Abiotic stresses such as drought, salt, cold, heat and exposure to toxic metals adversely affect growth and productivity of crop plants and are serious threats to agriculture. Members of Stress Associated Protein (SAP) family in rice have been shown to provide tolerance to multiple abiotic stresses. There are 18 and 14 reported members of SAP family in rice and Arabidopsis, respectively. These SAPs contain A20, AN1, or both A20/AN1 zinc finger domains at the N- or C-terminus. Some members of SAP family proteins also contain extra Cys2-His2 RING motifs on the C-terminus. We describe here the functional characterization of two novel SAP genes, AtSAP10 and AtSAP11, from Arabidopsis thaliana ecotype Columbia. AtSAP10 gene contains an A20 and AN1 zinc-finger domain at the N- and C-terminal, respectively. Arabidopsis SAP10 showed differential regulation by various abiotic stresses such as heavy metals and metalloids (Ni, Cd, Mn, Zn, and As), high and low temperatures, cold, and ABA. Overexpression of AtSAP10 in Arabidopsis conferred strong tolerance to heavy metals such as Ni, Mn, and Zn and to high temperature stress. AtSAP10 transgenic plants under these stress conditions grew green and healthy, attained several-fold more biomass, and had longer roots as compared to wild type plants. Further, while these transgenic plants accumulated significantly greater amounts of Ni and Mn in both shoots and root tissues, there was no significant difference in the accumulation of Zn. AtSAP10 promoter-GUS fusion studies revealed a root and floral organ-specific expression of AtSAP10. Overexpression of AtSAP10-GFP fusion protein showed the localization in both nucleus and cytoplasm. A second gene from AtSAP family, AtSAP11, contains two AN1 zinc finger domains at N-terminal and two C2H2 zinc finger domains at C-terminus. Arabidopsis SAP11 showed differential regulation by various abiotic stresses such as heavy metals and metalloids (As, Cd and Zn), high and low temperatures, cold, and salt. Overexpression of AtSAP11 in Arabidopsis conferred moderate tolerance to heavy metals As and Zn and slightly enhanced tolerance to drought stress. AtSAP11 overexpression plants did not accumulate significantly higher amounts arsenic in shoots or roots. AtSAP11 promoter-GUS fusion studies revealed a floral organ-specific and fruit specific expression of AtSAP11. AtSAP11-GFP fusion showed an ER like localization of the fusion protein. Thus these results showed that AtSAP10 and AtSAP11 are potentially useful candidate genes for engineering tolerance to heavy metals and to abiotic stress in cultivated plants.

Molecular chaperones are proteins found in virtually every organism and are essential to cell survival. When plants are heat stressed, they upregulate and downregulate multiple genes, many of which are associated with the heat shock response. Small heat shock proteins (sHSPs) are one class of molecular chaperones that are upregulated during heat shock. They are proposed to act as the first line of defense by binding to heat sensitive proteins and preventing their irreversible aggregation. However, many details of sHSP function remain to be discovered and exactly what proteins they protect is unresolved. In addition to cytosolic sHSPs found in other organisms, plants also produce sHSPs that are targeted to organelles. In this study, I focus on the mitochondria and chloroplast localizing sHSPs: HSP23.5-MTI/CP, HSP23.6-MTI/CP, HSP25.3-CP, and HSP26.5-MTII in Arabidopsis thaliana. The heat tolerance of knockout mutants of these different organelle-localized sHSPs, including single, double, triple, and quadruple knockouts was assessed through various stress assays. A hypocotyl elongation assay indicated a mild heat sensitive phenotype for many of the sHSP knockout mutants and plants lacking all four sHSPs showed the greatest reduction in hypocotyl elongation following heat stress. In an vi assay with light grown seedlings, I observed plants that lacked the chloroplast-localizing HSP25.3-CP were sensitive to acute heat stress. In stress assays involving arsenic, plants that did not express mitochondrial sHSPs were the most sensitive to excess arsenic. Interestingly, plants lacking the four sHSPs were more resistant to salt and cadmium stress. The phenotypes of these sHSPs will bring us closer to defining their mechanism of action during heat or heavy metal stress and the mutants will provide a platform for further studies of sHSP structure and function.

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The response of grass, forb, and shrub seeds to the subalpine environment during the fall and under winter snow and under laboratory conditions at 20/28 C was observed for 3 consecutive years at an elevation of about 3000m on the Wasatch Plateau in central Utah. Seeds in nylon sleeves were planted under 2 cm of soil before snowfall. Under snow plantings were made directly on the soil surface and under 2 cm of soil. The seeds planted before snowfall were removed each year on four occasions: when under snow plantings were made, after snow depth exceeded 130 cm (deep snow), just before spring snowmelt, and 10 or more days following snowmelt when soils had warmed. Seeds planted under snow or under snow and soil were removed on two occasions: after snow depth exceeded 130 cm and just before the snow melted in the spring. Environmental conditions which produced seed germination varied with species, origin of species, age of seed, and temperature. Fifty-four of the 60 species planted in September under 2 cm of soil before snowfall had some seed germinated when examined just before the snow melted in the spring. Fifty of the same species planted after winter snow covered the ground in November also had some seed germination just before the snow melted in the spring. Seed germination at the near 0 C temperatures on the soil surface under snow was usually similar to the comparable seed lots also placed under 2 cm of soil. Germinability of the grasses, forbs, and shrubs studied was placed in three broad classifications: (1) Little or no seed dormancy with germination at low and warm temperatures. Seeds germinated soon after harvest at temperatures near 0 C and also in the laboratory at 20/28 C. Examples are Agropyron desertorum, A. intermedium, Chrysothamnus viscidiflorus, and Lupinus alpestris. (2) Little or no seed dormancy with germination only at warm temperatures. Seeds did not germinate at the low temperatures present in the subalpine zone during the fall and winter but germinated readily in the laboratory at 20/28 C. Examples are Aquilegia caerulea , Potentilla gracilis var. pulcherrima, Rudbeckia occidentalis, and Valeriana edulis. (3) Dormancy at seed harvest. Germination was usually increased by near 0 C temperatures, by aging, and by other environmental conditions present under deep snow. Examples are Agropyron trachycaulum, Delphinium barbeyi, Madia glomerata, and Ribes cereum var. inebrians.