Virus-induced gene complementation reveals a transcription factor network in modulation of tomato fruit ripening

Plant virus technology, in particular virus-induced gene silencing, is a widely used reverse- and forward-genetics tool in plant functional genomics. However the potential of virus technology to express genes to induce phenotypes or to complement mutants in order to understand the function of plant genes is not well documented. Here we exploit Potato virus X as a tool for virus-induced gene complementation (VIGC). Using VIGC in tomato, we demonstrated that ectopic viral expression of LeMADS-RIN, which encodes a MADS-box transcription factor (TF), resulted in functional complementation of the non-ripening rin mutant phenotype and caused fruits to ripen. Comparative gene expression analysis indicated that LeMADS-RIN up-regulated expression of the SBP-box (SQUAMOSA promoter binding protein-like) gene LeSPL-CNR, but down-regulated the expression of LeHB-1, an HD-Zip homeobox TF gene. Our data support the hypothesis that a transcriptional network may exist among key TFs in the modulation of fruit ripening in tomato

Horticulture Research

Grafting is an ancient method that has been intensively used for the clonal propagation of vegetables and woody trees. Despite its importance in agriculture the physiological and molecular mechanisms underlying phenotypic changes of plants following grafting are still poorly understood. In the present study, we analyse the populations of small RNAs in homo and heterografts and take advantage of the sequence differences in the genomes of heterograft partners to analyse the possible exchange of small RNAs. We demonstrate that the type of grafting per se dramatically influences the small RNA populations independently of genotypes but also show genotype specific effects. In addition, we demonstrate that bilateral exchanges of small RNAs, mainly short interfering RNAs, may occur in heterograft with the preferential transfer of small RNAs from the scion to the rootstock. Altogether, the results suggest that small RNAs may have an important role in the phenotype modifications observed in heterografts

Limiting carbohydrates to trunk and roots improves bud fruitfulness, fruit set and yield in cv. Malbec

Many commercial vineyards of Malbec, the most cultivated grapevine in Argentina, show unstable yield because of variations in bud fruitfulness and the occurrence of “shatter”, characterised by poor fruit set and fruitlet abscission. Shatter can be due to plant material, growing conditions and meteorological events. Among the parameters that determine fruit set efficiency and vine yield, the availability of carbohydrates (CH) plays an essential role. We previously showed that controlling CH partitioning by removing part of the phloem tissue through an annular incision at the base of the fruit shoots (shoot girdling) reduced shatter in Malbec. The objective of this research was to evaluate the partitioning of CH for the different sink organs of the aerial part of the plant when an interruption of phloem flux from leaves to storage organs is imposed by a girdle. Shoot and trunk girdling trials were conducted during the 2018 and 2019 growing seasons, respectively. At flowering, girdling was performed on different plant lots either at the base of the shoot (Base G), above the distal cluster of the shoot (Top G), to the trunk (Trunk G) or no girdling (Control). Most of the yield components were increased by Base G and Trunk G with no significant impact on vegetative growth or fruit quality. Total shoot biomass was not affected in Base G, while a 39 % reduction was observed in Top G, as compared to Control. The partitioning pattern within the shoot was modified by shoot girdling, and Base G favoured the accumulation of CH towards clusters at the expense of lateral shoots. Shoot girdling increased node diameter, bud fruitfulness and inflorescence dry weight of the shoot. Trunk-girdled vines showed an increase in fruit set and total yield at harvest, with no significant impact on vegetative growth. The restriction of CH flow to the trunk and roots by Base G and Trunk G at flowering increased yield components at harvest without affecting vegetative growth or grape quality. These results reinforce our hypothesis that the root system of the vines under study constitutes a strong sink during the flowering period that competes for CH

Moving towards grapevine genotypes better adapted to abiotic constraints

Vitis spp., both in their cultivated and wild forms, have been growing in a large diversity of environments for thousands of years. As a result, they have developed many adaptive mechanisms controlled by a range of regulatory processes. The cultivated species, Vitis vinifera, is quite well adapted to semi-arid conditions and its cultivation can be used to produce crops on marginal lands. However, this is threatened by climate change, which is associated with increased temperature and CO2 atmospheric content, changes in water availability and an increased likelihood of extreme events, such as heat waves and early spring frosts. Indirect effects of climate change on solar radiation and soil minerals are also expected. Consequently, cultivated grapevines will presumably face more abiotic constraints occurring concomitantly or successively over one or more growing cycles. In addition to climate change, worldwide viticulture must reduce the use of pesticides. Adapting to climate change and reducing pesticide use are challenging, and increase the need to create new grapevine varieties that are more resistant to diseases and better adapted to abiotic constraints. For this purpose, the adaptive mechanisms of wild and cultivated Vitis spp. must be exploited. While major advances have already been made in exploiting wild alleles for disease resistance, the polygenic nature of adaptation to abiotic factors has slowed down research progress. To tackle this limitation, ambitious integrative strategies need to be undertaken from collection and characterization of genetic resources, investigations on genetic architecture and identification of underlying genes (including those involved in epigenetic regulation), to the implementation of new breeding technologies and the development of genomic selection. An update on the state-of-the-art regarding these aspects is presented

Epigenetics: an innovative lever for grapevine breeding in times of climatic changes

Climate change imposes numerous threats to viticulture. Different strategies have been developed to mitigate these effects that range from innovative vineyard management methods and precision viticulture to the breeding of new varieties and rootstocks better adapted to environmental challenges. Epigenetics refer to heritable changes in genome functioning that are not mediated by DNA sequence variations. The recent discovery that epigenetic memories can mediate acclimation and adaptation of plants to their environment now provides new levers for plant improvement facing climate changes without significant impact on the genetic information. This can be mediated either by using the epigenetic memories of stresses and/ or by creating epigenetic diversity in the form of new epialleles without changing the genetic information. Indeed, grapevine is a perennial grafted clonally propagated plant, and as such, presents epigenetic specificities. These specificities require adapting strategies that have already been developed in model plants but also offer opportunities to explore how epigenetic memories and diversity can be a major source of rapid adaptation to the environment for plants bearing similar properties. Among these strategies, both annual and trans-annual plant priming with different types of elicitors might provide efficient ways to better face (a)biotic stresses. The use of epigenetic exchanges between scion and rootstocks and/or the creation of non-targeted epigenetic variations at a genome-wide scale, or targeted using epigenetic editing, may provide innovative and promising avenues for grapevine improvement to face challenges imposed by climate changes. © This article is published under the Creative Commons licence (CC BY 4.0)

One prep to catch them all: “2 in 1”, an efficient method for the simultaneous extraction of DNA and RNA from Grapevine tissues

Recent advances in our understanding of plant physiology and adaptation to the environment are tightly related to the development of ‘omics’ technologies such as metabolomics, transcriptomics, genomics and epigenomics that allow a more comprehensive view of the plant functioning. In this context, the ability to extract DNA and RNA from small amounts of plant material can be a limiting factor, worse in the case of non-model plants for which efficient nucleic extraction procedures are lacking. In the case of grapevine, extraction of high-quality DNA is typically limited by the high polyphenolic and polysaccharide contents of the different tissues. Here, we propose an adaptation of the method of Reid et al. (2006) that allows the simultaneous and efficient extraction of DNA and RNA from grapevine vegetative and berry tissues from in vitro grown grapevine plants and cells and from other plants. The protocol allows the extraction of high-quality RNA and DNA for standard molecular biology methods as well as for Next Generation Sequencing (NGS). It also works with a limited amount of plant material, such as young developing buds, and provides the means to analyse “omics” data from a single plant sample

Molecular and functional characterization of SISPL-CNR in tomato fruit ripening and cell death

SlSPL-CNR, an SBP-box transcription factor (TF) gene residing at the epimutant Colourless non-ripening (Cnr) locus, is involved in tomato ripening. This epimutant provides a unique model to investigate the (epi)genetic basis of fruit ripening. Here we report that SlSPL-CNR is a nucleus-localized protein with a distinct monopartite nuclear localization signal (NLS). It consists of four consecutive residues ‘30KRKR33’ at the N-terminal of the protein. Mutation of the NLS abolishes SlSPL-CNR to localize into nucleus. SlSPL-CNR comprises two zinc-finger motifs (ZFMs) within the C-terminal SBP-box domain. Both ZFMs contribute to zinc-binding activity. SlSPL-CNR can induce cell death in tomato and tobacco. Induction of cell death by SlSPL-CNR is dependent on its nuclear localization. However, the two ZFMs have differential impacts on SlSPL-CNR to induce severe necrosis or mild necrotic ringspot. NLS and ZFM mutants cannot complement Cnr fruits to ripen. SlSPL-CNR interacts with SlSnRK1. Virus-induced SlSnRK1 silencing leads to reduction in expression of ripening-related genes and inhibits ripening in tomato. We conclude that SlSPL-CNR is a multifunctional protein that consists of a distinct monopartite NLS, binds to zinc and interacts with SlSnRK1 to affect cell death and tomato fruit ripening

A DEMETER-like DNA demethylase protein governs tomato fruit ripening

In plants, genomic DNA methylation which contributes to development and stress responses can be actively removed by DEMETER-like DNA demethylases (DML). Indeed, in Arabidopsis DMLs are important for maternal imprinting and endosperm demethylation, but only few studies demonstrate the developmental roles of active DNA demethylation conclusively in this plant. Here we show a direct cause and effect relationship between active DNA demethylation mainly mediated by the tomato DML, SlDML2, and fruit ripening; an important developmental process unique to plants. RNAi SlDML2 knock-down results in ripening inhibition via hypermethylation and repression of the expression of genes encoding ripening transcription factors and rate-limiting enzymes of key biochemical processes such as carotenoid synthesis. Our data demonstrate that active DNA demethylation is central to the control of ripening in tomat

Zebularine, a DNA Methylation Inhibitor, Activates Anthocyanin Accumulation in Grapevine Cells

Through its role in the regulation of gene expression, DNA methylation can participate in the control of specialized metabolite production. We have investigated the link between DNA methylation and anthocyanin accumulation in grapevine using the hypomethylating drug, zebularine and Gamay Teinturier cell suspensions. In this model, zebularine increased anthocyanin accumulation in the light, and induced its production in the dark. To unravel the underlying mechanisms, cell transcriptome, metabolic content, and DNA methylation were analyzed. The up-regulation of stress-related genes, as well as a decrease in cell viability, revealed that zebularine affected cell integrity. Concomitantly, the global DNA methylation level was only slightly decreased in the light and not modified in the dark. However, locus-specific analyses demonstrated a decrease in DNA methylation at a few selected loci, including a CACTA DNA transposon and a small region upstream from the UFGT gene, coding for the UDP glucose:flavonoid-3-O-glucosyltransferase, known to be critical for anthocyanin biosynthesis. Moreover, this decrease was correlated with an increase in UFGT expression and in anthocyanin content. In conclusion, our data suggest that UFGT expression could be regulated through DNA methylation in Gamay Teinturier, although the functional link between changes in DNA methylation and UFGT transcription still needs to be demonstrated