Genome-wide identification and analysis of grape aldehyde dehydrogenase (ALDH) gene superfamily

Background

The completion of the grape genome sequencing project has paved the way for novel gene discovery and functional analysis. Aldehyde dehydrogenases (ALDHs) comprise a gene superfamily encoding NAD(P)⁺-dependent enzymes that catalyze the irreversible oxidation of a wide range of endogenous and exogenous aromatic and aliphatic aldehydes. Although ALDHs have been systematically investigated in several plant species including Arabidopsis and rice, our knowledge concerning the ALDH genes, their evolutionary relationship and expression patterns in grape has been limited.

Methodology/Principal Findings

A total of 23 ALDH genes were identified in the grape genome and grouped into ten families according to the unified nomenclature system developed by the ALDH Gene Nomenclature Committee (AGNC). Members within the same grape ALDH families possess nearly identical exon-intron structures. Evolutionary analysis indicates that both segmental and tandem duplication events have contributed significantly to the expansion of grape ALDH genes. Phylogenetic analysis of ALDH protein sequences from seven plant species indicates that grape ALDHs are more closely related to those of Arabidopsis. In addition, synteny analysis between grape and Arabidopsis shows that homologs of a number of grape ALDHs are found in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes arose before the speciation of the grape and Arabidopsis. Microarray gene expression analysis revealed large number of grape ALDH genes responsive to drought or salt stress. Furthermore, we found a number of ALDH genes showed significantly changed expressions in responses to infection with different pathogens and during grape berry development, suggesting novel roles of ALDH genes in plant-pathogen interactions and berry development.

Conclusion

The genome-wide identification, evolutionary and expression analysis of grape ALDH genes should facilitate research in this gene family and provide new insights regarding their evolution history and functional roles in plant stress tolerance.     

Vacuolar Release of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid, the Conjugated Form of the Ethylene Precursor

The mechanisms underlying the vacuolar retention or release of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the conjugated form of the ethylene precursor, has been studied in grape (Vitis vinifera) cells grown in vitro using the technique of compartmental analysis of radioisotope elution. Following its accumulation in the vacuole, M[2,3-¹⁴C]ACC could be released from cells when the vacuolar pH was artificially lowered by external buffers from its initial value of 6.2 to below the critical pH of 5.5. Successive release and retention of vacuolar MACC could be achieved by switching the vacuolar pH from values lower and higher than 5.5. The rate constant of efflux was highly correlated with the vacuolar pH. In plant tissues having low vacuolar pH under natural conditions, e.g. apple fruits (pH 4.2) and mung bean hypocotyls (pH 5.3), an efflux of M[2,3-¹⁴C]ACC also occurred. Its rate constant closely corresponded to the theorical values derived from the correlation established for grape cells. Evidence is presented that the efflux proceeded by passive lipophilic membrane diffusion only when MACC was in the protonated form. In contrast to other organic anions like malic acid, the mono and diionic species could not permeate the tonoplast, thus indicating the strict dependence of MACC retention upon the ionic status of the molecule and the absence of carrier-mediated efflux.     

Genome-Wide Identification and Analysis of the TIFY Gene Family in Grape

Background

The TIFY gene family constitutes a plant-specific group of genes with a broad range of functions. This family encodes four subfamilies of proteins, including ZML, TIFY, PPD and JASMONATE ZIM-Domain (JAZ) proteins. JAZ proteins are targets of the SCF^COI1 complex, and function as negative regulators in the JA signaling pathway. Recently, it has been reported in both Arabidopsis and rice that TIFY genes, and especially JAZ genes, may be involved in plant defense against insect feeding, wounding, pathogens and abiotic stresses. Nonetheless, knowledge concerning the specific expression patterns and evolutionary history of plant TIFY family members is limited, especially in a woody species such as grape.

Methodology/Principal Findings

A total of two TIFY, four ZML, two PPD and 11 JAZ genes were identified in the Vitis vinifera genome. Phylogenetic analysis of TIFY protein sequences from grape, Arabidopsis and rice indicated that the grape TIFY proteins are more closely related to those of Arabidopsis than those of rice. Both segmental and tandem duplication events have been major contributors to the expansion of the grape TIFY family. In addition, synteny analysis between grape and Arabidopsis demonstrated that homologues of several grape TIFY genes were found in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes arose before the divergence of lineages that led to grape and Arabidopsis. Analyses of microarray and quantitative real-time RT-PCR expression data revealed that grape TIFY genes are not a major player in the defense against biotrophic pathogens or viruses. However, many of these genes were responsive to JA and ABA, but not SA or ET.

Conclusion

The genome-wide identification, evolutionary and expression analyses of grape TIFY genes should facilitate further research of this gene family and provide new insights regarding their evolutionary history and regulatory control.     

Attraction to Host Plant Volatiles and Feeding Performance of <Emphasis Type="Italic">Naupactus Xanthographus</Emphasis> (Coleoptera: Curculionidae) is Affected by Starvation

The grape weevil, Naupactus xanthographus Germar (Coleoptera: Curculionidae), is a polyphagous insect which is a cause of important damage to several economically relevant crops, including grape (Vitis vinifera) and avocado (Persea americana), in several countries of Latin America. The larvae cause damage to the roots and rootlets of plants, and adults feed on leaves of their host plant. Despite its economic importance, there are few reports on the behavioral and nutritional ecology of this weevil. In this context, laboratory feeding and olfactometer bioassays with N. xanthographus were performed. The feeding performance was evaluated by measuring the weight variation of the insects after 1 and 6 h of feeding on grape or avocado leaves, respectively. After 1 h of feeding, insects showed no significant differences in weight increase. However, after a period of 6 h of feeding, males had continued feeding on grape leaves, but not on avocado leaves. Bioassays using a Y-tube olfactometer showed that males are attracted to volatiles of both host plants. Furthermore, starved males and females showed no preference to volatiles of grape or avocado. However, non-starved males and females preferred grape volatiles over avocado volatiles. Based on the combined results of the assays, we conclude that grape is preferred over avocado for N. xanthographus. Furthermore, this is the first report on the effect of starvation on the attraction to host plant volatiles in Curculionidae.     

Ascorbic acid metabolism in geranium and grape

l-Ascorbic acid-[UL-¹⁴C] has been used to follow the appearance of ¹⁴C-labeled oxalic acid and tartaric acid as metabolic products of oxidative cleavage of ascorbic acid in geranium apices (Pelargonium crispum). The enantiomeric specificity of ascorbic acid metabolism was established in geranium by comparing the incorporation of d- and l-ascorbic acid-[6-¹⁴C] in the presence of l-ascorbic acid-[4-³H]. l-Ascorbic acid-[4-³H] has been used to demonstrate the retention of ³H during biosynthesis of l-(+)-tartaric acid in the geranium and its exchange with water during biosynthesis of l-( +)-tartaric acid in the grape.     

Characterization of Epiphytic Bacterial Communities from Grapes,Leaves, Bark and Soil of Grapevine Plants Grown,and Their Relations

Despite its importance in plant health and crop quality, the diversity of epiphytic bacteria on grape berries and other plant parts, like leaves and bark, remains poorly described, as does the role of telluric bacteria in plant colonization. In this study, we compare the bacterial community size and structure in vineyard soils, as well as on grapevine bark, leaves and berries. Analyses of culturable bacteria revealed differences in the size and structure of the populations in each ecosystem. The highest bacteria population counts and the greatest diversity of genera were found in soil samples, followed by bark, grapes and leaves. The identification of isolates revealed that some genera – Pseudomonas, Curtobacterium, and Bacillus – were present in all ecosystems, but in different amounts, while others were ecosystem-specific. About 50% of the genera were common to soil and bark, but absent from leaves and grapes. The opposite was also observed: grape and leaf samples presented 50% of genera in common that were absent from trunk and soil. The bacterial community structure analyzed by T-RFLP indicated similarities between the profiles of leaves and grapes, on the one hand, and bark and soil, on the other, reflecting the number of shared T-RFs. The results suggest an interaction between telluric bacterial communities and the epiphytic bacteria present on the different grapevine parts.     

Movement of grape mealybug,Pseudococcus maritimus,on and between host plants

The importance of the grape mealybug, Pseudococcus maritimus (Ehrhorn) (Homoptera: Pseudococcidae), as a pest of wine grapes, Vitis vinifera L. (Vitaceae), has increased as a result of recent reports that it is capable of transmitting at least one of the viruses associated with grapevine leafroll disease. However, its natural rate of movement between host plants – and hence its role in the epidemiology of this disease – is poorly understood. In order to better assess the risk of field spread of leafroll disease by this insect, several experiments were conducted to determine the extent to which P. maritimus can move between grape plants, both by walking and by airborne dispersal. In experiments with first instars, both field and shade‐house studies indicated that the mealybugs do not walk far and only very rarely reach adjacent plants by this means. Field trapping experiments showed that grape mealybugs can be dispersed by wind, but that there is a marked decline in numbers with increasing distance from the source plant. The implications of these observations for the field spread and management of grapevine leafroll disease are discussed.     

Effect of Grape Polyphenols on Blood Pressure: A Meta-Analysis of Randomized Controlled Trials

Background

The effect of grape polyphenols on blood pressure remains unclear, which we aimed to address via a meta-analysis study.

Methods

We conducted study trial searches in PubMed, Embase, and the Cochrane Library databases. Summary estimates of weighted mean differences and 95% confidence intervals were obtained by using fixed-effects models. Subgroup analyses were performed to identify the source of heterogeneity. The protocol details of our meta-analysis have been submitted to the international database of prospectively registered systematic reviews (registration number CRD42015019196).

Results

Ten studies were included in the present meta-analysis. Our results showed daily grape polyphenol intake could significantly reduce systolic blood pressure by 1.48 mmHg when compared to control subjects (12 comparisons; -1.48 [-2.79 to -0.16] mmHg; P = 0.03). Subgroup analyses indicated larger reduction was identified in the intake of low-dose of grape polyphenols (< 733 mg/day, median level of the included studies) or patients with metabolic syndrome. Contrarily, diastolic blood pressure was not significantly decreased in the grape polyphenols group as compared to controls. No significant heterogeneity or publication bias was detected in the meta-analysis of either systolic or diastolic blood pressure.

Conclusions

Daily grape polyphenol intake can significantly reduce the systolic blood pressure in humans, although the reduction is modest when compared with anti-hypertensive medications. Larger, better designed trials, that specifically include hypertensive subjects, are required to verify our results in the future.     

Biology and fertility life table of Bactrocera carambolae on grape and acerola

Bactrocera carambolae Drew & Hancock (Diptera: Tephritidae), native of Southeast Asia, is present in Brazil but restricted to Amapá, Pará and Roraima, where it has quarantine pest status. The possible dispersion to other fruit producing regions of Brazil could cause damages, including for exportation, due to quarantine restrictions imposed by fruit importing countries. The objective of this work was to describe the biological parameters and calculate the fertility life table of B. carambolae on grape (Vitis vinifera L.) and acerola (Malpighia emarginata DC.). The experiment was conducted in the laboratory under controlled conditions. The mean number of punctures and the mean number of eggs per female on grapes were 1.48 ± 0.05 and 9.87 ± 0.36, respectively. The mean number puparia per fruit was 0.1 ± 0.02 (grape) and 0.5 ± 0.10 (acerola). The pupal viability was 82.4% (grape) and 70.6% (acerola). The mean time ± SE of one generation, in days, was 25.8 ± 1.10 (grape) and 19.7 ± 0.21 (acerola). The mean fecundity was 1663.8 ± 501.01 (grape) and 206.9 ± 26.21 (acerola) with eggs viability of 5.6% (grape) and 12.5% (acerola). The mean longevity, in days, was 77.3 ± 12.13 on grape and 82.4 ± 4.24 on acerola. The study found that B. carambolae completes its biological cycle on grape and on acerola leaving offspring.     

Tracking cell wall changes in wine and table grapes undergoing Botrytis cinerea infection using glycan microarrays

Background and AimsThe necrotrophic fungus Botrytis cinerea infects a broad range of fruit crops including domesticated grapevine Vitis vinifera cultivars. Damage caused by this pathogen is severely detrimental to the table and wine grape industries and results in substantial crop losses worldwide. The apoplast and cell wall interface is an important setting where many plant–pathogen interactions take place and where some defence-related messenger molecules are generated. Limited studies have investigated changes in grape cell wall composition upon infection with B. cinerea, with much being inferred from studies on other fruit crops.MethodsIn this study, comprehensive microarray polymer profiling in combination with monosaccharide compositional analysis was applied for the first time to investigate cell wall compositional changes in the berries of wine (Sauvignon Blanc and Cabernet Sauvignon) and table (Dauphine and Barlinka) grape cultivars during Botrytis infection and tissue maceration. This was used in conjunction with scanning electron microscopy (SEM) and X-ray computed tomography (CT) to characterize infection progression.Key ResultsGrapes infected at veraison did not develop visible infection symptoms, whereas grapes inoculated at the post-veraison and ripe stages showed evidence of significant tissue degradation. The latter was characterized by a reduction in signals for pectin epitopes in the berry cell walls, implying the degradation of pectin polymers. The table grape cultivars showed more severe infection symptoms, and corresponding pectin depolymerization, compared with wine grape cultivars. In both grape types, hemicellulose layers were largely unaffected, as was the arabinogalactan protein content, whereas in moderate to severely infected table grape cultivars, evidence of extensin epitope deposition was present.ConclusionsSpecific changes in the grape cell wall compositional profiles appear to correlate with fungal disease susceptibility. Cell wall factors important in influencing resistance may include pectin methylesterification profiles, as well as extensin reorganization.     

Genomic Insights into the Evolutionary Origin of Xanthomonas axonopodis pv. citri and Its Ecological Relatives

Xanthomonas axonopodis pv. citri (Xac) is the causal agent of citrus bacterial canker (CBC) and is a serious problem worldwide. Like CBC, several important diseases in other fruits, such as mango, pomegranate, and grape, are also caused by Xanthomonas pathovars that display remarkable specificity toward their hosts. While citrus and mango diseases were documented more than 100 years ago, the pomegranate and grape diseases have been known only since the 1950s and 1970s, respectively. Interestingly, diseases caused by all these pathovars were noted first in India. Our genome-based phylogenetic studies suggest that these diverse pathogens belong to a single species and these pathovars may be just a group of rapidly evolving strains. Furthermore, the recently reported pathovars, such as those infecting grape and pomegranate, form independent clonal lineages, while the citrus and mango pathovars that have been known for a long time form one clonal lineage. Such an understanding of their phylogenomic relationship has further allowed us to understand major and unique variations in the lineages that give rise to these pathovars. Whole-genome sequencing studies including ecological relatives from their putative country of origin has allowed us to understand the evolutionary history of Xac and other pathovars that infect fruits.     

Abiotic and oxidative stress-dependent regulation of expression of the thioredoxin h multigenic family in grape Vitis vinifera

Thioredoxins (Trxs) are small ubiquitous oxidoreductases that participate in dithiol-disulphide exchange reactions. They are present in all organisms from prokaryotes to higher eukaryotes and involved in a variety of cellular processes in higher plants. The expression pattern of the multigenic family of grape Trxs_h was analysed in different tissues by semiquantitative RT-PCR. The grape Trx_h genes were approximately expressed in all tissues, but with different expression patterns than each other. The in silico analysis of the promoter regions of grape Trxs _h demonstrated the presence of a number of potential cis-acting elements to respond to environmental signals, suggesting that Trxs_h may respond to a variety of environmental signals, including dehydration, salinity, heat, cold, heavy metals, light, pathogens, and plant hormones. The grape Trx h genes were also found to be differentially induced under abiotic stress conditions due to the presence of different putative regulatory elements in their promoters. Collectively, the distinct expression patterns and different responses to environmental stress conditions suggest that each isoform of the multigenic family of VvTrxh may have a specific and non-redundant function in grape.     

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.     

An innovative approach to grape metabolomics: stilbene profiling by suspect screening analysis

Suspect screening analysis is a targeted metabolomics approach in which identification of compounds relies on specific available information such as their molecular formula and isotopic pattern. This method was applied to the study of grape metabolomics with an UPLC/MS high-resolution Q-TOF mass spectrometer (nominal resolution 40,000) coupled with a Jet Stream ionization source. The present paper describes the detailed qualitative and quantitative study of grape stilbenes, the principal polyphenols associated with the beneficial effects of drinking wine. For identification of compounds, a new database was expressly constructed from the molecular information of potential metabolites of grape and wine from the literature and other electronic databases. Currently, GrapeMetabolomics contains about a thousand putative grape compounds. If untargeted analysis of a sample provides identification of a new compound with a sufficiently confident score, it is added to the database. Thus, by increasing the number of samples studied, GrapeMetabolomics can be expanded. This method is effective for identification of the molecular formulae of several hundred metabolites in two runs (positive and negative ionization) with minimal sample preparation, and can also be used to analyse some single classes of compounds involved in cell and tissue metabolism. With this approach, a total of 18 stilbene derivatives was identified in two grape samples (Raboso Piave and Primitivo) on the basis of accurate mass measurements and isotopic patterns, and identification was confirmed by MS/MS analysis. The approach can also potentially be applied to the metabolomics of other plant varieties.     

Context-dependent transmission of a generalist plant pathogen: host species and pathogen strain mediate insect vector competence

The specificity of pathogen–vector–host interactions is an important element of disease epidemiology. For generalist pathogens, different pathogen strains, vector species, or host species may all contribute to variability in disease incidence. One such pathogen is Xylella fastidiosa Wells et al., a xylem-limited bacterium that infects dozens of crop, ornamental, and native plants in the USA. This pathogen also has a diverse vector complex and multiple biologically distinct strains. We studied the implications of diversity in this pathogen–vector–host system, by quantifying variability in transmission efficiency of different X. fastidiosa strains (isolates from almond and grape genetic groups) for different host plants (grape, almond, and alfalfa) by two of the most important vectors in California: glassy-winged sharpshooter [ Homalodisca vitripennis (Germar)] and green sharpshooter ( Draeculacephala minerva Ball) (both Hemiptera: Cicadellidae). Transmission of isolates of the almond strain by H. vitripennis did not differ significantly, whereas transmission varied significantly among isolates from the grape strain (15–90%). Host plant species did not affect H. vitripennis transmission. Conversely, D. minerva efficiency was mediated by both host plant species and pathogen strain. No acquisition of an almond isolate occurred regardless of plant type (0/122), whereas acquisition of a grape isolate from alfalfa was 10-fold higher than from grape or almond plants. These results suggest that pathogen, vector, and host diversity impose contingencies on the transmission ecology of this complex disease system. Studies aimed at the development of management strategies for X. fastidiosa diseases should consider the complexity of these interactions as they relate to disease spread.     

Molecular cloning and biochemical characterization of the UDP-glucose: Flavonoid 3-O-glucosyltransferase from Concord grape (Vitis labrusca)

Glucosylation of anthocyanidin substrates at the 3-O-position is crucial for the red pigmentation of grape berries and wine. The gene that encodes the enzyme involved in this reaction has been cloned from Vitis labrusca cv. Concord, heterologously expressed, and the recombinant enzyme (rVL3GT) was characterized. VL3GT has 96% amino acid sequence identity with Vitis vinifera VV3GT and groups phylogenetically with several other flavonoid 3-O-glycosyltransferases. In vitro substrate specificity studies and kinetic analyses of rVL3GT indicate that this enzyme preferentially glucosylates cyanidin as compared with quercetin. Crude protein extracts from several Concord grape tissues were assayed for glucosyltransferase activity with cyanidin and quercetin as acceptor substrates. A comparison of the VL3GT activities toward with these substrates showed that the 3GT enzyme activity is consistent with the expression of VL3GT in these tissues and is coincident with the biosynthesis of anthocyanins in both location and developmental stages. Enzyme activities in grape mesocarp, pre-veraison exocarp, leaf, flower bud, and flower tissues glucosylated quercetin but not cyanidin at high rates, suggesting the presence of additional enzymes which are able to glucosylate the 3-O-position of flavonols with higher specificity than anthocyanidins.