Carmen Espinoza

Gene expression associated with compatible viral diseases in grapevine cultivars

Viral diseases affect grapevine cultures without inducing any resistance response. Thus, these plants develop systemic diseases and are chronically infected. Molecular events associated with viral compatible infections responsible for disease establishment and symptoms development are poorly understood. In this study, we surveyed viral infection in grapevines at a transcriptional level. Gene expression in the Vitis vinifera red wine cultivars Carménère and Cabernet-Sauvignon naturally infected with GLRaV-3 were evaluated using a genome-wide expression profiling with the Vitis vinifera GeneChip® from Affymetrix. We describe numerous genes that are induced or repressed in viral infected grapevines leaves. Changes in gene expression involved a wide spectrum of biological functions, including processes of translation and protein targeting, metabolism, transport, and cell defense. Considering cellular localization, the membrane and endomembrane systems appeared with the highest number of induced genes, while chloroplastic genes were mostly repressed. As most induced genes associated with the membranous system are involved in transport, the possible effect of virus in this process is discussed. Responses of both cultivars are analyzed and the results are compared with published data from other species. This is the first study of global gene profiling in grapevine in response to viral infections using DNA microarray.

Cisgenesis and Intragenesis: New tools For Improving Crops

Genetically Modified Organisms (GMO) could be the answer for many relevant problems affecting crops. However, improving crops through GMO is also often associated with safety concerns, environmental risks and health issues due to the presence of foreign DNA. These limitations have prompted the development of alternative technologies. Recently, cisgenesis and intragenesis have been developed as new tools aimed to modify crops. While cisgenesis involves genetic modification using a complete copy of natural genes with their regulatory elements that belong exclusively to sexually compatible plants, intragenesis refers to the transference of new combinations of genes and regulatory sequences belonging to that particular species. So far, application of cisgenesis and intragenesis as alternatives to conventional transgenesis are limited to a few species, mainly due to the lack of knowledge of the regulatory sequences required. The grape is one of the most cultivated crops worldwide and is the most economically relevant crop in Chile. Its genomic sequence has been completed, making available new sources of information to improve grape traits by genetic manipulation. This review is focused on the current alternatives to transgenesis in plants, including new approaches to develop marker-free crops, their application to economically relevant crops and future perspectives in the area. Also, the identification of grapevine promoters with a wide range of expression profiles is shown. The expression pattern of these genes was analyzed in different tissues and developmental stages, as well as under several stresses and stimuli, giving a broad range of expression patterns, including genes expressed exclusively during ripening, in response to sugars, senescence and biotic stress, among others. Genes with strong and constitutive expression were also identified. Functional analysis using reporter genes has been conducted in order to confirm the promoters transcription activity, opening new possibilities for developing cisgenic/intragenic grapevines.

Inspection of the grapevine BURP superfamily highlights an expansion of RD22 genes with distinctive expression features in berry development and ABA-mediated stress responses

The RESPONSIVE TO DEHYDRATION 22 (RD22) gene is a molecular link between abscisic acid (ABA) signalling and abiotic stress responses. Its expression has been used as a reliable ABA early response marker. In Arabidopsis, the single copy RD22 gene possesses a BURP domain also located at the C-terminus of USP embryonic proteins and the beta subunit of polygalacturonases. In grapevine, a RD22 gene has been identified but putative paralogs are also found in the grape genome, possibly forming a large RD22 family in this species. In this work, we searched for annotations containing BURP domains in the Vitis vinifera genome. Nineteen proteins were defined by a comparative analysis between the two genome predictions and RNA-Seq data. These sequences were compared to other plant BURPs identified in previous genome surveys allowing us to reconceive group classifications based on phylogenetic relationships and protein motif occurrence. We observed a lineage-specific evolution of the RD22 family, with the biggest expansion in grapevine and poplar. In contrast, rice, sorghum and maize presented highly expanded monocot-specific groups. The Vitis RD22 group may have expanded from segmental duplications as most of its members are confined to a region in chromosome 4. The inspection of transcriptomic data revealed variable expression of BURP genes in vegetative and reproductive organs. Many genes were induced in specific tissues or by abiotic and biotic stresses. Three RD22 genes were further studied showing that they responded oppositely to ABA and to stress conditions. Our results show that the inclusion of RNA-Seq data is essential while describing gene families and improving gene annotations. Robust phylogenetic analyses including all BURP members from other sequenced species helped us redefine previous relationships that were erroneously established. This work provides additional evidence for RD22 genes serving as marker genes for different organs or stresses in grapevine.

Analysis of local and systemic spread of the crucifer-infecting TMV-Cg virus in tobacco and several Arabidopsis thaliana ecotypes

The crucifer-infecting tobacco mosaic virus, TMV-Cg, infects Arabidopsis thaliana (L.) Heynh. efficiently without causing severe symptoms. The systemic spread of TMV-Cg in Arabidopsis was evaluated in 14ecotypes. Five days after inoculation, TMV-Cg was detected in apical leaves of 8 out of 14 ecotypes. As expected, the spread of TMV-Cg in the ecotypes tested was considerably faster than that of tobacco mosaic virus (TMV-U1). To study the participation of viral proteins in the TMV-Cg-induced infection, a complete genomic cDNA of TMV-Cg was cloned. The role of TMV-Cg movement protein in systemic spread was tested with a hybrid virus, constructed from the TMV-U1 genome and the TMV-Cg movement protein gene. Contrary to expectations, the systemic spread of this hybrid in Arabidopsis was similar to that of TMV-U1. The failure of the hybrid virus to spread at rates similar to those of TMV-Cg was not due to restrictions in local movement. In tobacco (Nicotiana tabacum L.), the hybrid virus spread efficiently and induced systemic mosaic symptoms characteristic of TMV-U1. The TMV-Cg cDNA clone provides an attractive tool to study virus-host interactions.

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

BackgroundIndole-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.ResultsIn 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.ConclusionsThese 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.

Omics Approaches for Understanding Grapevine Berry Development: Regulatory Networks Associated with Endogenous Processes and Environmental Responses

Grapevine fruit development is a dynamic process that can be divided into three stages: formation (I), lag (II), and ripening (III), in which physiological and biochemical changes occur, leading to cell differentiation and accumulation of different solutes. These stages can be positively or negatively affected by multiple environmental factors. During the last decade, efforts have been made to understand berry development from a global perspective. Special attention has been paid to transcriptional and metabolic networks associated with the control of grape berry development, and how external factors affect the ripening process. In this review, we focus on the integration of global approaches, including proteomics, metabolomics, and especially transcriptomics, to understand grape berry development. Several aspects will be considered, including seed development and the production of seedless fruits; veraison, at which anthocyanin accumulation begins in the berry skin of colored varieties; and hormonal regulation of berry development and signaling throughout ripening, focusing on the transcriptional regulation of hormone receptors, protein kinases, and genes related to secondary messenger sensing. Finally, berry responses to different environmental factors, including abiotic (temperature, water-related stress and UV-B radiation) and biotic (fungi and viruses) stresses, and how they can significantly modify both, development and composition of vine fruit, will be discussed. Until now, advances have been made due to the application of Omics tools at different molecular levels. However, the potential of these technologies should not be limited to the study of single-level questions; instead, data obtained by these platforms should be integrated to unravel the molecular aspects of grapevine development. Therefore, the current challenge is the generation of new tools that integrate large-scale data to assess new questions in this field, and to support agronomical practices.

Grapevine Biotechnology: Molecular Approaches Underlying Abiotic and Biotic Stress Responses

Grapevine is one of the most abundant crops worldwide, with varieties destined for fresh and dry consumption, as well as wine production. Unfortunately, grapevine plants are affected by both biotic and abiotic stresses, generating significant economic losses. These conditions can negatively impact grape cultivation at different stages: plant and berry development during preand post-harvest, production, fresh fruit processing and export, along with wine quality. Most of the grapevine varieties are susceptible to several pathogens and within this chapter, particular attention is given to fungi (Botrytis cinerea and Erysiphe necator) and viruses, since they are a worldwide concern. Within the latter, special focus is given to the grapevine leafroll disease, a complex and destructive infection. On the other hand, abiotic stress is also relevant in grapevine, and in this chapter it will be exemplified by UV-B radiation and its impact on growth and fruit development, plant adaptive responses and its relationship with the quality of grape berries for winemaking. The main biotic and abiotic grapevine stress factors are reviewed in this chapter, considering a special focus on biotechnological approaches carried out in order to address them and minimize their detrimental consequences.