (SOD, CAT, and POD) were reduced.3. Changes in gibberellins content following GA3application. GA3application increasedsamples GA content from1h to24h, which then decreased or dropped to levels similar tocontrols72h after GA3application.4. Transcriptome changes in grapevine flowers induced by GA3. Floral clusters in‘Kyoho’ plants were treated with GA312d prior to full bloom, transcriptome sequencingusing the Illumina Hiseq2000sequencing technology was performed. The results showed thatcompared with controls,1975(1218up,757down) and2713(1360up,1353down) geneswere differentially expressed1h and24h after GA3treatment, respectively. Gene Ontology(GO) functional classification of differentially expressed genes and pathways that wereaffected by GA3treatment were identified. In the case of functional categories based onbiological process of GO, some differentially expressed genes were associated with the‘reproduction’,‘embryonic development’,‘post-embryonic development’,‘flowerdevelopment’,‘pollination’,‘abscission’,‘pollen-pistil interaction’, etc, that might be relatedto seed abortion in GA3-treated plants. In the case of significantly changed pathways, with theexception of amino acid metabolism, carbohydrate metabolism and hormone metabolism,there were other pathways, such as glutathione-mediated detoxification and removal ofsuperoxide radicals, which could be related to the changes in antioxidant activity induced byGA3application.5. Genome-wide analysis of grape rboh genes. In this study, a total of seven rboh geneswhich were related to ROS from grape were identified and characterized. Genomic structureand predicted protein sequence analysis indicated that the sequences of plant rboh genes arehighly conserved. Synteny analysis demonstrated that four paired homologs of Vvrboh andAtrboh genes were located in syntenic genomic regions, suggesting that these genes likely share a common ancestor. The expression pattern of Vvrboh genes in different tissues wasassessed by qRT-PCR and two were constitutively expressed in all tissues tested. Vvrbohtranscript levels were increased from6h to72h following GA3treatment (with the exceptionof rbohA), that could be related to the accumulation of ROS in GA3-treated samples.