SULFUR NUTRITION AND PARTITIONING IN RICE UNDER DIFFERENT STRESS CONDITIONS

Sulfur (S) is an essential macronutrient required by plants for their correct development. This element is fundamental for the biosynthesis of different compounds, such as the two amino acids, cysteine (Cys) and methionine (Met), vitamins (biotin and thiamine), peptides involved in the response to abiotic stresses (glutathione - GSH, and phytochelatines - PCs), lipids and co-factors. Sulfate (SO42-) is the main S form taken up from soil by root system and then assimilated inside the cells during the sulfur reductive pathway. The uptake and the systemic movements of this anion are accomplished by the SULfate TRansporter (SULTR) gene family, which encode for H+/SO42- membrane co-transporters with different localization, amino acidic sequences, and affinity to sulfate. Since has been demonstrated that S has a key role in the response to different abiotic stresses (such as sulfur deficiency, heavy metal exposure or salt stress), the expression of these genes must be finely regulated, according to the different environmental conditions and requests for S reduced compounds. The general aim of the present thesis is the description of S systemic fluxes in rice in different stress conditions, to obtain more information about the contribution of S in determining plant tolerance to abiotic stresses. To achieve the goal, we also took advantage of analysis performed with an elemental analyzer coupled with an isotope ratio mass spectrometer (EA-IRMS), a powerful instrument which utilizes stable isotopes of elements as tracers. The entire research has been divided in three different parts. In the first work, potential 32S/34S isotope effects occurring during SO42- uptake were investigated in a closed hydroponic system in which a limited amount of substrate (SO42- in the nutrient solution) was continuously removed from the solution by the activity of the sulfate transporters of the root and converted in a final product (total S of the plant). An isotope discrimination against 34S occurred during SO42- uptake: plants had a lighter S isotope composition, and the residual SO42- in the hydroponic solution was enriched in heavy stable isotope. Fractionation during uptake showed two phases characterized by different fractionation factors, reflecting changes in the expression of the OsSULTR deputed to the root uptake which may explain the different isotope phenotypes observed during plant sulfate acquisition. Moreover, the possible 32S/34S isotope effects associated to both S partitioning and metabolism were investigated by comparing plants pre-grown in complete nutrient solutions and then continuously maintained on media containing SO42- (steady-state) or deprived of SO42- for 72h. The SO42- pool of the steady-state shoot was significantly 32S depleted with respect to the SO42- pools of root, while the organic S (Sorg) pools were significantly depleted in 34S compared to both the SO42- pool of both the organs and the S source. These results suggested a higher S assimilation in the aerial part of plants which favor the lighter isotope. Under S starvation, S assimilation progressively enriched the Sorg pools in the lighter 32S isotope and the residual SO42- in both the organs in the heavier 34S isotope. Most pronounced isotope separations were again observed in the shoot, confirming the prominent role of this organ in SO42- assimilation and S allocation. No fractionation due to translocation activity was observed. In the second part of the work, to validate the results previously obtained, we performed a mass balance study in rice plants exposed for 72h to different Cd concentrations, to investigate possible changes in S stable isotope fractionation due to this stress: in fact, adaptation of S metabolism has a pivotal role in responses to heavy metal exposure. As expected, Cd treatment strongly enhanced SO42- uptake and assimilation, as indicated by the analyses of the S pools (Stot, SO42-, and Sorg). S isotope analyses performed on the whole plants revealed changes in the S metabolism associated to variations in the discrimination against 34S, which was less evident as Cd concentration in the external medium increased. Transcriptional analysis suggested again that change of the ratio between relative transcripts of OsSULTR1;1 and OsSULTR1;2, as observed for S starvation, may be responsible for the progressive decreased in 34S isotope discrimination. The important role of shoot in S assimilation was confirmed: isotope fractionation associated to sulfate assimilation was higher in shoot than in root, and progressively increased as Cd concentration did. The last part of work was focused on fully characterize, under hydroponics-controlled conditions in the absence or in the presence of salt stress (80 mM NaCl), the phenotypic behavior in the already available salt tolerant introgression line (IL) Onice 11 (O11), obtained by Marker-Assisted Back-Cross (MABC) selection starting from the cross between the Italian japonica elite cultivars Onice (sensitive recurrent parent) and the indica variety IR64-SalTol (tolerant), donor of the major QTL SalTol. Moreover, S acquisition and metabolism of O11 and both the parental lines were evaluated to investigate their possible implication in determining the different tolerance to salt stress. Results showed the beneficial effect of the introgression of the SalTol QTL from the indica variety into selected japonica rice line, based on different characteristics of selected phenotypic-biochemical-physiological parameters. However, salt stress strongly affected S uptake and assimilation, and we can reasonably suppose that these features do not justify the different salt tolerance in the considered IL O11. In conclusions, rice plants can discriminate against 34S during SO42- uptake and assimilation. Between plants organs, shoot represents the predominant one involved in S assimilation. Abiotic stresses, such as S starvation or Cd exposure, lead to changes in the ratio of relative transcripts between the OsSULTRs involved in the uptake of sulfate, and this may be the cause of the different isotope phenotypes observed. Finally, salt tolerance in the IL O11 appears to not be dependent on different S metabolism.