Multi-hormonal analysis and aquaporins regulation reveal new insights on drought tolerance in grapevine.

Disentangling the factors that foster the tolerance to water stress in plants could provide great benefits to crop productions. In a two-year experiment, two new PIWI (fungus resistant) grapevine varieties, namely Merlot Kanthus and Sauvignon Kretos (Vitis hybrids), grown in the field, were subjected to two different water regimes: weekly irrigated (IR) or not irrigated (NIR) for two months during the summer. The two varieties exhibited large differences in terms of performance under water-limiting conditions. In particular, Merlot Kanthus strongly decreased stem water potential (Ψs) under water shortage and Sauvignon Kretos maintained higher Ψs values accompanied by generally high stomatal conductance and net carbon assimilation, regardless of the treatment. We hypothesized differences in the hormonal profile that mediate most of the plant responses to stresses or in the regulation of the aquaporins that control the water transport in the leaves. In general, substantial differences were found in the abundance of different hormonal classes, with Merlot Kanthus reporting higher concentrations of cytokinins while Sauvignon Kretos higher concentrations of auxins, jasmonate and salicylic acid. Interestingly, under water stress conditions ABA modulation appeared similar between the two cultivars, while other hormones were differently modulated between the two varieties. Regarding the expression of aquaporin encoding genes, Merlot Kanthus showed a significant downregulation of VvPIP2;1 and VvTIP2;1 in leaves exposed to water stress. Both genes have probably a role in influencing leaf conductance, and VvTIP2;1 has been correlated with stomatal conductance values. This evidence suggests that the two PIWI varieties are characterized by different behaviour in response to drought. Furthermore, the findings of the study may be generalized, suggesting the involvement of a complex hormonal cross-talk and aquaporins in effectively influencing plant performance under water shortage.

The activation of iron deficiency responses of grapevine rootstocks is dependent to the availability of the nitrogen forms.

BACKGROUND: In viticulture, iron (Fe) chlorosis is a common abiotic stress that impairs plant development and leads to yield and quality losses. Under low availability of the metal, the applied N form (nitrate and ammonium) can play a role in promoting or mitigating Fe deficiency stresses. However, the processes involved are not clear in grapevine. Therefore, the aim of this study was to investigate the response of two grapevine rootstocks to the interaction between N forms and Fe uptake. This process was evaluated in a hydroponic experiment using two ungrafted grapevine rootstocks Fercal (Vitis berlandieri x V. vinifera) tolerant to deficiency induced Fe chlorosis and Couderc 3309 (V. riparia x V. rupestris) susceptible to deficiency induced Fe chlorosis. RESULTS: The results could differentiate Fe deficiency effects, N-forms effects, and rootstock effects. Interveinal chlorosis of young leaves appeared earlier on 3309 C from the second week of treatment with NO3-/NH4+ (1:0)/-Fe, while Fercal leaves showed less severe symptoms after four weeks of treatment, corresponding to decreased chlorophyll concentrations lowered by 75% in 3309 C and 57% in Fercal. Ferric chelate reductase (FCR) activity was by trend enhanced under Fe deficiency in Fercal with both N combinations, whereas 3309 C showed an increase in FCR activity under Fe deficiency only with NO3-/NH4+ (1:1) treatment. With the transcriptome analysis, Gene Ontology (GO) revealed multiple biological processes and molecular functions that were significantly regulated in grapevine rootstocks under Fe-deficient conditions, with more genes regulated in Fercal responses, especially when both forms of N were supplied. Furthermore, the expression of genes involved in the auxin and abscisic acid metabolic pathways was markedly increased by the equal supply of both forms of N under Fe deficiency conditions. In addition, changes in the expression of genes related to Fe uptake, regulation, and transport reflected the different responses of the two grapevine rootstocks to different N forms. CONCLUSIONS: Results show a clear contribution of N forms to the response of the two grapevine rootstocks under Fe deficiency, highlighting the importance of providing both N forms (nitrate and ammonium) in an appropriate ratio in order to ease the rootstock responses to Fe deficiency.

Berry shrivel in grapevine: a review considering multiple approaches.

Grapevine berry shrivel, a ripening disorder, causes significant economic losses in the worldwide wine and table grape industries. An early interruption in ripening leads to this disorder, resulting in shriveling and reduced sugar accumulation affecting yield and fruit quality. Loss of sink strength associated with berry mesocarp cell death is an early symptom of this disorder; however, potential internal or external triggers are yet to be explored. No pathogens have been identified that might cause the ripening syndrome. Understanding the underlying causes and mechanisms contributing to berry shrivel is crucial for developing effective mitigation strategies and finding solutions for other ripening disorders associated with climacteric and non-climacteric fruits. This review discusses alterations in the fruit ripening mechanism induced by berry shrivel disorder, focusing primarily on sugar transport and metabolism, cell wall modification and cell death, and changes in the phytohormone profile. The essential open questions are highlighted and analyzed, thus identifying the critical knowledge gaps and key challenges for future research.

Local conditions matter: Minimal and variable effects of soil disturbance on microbial communities and functions in European vineyards.

Soil tillage or herbicide applications are commonly used in agriculture for weed control. These measures may also represent a disturbance for soil microbial communities and their functions. However, the generality of response patterns of microbial communities and functions to disturbance have rarely been studied at large geographical scales. We investigated how a soil disturbance gradient (low, intermediate, high), realized by either tillage or herbicide application, affects diversity and composition of soil bacterial and fungal communities as well as soil functions in vineyards across five European countries. Microbial alpha-diversity metrics responded to soil disturbance sporadically, but inconsistently across countries. Increasing soil disturbance changed soil microbial community composition at the European level. However, the effects of soil disturbance on the variation of microbial communities were smaller compared to the effects of location and soil covariates. Microbial respiration was consistently impaired by soil disturbance, while effects on decomposition of organic substrates were inconsistent and showed positive and negative responses depending on the respective country. Therefore, we conclude that it is difficult to extrapolate results from one locality to others because microbial communities and environmental conditions vary strongly over larger geographical scales.

Expression Analyses in the Rachis Hint towards Major Cell Wall Modifications in Grape Clusters Showing Berry Shrivel Symptoms.

Berry shrivel (BS) is one of the prominent and still unresolved ripening physiological disorders in grapevine. The causes of BS are unclear, and previous studies focused on the berry metabolism or histological studies, including cell viability staining in the rachis and berries of BS clusters. Herein, we studied the transcriptional modulation induced by BS in the rachis of pre-symptomatic and symptomatic clusters with a custom-made microarray qPCR in relation to a previous RNASeq study of BS berries. Gene set analysis of transcript expression in symptomatic rachis tissue determined suppression of cell wall biosynthesis, which could also be confirmed already in pre-symptomatic BS rachis by CESA8 qPCR analyses, while in BS berries, a high number of SWITCH genes were suppressed at veraison. Additionally, genes associated with the cell wall were differently affected by BS in berries. A high percentage of hydrolytic enzymes were induced in BS grapes in rachis and berries, while other groups such as, e.g., xyloglucan endotransglucosylase/hydrolase, were suppressed in BS rachis. In conclusion, we propose that modulated cell wall biosynthesis and cell wall assembly in pre-symptomatic BS rachis have potential consequences for cell wall strength and lead to a forced degradation of cell walls in symptomatic grape clusters. The similarity to sugar starvation transcriptional profiles provides a link to BS berries, which are low in sugar accumulation. However, further studies remain necessary to investigate the temporal and spatial coordination in both tissues.

Comparative Transcriptome Analysis of Two Root-Feeding Grape Phylloxera (D. vitifoliae) Lineages Feeding on a Rootstock and V. vinifera.

Grape phylloxera is one of the most dangerous insect pests for worldwide viticulture. The leaf- and root-galling phylloxerid has been managed by grafting European grapevines onto American rootstock hybrids. Recent reports pinpoint the appearance of host-adapted biotypes, but information about the biomolecular characteristics underlying grape phylloxera biotypisation and its role in host performance is scarce. Using RNA-sequencing, we sequenced the transcriptome of two larval stages: L1 (probing) and L2-3 (feeding) larvae of two root-feeding grape phylloxera lineages feeding on the rootstock Teleki 5C (biotype C) and V. vinifera Riesling (biotype A). In total, 7501 differentially expressed genes (DEGs) were commonly modulated by the two biotypes. For the probing larvae, we found an increased number of DEGs functionally associated with insect chemoreception traits, such as odorant-binding proteins, chemosensory proteins, ionotropic, odorant, and gustatory receptors. The transcriptomic profile of feeding larvae was enriched with DEGs associated with the primary metabolism. Larvae feeding on the tolerant rootstock Teleki 5C exhibited higher numbers of plant defense suppression-associated DEGs than larvae feeding on the susceptible host. Based on the identified DEGs, we discuss their potential role for the compatible grape phylloxera-Vitis interaction belowground. This study was the first to compare the transcriptomes of two grape phylloxera lineages feeding on a tolerant and susceptible host, respectively, and to identify DEGs involved in the molecular interaction with these hosts. Our data provide a source for future studies on host adaptation mechanisms of grape phylloxera and help to elucidate grape phylloxera resistance further.

Phytohormone profiles are strongly altered during induction and symptom development of the physiological ripening disorder berry shrivel in grapevine.

The process of grape berry ripening follows three phases with distinct metabolic processes and complex regulations via phytohormones. The physiological ripening disorder berry shrivel (BS) is characterized by reduced sugar accumulation, low anthocyanin contents, and high acidity in affected berries. The processes leading to BS induction are unknown, but recent transcriptional data on reduced expression of switch genes hint towards a disturbed ripening onset. Herein we investigated the phytohormone composition throughout grape berry ripening in healthy and BS berries in Vitis vinifera L. cultivar Blauer Zweigelt. Thereby we hypothesize that phytohormones are key players for BS induction and suppress the expression of switch genes at veraison. The presented metabolomics and RNAseq data describe two distinct phytohormone profiles in BS berries, differing between pre- and post-veraison with a clear ethylene precursor (aminocyclopropane-1-carboxylic acid, ACC) peak before veraison. Exogenous application of ACC led to BS symptoms, while ethephone application led to berry abscission. During post-veraison, we observed high ABA-glucose ester (ABA-GE) and low indole-3-acetate aspartate (IAA-Asp) and isopentenyladenine (iP) contents in BS berries and the transcriptional induction of several phytohormone pathways. The presented descriptive data provide valuable knowledge to further decipher the role of phytohormones in BS induction and BS symptom development.

The salivary gland proteome of root-galling grape phylloxera (Daktulosphaira vitifoliae Fitch) feeding on Vitis spp.

The successful parasitisation of a plant by a phytophagous insect is dependent on the delivery of effector molecules into the host. Sedentary gall forming insects, such as grape phylloxera (Daktulosphaira vitifoliae Fitch, Phylloxeridae), secrete multiple effectors into host plant tissues that alter or modulate the cellular and molecular environment to the benefit of the insect. The identification and characterisation of effector proteins will provide insight into the host-phylloxera interaction specifically the gall-induction processes and potential mechanisms of plant resistance. Using proteomic mass spectrometry and in-silico secretory prediction, 420 putative effectors were determined from the salivary glands or the root-feeding D. vitifoliae larvae reared on Teleki 5C (V. berlandieri x V. riparia). Among them, 170 conserved effectors were shared between D. vitifoliae and fourteen phytophagous insect species. Quantitative RT-PCR analysis of five conserved effector candidates (protein disulfide-isomerase, peroxidoredoxin, peroxidase and a carboxypeptidase) revealed that their gene expression decreased, when larvae were starved for 24 h, supporting their assignment as effector molecules. The D. vitifoliae effectors identified here represent a functionally diverse group, comprising both conserved and unique proteins that provide new insight into the D. vitifoliae-Vitis spp. interaction and the potential mechanisms by which D. vitifoliae establishes the feeding site, suppresses plant defences and modulates nutrient uptake.

Grape phylloxera (D. vitifoliae) manipulates SA/JA concentrations and signalling pathways in root galls of Vitis spp.

Overcoming host defensive traits is a prerequisite to establish compatible plant-parasite interactions. Following parasite perception, jasmonic (JA) and salicylic acid (SA) signalling pathways mediate biotic stress signals resulting in the activation of host defence responses. Piercing-sucking grape phylloxera (Daktulosphaira vitifoliae) infests Vitis spp. by the formation of organoid root galls. This study aims to investigate whether host defensive SA/JA signalling pathways are affected during D. vitifoliae infestation. We hypothesize that the JA signalling pathway is induced during larval probing (14 hai). Compatible root gall formation (24 hai - 14 dai) involves the reduction of the JA, but the induction of the SA signaling pathway. T5C (V.berlandieri x V.riparia) cuttings are infested with a D. vitifoliae single founder lineage (biotype C). Phytohormone quantification (HPLC-MS) and transcriptional alterations of JA/SA marker genes (qRT-PCR) are determined in root tissues from larval probing (14 hai) until gall formation (>14 dai). Non-infested root tips are considered controls. Our results show a significant induction of all analysed JA marker genes during insect probing (14 hai), but their significant reduction during early gall formation (24 hai). Following gall formation (5-14 dai) SA and JA-Ile increase. However, only the analysed SA marker genes are induced, whereas JA marker gene expression levels are significantly reduced. Based on this data we conclude that the observed suppression of the JA signalling pathway might represent an important step for the compatible D. vitifoliae - Vitis spp. root interaction. We discuss whether the induced SA defences protect nutritive root galls against soil microbes.

Transcriptomics of the grape berry shrivel ripening disorder.

KEY MESSAGE: The lower expression at veraison of several ripening master regulators "switch genes" can play a central role in the induction of the berry shrivel ripening physiological disorder in grapevine. Berry shrivel (BS) is a ripening physiological disorder affecting grape berry with visible symptoms appearing after veraison. Berry shrivel leads to shrinking berries with a reduced weight and a lower content of sugars and anthocyanins. In this study, for the first time a transcriptomic analysis coupled with selected metabolites quantification was undertaken to understand the metabolic modifications induced by the disorder. Different stages of berry development were considered including pre- and symptomatic berries. No metabolic alterations in the berry transcriptome and in the metabolite content was observed in pre-symptomatic and pre-veraison samples. Interestingly, at veraison, with still not visible symptoms appearing on the berry, a subset of genes, called switch genes previously suggested as master regulators of the ripening onset in grape berries, were strongly lower expressed in BS. Later during the ripening phase and with visible symptoms of the disorder, more than 3000 genes were differentially expressed. The genes up-regulated were related to hormone biosynthesis, response to stress and the phenylpropanoid pathway, while the genes down-regulated during ripening belonged mainly to the flavonoid pathway, and the sugar metabolism. In agreement, BS berries showed lower content of sugars and anthocyanins from the onset of veraison onward, while the amount of acids was not significantly affected. In conclusion, these results highlight a pivotal role of the switch genes in grapevine ripening, as well as their possible contribution to induce the ripening disorder berry shrivel, although it remains unclear whether this is part of the cause or consequences of the BS disorder.

Polyphenolic profiling of roots (Vitis spp.) under grape phylloxera (D. vitifoliae Fitch) attack.

Many plants respond to herbivore attacks by the formation of secondary metabolites, such as polyphenols. Grape phylloxera (Daktulosphaira vitifoliaeFitch) induces organoid root galls on fibrous root tips of tolerant Vitis spp. rootstocks. We aim to understand if and how secondary metabolites are involved in the compatible interaction of D. vitifoliae and tolerant Vitis ssp. rootstocks belowground. We hypothise that D. vitifoliae infestation triggers the accumulation of phenolic key compounds in root gall tissue without preventing the compatible host-parasite interaction on two tolerant rootstocks with different genetic background: Teleki 5C (V. berlandieri x V. riparia) and Fercal (B.C. n°1B x 31 Richter). Plants and insects are grown in isolated climate chambers to sample root tips of non-infested plants (control) as well as root tips and galls of D. vitifoliae infested plants (5-14 dai). HPLC-MS-based analyses of phenolic key compounds are compared with gene expression levels of the biosynthetic phenylpropanoid pathway analysing temporal sequences of D. vitifoliae infested root tissue. The results show that the induction of the phenylpropanoid pathway by D. vitifoliae infestation plays an important role in the plant response. Concentrations of phenolic key compounds vary significantly among the rootstocks tested. Both rootstocks display an accumulation of flavan-3-ols and stilbenes in infested root gall tissue. Comparing the host responses of the two rootstocks Fercal shows a stronger accumulation of stilbenes locally in infested root galls, whereas Teleki 5C indicates elevated amounts of stilbenes in non-infested root tip tissue.

The role of the secondary phloem during the development of the grapevine Berry Shrivel ripening disorder.

Berry Shrivel (BS) is a post-veraison physiological ripening disorder of grapevine berries. Its symptoms encompass low pH, reduced content of sugars and anthocyanins, and loss of turgor leading to berries shriveling. Evidence for the primary causes of BS is still speculative and anatomical studies are scarce. So far, anatomical studies have determined necrotic cells, degraded primary phloem cells and hardening of secondary phloem cells in the rachis of BS affected grapes. The picture is far from being complete. Herein we report in-depth analyses of the ultrastructure, anatomy and spatial elementary analysis of rachis and pedicel tissues of BS symptomatic grape clusters with different symptom severity. We hypothesize that structural changes in the vascular system of BS affected grape clusters could alter transport functions of the phloem tissue and contribute to the appearance of BS symptoms. By applying different microscopic techniques (LM, SEM, TEM and EDS) we found a number of anatomical differences in both, rachis and pedicels, between H and BS symptomatic grapes, which include: (i) extended areas of collapsed cells and cell wall thickenings in the secondary phloem in BS samples; (ii) reduced number of cell layers in the cambium in BS samples; (iii) higher rate of callose deposition on sieve plates that are additionally covered with a carbohydrate-like material in BS samples; and (iv) reduced (up to 60%) estimated sieve tube conductivity in BS samples.

The ripening disorder berry shrivel affects anthocyanin biosynthesis and sugar metabolism in Zweigelt grape berries.

MAIN CONCLUSION: Timescale analyses suggest the berry shrivel (BS) disorder is induced before veraison with strong effects on anthocyanin biosynthesis, and minor effects on sugar transport and metabolism. Berry shrivel (BS)-affected grapes have low sugar contents, high acidity, less anthocyanins and flaccid berries. To date no pathogenic causes are known, and studies to elucidate the molecular basis leading to symptom induction and development are limited. Here we present a study on pre-symptomatic as well as symptomatic BS berries to characterize early metabolic changes, with focus on anthocyanin biosynthesis and sugars metabolism. Healthy and BS berries from six sampling time points were used (BBCH79-BBCH89). Our objectives are (1) to search for the beginning of BS-related physiological processes; (2) to search for key enzymes and sugar transporters involved in BS induction and development and (3) to understand the consequences on polyphenol biosynthesis. We employed high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology for sugar and polyphenol analyses, respectively. Additionally we conducted expression analyses (qPCR) of key genes and enzymatic activity assays. Our results show that BS-related processes start before veraison, as determined by slightly reduced hexose contents and reduced expression levels of a vacuolar invertase (VviGIN1), two monosaccharide transporters (VviTMT2, VviTMT3) and the anthocyanin biosynthesis (VviUFGT, VviMYBA1/2) genes. Lower amounts of delphinidin and cyanidin glycosidic forms were determined, while caftaric acid, quercetin-3-O-glucuronide and (+)-catechin were increased in BS berries. Although not all results were conclusive, especially for the sugar metabolism, our data provide important knowledge to improve the understanding of the highly complex berry shrivel ripening disorder.

Phylloxera (Daktulosphaira vitifoliae Fitch) alters the carbohydrate metabolism in root galls to allowing the compatible interaction with grapevine (Vitis ssp.) roots.

Gall forming phylloxera may compete for nutrients with meristematic tissues and develop heterotrophic structures that act as carbon sinks. In this work, we studied the underlying starch metabolism, sink-source translocation of soluble sugars towards and within root galls. We demonstrated that nodosities store carbohydrates by starch accumulation and monitored the expression of genes involved in the starch metabolic. Thereby we proved that the nodosity is symplastically connected to the source tissues through its development and that the starch metabolism is significantly affected to synthesize and degrade starch within the gall. Genes required for starch biosynthesis and degradation are up-regulated. Among the carbohydrate transporters the expression of a glucose-6-phosphate translocater, one sucrose transporter and two SWEET proteins were increases, whereas hexose transporters, tonoplast monosaccharide transporter and Erd6-like sugar transporters were decreased. We found general evidence for plant response to osmotic stress in the nodosity as previously suggested for gall induction processes. We conclude that nodosities are heterogenous plant organs that accumulate starch to serve as temporary storage structure that is gradually withdrawn by phylloxera. Phylloxera transcriptionally reprograms gall tissues beyond primary metabolism and included downstream secondary processes, including response to osmotic stress.

Severe drought stress is affecting selected primary metabolites, polyphenols, and volatile metabolites in grapevine leaves (Vitis vinifera cv. Pinot noir).

Extreme weather conditions with prolonged dry periods and high temperatures as well as heavy rain events can severely influence grapevine physiology and grape quality. The present study evaluates the effects of severe drought stress on selected primary metabolites, polyphenols and volatile metabolites in grapevine leaves. Among the 11 primary metabolites, 13 polyphenols and 95 volatiles which were analyzed, a significant discrimination between control and stressed plants of 7 primary metabolites, 11 polyphenols and 46 volatile metabolites was observed. As single parameters are usually not specific enough for the discrimination of control and stressed plants, an unsupervised (PCA) and a supervised (PLS-DA) multivariate approach were applied to combine results from different metabolic groups. In a first step a selection of five metabolites, namely citric acid, glyceric acid, ribose, phenylacetaldehyde and 2-methylbutanal were used to establish a calibration model using PLS regression to predict the leaf water potential. The model was strong enough to assign a high number of plants correctly with a correlation of 0.83. The PLS-DA provides an interesting approach to combine data sets and to provide tools for the specific evaluation of physiological plant stresses.

Two tomato alpha-expansins show distinct spatial and temporal expression patterns during development of nematode-induced syncytia.

Cyst nematodes induce specific syncytial feeding structures within the root which develop from an initial cell by successive incorporation of neighbouring cells through local cell wall dissolutions followed by hypertrophy of included cells. Expansins are known to induce cell wall relaxation and extension in acidic pH, and they are involved in many processes requiring wall modification from cell expansion to cell wall disassembly. We studied the expression pattern of tomato (Lycopersicon esculentum L., cv. Money Maker) expansins during development of syncytia induced by the potato cyst nematode (Globodera rostochiensis Woll.). Based on semi-quantitative reverse transcription-polymerase chain reaction, two expansin genes, LeEXPA4 and LeEXPA5, were selected for detailed examinations because their expression was either elevated in infected roots (LeEXPA4) or specifically induced in the root upon nematode infection (LeEXPA5). Both genes have distinct spatial and temporal expression patterns that may reflect their different roles in syncytium development. LeEXPA4 transcripts were localized predominantly in parenchymatous vascular cylinder cells surrounding syncytia. This finding suggests that LeEXPA4 might be involved in cell wall disassembly or relaxation, mediating syncytium expansion and/or development of conductive tissues. By contrast, LeEXPA5 transcripts were localized in enlarging syncytial elements. Similarly, in immunogold localization experiments, polyclonal antibodies localized the LeEXPA5 protein in cell walls of syncytial elements. This expression pattern suggests that LeEXPA5 gene is specifically involved in enlargement of cells incorporated into syncytium.

Expansins are among plant cell wall modifying agents specifically expressed during development of nematode-induced syncytia.

Cyst nematodes are economically important pests. As obligatory biotrophic endoparasites they invade host roots and induce formation of syncytia, structures that serve them as the only source of nutrients. During syncytium development, extensive cell wall modifications take place. Cell wall dissolution occurs during cell wall opening formation, cell walls expand during hypertrophy of syncytial elements and local cell wall synthesis leads to the thickening of syncytial cell wall and the formation of cell wall ingrowths. Numerous studies revealed that nematodes change expression of plant genes encoding cell wall modifying proteins including expansins. Expansins poses unique abilities to induce cell wall extension in acidic pH. Recently, we demonstrated that two alpha-expansin genes LeEXPA4 and LeEXPA5 are upregulated in tomato roots infected with potato cyst nematode (Globodera rostochiensis). In this addendum, we present the most recent results concerning involvement of plant cell wall modifying genes in syncytium development and discuss possible practical applications of this knowledge for developing plants with resistance against nematodes.