Deidre H. Blackmore

Contrast in chloride exclusion between two grapevine genotypes and its variation in their hybrid progeny

Potted grapevines of 140 Ruggeri (Vitis berlandieri × Vitis rupestris), a good Cl− excluder, and K 51-40 (Vitis champinii × Vitis riparia ‘Gloire’), a poor Cl− excluder, and of a family obtained by crossing the two genotypes, were used to examine the inheritance of Cl− exclusion. Rooted leaves were then used to further investigate the mechanism for Cl− exclusion in 140 Ruggeri. In both a potting mix trial (plants watered with 50 mM Cl−) and a solution culture trial (plants grown in 25 mM Cl−), the variation in Cl− accumulation was continuous, indicating multiple rather than single gene control for Cl− exclusion between hybrids within the family. Upper limits of 42% and 35% of the phenotypic variation in Cl− concentration could be attributed to heritable sources in the potting mix and solution culture trials, respectively. Chloride transport in roots of rooted leaves of both genotypes appeared to be via the symplastic pathway, since addition of 8-hydroxy-1,3,6-pyrenetrisulphonic acid (PTS), an apoplastic tracer, revealed no obvious PTS fluorescence in the laminae of either genotype, despite significant accumulation of Cl− in laminae of K 51-40 during the PTS uptake period. There was no significant difference in either unidirectional 36Cl− flux (10 min) or 36Cl− uptake (3 h) into roots of rooted leaves exposed to 5, 10, or 25 mM Cl−. However, the percentage of 36Cl− transported to the lamina (3 h) was significantly lower in 140 Ruggeri than in K 51-40, supporting reduced Cl− loading into xylem and implicating the root stele in the Cl− exclusion mechanism.

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

BackgroundSalt 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.ResultsWhen 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.ConclusionsThese 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.

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

A protein from grapevine that transports sodium, potassium, and chloride ions across endomembranes is important for normal growth and 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 36Cl–, 22Na+, and 86Rb+ 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.

Analysis of the salt exclusion phenotype in rooted leaves of grapevine (Vitis spp.): Salt exclusion in rooted leaves of grapevine

Background and Aims Rooted leaves were used to analyse the salt exclusion phenotype in grapevines. Genotypes included rootstocks 140 Ruggeri and K51‐40 and cultivar Cabernet Sauvignon – respectively, good, poor and intermediate chloride excluders. Methods and Results We investigated the effect of short‐term salt treatment on the chloride, sodium and potassium concentration of organs and whole rooted leaves and the time course of chloride accumulation in salt‐treated, whole rooted leaves. The effect of Control and salt plus low and high nitrate concentration on chloride and nitrate concentration in organs was assessed. Salt treatment increased chloride and sodium concentration in all organs but had no effect on potassium concentration. The chloride concentration of whole rooted leaves and the sodium concentration of lamina and petiole were similar between salt‐treated rooted leaves of 140 Ruggeri and K51‐40; 140 Ruggeri accumulated more chloride in roots and less in leaf (petiole and lamina) than K51‐40. Roots of salt‐treated 140 Ruggeri and K51‐40 responded to higher external nitrate by decreasing chloride concentration and increasing nitrate concentration. Conclusions Restricted transport of chloride to the leaf and greater storage in roots is a feature of the chloride exclusion phenotype in rooted leaves. The short‐term capacity of 140 Ruggeri for chloride exclusion from the leaf was reduced in the salt plus high nitrate treatment. This was linked to higher nitrate and reduced chloride accumulation in roots. Significance of the Study The chloride exclusion phenotype in rooted leaves was shown to involve greater partitioning to roots and less to the leaf, rather than differences in net chloride accumulation by whole rooted leaves.