Donald E. Nelson

Adaptations to Environmental Stresses.

Environmental stresses come in many forms, yet the most prevalent stresses have in common their effect on plant water status. The availability of water for its biological roles as solvent and transport medium, as electron donor in the Hill reaction, and as evaporative coolant is often impaired by environmental conditions. Although plant species vary in their sensitivity and response to the decrease in water potential caused by drought, low temperature, or high salinity, it may be assumed that all plants have encoded capability for stress perception, signaling, and response. First, most cultivated species have wild relatives that exhibit excellent tolerance to abiotic stresses. Second, biochemical studies have revealed similarities in processes induced by stress that lead to accumulated metabolites in vascular and nonvascular plants, algae, fungi, and bacteria (Csonka, 1989; Galinski, 1993; Potts, 1994). These metabolites include nitrogen-containing compounds (proline, other amino acids, quaternary amino compounds, and polyamines) and hydroxyl compounds (sucrose, polyols, and oligosaccharides) (McCue and Hanson, 1990). Accumulation of any single metabolite is not restricted to taxonomic groupings, indicating that these are evolutionarily old traits. Third, molecular studies have revealed that a wide variety of species express a common set of genes and similar proteins (for example, Rab-related proteins and dehydrins) when stressed (Skriver and Mundy, 1990; Vilardell et al., 1994). Although functions for many of these genes have not yet been unequivocally assigned, it is likely, based on their characteristics, that these proteins play active roles in the response to stress. Learning about the biochemical and molecular mechanisms by which plants tolerate environmentat stresses is necessary for genetic engineering approaches to improving crop performance under stress. By investigating plants under stress, we can learn about the plasticity of metabolic pathways and the limits to their functioning. Also, questions of an ecological and evolutionary nature need investigation. Are the genes that confer salt tolerance on halophytes and/or drought tolerance on xerophytes evolutionarily ancient genes that have been selected against in saltand drought-sensitive plants (glycophytes) for the sake of productivity? Or have some species obtained nove1 genes in their evolutionary history that have enabled them to occupy stressful environments? How will the

Regulation of Cell-Specific Inositol Metabolism and Transport in Plant Salinity Tolerance

myo-Inositol and its derivatives are commonly studied with respect to cell signaling and membrane biogenesis, but they also participate in responses to salinity in animals and plants. In this study, we focused on L-myo-inositol 1-phosphate synthase (INPS), which commits carbon to de novo synthesis, and myo-inositol O-methyltransferase (IMT), which uses myo-inositol for stress-induced accumulation of a methylinositol, D-ononitol. The Imt and Inps promoters are transcriptionally controlled. We determined that the transcription rates, transcript levels, and protein abundance are correlated. During normal growth, INPS is present in all cells, but IMT is repressed. After salinity stress, the amount of INPS was enhanced in leaves but repressed in roots. IMT was induced in all cell types. The absence of myo-inositol synthesis in roots is compensated by inositol/ononitol transport in the phloem. The mobilization of photosynthate through myo-inositol translocation links root metabolism to photosynthesis. Our model integrates the transcriptional control of a specialized metabolic pathway with physiological reactions in different tissues. The tissue-specific differential regulation of INPS, which leads to a gradient of myo-inositol synthesis, supports root growth and sodium uptake. By inducing expression of IMT and increasing myo-inositol synthesis, metabolic end products accumulate, facilitating sodium sequestration and protecting photosynthesis.

Regulation of the Osmotin Gene Promoter.

By introducing a chimeric gene fusion of the osmotin promoter and [beta]-glucuronidase into tobacco by Agrobacterium-mediated transformation, we have demonstrated a very specific pattern of temporal and spatial regulation of the osmotin promoter during normal plant development and after adaptation to NaCl. We have found that the osmotin promoter has a very high natural level of activity in mature pollen grains during anther dehiscence and in pericarp tissue at the final, desiccating stages of fruit development. GUS activity was rapidly lost after pollen germination. The osmotin promoter thus appears to be unique among active pollen promoters described to date in that it is active only in dehydrated pollen. The osmotin promoter was also active in corolla tissue at the onset of senescence. Adaptation of plants to NaCl highly stimulated osmotin promoter activity in epidermal and cortex parenchyma cells in the root elongation zone; in epidermis and xylem parenchyma cells in stem internodes; and in epidermis, mesophyll, and xylem parenchyma cells in developed leaves. The spatial and temporal expression pattern of the osmotin gene appears consistent with both osmotic and pathogen defense functions of the gene.

Analysis of structure and transcriptional activation of an osmotin gene.

A Nicotiana tabacum gene encoding the basic PR-like protein osmotin was isolated and characterized. The gene is derived from the N. sylvestris parent of N. tabacum. In cell suspension cultures of tobacco, the osmotin gene was shown to be transcriptionally activated by treatment with ABA.Transcriptional activation of the osmotin promoter was further investigated in transformed plants carrying copies of a fusion of the cloned promoter to the β-glucuronidase reporter gene. In these plants, the osmotin promoter is transcriptionally activated by the hormones ABA and ethylene. The sensitivity of the osmotin promoter to ABA applied exogenously decreased with age in both roots and shoots of young seedlings. NaCl shock also activated the promoter in plant tissues. The osmotin promoter is much more active in root tissues than in shoot tissues.

Abundant Accumulation of the Calcium-Binding Molecular Chaperone Calreticulin in Specific Floral Tissues of Arabidopsis thaliana

Calreticulin (CRT) is a calcium-binding protein in the endoplasmic reticulum (ER) with an established role as a molecular chaperone. An additional function in signal transduction, specifically in calcium distribution, is suggested but not proven. We have analyzed the expression pattern of Arabidopsis thaliana CRTs for a comparison with these proposed roles. Three CRT genes were expressed, with identities of the encoded proteins ranging from 54 to 86%. Protein motifs with established functions found in CRTs of other species were conserved. CRT was found in all of the cells in low amounts, whereas three distinct floral tissues showed abundant expression: secreting nectaries, ovules early in development, and a set of subepidermal cells near the abaxial surface of the anther. Localization in the developing endosperm, which is characterized by high protein synthesis rates, can be reconciled with a specific chaperone function. Equally, nectar production and secretion, a developmental stage marked by abundant ER, may require abundant CRT to accommodate the traffic of secretory proteins through the ER. Localization of CRT in the anthers, which are degenerating at the time of maximum expression of CRT, cannot easily be reconciled with a chaperone function but may indicate a role for CRT in anther maturation or dehiscence.

Induction of a ribosome-inactivating protein upon environmental stress.

Transcripts of altered abundance in RNA from unstressed and 500 mm salt-shocked Mesembryanthemum crystallinum (common ice plant) were detected by reverse-transcription differential display (RT-DD). One transcript, Rip1, was of very low abundance in unstressed plants and was strongly induced by stress. RNA blot hybridizations showed strong induction and a diurnal rhythm of transcript abundance with a maximum each day around the middle of the light phase. Rip1 encodes a reading frame of 289 amino acids (molecular mass 32652), RIP1, with homology to single-chain ribosome inactivating proteins (rRNA N-glycosidases). The deduced amino acid sequence is 31.7% identical to pokeweed antiviral protein RIP-C (overall similarity 66.5%) with highest identity in domains of documented functional importance. RT-DD also detected mRNA for pyruvate,orthophosphate dikinase (PPDK) which has already been shown to be stress-induced in the ice plant [16]. RIP1, expressed in Escherichia coli, showed rRNA N-glycosidase activity against ice plant and rabbit reticulocyte ribosomes. The induction of Rip1 coincides with the transition period during which global changes in translation lead to adaptation of the ice plant to salt stress.

Analysis of an osmotically regulated pathogenesis-related osmotin gene promoter

Osmotin is a small (24 kDa), basic, pathogenesis-related protein, that accumulates during adaptation of tobacco (Nicotiana tabacum) cells to osmotic stress. There are more than 10 inducers that activate the osmotin gene in various plant tissues. The osmotin promoter contains several sequences bearing a high degree of similarity to ABRE, as-1 and E-8 cis element sequences. Gel retardation studies indicated the presence of at least two regions in the osmotin promoter that show specific interactions with nuclear factors isolated from cultured cells or leaves. The abundance of these binding factors increased in response to salt, ABA and ethylene. Nuclear factors protected a 35 bp sequence of the promoter from DNase I digestion. Different 5′ deletions of the osmotin promoter cloned into a promoter-less GUS-NOS plasmid (pBI 201) were used in transient expression studies with a Biolistic gun. The transient expression studies revealed the presence of three distinct regions in the osmotin promoter. The promoter sequence from −108 to −248 bp is absolutely required for reporter gene activity, followed by a long stretch (up to −1052) of enhancer-like sequence and then a sequence upstream of −1052, which appears to contain negative elements. The responses to ABA, ethylene, salt, desiccation and wounding appear to be associated with the −248 bp sequence of the promoter. This region also contains a putative ABRE (CACTGTG) core element. Activation of the osmotin gene by various inducers is discussed in view of antifungal activity of the osmotin protein.

SALINITY TOLERANCE - MECHANISMS, MODELS AND THE METABOLIC ENGINEERING OF COMPLEX TRAITS

Soil salinity reduces plant productivity in many farming areas world-wide. Salinity affects dry-land farming and is found in irrigated areas where sodium accumulates over time. Reports have appeared about rising water tables, a result of deforestation, moving saline groundwater to the surface, and about intruding seawater in coastal areas, following increased removal of fresh water for human consumption. The United Nations Environment Program estimates approximately 20% of the world’s agricultural land s as salt-stressed (1– 3). While the extent of salt-affected land is highly variable on a local scale, salinity is a considerable problem in countries such as Pakistan, India, sub-Saharan Africa and Australia where as much as one-third of the land may be affected. Estimates of losses due to salinity in irrigated areas world-wide vary considerably (4, 5). Prohibitively high salinity already forces aband onment of about 10 × 106 ha. of irrigated land every year (4), and one-third to one-half of the land presently irrigated may to be heading towards this fate. Salt build-up in irrigated areas is particularly significant considering that the production capacity of irrigated crops is approximately three times that of dryland farming (3). Breeding programs are ongoing for generating varieties that could tolerate higher soil salinity while maintaining productivity, but the success of such programs has been marginal. Flowers and Yeo (3) have discussed the impact of various breeding strategies: (a) improving halophytes, (b) incorporation of genes from halophilic relatives of crop species, (c) selection within the species’ range of phenotypes, (d) generation, through mutation, of new phenotypes followed by selection for salt tolerance, and (e) selection based on yield potential only, disregarding salt resistance. The authors, summarizing a search for salt-tolerant varieties which have been released to farmers, conclude that the research and breeding efforts have not resulted in new varieties in the field in a substantial way. As the preferred strategy for future breeding, they suggested the “pyramiding” of established beneficial physiological traits by multiple crosses.

Osmotin: A Protein Associated with Osmotic Stress Adaptation in Plant Cells

Osmotin is a cationic protein which accumulates (up to 12% of total cell protein) in cells adapted to grow in the medium with low water potentials. The synthesis of osmotin is developmentally regulated and is induced by abscisic acid (ABA) in cultured cells. In whole plants, both the synthesis and accumulation of osmotin is tissue specific. The highest rate of synthesis occurs in outer stem tissue and the highest level of accumulation occurs in roots. ABA induced synthesis of osmotin is transient in cells and NaCl stabilizes its synthesis and accumulation. NaCl adapted tobacco cells exhibit a stable increase in both their ability to tolerate salt and to produce osmotin in the absence of NaCl. Osmotin is localized in vacuolar inclusions but also appears to be loosely associated with the tonoplast and plasma membrane. Osmotin is also found in the culture medium of adapted cells during all stages of cell growth. The molecular weight of mature osmotin deduced from the cDNA nucleotide sequence is 23,984 daltons. Osmotin is synthesized as a preprotein 2.5 kD larger than the mature protein. Three proteins, thaumatin, TPR and MAI, exhibit a very high level (52% to 61%) of sequence homology with osmotin. Osmotin mRNA synthesis is induced by ABA. The level of osmotin mRNA increases after NaCl adaptation.

Structure, Regulation and Function of the Osmotin Gene

Over the past years several genes have been reported to be osmotically regulated (Storey and Storey, 1981; Holtum and Winter, 1982; Singh et al., 1985; Singh et al., 1987a;Bedford et al., 1987; Close et al., 1989; Cushman et al., 1989; Singh et al., 1989a; 1989b; Delauney and Verma, 1990; Perez-Prat et al., 1990; Skriver and Mundy, 1990; Bartels et al., 1991; Dhindsa, 1991, Estragarcia et al., 1991; Narasimhan et al., 1991; Perez-Prat et al., 1992; Kononowicz et al., 1992; Nelson et al., 1992; Niu et al., 1993; Zhu et al., 1993b). These studies have been rationalized on the assumption that amongst these genes are molecular determinants of osmotic tolerance. Although the products of many of these genes still remain unidentified there are a number that have been well characterized and are of interest of several laboratories. One of these genes is osmotin (Singh et al., 1987a; LaRosa et al., 1989, Meeks-Wagner et al., 1989; Grosset et al., 1990; Roberts and Selitrennikoff, 1990; Stintzi et al., 1991; Woloshuk et al., 1991; Casas et al., 1992; LaRosa et al., 1992, Kononowicz et al., 1993).