Physiological and biochemical mechanisms in response to Fe deficiency in grape rootstocks differently tolerant to lime chlorosis

INTRODUCTION. Grapevine frequently grows in calcareous soils, which limit iron (Fe) availability inducing the typical yellowing of young leaves and dramatically decreasing yield and quality of grapes. The control of chlorosis by means of Fe-chelates implies high costs and potential environmental risks. Among the alternative strategies, the use of tolerant rootstocks may represent an economical and efficient method for preventing Fe chlorosis. Vitis ssp. belong to Strategy I plants and under Fe deficiency increase the following activities: i) Fe-chelate reductase (FC-R), to reduce Fe3+ to Fe2+; ii) plasma membrane (PM) H+-ATPase, to enhance net excretion of protons in the rhizosphere; iii) Fe2+ transport by IRT1. MATERIALS AND METHODS.We analyzed two grapevine rootstocks, characterized by different tolerance to Fe deficiency (M1, tolerant and 101.14, susceptible), grown in a hydroponic culture with (+Fe) or without Fe (-Fe) and with NaHCO3 (+FeBic). We investigated some of the main physiological parameters linked to leaf gas exchanges (CIRAS), the pigment content (DUALEX) and determined the macro-and microelement concentration (ICP-MS). The main mechanisms induced by Fe deficiency in Strategy I species (FC-R, H+-ATPase, IRT1, PEPC), some key enzymes of glycolysis, PPP pathway and shikimate pathway, were also investigated by enzymatic assay and western blot analysis. The electrophoretic band corresponding to H+- ATPase was sequenced through nHPLC-ESI-MS/MS analysis. RESULTS AND DISCUSSION. For what concerns the leaf physiological parameters, M1 grown in +FeBic exhibited a behaviour similar to the control, while in 101.14 the presence of bicarbonate exerted the same effects on the analyzed traits as those found in the absence of Fe. Moreover, M1 roots showed a stronger increase in all the tested enzymatic activities in both stress conditions, while 101.14 ones exhibited the same activities in all growth conditions. In a calcareous soil, the activation of plasma membrane- H+-ATPase, as found in M1, could lower the rhizosphere pH, providing more favourable conditions for Fe uptake. In Fe-deficient M1 root extract, we observed the presence of a band, lower than 112 kDa, that was identified as a H+- ATPase form with a deletion at the N-terminal. Immunological analysis carried out with antibodies against the C- and N-terminals of the H+-ATPase, other than PM-bound 14.3.3 proteins, Thr-P and MAPkinase revealed substantial differences between M1 and 101.14. CONCLUSIONS. The results obtained suggest that the tolerance to Fe deficiency exhibited by M1 on calcareous soils is linked to a stronger activation of the Strategy I mechanisms and more likely to the presence of a PM H+-ATPase form in its high-activity state and that not only the Cterminal but also the N-terminal could be involved in its regulation. The work is supported by Progetto AGER- SERRES 2010-210