Suppression of the D-class MADS-box AGL11 gene triggers seedlessness in fleshy fruits.

KEY MESSAGE: Seedlessness, one of the most desired traits in fleshy fruits, can be obtained altering solely AGL11 gene, a D -class MADS-box. Opposite to overlapping functions described for ovule identity. AGAMOUS like-11 (AGL11) is a D-class MADS-box gene that determines ovule identity in model species. In grapevine, VviAGL11 has been proposed as the main candidate gene responsible for seedlessness because ovules develop into seeds after fertilization. Here, we demonstrate that AGL11 has a direct role in the determination of the seedless phenotype. In grapevine, broad expression analysis revealed very low expression levels of the seedless allele compared to the seeded allele at the pea-size berry stage. Heterozygous genotypes have lower transcript accumulation than expected considering the diploid nature of grapevine, thereby revealing that the dominant phenotype previously described for seedlessness is based on its expression level. In a seeded somatic variant of Sultanina (Thompson Seedless) that has well-developed seeds, Sultanine Monococco, structural differences were identified in the regulatory region of VviAGL11. These differences affect transcript accumulation levels and explain the phenotypic differences between the two varieties. Functional experiments in tomato demonstrated that SlyAGL11 gene silencing produces seedless fruits and that the degree of seed development is proportional to transcript accumulation levels. Furthermore, the genes involved in seed coat development, SlyVPE1 and SlyVPE2 in tomato and VviVPE in grapevine, that are putatively controlled by SlyAGL11 and VviAGL11, respectively, are expressed at lower levels in silenced tomato lines and in seedless grapevine genotypes. In conclusion, this work provides evidence that the D-class MADS-box AGL11 plays a major and direct role in seed development in fleshy fruits, providing a valuable tool for further analysis of fruit development.

VviAGL11 self-regulates and targets hormone- and secondary metabolism-related genes during seed development.

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.

Unraveling the Deep Genetic Architecture for Seedlessness in Grapevine and the Development and Validation of a New Set of Markers for VviAGL11-Based Gene-Assisted Selection.

Seedless inheritance has been considered a quasi-monogenic trait based on the VvAGL11 gene. An intragenic simple sequence repeat (SSR) marker, p3_VvAGL11, is currently used to opportunely discard seeded progeny, which represents up to 50% of seedlings to be established in the field. However, the rate of false positives remains significant, and this lack of accuracy might be due to a more complex genetic architecture, some intrinsic flaws of p3_VvAGL11, or potential recombination events between p3_VvAGL11 and the causal SNP located in the coding region. The purpose of this study was to update the genetic architecture of this trait in order to better understand its implications in breeding strategies. A total of 573 F1 individuals that segregate for seedlessness were genotyped with a 20K SNP chip and characterized phenotypically during four seasons for a fine QTL mapping analysis. Based on the molecular diversity of p3_VvAGL11 alleles, we redesigned this marker, and based on the causal SNP, we developed a qPCR-HRM marker for high-throughput and a Tetra-ARMS-PCR for simple predictive analyses. Up to 10 new QTLs were identified that describe the complex nature of seedlessness, corresponding to small but stable effects. The positive predictive value, based on VvAGL11 alone (0.647), was improved up to 0.814 when adding three small-effect QTLs in a multi-QTL additive model as a proof of concept. The new SSR, 5U_VviAGL11, is more informative and robust, and easier to analyze. However, we demonstrated that the association can be lost by intragenic recombination and that the e7_VviAGL11 SNP-based marker is thus more reliable and decreases the occurrence of false positives. This study highlights the bases of prediction failure based solely on a major gene and a reduced set of candidate genes, in addition to opportunities for molecular breeding following further and larger validation studies.

The transcriptional factor ZEB1 represses Syndecan 1 expression in prostate cancer.

Syndecan 1 (SDC-1) is a cell surface proteoglycan with a significant role in cell adhesion, maintaining epithelial integrity. SDC1 expression is inversely related to aggressiveness in prostate cancer (PCa). During epithelial to mesenchymal transition (EMT), loss of epithelial markers is mediated by transcriptional repressors such as SNAIL, SLUG, or ZEB1/2 that bind to E-box promoter sequences of specific genes. The effect of these repressors on SDC-1 expression remains unknown. Here, we demonstrated that SNAIL, SLUG and ZEB1 expressions are increased in advanced PCa, contrarily to SDC-1. SNAIL, SLUG and ZEB1 also showed an inversion to SDC-1 in prostate cell lines. ZEB1, but not SNAIL or SLUG, represses SDC-1 as demonstrated by experiments of ectopic expression in epithelial prostate cell lines. Inversely, expression of ZEB1 shRNA in PCa cell line increased SDC-1 expression. The effect of ZEB1 is transcriptional since ectopic expression of this gene represses SDC-1 promoter activity and ZEB1 binds to the SDC-1 promoter as detected by ChIP assays. An epigenetic mark associated to transcription repression H3K27me3 was bound to the same sites that ZEB1. In conclusion, this study identifies ZEB1 as a key repressor of SDC-1 during PCa progression and point to ZEB1 as a potentially diagnostic marker for PCa.