Single berry development – a new phenotyping and transcriptomics paradigm

Most present knowledge on berry development has been obtained from a random sampling of hundreds of berries to average their diversity of the experimental plot. According to recent studies, such heterogeneous samples formed from non-synchronized berries of mixed developmental stages are unsuitable for detecting fast physiological and molecular changes. Thus, it is necessary to revisit the physiological and transcriptional bases of berry ripening. Here we report the in-depth study of the late-ripening program in three genotypes. Berry expansion during the second growth phase was characterized on-vine through image analysis. Hundreds of sampled berries were individually analyzed for primary metabolites to calculate their respective accumulation rates with high precision. These primary individual fluxes and the growth kinetics allowed us to distinguish targeted developmental stages further investigated through RNA profiling. Single berry monitoring evidenced sharp developmental phases during which specific genes or pathways are quickly switched ON or OFF. The comparison between Syrah and the two microvines showed phenotypic differences in late-ripening stages in vines grown in the field (Syrah) and microvines (MV032 and MV102) grown in the greenhouse. This study shows that new high-throughput single berry phenotyping methods are required to compare unambiguous developmental stages in physiological or genetic studies

Involvement of ethylene signalling in a non-climacteric fruit: new elements regarding the regulation of ADH expression in grapevine

Although grape berries have been classified as non climacteric fruits, ongoing studies on grape ethylene signalling lead to challenge the role of ethylene in their ripening. One of the significant molecular changes in berries is the up regulation of ADH (alcohol dehydrogenase, EC. 1.1.1.1) enzyme activity at the inception of fruit ripening and of VvADH2 transcript levels. This paper shows that the ethylene signal transduction pathway could be involved in the control of VvADH2 expression in grapevine berries and in cell suspensions. The induction of VvADH2 transcription, either in berries at the inception of ripening or in cell suspensions, was found to be partly inhibited by 1 methylcyclopropene (1 MCP), an inhibitor of ethylene receptors. Treatment of cell suspensions with 2 chloroethylphosphonic acid (2-CEPA), an ethylene releasing compound, also resulted in a significant increase of ADH activity and VvADH2 transcription under anaerobiosis, showing that concomitant ethylene and anaerobic treatments in cell suspensions could result in changes of VvADH2 expression. All these results, associated with the presence in the VvADH2 promoter of regulatory elements for ethylene and anaerobic response, suggest that ethylene transduction pathway and anaerobic stress could be in part involved in the regulation of VvADH2 expression in ripening berries and cell suspensions. These data open new aspects of the expression control of a ripening-related gene in a non climacteric fruit

The Microvine: A Versatile Plant Model to Boost Grapevine Studies in Physiology and Genetics

The microvine is a grapevine somatic variant. The Vvgai1 mutation results in a miniaturization of the vegetative organs of the plant keeping fruit size intact and a systematic conversion of tendrils into inflorescences. The physiological characterization of the vegetative and reproductive development of the microvine makes it possible to infer kinetic data from spatial phenotypes. This biological model allows experiments on vine and grape development in tightly controlled conditions, which greatly accelerate physiology, molecular biology, as well as genetic studies. After introducing the main biological properties of the microvine, main results from various research programs performed with the microvine model will be presented

Day and night heat stress trigger different transcriptomic responses in green and ripening grapevine (vitis vinifera) fruit

Background: Global climate change will noticeably affect plant vegetative and reproductive development. The recent increase in temperatures has already impacted yields and composition of berries in many grapevine-growing regions. Physiological processes underlying temperature response and tolerance of the grapevine fruit have not been extensively investigated. To date, all studies investigating the molecular regulation of fleshly fruit response to abiotic stress were only conducted during the day, overlooking possible critical night-specific variations. The present study explores the night and day transcriptomic response of grapevine fruit to heat stress at several developmental stages. Short heat stresses (2 h) were applied at day and night to vines bearing clusters sequentially ordered according to the developmental stages along their vertical axes. The recently proposed microvine model (DRCF-Dwarf Rapid Cycling and Continuous Flowering) was grown in climatic chambers in order to circumvent common constraints and biases inevitable in field experiments with perennial macrovines. Post-véraison berry heterogeneity within clusters was avoided by constituting homogenous batches following organic acids and sugars measurements of individual berries. A whole genome transcriptomic approach was subsequently conducted using NimbleGen 090818 Vitis 12X (30 K) microarrays. Results: Present work reveals significant differences in heat stress responsive pathways according to day or night treatment, in particular regarding genes associated with acidity and phenylpropanoid metabolism. Precise distinction of ripening stages led to stage-specific detection of malic acid and anthocyanin-related transcripts modulated by heat stress. Important changes in cell wall modification related processes as well as indications for heat-induced delay of ripening and sugar accumulation were observed at véraison, an effect that was reversed at later stages. Conclusions: This first day - night study on heat stress adaption of the grapevine berry shows that the transcriptome of fleshy fruits is differentially affected by abiotic stress at night. The present results emphasize the necessity of including different developmental stages and especially several daytime points in transcriptomic studies

Is transcriptomic regulation of berry development more important at night than during the day?

Diurnal changes in gene expression occur in all living organisms and have been studied on model plants such as Arabidopsis thaliana. To our knowledge the impact of the nycthemeral cycle on the genetic program of fleshly fruit development has been hitherto overlooked. In order to circumvent environmental changes throughout fruit development, young and ripening berries were sampled simultaneously on continuously flowering microvines acclimated to controlled circadian light and temperature changes. Gene expression profiles along fruit development were monitored during both day and night with whole genome microarrays (Nimblegen® vitis 12x), yielding a total number of 9273 developmentally modulated probesets. All day-detected transcripts were modulated at night, whereas 1843 genes were night-specific. Very similar developmental patterns of gene expression were observed using independent hierarchical clustering of day and night data, whereas functional categories of allocated transcripts varied according to time of day. Many transcripts within pathways, known to be up-regulated during ripening, in particular those linked to secondary metabolism exhibited a clearer developmental regulation at night than during the day. Functional enrichment analysis also indicated that diurnally modulated genes considerably varied during fruit development, with a shift from cellular organization and photosynthesis in green berries to secondary metabolism and stress-related genes in ripening berries. These results reveal critical changes in gene expression during night development that differ from daytime development, which have not been observed in other transcriptomic studies on fruit development thus far. © 2014 Rienth et al.This work is part of the DURAVITIS program which is financially supported by the ANR (Agence national de la recherche) -Genopole (project ANR-2010-GENM-004-01) and the Jean Poupelain foundation (30 Rue Gâte Chien, 16100 Javrezac, France).Peer Reviewe

Influence of P+ layer parameters on 4H-SiC UV PiN photodetector characteristics

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The sugarless grape trait characterised by single berry phenotyping

In grape production, the selection of varieties well-adapted to climate fluctuations, especially warming, is based on achieving a balance between fruit sugars and acidity. In recent decades, temperature has been constantly rising during ripening causing excessive sugar concentrations and insufficient acidity in wine grapes in the warmest regions. There is thus an increasing interest in breeding new cultivars able to ripen at lower sugar concentration while preserving fruit acidity. However, the phenotyping of berry composition challenges both methodological and conceptual issues. Indeed, most authors predetermine either average harvest date, ripening duration, thermal time or even the hexoses concentration threshold itself to compare accessions at a hopefully similar ripe stage. In this study, we phenotyped the fruit development and composition of 6 genotypes, including 3 new disease-tolerant varieties known to produce wines with low alcoholic contents. The study was performed at single berry level from the end of the green growth stage to the end of phloem unloading, when water and solute contents reach a maximum per berry. The results confirm that sugarless genotypes achieve fruit ripening with 20-30 % less hexoses than the classical varieties, Grenache N and Merlot N, without impacting berry growth, total acidity or cation accumulation. The sugarless genotypes displayed a higher malic acid/tartaric acid balance than the other genotypes, but similar sucrose/H+ exchanges at the onset of ripening. Data suggest that the sugarless phenotype results from a specific plasticity in the relationship between growth and the turgor imposed by organic acid accumulation and sugar loading. This opens interesting perspectives for the understanding of the mechanism of grapevine berry growth and for breeding varieties that will cope better with climate warming

Patterns of sequence polymorphism in the fleshless berry locus in cultivated and wild Vitis vinifera accessions

<p>Abstract</p> <p>Background</p> <p>Unlike in tomato, little is known about the genetic and molecular control of fleshy fruit development of perennial fruit trees like grapevine (<it>Vitis vinifera </it>L.). Here we present the study of the sequence polymorphism in a 1 Mb grapevine genome region at the top of chromosome 18 carrying the <it>fleshless berry </it>mutation (<it>flb</it>) in order, first to identify SNP markers closely linked to the gene and second to search for possible signatures of domestication.</p> <p>Results</p> <p>In total, 62 regions (17 SSR, 3 SNP, 1 CAPS and 41 re-sequenced gene fragments) were scanned for polymorphism along a 3.4 Mb interval (85,127-3,506,060 bp) at the top of the chromosome 18, in both <it>V. vinifera cv</it>. Chardonnay and a genotype carrying the <it>flb </it>mutation, <it>V. vinifera cv</it>. Ugni Blanc mutant. A nearly complete homozygosity in Ugni Blanc (wild and mutant forms) and an expected high level of heterozygosity in Chardonnay were revealed. Experiments using qPCR and BAC FISH confirmed the observed homozygosity. Under the assumption that <it>flb </it>could be one of the genes involved into the domestication syndrome of grapevine, we sequenced 69 gene fragments, spread over the <it>flb </it>region, representing 48,874 bp in a highly diverse set of cultivated and wild <it>V. vinifera </it>genotypes, to identify possible signatures of domestication in the cultivated <it>V. vinifera </it>compartment. We identified eight gene fragments presenting a significant deviation from neutrality of the Tajima's D parameter in the cultivated pool. One of these also showed higher nucleotide diversity in the wild compartments than in the cultivated compartments. In addition, SNPs significantly associated to berry weight variation were identified in the <it>flb </it>region.</p> <p>Conclusions</p> <p>We observed the occurrence of a large homozygous region in a non-repetitive region of the grapevine otherwise highly-heterozygous genome and propose a hypothesis for its formation. We demonstrated the feasibility to apply BAC FISH on the very small grapevine chromosomes and provided a specific probe for the identification of chromosome 18 on a cytogenetic map. We evidenced genes showing putative signatures of selection and SNPs significantly associated with berry weight variation in the <it>flb </it>region. In addition, we provided to the community 554 SNPs at the top of chromosome 18 for the development of a genotyping chip for future fine mapping of the <it>flb </it>gene in a F2 population when available.</p

Identification of stable QTLs for vegetative and reproductive traits in the microvine (Vitis vinifera L.) using the 18 K Infinium chip

UMR AGAP - équipe DAAV - Diversité, adaptation et amélioration de la vigne[b]Background[/b] [br/]The increasing temperature associated with climate change impacts grapevine phenology and development with critical effects on grape yield and composition. Plant breeding has the potential to deliver new cultivars with stable yield and quality under warmer climate conditions, but this requires the identification of stable genetic determinants. This study tested the potentialities of the microvine to boost genetics in grapevine. A mapping population of 129 microvines derived from Picovine x Ugni Blanc flb, was genotyped with the Illumina® 18 K SNP (Single Nucleotide Polymorphism) chip. Forty-three vegetative and reproductive traits were phenotyped outdoors over four cropping cycles, and a subset of 22 traits over two cropping cycles in growth rooms with two contrasted temperatures, in order to map stable QTLs (Quantitative Trait Loci). [br/][b]Results[/b] [br/]Ten stable QTLs for berry development and quality or leaf area were identified on the parental maps. A new major QTL explaining up to 44 % of total variance of berry weight was identified on chromosome 7 in Ugni Blanc flb, and co-localized with QTLs for seed number (up to 76 % total variance), major berry acids at green lag phase (up to 35 %), and other yield components (up to 25 %). In addition, a minor QTL for leaf area was found on chromosome 4 of the same parent. In contrast, only minor QTLs for berry acidity and leaf area could be found as moderately stable in Picovine. None of the transporters recently identified as mutated in low acidity apples or Cucurbits were included in the several hundreds of candidate genes underlying the above berry QTLs, which could be reduced to a few dozen candidate genes when a priori pertinent biological functions and organ specific expression were considered. [br/][b]Conclusions[/b] [br/]This study combining the use of microvine and a high throughput genotyping technology was innovative for grapevine genetics. It allowed the identification of 10 stable QTLs, including the first berry acidity QTLs reported so far in a Vitis vinifera intra-specific cross. Robustness of a set of QTLs was assessed with respect to temperature variatio