Genome-Wide Analysis of the Expansin Gene Superfamily Reveals Grapevine-Specific Structural and Functional Characteristics

Background

Expansins are proteins that loosen plant cell walls in a pH-dependent manner, probably by increasing the relative movement among polymers thus causing irreversible expansion. The expansin superfamily (EXP) comprises four distinct families: expansin A (EXPA), expansin B (EXPB), expansin-like A (EXLA) and expansin-like B (EXLB). There is experimental evidence that EXPA and EXPB proteins are required for cell expansion and developmental processes involving cell wall modification, whereas the exact functions of EXLA and EXLB remain unclear. The complete grapevine (Vitis vinifera) genome sequence has allowed the characterization of many gene families, but an exhaustive genome-wide analysis of expansin gene expression has not been attempted thus far.

Methodology/Principal Findings

We identified 29 EXP superfamily genes in the grapevine genome, representing all four EXP families. Members of the same EXP family shared the same exon–intron structure, and phylogenetic analysis confirmed a closer relationship between EXP genes from woody species, i.e. grapevine and poplar (Populus trichocarpa), compared to those from Arabidopsis thaliana and rice (Oryza sativa). We also identified grapevine-specific duplication events involving the EXLB family. Global gene expression analysis confirmed a strong correlation among EXP genes expressed in mature and green/vegetative samples, respectively, as reported for other gene families in the recently-published grapevine gene expression atlas. We also observed the specific co-expression of EXLB genes in woody organs, and the involvement of certain grapevine EXP genes in berry development and post-harvest withering.

Conclusion

Our comprehensive analysis of the grapevine EXP superfamily confirmed and extended current knowledge about the structural and functional characteristics of this gene family, and also identified properties that are currently unique to grapevine expansin genes. Our data provide a model for the functional characterization of grapevine gene families by combining phylogenetic analysis with global gene expression profiling.     

Genome-wide and molecular evolution analysis of the subtilase gene family in Vitis vinifera

Background

Vitis vinifera (grape) is one of the most economically significant fruit crops in the world. The availability of the recently released grape genome sequence offers an opportunity to identify and analyze some important gene families in this species. Subtilases are a group of subtilisin-like serine proteases that are involved in many biological processes in plants. However, no comprehensive study incorporating phylogeny, chromosomal location and gene duplication, gene organization, functional divergence, selective pressure and expression profiling has been reported so far for the grape.

Results

In the present study, a comprehensive analysis of the subtilase gene family in V. vinifera was performed. Eighty subtilase genes were identified. Phylogenetic analyses indicated that these subtilase genes comprised eight groups. The gene organization is considerably conserved among the groups. Distribution of the subtilase genes is non-random across the chromosomes. A high proportion of these genes are preferentially clustered, indicating that tandem duplications may have contributed significantly to the expansion of the subtilase gene family. Analyses of divergence and adaptive evolution show that while purifying selection may have been the main force driving the evolution of grape subtilases, some of the critical sites responsible for the divergence may have been under positive selection. Further analyses of real-time PCR data suggested that many subtilase genes might be important in the stress response and functional development of plants.

Conclusions

Tandem duplications as well as purifying and positive selections have contributed to the functional divergence of subtilase genes in V. vinifera. The data may contribute to a better understanding of the grape subtilase gene family.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1116) contains supplementary material, which is available to authorized users.     

A high quality draft consensus sequence of the genome of a heterozygous grapevine variety

Background

Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented.

Principal Findings

We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before).

Conclusions

Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.     

Candidate genes within a 143 kb region of the flower sex locus in <Emphasis Type="Italic">Vitis</Emphasis>

Wild Vitis species are dioecious plants, while the cultivated counterpart, Vitis vinifera subspec. vinifera, generally shows hermaphroditic flowers. In Vitis the genetic determinants of flower sex have previously been mapped to a region on chromosome 2. In a combined strategy of map-based cloning and the use of the publicly available grapevine reference genome sequence, the structure of the grapevine flower sex locus has been elucidated with the subsequent identification of candidate genes which might be involved in the development of the different flower sex types. In a fine mapping approach, the sex locus in grapevine was narrowed down using a population derived from a cross of a genotype with a Vitis vinifera background (‘Schiava Grossa’ × ‘Riesling’) with the male rootstock cv. ‘Börner’ (V. riparia × V. cinerea). A physical map of 143 kb was established from BAC clones spanning the 0.5 cM region defined by the closest flanking recombination break points. Sequencing and gene annotation of the entire region revealed several candidate genes with a potential impact on flower sex formation. One of the presumed candidate genes, an adenine phosphoribosyltransferase, was analysed in more detail. The results led to the development of a marker for the presence or absence of the female alleles, while the male and hermaphroditic alleles are still to be differentiated. The impact of other candidate genes is discussed, especially with regard to plant hormone actions. The markers developed will permit the selection of female breeding lines which do not require laborious emasculation thus considerably simplifying grapevine breeding. The genetic finger prints displayed that our cultivated grapevines frequently carry a female allele while homozygous hermaphrodites are rare.     

Profiling of sugar transporter genes in grapevine coping with water deficit

The profiling of grapevine (Vitis vinifera L.) genes under water deficit was specifically targeted to sugar transporters. Leaf water status was characterized by physiological parameters and soluble sugars content. The expression analysis provided evidence that VvHT1 hexose transporter gene was strongly down-regulated by the increased sugar content under mild water-deficit. The genes of monosaccharide transporter VvHT5, sucrose carrier VvSUC11, vacuolar invertase VvGIN2 and grape ASR (ABA, stress, ripening) were up-regulated under severe water stress. Their regulation in a drought-ABA signalling network and possible roles in complex interdependence between sugar subcellular partitioning and cell influx/efflux under Grapevine acclimation to dehydration are discussed.     

Deep Sequencing of Viroid-Derived Small RNAs from Grapevine Provides New Insights on the Role of RNA Silencing in Plant-Viroid Interaction

Background

Viroids are circular, highly structured, non-protein-coding RNAs that, usurping cellular enzymes and escaping host defense mechanisms, are able to replicate and move through infected plants. Similarly to viruses, viroid infections are associated with the accumulation of viroid-derived 21–24 nt small RNAs (vd-sRNAs) with the typical features of the small interfering RNAs characteristic of RNA silencing, a sequence-specific mechanism involved in defense against invading nucleic acids and in regulation of gene expression in most eukaryotic organisms.

Methodology/Principal Findings

To gain further insights on the genesis and possible role of vd-sRNAs in plant-viroid interaction, sRNAs isolated from Vitis vinifera infected by Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1) were sequenced by the high-throughput platform Solexa-Illumina, and the vd-sRNAs were analyzed. The large majority of HSVd- and GYSVd1-sRNAs derived from a few specific regions (hotspots) of the genomic (+) and (−) viroid RNAs, with a prevalence of those from the (−) strands of both viroids. When grouped according to their sizes, vd-sRNAs always assumed a distribution with prominent 21-, 22- and 24-nt peaks, which, interestingly, mapped at the same hotspots.

Conclusions/Significance

These findings show that different Dicer-like enzymes (DCLs) target viroid RNAs, preferentially accessing to the same viroid domains. Interestingly, our results also suggest that viroid RNAs may interact with host enzymes involved in the RNA-directed DNA methylation pathway, indicating more complex scenarios than previously thought for both vd-sRNAs genesis and possible interference with host gene expression.     

Characterization of genome-wide long terminal repeat retrotransposons provide insights into trait evolution of four grapevine species

Grapevine is one of the most economically important crops in the world. Although long terminal repeat (LTR) retrotransposons are thought to have played an important role in plants, its distribution in grapevine is not clear. Here, we identified genome-wide intact LTR retrotransposons in a total of six high-quality grapevine genomes from Vitis vinifera L., Vitis sylvestris C.C. Gmel., Vitis riparia Michx. and Vitis amurensis Rupr. with an average of 2938 per genome. Among them, the Copia superfamily (particularly for Ale) is a major component of the LTR retrotransposon in grapevine. Insertion time and copy number analysis revealed that the expansion of 70% LTR retrotransposons concentrating on approximately 2.5 Ma was able to drive genome size variation. Phylogenetic tree and syntenic analyses showed that most LTR retrotransposons in these genomes formed and evolved after species divergence. Furthermore, the function and expression of genes inserted by LTR retrotransposons in V. vinifera (Pinot noir) and V. riparia were explored. The length and expression of genes related to starch metabolism and quinone synthesis pathway in Pinot noir and environmental adaptation pathway in V. riparia were significantly affected by LTR retrotransposon insertion. The results improve the understanding of LTR retrotransposons in grapevine genomes and provide insights for its potential contribution to grapevine trait evolution.     

Xylem structure of four grape varieties and 12 alternative hosts to the xylem-limited bacterium Xylella fastidious

Background and Aims

The bacterium Xylella fastidiosa (Xf), responsible for Pierce''s disease (PD) of grapevine, colonizes the xylem conduits of vines, ultimately killing the plant. However, Vitis vinifera grapevine varieties differ in their susceptibility to Xf and numerous other plant species tolerate Xf populations without showing symptoms. The aim of this study was to examine the xylem structure of grapevines with different susceptibilities to Xf infection, as well as the xylem structure of non-grape plant species that support or limit movement of Xf to determine if anatomical differences might explain some of the differences in susceptibility to Xf.

Methods

Air and paint were introduced into leaves and stems to examine the connectivity between stem and leaves and the length distribution of their vessels. Leaf petiole and stem anatomies were studied to determine the basis for the free or restricted movement of Xf into the plant.

Key Results

There were no obvious differences in stem or petiole vascular anatomy among the grape varieties examined, nor among the other plant species that would explain differences in resistance to Xf. Among grape varieties, the more tolerant ‘Sylvaner’ had smaller stem vessel diameters and 20 % more parenchyma rays than the other three varieties. Alternative hosts supporting Xf movement had slightly longer open xylem conduits within leaves, and more connection between stem and leaves, when compared with alternative hosts that limit Xf movement.

Conclusions

Stem–leaf connectivity via open xylem conduits and vessel length is not responsible for differences in PD tolerance among grape varieties, or for limiting bacterial movement in the tolerant plant species. However, it was found that tolerant host plants had narrower vessels and more parenchyma rays, possibly restricting bacterial movement at the level of the vessels. The implications of xylem structure and connectivity for the means and regulation of bacterial movement are discussed.