Regulation of polar auxin transport in grapevine fruitlets (Vitis vinifera L.) and the proposed role of auxin homeostasis during fruit abscission.

BACKGROUND: Indole-3-acetic acid (IAA), the most abundant auxin, is a growth promoter hormone involved in several developmental processes. Auxin homeostasis is very important to its function and this is achieved through the regulation of IAA biosynthesis, conjugation, degradation and transport. In grapevine, IAA plays an essential role during initial stages of berry development, since it delays fruitlet abscission by reducing the ethylene sensitivity in the abscission zone. For this reason, Continuous polar IAA transport to the pedicel is required. This kind of transport is controlled by IAA, which regulates its own movement by modifying the expression and localization of PIN-FORMED (PIN) auxin efflux facilitators that localize asymmetrically within the cell. On the other hand, the hormone gibberellin (GA) also activates the polar auxin transport by increasing PIN stability. In Vitis vinifera, fruitlet abscission occurs during the first two to three weeks after flowering. During this time, IAA and GA are present, however the role of these hormones in the control of polar auxin transport is unknown. RESULTS: In this work, the use of radiolabeled IAA showed that auxin is basipetally transported during grapevine fruitlet abscission. This observation was further supported by immunolocalization of putative VvPIN proteins that display a basipetal distribution in pericarp cells. Polar auxin transport and transcripts of four putative VvPIN genes decreased in conjunction with increased abscission, and the inhibition of polar auxin transport resulted in fruit drop. GA3 and IAA treatments reduced polar auxin transport, but only GA3 treatment decreased VvPIN transcript abundance. When GA biosynthesis was blocked, IAA was capable to increase polar auxin transport, suggesting that its effect depends on GA content. Finally, we observed significant changes in the content of several IAA-related compounds during the abscission period. CONCLUSIONS: These results provide evidence that auxin homeostasis plays a central role during grapevine initial fruit development and that GA and IAA controls auxin homeostasis by reducing polar auxin transport.

The contribution of flowering time and seed content to uneven ripening initiation among fruits within Vitis vinifera L. cv. Pinot noir clusters.

MAIN CONCLUSION: Ripening initiation-associated hormonal changes and sugar accumulation for individual fruits differed by seed content and did not depend heavily on flowering time or duration from anthesis to clusters' onset of ripening. For Vitis vinifera, the ripening initiation of individual fruits in a cluster occurs unevenly. This developmental period is called véraison. Why individual fruits initiate ripening at different times is not well studied, though differences in seed content and unequal developmental durations that arise from asynchronous flowering within a cluster have been proposed. This study examined how much both variables contribute to individual fruits' ripening progress by mid-véraison, when half of berries in a cluster have initiated ripening, and whether either or both factors affect the timing of characteristic, ripening-initiation associated changes in abscisic acid and auxin before, at, and after véraison. Overall, developmental duration and flowering time did not sufficiently explain how far berries had progressed into the ripening stage because fruits did not require a fixed amount of time to initiate ripening. Fruits from early and late flowers but of similar seed content were able to initiate ripening at the same time despite differences in chronological age. This suggests either an early developmental enhancement occurred for late-initiated fruits or that flowering time is an inappropriate "day zero". Ultimately, only seed content was linked to the timing and magnitude of ripening-related hormone changes, supporting that seeds have a comparatively larger influence than flowering time on the ripening initiation of individual berries. More specifically, if the fraction of berry weight occupied by seed was high, then the initiation of ripening for that berry and its associated hormone changes were delayed relative to berries with less seed weight versus total berry weight.

Timing of ripening initiation in grape berries and its relationship to seed content and pericarp auxin levels.

BACKGROUND: Individual berries in a grape (Vitis vinifera L.) cluster enter the ripening phase at different times leading to an asynchronous cluster in terms of ripening. The factors causing this variable ripening initiation among berries are not known. Because the influence via hormonal communication of the seed on fruit set and growth is well known across fruit species, differences in berry seed content and resultant quantitative or qualitative differences in the hormone signals to the pericarp likely influence the relative timing of ripening initiation among berries of the cluster. RESULTS: At the time of the initiation of cluster ripening (véraison), underripe green berries have higher seed content compared to the riper berries and there is a negative correlation between the seed weight-to-berry weight ratio (SB) and the sugar level in berries of a cluster. Auxin levels in seeds relative to the pericarp tissues are two to 12 times higher at pre-ripening stages. The pericarp of berries with high-SB had higher auxin and lower abscisic acid (ABA) levels compared to those with low-SB from two weeks before véraison. In the prevéraison cluster, the expression of auxin-response factor genes was significantly higher in the pericarp of high-SB berries and remained higher until véraison compared to low-SB berries. The expression level of auxin-biosynthetic genes in the pericarp was the same between both berry groups based upon similar expression activity of YUC genes that are rate-limiting factors in auxin biosynthesis. On the other hand, in low-SB berries, the expression of ABA-biosynthetic and ABA-inducible NCED and MYB genes was higher even two weeks before véraison. CONCLUSIONS: Differences in the relative seed content among berries plays a major role in the timing of ripening initiation. Towards the end of berry maturation phase, low and high levels of auxin are observed in the pericarp of low- and high-SB berries, respectively. This results in higher auxin-signaling activity that lasts longer in the pericarp of high-SB berries. In contrast, in low-SB berries, concomitant with an earlier decrease of auxin level, the features of ripening initiation, such as increases in ABA and sugar accumulation begin earlier.

A comparative study of ripening among berries of the grape cluster reveals an altered transcriptional programme and enhanced ripening rate in delayed berries.

Transcriptional studies in relation to fruit ripening generally aim to identify the transcriptional states associated with physiological ripening stages and the transcriptional changes between stages within the ripening programme. In non-climacteric fruits such as grape, all ripening-related genes involved in this programme have not been identified, mainly due to the lack of mutants for comparative transcriptomic studies. A feature in grape cluster ripening (Vitis vinifera cv. Pinot noir), where all berries do not initiate the ripening at the same time, was exploited to study their shifted ripening programmes in parallel. Berries that showed marked ripening state differences in a véraison-stage cluster (ripening onset) ultimately reached similar ripeness states toward maturity, indicating the flexibility of the ripening programme. The expression variance between these véraison-stage berry classes, where 11% of the genes were found to be differentially expressed, was reduced significantly toward maturity, resulting in the synchronization of their transcriptional states. Defined quantitative expression changes (transcriptional distances) not only existed between the véraison transitional stages, but also between the véraison to maturity stages, regardless of the berry class. It was observed that lagging berries complete their transcriptional programme in a shorter time through altered gene expressions and ripening-related hormone dynamics, and enhance the rate of physiological ripening progression. Finally, the reduction in expression variance of genes can identify new genes directly associated with ripening and also assess the relevance of gene activity to the phase of the ripening programme.

Transgene-free genome editing and RNAi ectopic application in fruit trees: Potential and limitations.

For the past fifteen years, significant research advances in sequencing technology have led to a substantial increase in fruit tree genomic resources and databases with a massive number of OMICS datasets (transcriptomic, proteomics, metabolomics), helping to find associations between gene(s) and performance traits. Meanwhile, new technology tools have emerged for gain- and loss-of-function studies, specifically in gene silencing and developing tractable plant models for genetic transformation. Additionally, innovative and adapted transformation protocols have optimized genetic engineering in most fruit trees. The recent explosion of new gene-editing tools allows for broadening opportunities for functional studies in fruit trees. Yet, the fruit tree research community has not fully embraced these new technologies to provide large-scale genome characterizations as in cereals and other staple food crops. Instead, recent research efforts in the fruit trees appear to focus on two primary translational tools: transgene-free gene editing via Ribonucleoprotein (RNP) delivery and the ectopic application of RNA-based products in the field for crop protection. The inherent nature of the propagation system and the long juvenile phase of most fruit trees are significant justifications for the first technology. The second approach might have the public favor regarding sustainability and an eco-friendlier environment for a crop production system that could potentially replace the use of chemicals. Regardless of their potential, both technologies still depend on the foundational knowledge of gene-to-trait relationships generated from basic genetic studies. Therefore, we will discuss the status of gene silencing and DNA-based gene editing techniques for functional studies in fruit trees followed by the potential and limitations of their translational tools (RNP delivery and RNA-based products) in the context of crop production.

The role of auxin during early berry development in grapevine as revealed by transcript profiling from pollination to fruit set.

Auxin is a key phytohormone that modulates fruit formation in many fleshy fruits through the regulation of cell division and expansion. Auxin content rapidly increases after pollination and the manipulation in its levels may lead to the parthenocarpic development. ln Vitis vinifera L., little is known about the early fruit development that encompasses from pollination to fruit set. Pollination/fertilization events trigger fruit formation, and auxin treatment mimics their effect in grape berry set. However, the role of auxin in this process at the molecular level is not well understood. To elucidate the participation of auxin in grapevine fruit formation, morphological, reproductive, and molecular events from anthesis to fruit set were described in sequential days after pollination. Exploratory RNA-seq analysis at four time points from anthesis to fruit set revealed that the highest percentage of genes induced/repressed within the hormone-related gene category were auxin-related genes. Transcript profiling showed significant transcript variations in auxin signaling and homeostasis-related genes during the early fruit development. Indole acetic acid and several auxin metabolites were present during this period. Finally, application of an inhibitor of auxin action reduced cell number and the mesocarp diameter, similarly to unpollinated berries, further confirming the key role of auxin during early berry development. This work sheds light into the molecular features of the initial fruit development and highlights the auxin participation during this stage in grapevine.

Charting plant interactomes: possibilities and challenges.

Protein-protein interactions are essential for nearly all cellular processes. Therefore, an important goal of post-genomic research for defining gene function and understanding the function of macromolecular complexes involves creating 'interactome' maps from empirical or inferred datasets. Systematic efforts to conduct high-throughput surveys of protein-protein interactions in plants are needed to chart the complex and dynamic interaction networks that occur throughout plant development. However, no single approach can build a complete map of the interactome. Here, we review the utility and potential of various experimental approaches for creating large-scale protein-protein interaction maps in plants. Bioinformatics approaches for curating and assessing the confidence of these datasets through inter-species comparisons will be crucial in achieving a complete understanding of protein interaction networks in plants.

Short day transcriptomic programming during induction of dormancy in grapevine.

Bud dormancy in grapevine is an adaptive strategy for the survival of drought, high and low temperatures and freeze dehydration stress that limit the range of cultivar adaptation. Therefore, development of a comprehensive understanding of the biological mechanisms involved in bud dormancy is needed to promote advances in selection and breeding, and to develop improved cultural practices for existing grape cultivars. The seasonally indeterminate grapevine, which continuously develops compound axillary buds during the growing season, provides an excellent system for dissecting dormancy, because the grapevine does not transition through terminal bud development prior to dormancy. This study used gene expression patterns and targeted metabolite analysis of two grapevine genotypes that are short photoperiod responsive (Vitis riparia) and non-responsive (V. hybrid, Seyval) for dormancy development to determine differences between bud maturation and dormancy commitment. Grapevine gene expression and metabolites were monitored at seven time points under long (LD, 15 h) and short (SD, 13 h) day treatments. The use of age-matched buds and a small (2 h) photoperiod difference minimized developmental differences and allowed us to separate general photoperiod from dormancy specific gene responses. Gene expression profiles indicated three distinct phases (perception, induction and dormancy) in SD-induced dormancy development in V. riparia. Different genes from the NAC DOMAIN CONTAINING PROTEIN 19 and WRKY families of transcription factors were differentially expressed in each phase of dormancy. Metabolite and transcriptome analyses indicated ABA, trehalose, raffinose and resveratrol compounds have a potential role in dormancy commitment. Finally, a comparison between V. riparia compound axillary bud dormancy and dormancy responses in other species emphasized the relationship between dormancy and the expression of RESVERATROL SYNTHASE and genes associated with C3HC4-TYPE RING FINGER and NAC DOMAIN CONTAINING PROTEIN 19 transcription factors.

VitisCyc: a metabolic pathway knowledgebase for grapevine (Vitis vinifera).

We have developed VitisCyc, a grapevine-specific metabolic pathway database that allows researchers to (i) search and browse the database for its various components such as metabolic pathways, reactions, compounds, genes and proteins, (ii) compare grapevine metabolic networks with other publicly available plant metabolic networks, and (iii) upload, visualize and analyze high-throughput data such as transcriptomes, proteomes, metabolomes etc. using OMICs-Viewer tool. VitisCyc is based on the genome sequence of the nearly homozygous genotype PN40024 of Vitis vinifera "Pinot Noir" cultivar with 12X v1 annotations and was built on BioCyc platform using Pathway Tools software and MetaCyc reference database. Furthermore, VitisCyc was enriched for plant-specific pathways and grape-specific metabolites, reactions and pathways. Currently VitisCyc harbors 68 super pathways, 362 biosynthesis pathways, 118 catabolic pathways, 5 detoxification pathways, 36 energy related pathways and 6 transport pathways, 10,908 enzymes, 2912 enzymatic reactions, 31 transport reactions and 2024 compounds. VitisCyc, as a community resource, can aid in the discovery of candidate genes and pathways that are regulated during plant growth and development, and in response to biotic and abiotic stress signals generated from a plant's immediate environment. VitisCyc version 3.18 is available online at http://pathways.cgrb.oregonstate.edu.