Aquaporin-Regulated Response of Grapevine Roots to Salinity and Role in Inherent Differences Among Rootstocks

Soil salinization is an emerging problem in California vineyards. Research is needed to more fully understand the physiological response of grapevine roots to salt stress in order to develop cultural strategies that improve in-field management and to facilitate breeding of tolerance. Upon exposure to salinity, roots often exhibit a rapid decrease of water uptake capacity caused by inhibition of water-channel proteins called aquaporins. Aquaporins are found throughout fine root cellular membranes and can control the efficiency of water extraction from the soil. Prevention and/or alleviation of salinity-induced aquaporin inhibition have been demonstrated for some plants using calcium supplements in experimental conditions.

Such a mechanism may contribute to the success of gypsum (i.e. calcium sulfate) applications used to lessen the detrimental effects of vineyard salinity. In the original grant, we proposed to address the following short-intermediate term goals: 1) to quantify aquaporin response to salinity and the ameliorative effects of calcium in a suite of grapevine rootstocks using both hydraulic physiology and molecular probes under hydroponic and soil growth conditions; and 2) to investigate the role that aquaporins play in grapevine rootstock physiological responses to other abiotic factors (i.e. drought) and their contribution to vine vigor.

Our results indicate that aquaporins play a role in water uptake across numerous Vitis rootstocks. We documented significantly higher inherent aquaporin expression in high vigor and drought resistant rootstocks (e.g. 140Ru, 1103P and 110R) compared to those with low vigor and drought intolerance ratings (e.g. 420A and 101-14). These inherent differences may explain the known variation in vigor among these rootstocks, likely play a role in divergent patterns of drought tolerance, and represent potential target genes for breeding similar traits. In more recent efforts, we characterized anatomical, molecular, and biophysical aspects of fine roots impacting water uptake in Vitis, a woody perennial. This study provides one of the few quantitative analyses of tissues specific aquaporin expression in roots, and the first in a woody species.

The study revealed strong parallels in developmental anatomy, distribution of aquaporins, and relationships with Lpr between herbaceous and woody fine roots within the meristematic/elongation and maturation zones. These similarities suggest a common foundation likely underlies the integration of root development and water uptake across plants. Fine root hydraulic permeability along the root length was positively correlated with aquaporin gene expression and negatively correlated with suberin deposition. For the salinity response of aquaporins, we consistently found a dramatic upregulation of aquaporin gene expression for both the PIP1 and PIP2 aquaporin families. This initial response dampened over time as expression patterns returned to near pre stress conditions. Patterns of expression were similar across rootstocks (i.e. patterns of response were not clearly and consistently associated with resistance or susceptibility to salinity stress among rootstocks).

Salinity experiments were done using a variety of experimental procedures and always showed similar results. Hydraulic conductivity of fine roots did not show a concomitant increase after salt stress initiation. This suggests that the increase in aquaporin expression increase following salt stress likely played a more local role on the cellular basis (i.e. by affecting local cell to cell water relations) rather than affecting the bulk tissue conductivity. Similar gene expression patterns were found in our newly developed tissue culture method when roots were transferred to saline media. This method was very successful in enabling us to track growth rate of individual roots over time and after exposure to salt. Fine root growth slowed abruptly upon transfer to saline media, but this effect was ameliorated if gypsum was present in either the establishment media or in the transfer media. These results suggest that gypsum applications commonly used in vineyards to lessen the effects of soil salinity are not only affecting the ion exchange of the soil column, but also having a direct impact on grapevine physiology by enabling the roots to maintain growth despite the saline conditions. More work is needed to explore this in detail and Drs. Walker and McElrone have recently initiated efforts to use the tissue culture system for evaluating Boron toxicity.

Rootstock Tolerance to Soil Salinity: Impact of Salinity

During Years 1 thru 3 of this project, we made progress towards all three Objectives. For Objective 1, we identified two suitable vineyard plots containing contrasting soils, then sampled and characterized the soil profiles present within these plots. The results of these analyses confirmed the differences in morphology and texture between the two soils, and also revealed differences in cation exchange capacity, hydraulic conductivity, and plant available water. These differences will likely have a significant impact on plant growth and nutrient uptake. For Objective 2, we grafted and planted the various rootstock-scion combinations at both sites. By the end of Year 2, the plants were sufficiently large and well established that we were able to sample petioles and blades at harvest time. Following discussions with the vineyard management staff, the research team decided collectively that the vines were still too small to safely begin treatment with saline water conditions during Year 3. Accordingly, during Year 3 we proceeded with Objective 3, continuing to study baseline differences in plant nutrient uptake, comparing the 10 different rootstocks to one another within a single soil type, and comparing vine replicates grown on identical rootstocks in the two contrasting soil types. There were significant differences between rootstocks for all nutrients studied. In most cases, the rootstock rank order was similar for vines grown on the Alfisol and those grown on the Entisol; however, there were many exceptions to this trend. For the coming year, as described in the renewal application for Year 4, saline irrigation will begin in spring 2011. In Years 4 and 5, plant nutrient uptake and soil solution chemistry will be monitored concurrently in order to determine the relationship 2 between soil salinity and plant nutrition, and to compare rootstocks. This study constitutes a limited rootstock trial employing a panel of 10 rootstocks commonly used in California, comparing two soil types that are also common in California winegrowing regions. This project has high potential for generating information of relevance to California winegrape growers, as it will be one of the most comprehensive studies to date analyzing the effects of soil salinity on specific rootstocks.

Aquaporin-Regulated Response of Grapevine Roots to Salinity and Role in Inherent Differences Among Rootstocks

Soil salinization is an emerging problem in California vineyards. Research is needed to more fully understand the physiological response of grapevine roots to salt stress in order to develop cultural strategies that improve in-field management and to facilitate breeding of tolerance. Upon exposure to salinity, roots often exhibit a rapid decrease of water uptake capacity caused by inhibition of water-channel proteins called aquaporins. Aquaporins are found throughout fine root cellular membranes and can control the efficiency of water extraction from the soil. Prevention and/or alleviation of salinity-induced aquaporin inhibition have been demonstrated for some plants using calcium supplements in experimental conditions. Such a mechanism may contribute to the success of gypsum (i.e. calcium sulfate) applications used to lessen the detrimental effects of vineyard salinity.

In the original grant, we proposed to address the following short-intermediate term goals: 1) to quantify aquaporin response to salinity and the ameliorative effects of calcium in a suite of grapevine rootstocks using both hydraulic physiology and molecular probes under hydroponic and soil growth conditions; and 2) to investigate the role that aquaporins play in grapevine rootstock physiological responses to other abiotic factors (i.e. drought) and their contribution to vine vigor.

Our results indicate that aquaporins play a role in water uptake across numerous Vitis rootstocks. We documented significantly higher inherent aquaporin expression in high vigor and drought resistant rootstocks (e.g. 140Ru, 1103P and 110R) compared to those with low vigor and drought intolerance ratings (e.g. 420A and 101-14). These inherent differences may explain the known variation in vigor among these rootstocks, likely play a role in divergent patterns of drought tolerance, and represent potential target genes for breeding similar traits. In more recent efforts, we characterized anatomical, molecular, and biophysical aspects of fine roots impacting water uptake in Vitis, a woody perennial. This study provides one of the few quantitative analyses of tissues specific aquaporin expression in roots, and the first in a woody species. The study revealed strong parallels in developmental anatomy, distribution of aquaporins, and relationships with Lpr between herbaceous and woody fine roots within the meristematic/elongation and maturation zones. These similarities suggest a common foundation likely underlies the integration of root development and water uptake across plants. Fine root hydraulic permeability along the root length was positively correlated with aquaporin gene expression and negatively correlated with suberin deposition.

For the salinity response of aquaporins, we consistently found a dramatic upregulation of aquaporin gene expression for both the PIP1 and PIP2 aquaporin families. This initial response dampened over time as expression patterns returned to near pre stress conditions. Patterns of expression were similar across rootstocks (i.e. patterns of response were not clearly and consistently associated with resistance or susceptibility to salinity stress among rootstocks). Salinity experiments were done using a variety of experimental procedures and always showed similar results. Hydraulic conductivity of fine roots did not show a concomitant increase after salt stress initiation. This suggests that the increase in aquaporin expression increase following salt stress likely played a more local role on the cellular basis (i.e. by affecting local cell to cell water relations) rather than affecting the bulk tissue conductivity. Similar gene expression patterns were found in our newly developed tissue culture method when roots were transferred to saline media. This method was very successful in enabling us to track growth rate of individual roots over time and after exposure to salt. Fine root growth slowed abruptly upon transfer to saline media, but this effect was ameliorated if gypsum was present in either the establishment media or in the transfer media. These results suggest that gypsum applications commonly used in vineyards to lessen the effects of soil salinity are not only affecting the ion exchange of the soil column, but also having a direct impact on grapevine physiology by enabling the roots to maintain growth despite the saline conditions. More work is needed to explore this in detail and Drs. Walker and McElrone have recently initiated efforts to use the tissue culture system for evaluating Boron toxicity.

Aquaporin-regulated response of grapevine roots to salinity

Soil salinization is an emerging problem in California vineyards. Research is needed to more fully understand the physiological response of grapevine roots to salt stress in order to develop cultural strategies that improve in-field management and to facilitate breeding of tolerance. Upon exposure to salinity, roots often exhibit a rapid decrease of water uptake capacity caused by inhibition of water-channel proteins called aquaporins. Aquaporins are found throughout fine root cellular membranes and can control the efficiency of water extraction from the soil. Prevention and/or alleviation of salinity-induced aquaporin inhibition have been demonstrated for some plants using calcium supplements in experimental conditions. Such a mechanism may contribute to the success of gypsum (i.e. calcium sulfate) applications used to lessen the detrimental effects of vineyard salinity. In our grant, we proposed to address the following short-intermediate term goals: 1) to quantify aquaporin response to salinity and the ameliorative effects of calcium in a suite of grapevine rootstocks using both hydraulic physiology and molecular probes under hydroponic and soil growth conditions; and 2) to investigate the role that aquaporins play in grapevine rootstock physiological responses to other abiotic factors (i.e. drought, anoxia, nutrient status) and their contribution to vine vigor. We are on target to achieve these goals over a two year funding period (the current Research Support Agreement between USDA-ARS and UCDavis expires 31 September 2010). Our results from 2008 and 2009 indicate that aquaporins play an integral role in water uptake across numerous Vitis rootstocks and exhibit a strong and rapid response to salinity stress. In 2008, we documented significantly higher inherent aquaporin expression in high vigor and drought resistant rootstocks (1103P and 110R) compared to those with low vigor and drought intolerance ratings (420A and 101-14). In additional experiments conducted during 2009, we continued to find this pattern regardless of the growing conditions (i.e. soil or hydroponics). These inherent differences may contribute to the known variation in vigor and drought tolerance among these rootstocks, and represent potential target genes for breeding similar traits. In 2009, we continued to assess the response of roots to salinity stress and the ameliorative affects of calcium. Aquaporin gene expression in numerous rootstocks (Ramsey, Riparia, French Colombard, Thompson seedless, 420A, 101-14, 110R, 039-16, 5BB, 1103P) was highly responsive to salt stress. In most of the rootstocks studied, expression increased significantly under salinity stress across all PIP1 and PIP2 aquaporin genes. In a subsequent hydroponic study, we tracked the hydraulic conductivity of fine roots under control, NaCl, or NaCl plus calcium treatments. We found no ameliorative effects of calcium, but did find that conductivity under salinity was maintained similarly to controls for some of the rootstocks. An up-regulation of aquaporin gene expression under salinity conditions likely plays a role in this response (gene expression for these roots will be completed by Spring 2010). We have begun additional experiments to determine if long term growth in calcium enables tolerance to future exposure to salt stress.

Grapevine Canker Diseases

Until recently, grapevine dieback in California grapevines was attributed mainly to the fungus Eutypa lata in the family Diatrypaceae. This fungus has been known as the causal agent for Eutypa dieback. However, we have shown that dieback of grapevines in California is also caused by other fungi in the family Diatrypaceae (Eutypa leptoplacaEutypelland Diatrypella) as well as by several Botryosphaeria spp. To date, we have identified at least nine species of Diatrypaceous fungi associated with grapevine cankers as well as nine different species of Botryosphaeria, which appeared to constitute the main pathogens isolated from grapevine cankers statewide (Úrbez-Torres, et al., 2006; Úrbez-Torres, et al., 2007). Isolation from cankers and spore trapping studies in the table grape areas of the Coachella Valley have revealed the high incidence of Eutypella vitis (Diatrypaceae). This is a new pathogen in Coachella Valley table grape area. In addition, isolations from cankers have shown Phomopsis viticola to be the most common pathogen isolated from grapevine cankers in table and raisin cultivars in Fresno and Tulare Cos (Úrbez-Torres, et al., 2006). Results of spore trapping studies have shown that Botryosphaeria spores were mainly trapped following rainfall events in Napa Valley, Arbuckle, Lodi and San Luis Obispo, and following overhead irrigation in the Coachella Valley. Botryosphaeria spores were trapped from September 2006 to Jan., 2007. Interestingly, Botryosphaeria spores were also trapped without rainfall or irrigation in Arbuckle, suggesting that other environmental factors may contribute to spore release. Temperature studies for mycelial growth and pycnidial formation have shown different optimum temperature regimes among Botryosphaeria spp found in California, which agree with the actual differences in terms of geographical distribution of Botryosphaeria spp in California. We have developed new assays to evaluate the pathogenicity or virulence of the various fungi associated with grapevine dieback. Indeed, pathogenicity testing using green shoots in the laboratory as well as in the field appeared as a reliable method to rapidly assess fungal pathogenicity. Surveys for the host range as well as perithecia of Botryosphaeria spp in California have revealed additional host plants and sources of inoculum for these pathogens. These findings are providing a better understanding of the disease cycle of Botryosphaeria canker disease. Also, our work has stressed the importance of riparian and forest systems adjacent to vineyards in understanding the epidemiology, disease cycle and development of canker diseases in grapevines. Double pruning was shown to be an effective cultural practice which completely eliminates canker formation by Eutypa spp (Weber, E. et al., 2007) and we are now testing this practice against the more rapidly moving Botryosphaeria spp. Finally, our research has offered alternative control methods for E. lata using boron based chemicals. The use of 3-5{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} boron mixed with a commercial tree wound paste gave excellent disease control (91{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}). Interestingly, 1{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} boric acid also gave excellent disease control (82{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}).

Influence of rootstock and vine spacing on root distribution, vine growth, crop yield, fruit and wine composition, canopy microclimate and wine quality of Cabernet Sauvignon

In 1991 and 1992, the effect of seven rootstocks (AxR#l, 110R, 5C, 3309, 420A, 1616, and 039-16) grafted to Cabernet Sauvignon in combination with three between row spacings (2, 3, and 4 m) and two in-row spacings (1 and 2 m) on root and shoot growth, water utilization, leaf, fruit and wine composition, crop yield and wine quality were evaluated in a field plot trial established at the Oakville Experimental Vineyard in 1987. The data revealed that the above seven rootstocks can be divided into three groups based on rooting depth, pruning weights and crop yield. 110R, AxR, and 039-16 rootstocks had the deepest roots, made the most vegetative growth and had the highest crop yield; 5C, 3309, and 1616 were intermediate in growth and yield, and 420A had the least amount of shoot growth and lowest yield. Neutron probe measurements also showed that AxR, 039-16, and 110R were able to utilize water down to depths of 210 cm (-7 ft); 3309, 5C, and 1616 mainly used water at depths between 30 and 150 cm and 420A mostly used water at depths less than 120 cm. In another field trial comparing St. George, AxR, and 110R it was shown that St. George had greater root density and deeper roots than 110R and AxR#l. There were relatively little differences in fruit composition at harvest between the seven rootstocks. 039-16 consistently had the highest pH, K+, and malic acid of the seven rootstocks. 110R fruit was the first to ripen and had the highest titratable acidity and anthocyanin pigment per berry, however, on a per gram basis, the level of anthocyanin did not differ significantly between rootstocks. In leaf petiole analyses, 039-16 had the highest level of K\ Ca*+, NO;, and Ca/mg ratio, and 420A was consistently the lowest in K at both bloom and veraison. The average crop yield of vines from rows spaced 2, 3, and 4 m apart was 7.7, 5.9, and 5.1 tons/ac, respectively, and for in-row spacings of 1 and 2 m, 7.0 and 5.5 tons/ac, respectively. Fruits from vines spaced 3 and 4 m apart between rows were significantly higher in sugar and pH than 2 m row spacing, however, TA, malic acid, K, and anthocyanin in fruits did not differ between row spacing treatments. Fruits from vines spaced 1 m apart within rows were significantly higher in pH, K, malic acid, and anthocyanin/berry than fruits from vines spaced 2 m apart. Wines made from Cabernet Sauvignon grafted onto different rootstocks differed in composition. 039-16 wines had the highest pH and hue and less red and total coloration than 5C, 3309 and 110R. 5C wine had the lowest pH and hue and the highest level of anthocyanin and total coloration of the above four stocks. Wines made from vines at two meter spacing generally had higher titratable acidity and level of anthocyanin than four meter row spacing wines. Within row vine spacing had little effect on wine composition. Duo-trio tasting of wines made from the 1992 vintage showed that 5C and 3309 wines could generally be distinguished from 110R and 039-16 wines. 5C wine had the most fruity character, whereas 110R and 039-16 wines were more vegetative and astringent in character. 3309 wine had the most herbal/spicy character of the four rootstocks. For 5C rootstock, closer vine spacing (2 x 1 and 2 x 2 m) wines could be distinguished from wider vine spacing (4 x 1 and 4 x 2 m) with a preference for the closer vine spacing wines. However, for 110R rootstock, wines did not differ between row and vine spacing in duo-trio taste comparisons.