A comparative study of Cl⁻ transport across the roots of two grapevine rootstocks, K 51-40 and Paulsen, differing in salt tolerance.

Abstract

Soil salinity is one of the major abiotic stresses that decreases agricultural crop production through imposition of both ionic and osmotic stresses. The accumulation of Na⁺ and Cl⁻ in the cytosol to toxic levels inhibits metabolism. Unlike Na⁺, less is known about Cl⁻ uptake and transport in plants. Grapevine is moderately sensitive to salinity and accumulation of toxic levels of Cl⁻ in leaves is the major reason for salt-induced symptoms. In this study Cl⁻ uptake and transport mechanism(s) were investigated in two grapevine (Vitis sp.) rootstock hybrids differing in salt tolerance: 1103 Paulsen (salt tolerant) and K 51-40 (salt sensitive). Increased external salinity caused high Cl⁻ accumulation in shoots of the salt sensitive K 51-40 in comparison to Paulsen. Measurement of ¹ ⁵ NO₃⁻ net fluxes under high salinity showed that by increasing external Cl⁻ concentrations K 51-40 roots showed reduced NO₃⁻ accumulation. This was associated with increased accumulation of Cl⁻. In comparison to Paulsen, K 51-40 showed reduced NO₃⁻ / Cl⁻ root selectivity with increased salinity, but Paulsen had lower selectivity over the whole salinity range (0-45 mM). In order to examine if root hydraulic and permeability characterisations accounted for differences between varieties, the root pressure probe was used on excised roots. This showed that the osmotic Lpr was significantly smaller than hydrostatic Lpr, but no obvious difference was observed between the rootstocks. The reflection coefficient (σ) values (0.48-0.59) were the same for both rootstocks, and root anatomical studies showed no obvious difference in apoplastic barriers of the main and lateral roots. Comparing the uptake of Cl⁻ with an apoplastic tracer, PTS (3-hydroxy-5, 8, 10-pyrentrisulphonic acid), showed that there was no correlation between Cl⁻ and PTS transport. These results indicated that by-pass flow of salts to the xylem is the same for both rootstocks (10.01±3.03 % and 12.1±1.21 %) and hence pointed to differences in membrane transport to explain difference in Cl⁻ transport to the shoot. ³ ⁶Cl⁻ fluxes across plasma membrane and tonoplast of K 51-40 and Paulsen roots showed that ³ ⁶Cl⁻ influx in root segments of Paulsen was greater than K 51-40 over the first 10 minutes. Unidirectional influx within 10 min loading time showed increases with increases in the external concentrations in both rootstocks but Paulsen had higher influx rate when compared to K 51-40. This appeared to be due to a greater Vmax. There was no significant difference in Km. It was shown that ³ ⁶Cl⁻ accumulation and transport rate to the shoot of K 51-40 was higher than that of Paulsen. Compartmental analysis of ³ ⁶Cl⁻ efflux from intact roots confirmed that the difference in influx observed between the rootstocks was consistent with the results obtained for excised roots, although the values were not exactly the same. It was also shown that the main root of Paulsen had greater contribution to ³ ⁶Cl⁻ uptake than lateral roots. ³ ⁶Cl⁻ fluxes by lateral roots were not significantly different between the rootstocks. Cl⁻ and Na⁺ distribution patterns in different root cell types were determined using the X-ray microanalysis technique. It was shown that Cl⁻ content in the hypodermis and cortical cells was higher than the other cell types in both rootstocks, but overall Cl⁻ content in the root of Paulsen was higher than K 51-40. The pericycle of the main root of Paulsen accumulated more Cl⁻ than K 51-40. It was concluded that Cl⁻ loading to the xylem was different in the rootstocks and Paulsen tended to prevent the xylem Cl⁻ loading process. Lateral roots also displayed opposite behaviour consistent with flux analysis. Membrane potential difference (PD) of the cortical cells showed a rapid and transient depolarization by adding 30 mM NaCl in both rootstocks that was followed by a gradual hyperpolarization. Depolarizations caused by 30 mM Choline-Cl, Na-MES and NaCl measured by the root surface potential method showed that Choline-Cl in K 51-40 and Na-MES in Paulsen caused greater depolarization than that of Na-MES in K 51-40 and Choline-Cl in Paulsen respectively. Assuming that PD measured in this method was the trans-root potential (TRP), it was concluded that the higher depolarization by Choline-Cl in K 51-40 can be due to higher Cl⁻ efflux rate to the xylem. Two different mechanisms were also detected for Cl⁻ transport: HATS which was observed in the range of 0.5-5 mM and a LATS in the range of 10-30 mM of the external NaCl concentration. This was consistent with the concentration dependence of Cl⁻ influx. In conclusion, evidence obtained from different experiments of this study indicated that in the grapevine rootstocks (Paulsen and K 51-40) Cl⁻ was mostly transported through the symplastic pathway. From ECl values determined for the rootstocks by the Nernst equation, a proton-driven transport system was responsible for Cl⁻ transport in both the HATS and LATS range of external NaCl concentrations. The rate of Cl⁻ transport from the root to shoot (xylem loading) was the major difference in Cl⁻ transport between the rootstocks in terms of salinity tolerance.