Drought, the most prominent threat to agricultural production worldwide, accelerates leaf senescence, leading to a decrease in canopy size, loss in photosynthesis and reduced yields. On the basis of the assumption that senescence is a type of cell death program that could be inappropriately activated during drought, we hypothesized that it may be possible to enhance drought tolerance by delaying drought-induced leaf senescence. We generated transgenic plants expressing an isopentenyltransferase gene driven by a stress- and maturation-induced promoter. Remarkably, the suppression of drought-induced leaf senescence resulted in outstanding drought tolerance as shown by, among other responses, vigorous growth after a long drought period that killed the control plants. The transgenic plants maintained high water contents and retained photosynthetic activity (albeit at a reduced level) during the drought. Moreover, the transgenic plants displayed minimal yield loss when watered with only 30% of the amount of water used under control conditions. The production of drought-tolerant crops able to grow under restricted water regimes without diminution of yield would minimize drought-related losses and ensure food production in water-limited lands.

TransgenicBrassica napusplants overexpressingAtNHX1,a vacuolar Na⁺/H⁺antiport fromArabidopsis thaliana,were able to grow, flower, and produce seeds in the presence of 200 mM sodium chloride. Although the transgenic plants grown in high salinity accumulated sodium up to 6% of their dry weight, growth of the these plants was only marginally affected by the high salt concentration. Moreover, seed yields and the seed oil quality were not affected by the high salinity of the soil. Our results demonstrate the potential use of these transgenic plants for agricultural use in saline soils. Our findings, showing that the modification of a single trait significantly improved the salinity tolerance of this crop plant, suggest that with a combination of breeding and transgenic plants it could be possible to produce salt-tolerant crops with far fewer target traits than had been anticipated.

Intracellular Na+/H+ (NHX) antiporters have important roles in cellular pH and Na+, K+ homeostasis. The six Arabidopsis thaliana intracellular NHX members are divided into two groups, endosomal (NHX5 and NHX6) and vacuolar (NHX1 to NHX4). Of the vacuolar members, NHX1 has been characterized functionally, but the remaining members have largely unknown roles. Using reverse genetics, we show that, unlike the single knockouts nhx1 or nhx2, the double knockout nhx1 nhx2 had significantly reduced growth, smaller cells, shorter hypocotyls in etiolated seedlings and abnormal stamens in mature flowers. Filaments of nhx1 nhx2 did not elongate and lacked the ability to dehisce and release pollen, resulting in a near lack of silique formation. Pollen viability and germination was not affected. Quantification of vacuolar pH and intravacuolar K+ concentrations indicated that nhx1 nhx2 vacuoles were more acidic and accumulated only 30% of the wild-type K+ concentration, highlighting the roles of NHX1 and NHX2 in mediating vacuolar K+/H+ exchange. Growth under added Na+, but not K+, partly rescued the flower and growth phenotypes. Our results demonstrate the roles of NHX1 and NHX2 in regulating intravacuolar K+ and pH, which are essential to cell expansion and flower development.

Summary: Drought is the major environmental factor limiting crop productivity worldwide. We hypothesized that it is possible to enhance drought tolerance by delaying stress‐induced senescence through the stress‐induced synthesis of cytokinins in crop‐plants. We generated transgenic rice (Oryza sativa) plants expressing an isopentenyltransferase (IPT) gene driven by P_SARK, a stress‐ and maturation‐induced promoter. Plants were tested for drought tolerance at two yield‐sensitive developmental stages: pre‐ and post‐anthesis. Under both treatments, the transgenic rice plants exhibited delayed response to stress with significantly higher grain yield (GY) when compared to wild‐type plants. Gene expression analysis revealed a significant shift in expression of hormone‐associated genes in the transgenic plants. During water‐stress (WS), P_SARK::IPT plants displayed increased expression of brassinosteroid‐related genes and repression of jasmonate‐related genes. Changes in hormone homeostasis were associated with resource(s) mobilization during stress. The transgenic plants displayed differential expression of genes encoding enzymes associated with hormone synthesis and hormone‐regulated pathways. These changes and associated hormonal crosstalk resulted in the modification of source/sink relationships and a stronger sink capacity of the P_SARK::IPT plants during WS. As a result, the transgenic plants had higher GY with improved quality (nutrients and starch content).

Intracellular Na⁺/H⁺ antiporters (NHXs) play important roles in cellular pH and Na⁺ and K⁺ homeostasis in all eukaryotes. Based on sequence similarity, the six intracellular Arabidopsis thaliana members are divided into two groups. Unlike the vacuolar NHX1-4, NHX5 and NHX6 are believed to be endosomal; however, little data exist to support either their function or localization. Using reverse genetics, we show that whereas single knockouts nhx5 or nhx6 did not differ from the wild type, the double knockout nhx5 nhx6 showed reduced growth, with smaller and fewer cells and increased sensitivity to salinity. Reduced growth of nhx5 nhx6 was due to slowed cell expansion. Transcriptome analysis indicated that nhx5, nhx6, and the wild type had similar gene expression profiles, whereas transcripts related to vesicular trafficking and abiotic stress were enriched in nhx5 nhx6. We show that unlike other intracellular NHX proteins, NHX5 and NHX6 are associated with punctate, motile cytosolic vesicles, sensitive to Brefeldin A, that colocalize to known Golgi and trans-Golgi network markers. We provide data to show that vacuolar trafficking is affected in nhx5 nhx6. Possible involvements of NHX5 and NHX6 in maintaining organelle pH and ion homeostasis with implications in endosomal sorting and cellular stress responses are discussed.

Summary: The function of vacuolar Na⁺/H⁺ antiporter(s) in plants has been studied primarily in the context of salinity tolerance. By facilitating the accumulation of Na⁺ away from the cytosol, plant cells can avert ion toxicity and also utilize vacuolar Na⁺ as osmoticum to maintain turgor. As many genes encoding these antiporters have been cloned from salt‐sensitive plants, it is likely that they function in some capacity other than salinity tolerance. The wide expression pattern of Arabidopsis thaliana sodium proton exchanger 1 (AtNHX1) in this study supports this hypothesis. Here, we report the isolation of a T‐DNA insertional mutant of AtNHX1, a vacuolar Na⁺/H⁺ antiporter in Arabidopsis. Vacuoles isolated from leaves of the nhx1 plants had a much lower Na⁺/H⁺ and K⁺/H⁺ exchange activity. nhx1 plants also showed an altered leaf development, with reduction in the frequency of large epidermal cells and a reduction in overall leaf area compared to wild‐type plants. The overexpression of AtNHX1 in the nhx1 background complemented these phenotypes. In the presence of NaCl, nhx1 seedling establishment was impaired. These results place AtNHX1 as the dominant K⁺ and Na⁺/H⁺ antiporter in leaf vacuoles in Arabidopsis and also suggest that its contribution to ion homeostasis is important for not only salinity tolerance but development as well.