Rice marks second among the agricultural crop plants in the world (FAO, 2004). This work aimed at identifying the molecular mechanisms implicated in tolerance to salt. Salinity is a major environmental threat for agricultural production that affects ionic and osmotic as well as nutritional relation of plants. Ion channels are key players in maintaining ion homeostasis also under salinity. Cl- content was very low in control conditions but under 150 mM NaCl, Cl- was abundantly accumulated in leaves of the salt sensitive rice line IR29, whereas the salt tolerant line Pokkali excluded it from the leaves. Transcript of OsCLC1 i.e. voltage-dependent Cl- channels was found in both lines in roots and leaves under normal growth conditions and was repressed in IR29 and induced transiently in Pokkali upon salt treatment. Simultaneous, transcript amounts of the Na+/H+ antiporter OsNHX1 and the vacuolar H+-ATPase subunit OsVHA-B decreased in IR29, whereas Pokkali showed transient increase of OsVHA-B. Subsequent analysis of the water channel aquaporin OsPIP2;1 and the cell-specificity of OsCLC1 transcript distribution by in situ PCR showed coordinated regulation of OsCLC1, OsVHA-B, OsNHX1 and OsPIP2;1 on the one hand and suggest that OsCLC1 functions in osmotic adjustment at high salinity on the second hand.

Transcript of the K+ transporter OsHAK7 that belongs to the HAK/KT/KUP family were also analysed in relation to K+ homeostasis. K+ content was high in plant tissues under normal conditions, however salt stress decreased root levels and strongly increased its accumulation in leaf cells in both IR29 and Pokkali. OsHAK7 showed high transcript abundance only during the first 6 h of the salt treatment in leaves, whereas in roots the induction was maintained up to 48 h in both lines. Tissue and cell-specificity distribution of OsHAK7 transcript by in situ PCR revealed expression in plant tissues under normal conditions. Strong signals in the mesophyll of both rice lines were detected in leaves, whereas expression in the vasculature cells was specific to Pokkali. In response to salt stress, transcript amounts were reduced in the mesophyll and were detectable in phloem and xylem parenchyma cells of both lines. Analyses of these results demonstrated transcriptional regulation of OsHAK7 under salinity stress and suggest that the K+ transporter functions in salt-dependent K+ homeostasis in rice.

A comparative analysis of salt stress responses in the monocotyledonous halophyte Festuca rubra ssp littoralis and the salt sensitive crop species wheat (Triticum aestivum) were investigated for better understanding strategies of salt tolerance. Ion accumulation was similar in both species except for Ca, Mg, Fe and Na, whose contents were higher in Festuca than in wheat in control conditions. In response to 125 mM NaCl (which characterised severe stress for wheat), the crop species (Triticum aestivum) limited the uptake of Na+ in leaves whereas Festuca significantly accumulated it in root and leaves. In addition, Mg and Fe content increased in Festuca. At 500 mM NaCl, Festuca accumulated Na+ in both tissues. Expression of genes with important function in the regulation of ion homeostasis was also analysed. In root tissue treatment of 125 mM NaCl improved the transcript level of Festuca FrPIP2;1, FrVHA-B and FrNHX, whereas in wheat the expression of TaPIP2;1 and TaVHA-B was down regulated. FrPIP2;1, FrVHA-B and FrNHX cell-specificity analysis indicated expression in root epidermis, cortex cells, endodermis and in the vasculature tissue. Treatment of 500 mM NaCl showed repression in the epidermis and the outer cortex cells whereas strong signals were observed in the endodermis and the vasculature. These results indicated divergent transcriptional regulation of the aquaporin PIP2;1, V-ATPase and the Na+/H+ antiporter NHX and seems to be correlated with salt tolerance and salt sensitivity in Festuca, in the rice lines Pokkali, IR29 and wheat and suggested coordinated control of ion homeostasis and water status at high salinity in plants.

As reported in many studies, salinity is a complex constraint that induced the regulation of many of other genes with significant function in the mechanism of salt tolerance. Identification of probable salt induced genes was investigated by using rice and Festuca cDNA-arrays to identify 192 and 480 salt responsive expressed sequence tags (ESTs) from a rice and Festuca salt stress-cDNA-library. The rice cDNA-array hybridizations compared between the salt sensitive line IR29 and the salt tolerant line Pokkali showed no significant difference. Considering the number of salt regulated genes, more induced genes could be showed in Pokkali leaf than in IR29 under 150 mM NaCl 6 h. IR29 recovered slowly according to the duration of the treatment and at 48 h, more genes were regulated in IR29 than in Pokkali. While more genes were up and down-regulated under NaCl and LiCl stress, salt stress under K+ starvation induced more regulated genes in Pokkali than in IR29. Salt-induced gene expression was compared between the salt sensitive line IR29 and the halotolerant Festuca using Festuca cDNA-arrays. Treatment of 125mM NaCl during 6 h indicated no significant difference in the number of upregulated genes in both species, however, several genes were repressed in Festuca. Festuca showed only a high rate of upregulated genes at high salt concentration (500 mM NaCl). Functional classification of salt-induced genes identified gene products related to metabolism such as the NADP-dependent oxidoreductase that is a component of the antioxidative system. Second large group corresponded to genes with unknown function. In these groups as well as in the group of defence, many of the induced genes were only observed at 500 mM NaCl. These results suggest a small rate of genes were needed to maintain normal growth under low salinity in the halophyte Festuca. This number increased and reached the maximum at 500 mM NaCl, whereas in the salt sensitive rice line IR29 the maximum was reached at low salt concentration. Transcription factors, translation and signal transduction constituted a small group with a slight increase in Festuca treated for 6 h with 125 mM NaCl and 500 mM NaCl. The expression of the translation initiation factor SUI1 as well as the signalling transduction element protein kinase SPK3 seemed to be moderate in the Festuca-cDNA-array. However Northern blot expression of the rice translation initiation factor OsTIF (SUI1) and the rice serine-theonine protein kinase OsSPK3 showed clear improvement in the halophythe Festuca at 500 mM NaCl. In IR29, Northern blot analysis showed a decrease in the transcript abundance of the genes. According to their induced expression in Festuca to high salinity, sequences of OsTIF as well as the sequence of OsSPK3 inserted and analyzed in the salt sensitive rice IR29. Under salt stress conditions, transgenic plants overexpressing OsTIF or OsSPK3 increased the transcript level of both genes and improved the tolerance to salinity compared to the wild-type. In addition, expression of the V-ATPase in transgenic plants was significantly induced under salt stress. These results suggest that the translation initiation factor OsTIF and the protein kinase OsSPK3 are useful for improvement of salt tolerance in rice.