Modifying sodium transport to improve salinity tolerance of commercial rice cultivars (Oryza sativa L.).

Abstract

Salinity tolerance in rice is negatively correlated with sodium accumulation in the shoot. Therefore, one approach to improve rice salinity tolerance is through the modification of sodium transport pathways within the plant, either by constitutive or cell type-specific expression of genes encoding proteins important for sodium homeostasis. In rice, work so far has predominately been limited to poorly adapted cultivars or has used technologies incompatible with future breeding programs. It is therefore important to transfer the knowledge obtained from the modification of Na⁺ transport processes in other plants and test the validity of this approach in commercially relevant rice cultivars, using compatible technologies for further application of the approach in the field. Five candidate commercial rice cultivars were selected from Indonesia. The salt tolerance of these rice cultivars were studied in hydroponics. Variation existed in the salinity tolerance mechanisms among the rice cultivars, offering the potential to use different approaches for improving salinity tolerance. Agrobacterium-mediated transformation efficiency of the cultivars was evaluated using calli derived from the scutellum of mature seeds. The study revealed only two cultivars, Fatmawati and IR64, could regenerate transgenics. A non-destructive image based phenotyping protocol was developed for screening rice undergoing salt stress and was used to further examine the salinity tolerance of Fatmawati and IR64. The two cultivars showed differences in both their salinity tolerance and in the salinity tolerance mechanisms they used. Due to the differences in their salinity tolerance and due to their amenability for Agrobacterium-mediated transformation, Fatmawati and IR64 were selected for transformation with salinity tolerance genes using constitutive and cell type-specific promoters. The maize Ubiquitin-1 and cauliflower mosaic virus 35S promoters were used as constitutive promoters. Cell type-specific promoters were identified from either the literature or rice databases and used to drive the genes in specific cells in the root. The cell type-specific alterations are targeted to minimize net sodium influx into the root from the soil, maximise sodium retrieval from the xylem, or increase sodium compartmentalization in the root tissue. Rice lines were generated which constitutively expressed the genes encoding the vacuolar H⁺-pyrophosphatases AtAVP1 and OsOVP4 and the protein kinase AtCIPK16. Transgenic rice lines were also developed which expressed Na⁺ transporter OsHKT1;5 driven by a stelar specific promoter and Na⁺ pumping ATPase from Physcomitrella patens (PpENA1) driven by an epidermal specific promoter. The salinity tolerance of the transgenic rice lines was characterized in the T₁ generation using either non-destructive image based phenotyping or destructive analysis in hydroponic experiments. Results from this study showed that constitutive expression of AtAVP1 lead to increased biomass of transgenic rice both under salt stress and non-stress conditions. The present study demonstrated the expression of OsHKT1;5 in the root stele reduced shoot Na⁺ accumulation, while the expression of PpENA1 in the root epidermis reduced root Na⁺ concentration. However, the effect of the alteration on the whole plant salinity tolerance of the transgenic rice still requires further characterization. Further assessment of these transgenic lines in later generations is necessary.