Salinity tolerance of two potato cultivars (Solanum tuberosum) correlates with differences in vacuolar transport activity

Potato is an important cultivated crop species and since it is moderately salt sensitive there is a need to develop more salt tolerant cultivars. A high activity of Na+ transport across the tonoplast in exchange for H+ is essential to reduce Na+ toxicity. The proton motive force (PMF) generated by the V-H+-ATPase and the V-H+-PPase energizes the Na+ (K+)/H+ antiport. We compared the activity, gene expression, and protein levels of the vacuolar proton pumps and the Na+ /H+ antiporters in two potato cultivars (Solanum tuberosum) contrasting in their salt tolerance (cv. Desiree; tolerant and Mozart; sensitive) grown at 0 and 60 mM NaCl. Tonoplast-enriched vesicles were used to study the pump activity and protein levels of the V-H+-ATPase and the V-H+-PPase and the activity of the Na+ /H+ antiporter. Although salt stress reduced the V-H+-ATPase and the V-H+-PPase activity in both cultivars, the decline in H+ pump activity was more severe in the salt-sensitive cultivar Mozart. After salt treatment, protein amounts of the vacuolar H+ pumps decreased in Mozart but remained unchanged in the cultivar Desiree. Decreased protein amounts of the V-H+-PPase found in Mozart may explain the reduced V-H+-PPase activity found for Mozart after salt stress. Under non-stress conditions, protein amounts of V-H+-PPase were equal in both cultivars while the V-H+-PPase activity was already twice as high and remained higher after salt treatment in the cultivar Desiree as compared to Mozart. This cultivar-dependent V-H+-PPase activity may explain the higher salt tolerance of Desiree. Moreover, combined with reduced vacuolar H+ pump activity, Mozart showed a lower Na+ /H+ exchange activity and the Km for Na+ is at least twofold lower in tonoplast vesicles from Desiree, what suggests that NHXs from Desiree have a higher affinity for Na+ as compared to Mozart. From these results, we conclude that the higher capacity in combination with the higher affinity for Na+ uptake can be an important factor to explain the differences in salt tolerance of these two potato cultivars

Assessment of natural variation in the first pore domain of the tomato HKT1;2 transporter and characterization of mutated versions of SlHKT1;2 expressed in Xenopus laevis oocytes and via complementation of the salt sensitive athkt1;1 mutant

Single Nucleotide Polymorphisms (SNPs) within the coding sequence of HKT transporters are important for the functioning of these transporters in several plant species. To unravel the functioning of HKT transporters analysis of natural variation and multiple site-directed mutations studies are crucial. Also the in vivo functioning of HKT proteins, via complementation studies performed with athkt1;1 plants, could provide essential information about these transporters. In this work, we analysed the natural variation present in the first pore domain of the HKT1;2 coding sequence of 93 different tomato accessions, which revealed that this region was conserved among all accessions analysed. Analysis of mutations introduced in the first pore domain of the SlHKT1;2 gene showed, when heterologous expressed in Xenopus laevis oocytes, that the replacement of S70 by a G allowed SlHKT2;1 to transport K+, but also caused a large reduction in both Na+ and K+ mediated currents. The study of the transport characteristics of SlHKT1;2 revealed that Na+-transport by the tomato SlHKT1;2 protein was inhibited by the presence of K+ at the outside of the membrane. GUS expression under the AtHKT1;1 promoter gave blue staining in the vascular system of transgenic Arabidopsis. athkt1;1 mutant plants transformed with AtHKT1;1, SlHKT1;2, AtHKT1;1S68G and SlHKT1;2S70G indicated that both AtHKT1;1 and SlHKT1;2 were able to restore the accumulation of K+ in the shoot, although the low accumulation of Na+ as shown by WT plants was only partially restored. The inhibition of Na+ transport by K+, shown by the SlHKT1;2 transporter in oocytes (and not by AtHKT1;1), was not reflected in Na+ accumulation in the plants transformed with SlHKT1;2. Both AtHKT1;1-S68G and SlHKT1;2-S70G were not able to restore the phenotype of athkt1;1 mutant plants

Data_Sheet_1_Salinity Tolerance of Two Potato Cultivars (Solanum tuberosum) Correlates With Differences in Vacuolar Transport Activity.xlsx

<p>Potato is an important cultivated crop species and since it is moderately salt sensitive there is a need to develop more salt tolerant cultivars. A high activity of Na⁺ transport across the tonoplast in exchange for H⁺ is essential to reduce Na⁺ toxicity. The proton motive force (PMF) generated by the V-H⁺-ATPase and the V-H⁺-PPase energizes the Na⁺(K⁺)/H⁺ antiport. We compared the activity, gene expression, and protein levels of the vacuolar proton pumps and the Na⁺/H⁺ antiporters in two potato cultivars (Solanum tuberosum) contrasting in their salt tolerance (cv. Desiree; tolerant and Mozart; sensitive) grown at 0 and 60 mM NaCl. Tonoplast-enriched vesicles were used to study the pump activity and protein levels of the V-H⁺-ATPase and the V-H⁺-PPase and the activity of the Na⁺/H⁺ antiporter. Although salt stress reduced the V-H⁺-ATPase and the V-H⁺-PPase activity in both cultivars, the decline in H⁺ pump activity was more severe in the salt-sensitive cultivar Mozart. After salt treatment, protein amounts of the vacuolar H⁺ pumps decreased in Mozart but remained unchanged in the cultivar Desiree. Decreased protein amounts of the V-H⁺-PPase found in Mozart may explain the reduced V-H⁺-PPase activity found for Mozart after salt stress. Under non-stress conditions, protein amounts of V-H⁺-PPase were equal in both cultivars while the V-H⁺-PPase activity was already twice as high and remained higher after salt treatment in the cultivar Desiree as compared to Mozart. This cultivar-dependent V-H⁺-PPase activity may explain the higher salt tolerance of Desiree. Moreover, combined with reduced vacuolar H⁺ pump activity, Mozart showed a lower Na⁺/H⁺ exchange activity and the K_m for Na⁺ is at least twofold lower in tonoplast vesicles from Desiree, what suggests that NHXs from Desiree have a higher affinity for Na⁺ as compared to Mozart. From these results, we conclude that the higher capacity in combination with the higher affinity for Na⁺ uptake can be an important factor to explain the differences in salt tolerance of these two potato cultivars.</p

Ion Homeostasis and Metabolome Analysis of Arabidopsis 14-3-3 Quadruple Mutants to Salt Stress

Salinity is one of the major abiotic stresses that limits agricultural productivity worldwide. Many proteins with defined functions in salt stress adaptation are controlled through interactions with members of the 14-3-3 family. In the present study, we generated three 14-3-3 quadruple knockout mutants (qKOs: klpc, klun, and unpc) to study the role of six non-epsilon group 14-3-3 proteins for salt stress adaptation. The relative growth inhibition under 100 mM of NaCl stress was the same for wild-type (Wt) and qKOs, but the accumulation of Na(+) in the shoots of klpc was significantly lower than that in Wt. This difference correlated with the higher expression of the HKT1 gene in klpc. Considering the regulatory role of 14-3-3 proteins in metabolism and the effect of salt stress on metabolite accumulation, we analyzed the effect of a 24-h salt treatment on the root metabolome of nutrient solution-grown genotypes. The results indicated that the klpc mutant had metabolome responses that were different from those of Wt. Notably, the reducing sugars, glucose and fructose, were lower in klpc under control and salt stress. On the other hand, their phosphorylated forms, glucose-6P and fructose-6P, were lower under salt stress as compared to Wt. This study provided insight into the functions of the 14-3-3 proteins from non-epsilon group members. In summary, it was found that these proteins control ion homeostasis and metabolite composition under salt stress conditions and non-stressed conditions. The analyses of single, double, and triple mutants that modify subsets from the most effective qKO mutant (klpc) may also reveal the potential redundancy for the observed phenotypes

Analysis of Arabidopsis thaliana HKT1 and Eutrema salsugineum/botschantzevii HKT1;2 Promoters in Response to Salt Stress in Athkt1:1 Mutant

Soil salinity imposes a serious threat to the productivity of agricultural crops. Among several other transporters, high-affinity K + transporter (HKT)’s play an important role in reducing the phytotoxicity of Na + . Expression of Eutrema salsugineum (a halophyte) HKT1;2 is induced upon salt exposure. To elucidate the role of its promoter, we compared the sequences of HKT1;2 promoters from E. salsugineum (1822 bp) and E. botschantzevii (1811 bp) with Arabidopsis thaliana HKT1;1 (846 bp) promoter. In silico analysis predicted several cis-acting regulatory elements (GT-1 elements, core motifs of DRE/CRT, MYC/MYB-recognition sites and ACGT elements). Activities of the three promoters were analyzed by measuring HKT1;1 and/or HKT1;2 transcript level in the Athkt1;1 mutant plants. NaCl tolerance of the transgenics was also assessed. Our results depicted that expressing either AtHKT1;1 or EsHKT1;2 coding regions under the control of AtHKT1;1 promoter, almost reversed the hypersensitivity of the mutant for salt, on contrarily, when AtHKT1;1 coding sequence expressed under either Es or EbHKT1;2 promoters did not. Changes in shoot Na + /K + concentrations under salt exposure is significantly consistent with the complementation ability of the mutant. The transcript concentration for genes under the control of either of Eutrema promoters, at control level was very less. This may suggest that either an important upstream response motif is missed or that A. thaliana misses a transcriptional regulator that is essential for salt-inducible HKT1 expression in Eutrema

The effect of NaCl treatments on proline concentrations in (A)leaves, (B) stems and (C) roots of six potato cultivars. Data represents the means±SE for three biological replicates.

<p>The effect of NaCl treatments on proline concentrations in (A)leaves, (B) stems and (C) roots of six potato cultivars. Data represents the means±SE for three biological replicates.</p