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Mechanisms and Candidate Genes for Seed and Fruit Set in Grapevine
Abstract
Background: Grapevine reproductive development has direct implications on yield. It also impacts on berry and wine quality by affecting traits like cluster compactness, bunch and berry size, berry skin to pulp ratio or seedlessness. Seasonal fluctuations in yield, fruit composition and wine attributes, which are largely driven by climatic factors, are major challenges for worldwide table grape and wine industry. Accordingly, a better understanding of reproductive processes such as gamete development, fertilization, seed and fruit set is of paramount relevance for managing yield and quality. With the aim of providing new insights into this field, we searched for clones with contrasting seed content in two germplasm collections.
Results: We identified eight variant pairs that seemingly differ only in seed-related characteristics while showing identical genotype when tested with the GrapeReSeq_Illumina_20K_SNP_chip and several microsatellites. We performed multi-year observations on fruit and seed set deriving from different pollination treatments, with special emphasis on the pair composed by Sangiovese and its seedless variant locally named Corinto Nero. The pollen of Corinto Nero failed to germinate in vitro and gave poor berry set when used to pollinate other varieties. Most berries from both open- and cross-pollinated Corinto Nero inflorescences did not contain seeds. The genetic analysis of seedlings derived from occasional Corinto Nero normal seeds revealed that the few Corinto Nero functional gametes are mostly unreduced. A number of genes potentially involved in sporogenesis and gametogenesis showed contrasting expression between Corinto Nero and Sangiovese and five missense single nucleotide polymorphisms were identified from transcriptomic data. The above findings suggest that the seedless phenotype of Corinto Nero is driven by pollen and/or embryo sac defects, and both events likely arise from meiotic anomalies. Finally, three genotypes, including Sangiovese and Corinto Nero, were unexpectedly found to develop fruits without pollen contribution and occasionally showed normal-like seeds.
Conclusions: Our collective results suggest that parthenocarpy and stenospermocarpy are not restricted to Black Corinth (alias Korinthiaki) and Sultanina-derived cultivars. The single nucleotide polymorphisms identified between Sangiovese and its parthenocarpic variant Corinto Nero are suitable for testing as traceability markers for propagated material and as functional candidates for the seedless phenotype.
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In flowering plants, pollen development is under a dynamic and well‐orchestrated transcriptional control, characterized by an early phase with high transcript diversity and a late post‐mitotic phase skewed to a cell‐type specific‐transcriptome. Such transcriptional changes require a balance between synthesis and degradation of mRNA transcripts, the latter being initiated by deadenylation. The CCR4‐NOT complex is the main evolutionary conserved deadenylase complex in eukaryotes, and its function is essential during germline specification in animals. We hypothesised that the CCR4‐NOT complex might play a central role in mRNA turnover during microgametogenesis in Arabidopsis. Disruption of NOT1 gene, which encodes the scaffold protein of the CCR4‐NOT complex, showed abnormal seed set. Genetic analysis failed to recover homozygous progeny and reciprocal crosses confirmed reduced transmission through the male and female gametophytes. Concordantly not1 embryo sacs showed delayed development and defects in embryogenesis. not1 pollen grains exhibited abnormal male germ unit configurations and failed to germinate. Transcriptome analysis of pollen from not1/+ mutants revealed that lack of NOT1 leads to an extensive transcriptional deregulation during microgametogenesis. Therefore, our work establishes NOT1 as an important player during gametophyte development in Arabidopsis.
Vegetative propagation of Vitis vinifera cultivars over hundreds of years has led to the accumulation of a large number of somatic variants of the same grapevine variety. These variants are now considered a working tool to cope with changing environmental conditions as a result of, among others, global warming. In this work, three somatic variants of the major grapevine variety of the South West (SW) of Andalusia (Spain), Palomino Fino, have been genetically and morphologically characterized, as well as their grape musts from two different vintages. The genetic analysis at 22 microsatellite loci confirmed the identity of the three somatic variants that presented the same genotype as Palomino Fino, while the morphological study showed differences between the three somatic variants and Palomino Fino, highlighting the somatic variant Palomino Pelusón. Regarding the physicochemical analysis of its musts, differences were also observed between the somatic variants and Palomino Fino. As a result of all of the above, the use of grapes from somatic variants can be a viable and natural alternative for the production of quality wines in warm climate areas. On the other hand, promoting the cultivation of the somatic variants could contribute to preventing the loss of Palomino Fino intraspecific variability.
Background
Gibberellins (GA 3 ) are the most sprayed growth regulator for table grape production worldwide, increasing berry size of seedless varieties through pericarp cell expansion. However, these treatments also exacerbate berry drop, which has a detrimental effect on the postharvest quality of commercialized clusters. Several studies have suggested that pedicel stiffening caused by GA 3 would have a role in this disorder. Nevertheless, transcriptional and phenotypic information regarding pedicel responses to GA 3 is minimal.
Results
Characterization of responses to GA 3 treatments using the lines L23 and Thompson Seedless showed that the former was up to six times more susceptible to berry drop than the latter. GA 3 also increased the diameter and dry matter percentage of the pedicel on both genotypes. Induction of lignin biosynthesis-related genes by GA 3 has been reported, so the quantity of this polymer was measured. The acetyl bromide method detected a decreased concentration of lignin 7 days after GA 3 treatment, due to a higher cell wall yield of the isolated fractions of GA 3 -treated pedicel samples which caused a dilution effect. Thus, an initial enrichment of primary cell wall components in response to GA 3 was suggested, particularly in the L23 background. A transcriptomic profiling was performed to identify which genes were associated with these phenotypic changes. This analysis identified 1281 and 1787 genes differentially upregulated by GA 3 in L23 and cv. Thompson Seedless, respectively. Concomitantly, 1202 and 1317 downregulated genes were detected in L23 and cv. Thompson Seedless (FDR < 0.05). Gene ontology analysis of upregulated genes showed enrichment in pathways including phenylpropanoids, cell wall metabolism, xylem development, photosynthesis and the cell cycle at 7 days post GA 3 application. Twelve genes were characterized by qPCR and striking differences were observed between genotypes, mainly in genes related to cell wall synthesis.
Conclusions
High levels of berry drop are related to an early strong response of primary cell wall synthesis in the pedicel promoted by GA 3 treatment. Genetic backgrounds can produce similar phenotypic responses to GA 3 , although there is considerable variation in the regulation of genes in terms of which are expressed, and the extent of transcript levels achieved within the same time frame.
Members of the plant-specific GASA (gibberellic acid-stimulated Arabidopsis) gene family have multiple potential roles in plant growth and development, particularly in flower induction and seed development. However, limited information is available about the functions of these genes in fruit plants, particularly in grapes. We identified 14 GASA genes in grapevine (Vitis vinifera L.) and performed comprehensive bioinformatics and expression analyses. In the bioinformatics analysis, the locations of genes on chromosomes, physiochemical properties of proteins, protein structure, and subcellular positions were described. We evaluated GASA proteins in terms of domain structure, exon-intron distribution, motif arrangements, promoter analysis, phylogenetic, and evolutionary history. According to the results, the GASA domain is conserved in all proteins and the proteins are divided into three well-conserved subgroups. Synteny analysis proposed that segmental and tandem duplication have played a role in the expansion of the GASA gene family in grapes, and duplicated gene pairs have negative selection pressure. Most of the proteins were predicted to be in the extracellular region, chloroplasts, and the vacuole. In silico promoter analysis suggested that the GASA genes may influence different hormone signaling pathways and stress-related mechanisms. Additionally, we performed a comparison of the expression between seedless (Thompson seedless) and seeded (Red globe) cultivars in different plant parts, including the ovule during different stages of development. Furthermore, some genes were differentially expressed in different tissues, signifying their role in grapevine growth and development. Several genes (VvGASA2 and 7) showed different expression levels in later phases of seed development in Red globe and Thompson seedless, suggesting their involvement in seed development. Our study presents the first genome-wide identification and expression profiling of grapevine GASA genes and provides the basis for functional characterization of GASA genes in grapes. We surmise that this information may provide new potential resources for the molecular breeding of grapes.
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.
Grape is one of the most economically important fruits worldwide, and seedless fruit is a main target in grape breeding. The MADS-box genes encode a large family of transcription factors in higher plants, and serve various developmental roles including flower, fruit and seed development. However, little is known about function of MADS-box genes in grapevine, especially related to seed development. Here, we characterized a role for the grapevine MADS-box gene, VvMADS45, in seed development. Heterologous expression of VvMADS45 (VvMADS45-OE) in tomato resulted in larger flowers, fruit and seeds. In contrast, disruption of a tomato homolog of VvMADS45 (SlAGL104-KO) resulted in smaller flowers, fruit and seeds. In addition, both VvMADS45-OE and SlAGL104-KO lines produced fewer seeds than control. The fraction of pollen grains that appeared abnormal or failed to rehydrate was greater in VvMADS45-OE lines than in non-transgenic control plants, while relatively few abnormal pollen grains were formed in SlAGL104-KO lines. Expression of several genes related to flower, fruit and seed development was altered in both VvMADS45-OE and SlAGL104-KO lines. These results revealed that VvMADS45 participates in flower, fruit and seed development, especially seed development, which may provide new insight into the genetic basis of seedlessness in grapes.
Background: There are thousands of grapevine varieties that display a wide range of variation for traits like grape use (wine, table grape or both), color or ripening time, but little is known about their reproductive performance, especially flowering and fruitset (conversion from flower to fruit). Works focused at the study of these traits in grapevine evaluated one or few varieties and used different methodologies making comparisons difficult. This study aimed to characterize the reproductive performance of 120 varieties and its stability over two seasons using a precise methodology.
Methods: Reproductive performance was determined through counting flowers and berries in the same inflorescences/bunches (10 per variety), for which a new methodology of image analysis of scanned calyptras was developed. Varieties were classified according to their reproductive performance.
Results: A great diversity was found for most variables including fruitset and number of flowers. Large differences between varieties were observed both in values and in stability among seasons. The varieties clustered in three main classes that displayed significant differences not only for the reproductive performance variables used for the clustering but for most the variables studied. Varieties in these classes showed a non-random distribution regarding the grape use and the genetic structure based on molecular markers.
Conclusions: This is the largest study of reproductive performance variables such as fruitset ever done in grapevine. It provides specific values for many varieties for the first time, useful for breeding programs. The clustering based on these variables is related with those based on use and geographical origin.
Development of pollen, the male gametophyte of flowering plants, is tightly controlled by dynamic changes in gene expression. Recent research to clarify the molecular aspects of pollen development has revealed the involvement of several transcription factors in the induction of gene expression. However, limited information is available about the factors involved in the negative regulation of gene expression to eliminate unnecessary transcripts during pollen development. In this study, we revealed that AtNOT1 is an essential protein for proper pollen development and germination capacity. AtNOT1 is a scaffold protein of the AtCCR4-NOT complex, which includes multiple components related to mRNA turnover control in Arabidopsis. Phenotypic analysis using atnot1 heterozygote mutant pollen showed that the mature mutant pollen failed to germinate, and also revealed abnormal localization of nuclei and a specific protein at the tri-cellular pollen stage. Furthermore, transcriptome analysis of atnot1 heterozygote mutant pollen demonstrated that the down-regulation of a large number of transcripts, along with the up-regulation of specific transcripts required for pollen tube germination by AtNOT1 during late microgametogenesis, is important for proper pollen development and germination. Overall, our findings provide new insights into the negative regulation of gene expression during pollen development, by demonstrating the severely defective phonotype of atnot1 heterozygote mutant pollen.
Background:
The homeobox transcription factor has a diversity of functions during plant growth and development process. Previous transcriptome analyses of seed development in grape hybrids suggested that specific homeodomain transcription factors are involved in seed development in seedless cultivars. However, the molecular mechanism of homeobox gene regulating seed development in grape is rarely reported.
Results:
Here, we report that the grapevine VvHB58 gene, encoding a homeodomain-leucine zipper (HD-Zip I) transcription factor, participates in regulating fruit size and seed number. The VvHB58 gene was differentially expressed during seed development between seedless and seeded cultivars. Subcellular localization assays revealed that the VvHB58 protein was located in the nucleus. Transgenic expression of VvHB58 in tomato led to loss of apical dominance, a reduction in fruit pericarp expansion, reduced fruit size and seed number, and larger endosperm cells. Analysis of the cytosine methylation levels within the VvHB58 promoter indicated that the differential expression during seed development between seedless and seeded grapes may be caused by different transcriptional regulatory mechanisms rather than promoter DNA methylation. Measurements of five classic endogenous hormones and expression analysis of hormone-related genes between VvHB58 transgenic and nontransgenic control plants showed that expression of VvHB58 resulted in significant changes in auxin, gibberellin and ethylene signaling pathways. Additionally, several DNA methylation-related genes were expressed differentially during seed development stages in seedless and seeded grapes, suggesting changes in methylation levels during seed development may be associated with seed abortion.
Conclusion:
VvHB58 has a potential function in regulating fruit and seed development by impacting multiple hormonal pathways. These results expand understanding of homeodomain transcription factors and potential regulatory mechanism of seed development in grapevine, and provided insights into molecular breeding for grapes.
