Root-Specific Expression of Vitis vinifera VviNPF2.2 Modulates Shoot Anion Concentration in Transgenic Arabidopsis.

Grapevines (Vitis vinifera L., Vvi) on their roots are generally sensitive to salt-forming ions, particularly chloride (Cl-) when grown in saline environments. Grafting V. vinifera scions to Cl--excluding hybrid rootstocks reduces the impact of salinity. Molecular components underlying Cl--exclusion in Vitis species remain largely unknown, however, various anion channels and transporters represent good candidates for controlling this trait. Here, two nitrate/peptide transporter family (NPF) members VviNPF2.1 and VviNPF2.2 were isolated. Both highly homologous proteins localized to the plasma membrane of Arabidopsis (Arabidopsis thaliana) protoplasts. Both were expressed primarily in grapevine roots and leaves and were more abundant in a Cl--excluding rootstock compared to a Cl--includer. Quantitative PCR of grapevine roots revealed that VviNPF2.1 and 2.2 expression was downregulated by high [NO3 -] resupply post-starvation, but not affected by 25 mM Cl-. VviNPF2.2 was functionally characterized using an Arabidopsis enhancer trap line as a heterologous host which enabled cell-type-specific expression. Constitutive expression of VviNPF2.2 exclusively in the root epidermis and cortex reduced shoot [Cl-] after a 75 mM NaCl treatment. Higher expression levels of VviNPF2.2 correlated with reduced Arabidopsis xylem sap [NO3 -] when not salt stressed. We propose that when expressed in the root epidermis and cortex, VviNPF2.2 could function in passive anion efflux from root cells, which reduces the symplasmic Cl- available for root-to-shoot translocation. VviNPF2.2, through its role in the root epidermis and cortex, could, therefore, be beneficial to plants under salt stress by reducing net shoot Cl- accumulation.

The grapevine NaE sodium exclusion locus encodes sodium transporters with diverse transport properties and localisation.

Grapevine (Vitis vinifera L.) is a valuable crop for human consumption and wine production, and is prone to suffering from salinity stress in arid regions or when exposed to low quality irrigation water. A previous study identified a quantitative trait locus (QTL) NaE, containing six High-affinity Potassium Transporter 1 genes, that was associated with shoot Na+ exclusion in grapevine. While HKT1;1 was predicted to be the most likely gene within this QTL to encode for this important salinity tolerance sub-trait, four other HKTs within the QTL remained uncharacterised; VviHKT1;2 encodes a truncated transcript unlikely to form a functional transporter. In this study, two allelic variants for each of VviHKT1;6, VviHKT1;7 and VviHKT1;8 from the heterozygous grapevine variety Cabernet Sauvignon were functionally characterised. Using the Xenopus laevis oocyte heterologous expression system, as well as transient expression in tobacco leaves, we found that the VviHKT1;6 and VviHKT1;7 alleles encoded plasma membrane localised proteins that facilitated significant non-rectifying Na+ transport. Conversely, proteins encoded by the VviHKT1;8 alleles were inwardly-rectifying, weak Na+ transporters that localised to intracellular organelles. Mining of previous RNA-seq gene expression data suggested that VviHKT1;6-8 are weakly expressed in grapevine roots, flower buds, and seeds under normal conditions and different nutrient regimes. We propose that VviHKT1;6 and VviHKT1;7 are likely to have a less significant role in grapevine leaf Na+ exclusion than VviHKT1;1, and that VviHKT1;8 is involved in endomembrane Na+ transport.

Expression Patterns of Genes Encoding Sugar and Potassium Transport Proteins Are Simultaneously Upregulated or Downregulated When Carbon and Potassium Availability Is Modified in Shiraz (Vitis vinifera L.) Berries.

A link between the accumulation of sugar and potassium has previously been described for ripening grape berries. The functional basis of this link has, as of yet, not been elucidated but could potentially be associated with the integral role that potassium has in phloem transport. An experiment was conducted on Shiraz grapevines in a controlled environment. The accumulation of berry sugar was curtailed by reducing the leaf photoassimilation rate, and the availability of potassium was increased through soil fertilization. The study characterizes the relationship between the accumulation of sugar and potassium into the grape berry and describes how their accumulation patterns are related to the expression patterns of their transporter proteins. A strong connection was observed between the accumulation of sugar and potassium in the grape berry pericarp, irrespective of the treatment. The relative expression of proteins associated with sugar and potassium transport across the tonoplast and plasma membrane was closely correlated, suggesting transcriptional coregulation leading to the simultaneous translocation and storage of potassium and sugar in the grape berry cell.

Fast Phenomics in Vineyards: Development of GRover, the Grapevine Rover, and LiDAR for Assessing Grapevine Traits in the Field.

This paper introduces GRover (the grapevine rover), an adaptable mobile platform for the deployment and testing of proximal imaging sensors in vineyards for the non-destructive assessment of trunk and cordon volume and pruning weight. A SICK LMS-400 light detection and ranging (LiDAR) radar mounted on GRover was capable of producing precise (±3 mm) 3D point clouds of vine rows. Vineyard scans of the grapevine variety Shiraz grown under different management systems at two separate locations have demonstrated that GRover is able to successfully reproduce a variety of vine structures. Correlations of pruning weight and vine wood (trunk and cordon) volume with LiDAR scans have resulted in high coefficients of determination (R² = 0.91 for pruning weight; 0.76 for wood volume). This is the first time that a LiDAR of this type has been extensively tested in vineyards. Its high scanning rate, eye safe laser and ability to distinguish tissue types make it an appealing option for further development to offer breeders, and potentially growers, quantified measurements of traits that otherwise would be difficult to determine.

Functional differences in transport properties of natural HKT1;1 variants influence shoot Na+ exclusion in grapevine rootstocks.

Under salinity, Vitis spp. rootstocks can mediate salt (NaCl) exclusion from grafted V. vinifera scions enabling higher grapevine yields and production of superior wines with lower salt content. Until now, the genetic and mechanistic elements controlling sodium (Na+ ) exclusion in grapevine were unknown. Using a cross between two Vitis interspecific hybrid rootstocks, we mapped a dominant quantitative trait locus (QTL) associated with leaf Na+ exclusion (NaE) under salinity stress. The NaE locus encodes six high-affinity potassium transporters (HKT). Transcript profiling and functional characterization in heterologous systems identified VisHKT1;1 as the best candidate gene for controlling leaf Na+ exclusion. We characterized four proteins encoded by unique VisHKT1;1 alleles from the parents, and revealed that the dominant HKT variants exhibit greater Na+ conductance with less rectification than the recessive variants. Mutagenesis of VisHKT1;1 and TaHKT1.5-D from bread wheat, demonstrated that charged amino acid residues in the eighth predicted transmembrane domain of HKT proteins reduces inward Na+ conductance, and causes inward rectification of Na+ transport. The origin of the recessive VisHKT1;1 alleles was traced to V. champinii and V. rupestris. We propose that the genetic and functional data presented here will assist with breeding Na+ -tolerant grapevine rootstocks.

Potassium in the Grape (Vitis vinifera L.) Berry: Transport and Function.

K+ is the most abundant cation in the grape berry. Here we focus on the most recent information in the long distance transport and partitioning of K+ within the grapevine and postulate on the potential role of K+ in berry sugar accumulation, berry water relations, cellular growth, disease resistance, abiotic stress tolerance and mitigating senescence. By integrating information from several different plant systems we have been able to generate new hypotheses on the integral functions of this predominant cation and to improve our understanding of how these functions contribute to grape berry growth and ripening. Valuable contributions to the study of K+ in membrane stabilization, turgor maintenance and phloem transport have allowed us to propose a mechanistic model for the role of this cation in grape berry development.

Impact of reduced atmospheric CO2 and varied potassium supply on carbohydrate and potassium distribution in grapevine and grape berries (Vitis vinifera L.).

To assess the robustness of the apparent sugar-potassium relationship during ripening of grape berries, a controlled-environment study was conducted on Shiraz vines involving ambient and reduced (by 34%) atmospheric CO2 concentrations, and standard and increased (by 67%) soil potassium applications from prior to the onset of ripening. The leaf net photoassimilation rate was decreased by 35% in the reduced CO2 treatment. The reduction in CO2 delayed the onset of ripening, but at harvest the sugar content of the berry pericarp was similar to that of plants grown in ambient conditions. The potassium content of the berry pericarp in the reduced CO2 treatment was however higher than for the ambient CO2. Berry potassium, sugar and water content were strongly correlated, regardless of treatments, alluding to a ternary link during ripening. Root starch content was lower under reduced CO2 conditions, and therefore likely acted as a source of carbohydrates during berry ripening. Root carbohydrate reserve replenishment could also have been moderated under reduced CO2 at the expense of berry ripening. Given that root potassium concentration was less in the vines grown in the low CO2 atmosphere, these results point toward whole-plant fine-tuning of carbohydrate and potassium partitioning aimed at optimising fruit ripening.

Grapevine and Arabidopsis Cation-Chloride Cotransporters Localize to the Golgi and Trans-Golgi Network and Indirectly Influence Long-Distance Ion Transport and Plant Salt Tolerance.

Plant cation-chloride cotransporters (CCCs) have been implicated in conferring salt tolerance. They are predicted to improve shoot salt exclusion by directly catalyzing the retrieval of sodium (Na(+)) and chloride (Cl(-)) ions from the root xylem. We investigated whether grapevine (Vitis vinifera [Vvi]) CCC has a role in salt tolerance by cloning and functionally characterizing the gene from the cultivar Cabernet Sauvignon. Amino acid sequence analysis revealed that VviCCC shares a high degree of similarity with other plant CCCs. A VviCCC-yellow fluorescent protein translational fusion protein localized to the Golgi and the trans-Golgi network and not the plasma membrane when expressed transiently in tobacco (Nicotiana benthamiana) leaves and Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts. AtCCC-green fluorescent protein from Arabidopsis also localized to the Golgi and the trans-Golgi network. In Xenopus laevis oocytes, VviCCC targeted to the plasma membrane, where it catalyzed bumetanide-sensitive (36)Cl(-), (22)Na(+), and (86)Rb(+) uptake, suggesting that VviCCC (like AtCCC) belongs to the Na(+)-K(+)-2Cl(-) cotransporter class of CCCs. Expression of VviCCC in an Arabidopsis ccc knockout mutant abolished the mutant's stunted growth phenotypes and reduced shoot Cl(-) and Na(+) content to wild-type levels after growing plants in 50 mm NaCl. In grapevine roots, VviCCC transcript abundance was not regulated by Cl(-) treatment and was present at similar levels in both the root stele and cortex of three Vitis spp. genotypes that exhibit differential shoot salt exclusion. Our findings indicate that CCC function is conserved between grapevine and Arabidopsis, but neither protein is likely to directly mediate ion transfer with the xylem or have a direct role in salt tolerance.

Shoot chloride exclusion and salt tolerance in grapevine is associated with differential ion transporter expression in roots.

BACKGROUND: Salt tolerance in grapevine is associated with chloride (Cl-) exclusion from shoots; the rate-limiting step being the passage of Cl- between the root symplast and xylem apoplast. Despite an understanding of the physiological mechanism of Cl- exclusion in grapevine, the molecular identity of membrane proteins that control this process have remained elusive. To elucidate candidate genes likely to control Cl- exclusion, we compared the root transcriptomes of three Vitis spp. with contrasting shoot Cl- exclusion capacities using a custom microarray. RESULTS: When challenged with 50 mM Cl-, transcriptional changes of genotypes 140 Ruggeri (shoot Cl- excluding rootstock), K51-40 (shoot Cl- including rootstock) and Cabernet Sauvignon (intermediate shoot Cl- excluder) differed. The magnitude of salt-induced transcriptional changes in roots correlated with the amount of Cl- accumulated in shoots. Abiotic-stress responsive transcripts (e.g. heat shock proteins) were induced in 140 Ruggeri, respiratory transcripts were repressed in Cabernet Sauvignon, and the expression of hypersensitive response and ROS scavenging transcripts was altered in K51-40. Despite these differences, no obvious Cl- transporters were identified. However, under control conditions where differences in shoot Cl- exclusion between rootstocks were still significant, genes encoding putative ion channels SLAH3, ALMT1 and putative kinases SnRK2.6 and CPKs were differentially expressed between rootstocks, as were members of the NRT1 (NAXT1 and NRT1.4), and CLC families. CONCLUSIONS: These results suggest that transcriptional events contributing to the Cl- exclusion mechanism in grapevine are not stress-inducible, but constitutively different between contrasting varieties. We have identified individual genes from large families known to have members with roles in anion transport in other plants, as likely candidates for controlling anion homeostasis and Cl- exclusion in Vitis species. We propose these genes as priority candidates for functional characterisation to determine their role in chloride transport in grapevine and other plants.

Whole-plant transpiration efficiency of Sultana grapevine grown under saline conditions is increased through the use of a Cl--excluding rootstock.

The aim of this study was to test the influence of salinity (1, 20, 40 and 80 mol m-3) on the transpiration efficiency (W = biomass / water transpired), lamina gas exchange and carbon isotope discrimination (Δ) of grapevine (Vitis vinifera L. cv. Sultana) grown on own roots or grafted to a Cl--excluding rootstock (Ramsey; Vitis champiniL.). Growth of own-rooted and Ramsey-rooted vines irrigated with a salinity of 40 mol m-3 was reduced by 55 and 12%, respectively, compared with vines irrigated with 1 mol m-3. At 1 mol m-3 W of Ramsey-rooted vines was 1.3-fold higher than own-rooted vines (3.9 and 3.0 g L-1, respectively). Salinity resulted in a decrease in W of own-rooted vines (31% reduction at both 40 and 80 mol m-3). In contrast, W of Ramsey-rooted vines increased by up to 1.25-fold under saline conditions. Consequently, at 80 mol m-3 W of Ramsey-rooted vines was 2-fold higher than own-rooted vines. To our knowledge this is the first demonstration of the potential of a rootstock to increase W of a crop species under saline conditions. The rootstock-dependent differences in grapevine W under saline conditions were not determined by differences in lamina gas exchange. Differences in W associated with rootstock may be attributed to differences in ion uptake and the energy requirements associated with ion partitioning and the formation of compatible solutes.

Regulation of canopy conductance and transpiration and their modelling in irrigated grapevines.

Whole-vine transpiration was estimated for well-watered nine-year-old Sultana grapevines (Vitis vinifera L. cv. Sultana) from xylem sap flow measured with Granier's heat-dissipation probes. Canopy conductance of the grapevine was calculated by inverting the Penman-Monteith equation. Transpiration from grapevine canopies was strongly controlled by the canopy conductance. Canopy conductance decreased exponentially with increasing vapour pressure deficit (VPD) except in the morning when solar radiation was less than 200 W m-2 and the canopy conductance was predominantly limited by the solar radiation. A non-linear model of canopy conductance as a function of the solar radiation and VPD explained > 90% of the variation observed in canopy conductance. Under contrasting VPD conditions (daytime maximum of 3 kPa vs 8 kPa), grapevines were able to regulate their canopy conductance from 0.006 to 0.001 m s-1 to maintain a near constant transpiration. Whole-canopy transpiration calculated from modelled canopy conductance using the Penman-Monteith equation was highly correlated with the measured transpiration (sap flow) values over the range of 0-0.20 mm h-1 (R2 > 0.85). Cross-validation shows that these mechanistic models based on solar radiation and VPD provide good predictions of canopy conductance and transpiration under the conditions of the study.