Total transcriptome assays were performed on ovules and developing seeds derived from two seeded (Italia and Red globe) and two seedless (Thompson Seedless and Autumn Royal) table grape varieties. Total transcriptome assays were performed on ovules and developing seeds derived from two seeded (Italia and Red globe) and two seedless (Thompson Seedless and Autumn Royal) table grape varieties

VviAGL11, the Arabidopsis SEEDSTICK homolog, has been proposed to have a causative role in grapevine stenospermocarpy. An association between a mutation in the coding sequence (CDS) and the seedless phenotype was reported, however, no working mechanisms have been demonstrated yet. We performed a deep investigation of the full VviAGL11 gene sequence in a collection of grapevine varieties belonging to several seedlessness classes that revealed three different promoter-CDS combinations. By investigating the expression of the three VviAGL11 alleles, and by evaluating their ability to activate the promoter region, we observed that VviAGL11 self-activates in a specific promoter-CDS combination manner. Furthermore, by transcriptomic analyses on ovule and developing seeds in seeded and seedless varieties and co-expression approaches, candidate VviAGL11 targets were identified and further validated through luciferase assay and in situ hybridization. We demonstrated that VviAGL11 Wild Type CDS activates Methyl jasmonate esterase and Indole-3-acetate beta-glucosyltransferase, both involved in hormone signaling and Isoflavone reductase, involved in secondary metabolism. The dominant-negative effect of the mutated CDS was also functionally ectopically validated in target induction. VviAGL11 was shown to co-localize with its targets in the outer seed coat integument, supporting its direct involvement in seed development, possibly by orchestrating the crosstalk among MeJA, auxin, and isoflavonoids synthesis. In conclusion, the VviAGL11 expression level depends on the promoter-CDS allelic combination, and this will likely affect its ability to activate important triggers of the seed coat development. The dominant-negative effect of the mutated VviAGL11 CDS on the target genes activation was molecularly validated. A new regulatory mechanism correlating VviAGL11 haplotype assortment and seedlessness class in grapevine is proposed. Overall design: Ovules and developing seeds transcriptome of two seeded (Italia and Red globe) and two seedless (Thompson seedless and Autumn royal) table grape varietas. Four developing stages were considered (S1-S4; sheet 2) and each sample have been tested in biological triplicate (48 samples in total). For each probe, the fluorescence values is indicated.

VviAGL11作为拟南芥SEEDSTICK的同源基因，被认为在葡萄种子败育型无核（stenospermocarpy）过程中发挥因果性作用。此前有研究报道编码区（coding sequence, CDS）的突变与无籽表型存在关联，但尚未阐明其具体作用机制。我们针对涵盖多个无核等级的葡萄品种集合，对VviAGL11的全基因序列开展了深度分析，共鉴定出三种不同的启动子-编码区组合。通过分析这三种VviAGL11等位基因的表达模式，并评估它们激活启动子区域的能力，我们发现VviAGL11以特定的启动子-编码区组合方式实现自我激活。此外，通过对有籽与无籽品种的胚珠及发育中种子进行转录组分析，结合共表达分析策略，我们筛选得到候选的VviAGL11靶基因，并通过荧光素酶测定（luciferase assay）与原位杂交（in situ hybridization）完成了验证。我们证实，野生型VviAGL11 CDS可激活茉莉酸甲酯酯酶、吲哚-3-乙酸β-葡萄糖基转移酶（二者均参与激素信号通路）以及异黄酮还原酶（参与次生代谢）的表达。突变型CDS的显性负效应也在靶基因诱导实验中得到了异位功能验证。研究发现VviAGL11与其靶基因共同定位于种子外珠被，支持其直接参与种子发育过程，可能通过协调茉莉酸甲酯（methyl jasmonate, MeJA）、生长素与异黄酮合成之间的信号串扰发挥功能。综上，VviAGL11的表达水平取决于其启动子-编码区等位基因组合，这或将影响其激活种皮发育关键调控因子的能力。突变型VviAGL11 CDS对靶基因激活的显性负效应已得到分子层面的验证。本研究提出了一种全新的调控机制，将葡萄VviAGL11单倍型组合与无核等级相关联。总体实验设计：选取2个有籽鲜食葡萄品种——意大利葡萄（Italia）与红地球（Red globe），以及2个无籽鲜食葡萄品种——汤姆逊无核（Thompson seedless）与秋皇家（Autumn royal）的胚珠及发育中种子进行转录组分析。设置4个发育阶段（S1-S4；详见附表2），每个样本设置3次生物学重复，总计48个样本。每个探针的荧光信号值均已标注。