Margherita Lucchin

Genome-wide analysis of the grapevine stilbene synthase multigenic family: genomic organization and expression profiles upon biotic and abiotic stresses

BackgroundPlant stilbenes are a small group of phenylpropanoids, which have been detected in at least 72 unrelated plant species and accumulate in response to biotic and abiotic stresses such as infection, wounding, UV-C exposure and treatment with chemicals. Stilbenes are formed via the phenylalanine/polymalonate-route, the last step of which is catalyzed by the enzyme stilbene synthase (STS), a type III polyketide synthase (PKS). Stilbene synthases are closely related to chalcone synthases (CHS), the key enzymes of the flavonoid pathway, as illustrated by the fact that both enzymes share the same substrates. To date, STSs have been cloned from peanut, pine, sorghum and grapevine, the only stilbene-producing fruiting-plant for which the entire genome has been sequenced. Apart from sorghum, STS genes appear to exist as a family of closely related genes in these other plant species.ResultsIn this study a complete characterization of the STS multigenic family in grapevine has been performed, commencing with the identification, annotation and phylogenetic analysis of all members and integration of this information with a comprehensive set of gene expression analyses including healthy tissues at differential developmental stages and in leaves exposed to both biotic (downy mildew infection) and abiotic (wounding and UV-C exposure) stresses. At least thirty-three full length sequences encoding VvSTS genes were identified, which, based on predicted amino acid sequences, cluster in 3 principal groups designated A, B and C. The majority of VvSTS genes cluster in groups B and C and are located on chr16 whereas the few gene family members in group A are found on chr10. Microarray and mRNA-seq expression analyses revealed different patterns of transcript accumulation between the different groups of VvSTS family members and between VvSTSs and VvCHSs. Indeed, under certain conditions the transcriptional response of VvSTS and VvCHS genes appears to be diametrically opposed suggesting that flow of carbon between these two competing metabolic pathways is tightly regulated at the transcriptional level.ConclusionsThis study represents an overview of the expression pattern of each member of the STS gene family in grapevine under both constitutive and stress-induced conditions. The results strongly indicate the existence of a transcriptional subfunctionalization amongst VvSTSs and provide the foundation for further functional investigations about the role and evolution of this large gene family. Moreover, it represents the first study to clearly show the differential regulation of VvCHS and VvSTS genes, suggesting the involvement of transcription factors (TFs) in both the activation and repression of these genes.

A deep survey of alternative splicing in grape reveals changes in the splicing machinery related to tissue, stress condition and genotype

BackgroundAlternative splicing (AS) significantly enhances transcriptome complexity. It is differentially regulated in a wide variety of cell types and plays a role in several cellular processes. Here we describe a detailed survey of alternative splicing in grape based on 124 SOLiD RNAseq analyses from different tissues, stress conditions and genotypes.ResultsWe used the RNAseq data to update the existing grape gene prediction with 2,258 new coding genes and 3,336 putative long non-coding RNAs. Several gene structures have been improved and alternative splicing was described for about 30% of the genes. A link between AS and miRNAs was shown in 139 genes where we found that AS affects the miRNA target site. A quantitative analysis of the isoforms indicated that most of the spliced genes have one major isoform and tend to simultaneously co-express a low number of isoforms, typically two, with intron retention being the most frequent alternative splicing event.ConclusionsAs described in Arabidopsis, also grape displays a marked AS tissue-specificity, while stress conditions produce splicing changes to a minor extent. Surprisingly, some distinctive splicing features were also observed between genotypes. This was further supported by the observation that the panel of Serine/Arginine-rich splicing factors show a few, but very marked differences between genotypes. The finding that a part the splicing machinery can change in closely related organisms can lead to some interesting hypotheses for evolutionary adaptation, that could be particularly relevant in the response to sudden and strong selective pressures.

The R2R3-MYB Transcription Factors MYB14 and MYB15 Regulate Stilbene Biosynthesis in Vitis vinifera

This study reports the identification and functional characterization of two stress-inducible R2R3-MYB–type transcription factors, termed MYB14 and MYB15, which regulate the stilbene biosynthetic pathway in grapevine. Plant stilbenes are phytoalexins that accumulate in a small number of plant species, including grapevine (Vitis vinifera), in response to biotic and abiotic stresses and have been implicated in many beneficial effects on human health. In particular, resveratrol, the basic unit of all other complex stilbenes, has received widespread attention because of its cardio-protective, anticarcinogenic, and antioxidant properties. Although stilbene synthases (STSs), the key enzymes responsible for resveratrol biosynthesis, have been isolated and characterized from several plant species, the transcriptional regulation underlying stilbene biosynthesis is unknown. Here, we report the identification and functional characterization of two R2R3-MYB–type transcription factors (TFs) from grapevine, which regulate the stilbene biosynthetic pathway. These TFs, designated MYB14 and MYB15, strongly coexpress with STS genes, both in leaf tissues under biotic and abiotic stress and in the skin and seed of healthy developing berries during maturation. In transient gene reporter assays, MYB14 and MYB15 were demonstrated to specifically activate the promoters of STS genes, and the ectopic expression of MYB15 in grapevine hairy roots resulted in increased STS expression and in the accumulation of glycosylated stilbenes in planta. These results demonstrate the involvement of MYB14 and MYB15 in the transcriptional regulation of stilbene biosynthesis in grapevine.

Comprehensive transcript profiling of two grapevine rootstock genotypes contrasting in drought susceptibility links the phenylpropanoid pathway to enhanced tolerance

Highlights Drought tolerance in the M4 grapevine rootstock genotype could be associated with a higher capability to counteract oxidative stresses by enhancing the accumulation of resveratrol in roots.

Production of asymmetric somatic hybrid plants between Cichorium intybus L. and Helianthus annuus L.

Abstract In order to obtain male-sterile asymmetric somatic hybrids between chicory (Cichorium intybus L.) and a sunflower (Helianthus annuus L.) male-sterile cytoplasmic line, mesophyll chicory protoplasts inactivated with iodoacetic acid and hypocotyl sunflower protoplasts irradiated with γ-rays have been fused, using PEG and applying two different procedures. Thirty three plants were regenerated from putative hybrid calli. A cytological analysis of their root-tip cells indicated that most of them had 18 chromosomes, the same number as chicory. Through Southern hybridisation on total DNA using the maize mitochondrial specific gene probes Cox I, Cox II and Cob, three plants were identified as cytoplasmic asymmetric hybrids, as shown by hybridisation bands specific for both chicory and sunflower. One of the regenerated plants produced a novel pattern of hybridisation that was not detected in either parent. When hybridisation of total DNA was carried out with an atpA mitochondrial gene probe the same three cybrids presented both the fertile chicory fragment and the male-sterile sunflower fragment. Finally, Southern hybridisation with an ORF 522 probe, which in sunflower is co-transcribed with the atpA gene, confirmed the hybrid nature of the three plants. The morphology of the cybrids resembled the parental chicory phenotype, and at anthesis their anthers produced fewer pollen grains which could not germinate either ”in vitro” or ”in situ.” Cybrid plants grown in the field produced seeds when free-pollination occurred.

Analysis of gene expression during flowering in apomeiotic mutants of Medicago spp.: cloning of ESTs and candidate genes for 2n eggs

Mutants showing features of apomixis have been documented in alfalfa (Medicago sativa L.), a natural outcrossing sexual species. A differential display of mRNAs that combines cDNA-AFLP markers and bulked segregant analysis was carried out with the aim of selecting expressed sequence tags (ESTs) and cloning candidate genes for apomeiosis in mutants of alfalfa characterized by 2n egg formation at high frequencies. The approach enabled us to select either mutant- or wild type-specific transcript derived-fragments and to detect transcriptional changes potentially related to 2n eggs. Sequence alignments of a subset of 40 polymorphic clones showed significant homologies to genes of known function. An EST with identity to a β-tubulin gene, highly expressed in the wild type and poorly expressed in the apomeiotic mutants, and an EST with identity to a Mob1-like gene, qualitatively polymorphic between pre- and post-meiotic stages, were selected as candidate genes for apomeiosis because of their putative roles in the cell cycle. A number of clone-specific primers were designed for performing both 5′ and 3′ rapid amplification of cDNA ends to obtain the full-length clones. Southern blot hybridization revealed that both clones belong to a multi-gene family with a minimum of three genomic DNA members each. Northern blot hybridization of total RNA samples and in situ hybridization of whole buds enabled the definition of their temporal and spatial expression patterns in reproductive organs. Experimental achievements towards the elucidation of apomeiotic megasporogenesis in alfalfa are presented and discussed

A first linkage map of Cichorium intybus L. using a one-way pseudo-testcross and PCR-derived markers

We have used a one-way pseudo-testcross mapping strategy in combination with different types of PCR-based markers (RAPD, AFLP, SAMPL) to construct a first linkage map for variegated chicory (Cichorium intybus L. var. silvestre Biskoff, n=9), a self-incompatible vegetable species. The success of such a strategy depends on the presence of sufficiently high levels of heterozygosity in the individual plant which is being mapped and on the informativeness of the marker system that is used. A total of 371 markers, comprising 16 RAPDs, 72 SAMPLs and 283 AFLPs, were scored in 46 F1 individuals obtained from an interspecific cross between a C. intybus outbred individual and a C. endivia inbred line. Grouping of the markers at a LOD score of 4.0 resulted in 13 linkage groups covering 1330 cM. A framework map covering 1201.4 cM was assembled by using all markers that could be ordered with a LOD greater than 2.0. We estimate the total genome size of chicory to be ca. 1405 cM, thus considerably smaller than that estimated for lettuce (1950 cM). The usefulness of the different marker systems that were applied is analysed in terms of level of heterozygosity and marker index, i.e. number of different genetic loci that may be simultaneously analysed per experiment. Out of the 371 markers, 50 of them showed segregation distortion which is discussed in terms of the hybrid origin of the variegated chicory.

Alfalfa Mob1-like Genes are Expressed in Reproductive Organs during Meiosis and Gametogenesis

Mps-one-binder (Mob) proteins play an important role in chromosome separation and cell plate formation in yeast. We cloned two Mob1-like genes from alfalfa (Medicago sativa L.) and show that one gene is constitutively expressed while the other is expressed only in flower buds during sporogenesis and gametogenesis. For the analysis of gene expression during reproduction in alfalfa wild-types and apomeiotic mutants, a specific antisense riboprobe was designed for MsMob1 transcripts and a polyclonal antibody was raised against MsMob1 proteins. In situ mRNA localization as well as protein immunolocalization proved that MsMob1-like genes are specifically expressed in degenerating megaspores of normal ovules and in enlarged megaspore mother cells and embryo sacs of apomeiotic ovules. Gene products were also found in microspore tetrads at the beginning of pollen development as well as in tapetum cells of anthers undergoing programmed cell death to allow pollen dispersal at maturity. Overall results suggest that MsMob1-like genes can play a key role during the reproductive pathway in plants.

Characterization of a flint maize (Zea mays var. indurata) Italian landrace, II. Genetic diversity and relatedness assessed by SSR and Inter-SSR molecular markers

A comparative characterization of 10 field populations of the maize (Zea mays var. indurata) landrace “Nostrano di Storo” was carried out using different types of PCR-based markers. The inbred line B73 and three synthetics (VA143, VA154 and VA157) selected from as many landraces were also used. Genetic diversity and relatedness were evaluated over 84 SSR and 53 I-SSR marker alleles using a total of 253 individual DNAs. Up to 23 alleles per SSR locus were scored while the average effective number of alleles per population was 6.99. Neis total genetic diversity as assessed with SSR markers was HT = 0.851 while the average diversity within populations was HS = 0.795. The overall Wrights fixation index FST was as low as 0.066. Thus, more than 93% of the total variation was within population. Unique alleles over all SSR loci were found for six populations. An average of 17.7 marker alleles per I-SSR primer were scored with an effective number of marker alleles per locus of 1.34. The Shannons diversity information index over all populations and I-SSR loci was 0.332, varying from 0.286 to 0.391. The extent of differentiation between populations was as low as GST = 0.091. Dices genetic similarity matrices were estimated for both SSR and I-SSR markers. The mean genetic similarity coefficients within and between populations were respectively 0.269 and 0.217, for SSR markers, and 0.591 and 0.564, for I-SSR markers. UPGMA dendrograms displayed all field populations but one clustered into a distinct group, in which the synthetic VA154, selected from the “Marano Vicentino” landrace, was also included. One field population and the other two synthetics were clustered separately as well B73. The matrix correlation assayed by the Mantels correspondence test was as high as 0.908. Findings suggest that, although a high variability can be found among plants, most plant genotypes belong to the same landrace called “Nostrano di Storo”. Although gene flow from commercial hybrids might have occurred, the large number of polymorphisms and the presence of both unique alleles and alleles unshared with B73 and synthetics are the main factors underlying the value of this flint maize landrace as a source of genetic variation and peculiar germplasm traits. Because of its exclusive utilization for human consumption, such a molecular marker characterization will be a key step for obtaining the IGP mark and so promote the in situ conservation and protection of the landrace “Nostrano di Storo”.

Grapevine Rootstocks Differentially Affect the Rate of Ripening and Modulate Auxin-Related Genes in Cabernet Sauvignon Berries.

In modern viticulture, grafting commercial grapevine varieties on interspecific rootstocks is a common practice required for conferring resistance to many biotic and abiotic stresses. Nevertheless, the use of rootstocks to gain these essential traits is also known to impact grape berry development and quality, although the underlying mechanisms are still poorly understood. In grape berries, the onset of ripening (véraison) is regulated by a complex network of mobile signals including hormones such as auxins, ethylene, abscisic acid, and brassinosteroids. Recently, a new rootstock, designated M4, was selected based on its enhanced tolerance to water stress and medium vigor. This study investigates the effect of M4 on Cabernet Sauvignon (CS) berry development in comparison to the commercial 1103P rootstock. Physical and biochemical parameters showed that the ripening rate of CS berries is faster when grafted onto M4. A multifactorial analysis performed on mRNA-Seq data obtained from skin and pulp of berries grown in both graft combinations revealed that genes controlling auxin action (ARF and Aux/IAA) represent one of main categories affected by the rootstock genotype. Considering that the level of auxin tightly regulates the transcription of these genes, we investigated the behavior of the main gene families involved in auxin biosynthesis and conjugation. Molecular and biochemical analyses confirmed a link between the rate of berry development and the modulation of auxin metabolism. Moreover, the data indicate that this phenomenon appears to be particularly pronounced in skin tissue in comparison to the flesh.