Björn Welin

Stress-induced accumulation and tissue-specific localization of dehydrins in Arabidopsis thaliana

Stress-induced accumulation of five (COR47, LTI29, ERD14, LTI30 and RAB18) and tissue localization of four (LTI29, ERD14, LTI30 and RAB18) dehydrins in Arabidopsis were characterized immunologically with protein-specific antibodies. The five dehydrins exhibited clear differences in their accumulation patterns in response to low temperature, ABA and salinity. ERD14 accumulated in unstressed plants, although the protein level was up-regulated by ABA, salinity and low temperature. LTI29 mainly accumulated in response to low temperature, but was also found in ABA- and salt-treated plants. LTI30 and COR47 accumulated primarily in response to low temperature, whereas RAB18 was only found in ABA-treated plants and was the only dehydrin in this study that accumulated in dry seeds.Immunohistochemical localization of LTI29, ERD14 and RAB18 demonstrated tissue and cell type specificity in unstressed plants. ERD14 was present in the vascular tissue and bordering parenchymal cells, LTI29 and ERD14 accumulated in the root tip, and RAB18 was localized to stomatal guard cells. LTI30 was not detected in unstressed plants. The localization of LTI29, ERD14 and RAB18 in stress-treated plants was not restricted to certain tissues or cell types. Instead these proteins accumulated in most cells, although cells within and surrounding the vascular tissue showed more intense staining. LTI30 accumulated primarily in vascular tissue and anthers of cold-treated plants.This study supports a physiological function for dehydrins in certain plant cells during optimal growth conditions and in most cell types during ABA or cold treatment. The differences in stress specificity and spatial distribution of dehydrins in Arabidopsis suggest a functional specialization for the members of this protein family.

Characterization and differential expression of dhn/lea/rab-like genes during cold acclimation and drought stress in Arabidopsis thaliana

We have characterized cDNAs for two new dhn/lea/rab (dehydrin, late embryogenesis-abundant, responsive to ABA)-related genes from Arabidopsis thaliana. The two genes were strongly induced in plants exposed to low temperature (4 °C) and were accordingly designated lti45 and lti30 (low temperature-induced). The lti45 gene product contains the conserved serine stretch and three lysine-rich repeats characteristic of DHN/LEA/RAB proteins and is very similar to another low temperature-responsive protein of A. thaliana, COR47 [17]. Both proteins have the same repeat structure and an overall amino acid identity of 64%. This structural similarity of the proteins and the tandem array of the genes suggest that this gene pair arose through a duplication. The other polypeptide, LTI30, consists of several lysine-rich repeats, a structure found in CAP85, a low temperature-and water stress-responsive protein in spinach [41] and similar proteins found in wheat [20].The expression pattern of the five dhn/lea/rab-related genes (cor47, dhnX, lti30, lti45 and rab18) identified so far in A. thaliana, was characterized in plants exposed to low temperature, drought and abscisic acid (ABA). Expression of both lti30 and lti45 was mainly responsive to low temperature similar to cor47. The lti45 and lti30 genes show only a weak response to ABA in contrast to cor47, which is moderately induced by this hormone. The three genes were also induced in severely water-stressed plants although the expression of lti30 and lti45 was rather low. In contrast to these mainly low temperature-induced genes, the expression of rab18 was strongly induced both in water-stressed and ABA-treated plants but was only slightly responsive to cold. The dhnX gene showed a very different expression pattern. It was not induced with any of the treatments tested but exhibited a significant constitutive expression. The low-temperature induction of the genes in the first group, lti30 and lti45, is ABA-independent, deduced from experiments with the ABA-deficient (aba-1) and ABA-insensitive (abi1) mutants of A. thaliana, whereas the induction of rab18 is ABA-mediated. The expression of dhnX was not significantly affected in the ABA mutants.

Improved drought tolerance without undesired side effects in transgenic plants producing trehalose

Most organisms naturally accumulating trehalose upon stress produce the sugar in a two-step process by the action of the enzymes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). Transgenic plants overexpressing TPS have shown enhanced drought tolerance in spite of minute accumulation of trehalose, amounts believed to be too small to provide a protective function. However, overproduction of TPS in plants has also been found combined with pleiotropic growth aberrations. This paper describes three successful strategies to circumvent such growth defects without loosing the improved stress tolerance. First, we introduced into tobacco a double construct carrying the genes TPS1 and TPS2 (encoding TPP) from Saccharomyces cerevisiae. Both genes are regulated by an Arabidopsis RuBisCO promoter from gene AtRbcS1A giving constitutive production of both enzymes. The second strategy involved stress-induced expression by fusing the coding region of ScTPS1 downstream of the drought-inducible ArabidopsisAtRAB18 promoter. In transgenic tobacco plants harbouring genetic constructs with either ScTPS1 alone, or with ScTPS1 and ScTPS2 combined, trehalose biosynthesis was turned on only when the plants experienced stress. The third strategy involved the use of AtRbcS1A promoter together with a transit peptide in front of the coding sequence of ScTPS1, which directed the enzyme to the chloroplasts. This paper confirms that the enhanced drought tolerance depends on unknown ameliorated water retention as the initial water status is the same in control and transgenic plants and demonstrates the influence of expression of heterologous trehalose biosynthesis genes on Arabidopsis root development.

Structure and organization of two closely related low-temperature-induced dhn/lea/rab-like genes in Arabidopsis thaliana L. Heynh

We have isolated a 7 kb EcoRI genomic fragment from Arabidopsis thaliana which contains, in a tandem arrangement, two closely related dhn/lea/rab-like genes, lti29 (formerly named lti45) and cor47, corresponding to previously isolated cDNA clones. Both transcripts have been shown to accumulate in response to low temperature (LT), abscisic acid (ABA) and dehydration. Alignment of the amino acid sequences of the deduced polypeptides showed that they are 67% identical. The calculated molecular masses of the two polypeptides were 29 kDa for LTI29 and 30 kDa for COR47. Both polypeptides contain one conserved serine-stretch and three lysine-rich repeats characteristic of DHN/LEA/RAB-like proteins. In addition, both LTI29 and COR47 harbour and N-terminal acidic repeat only found in a few members amongst the DHN/LEA/RAB proteins. The close distance between the two genes (separated by 2.7 kb) and their tandem organization in the A. thaliana genome as well as the overall homology at the nucleotide sequence level of the coding region suggest that the two genes have evolved through a duplication event. This seems to be a common feature among A. thaliana LT-reponsive genes.

Enhanced Desiccation Survival by Engineering Osmolyte Biosynthesis in Plants

Plant growth, productivity and distribution are severely limited by environmental stress factors such as drought, salinity and freezing temperatures, all of which disturb the water balance of the cell. Plants as well as other organisms have evolved different strategies to alleviate the adverse effects of these stresses. A common adaptive response to stresses that results in water deficit is the accumulation of osmolytes or osmoprotectants that help cells to maintain their water balance and, in addition, protect macromolecules in stressed cells. The simplicity of the metabolic pathways leading to osmolyte biosynthesis makes them amenable to genetic engineering. We will discuss engineering the biosynthetic pathways of two different types of osmoprotectants, the quaternary ammonium compound glycine betaine, and the nonreducing disaccharide trehalose. Biosynthesis of both compounds is a two-step process in prokaryotes as well as in eukaryotes. For biosynthesis of glycine betaine we employed the bacterial genes betA and betB encoding choline dehydrogenase and betaine aldehyde dehydrogenase, respectively. These genes have been expressed in transgenic tobacco and were shown to result in biosynthesis of glycine betaine. For biosynthesis of trehalose we used the TPS1 gene from yeast encoding the first enzyme of the pathway, trehalose-6-phosphate synthase. Transgenic tobacco plants expressing TPS1 were shown to produce active TPS1 and accumulate trehalose in the leaves. Production of trehalose was associated with enhanced desiccation survival both in primary transformants and TPS1- positive progeny. The results suggest that engineering osmolyte biosynthesis may provide an efficient strategy to generate crop plants with enhanced tolerance to water deficit as well as improved post harvest storage properties.

Regulation of Low Temperature-Induced Genes during Cold Acclimation of Arabidopsis Thaliana

Arabidopsis thaliana provides an ideal model system for molecular analysis of plant cold acclimation. This small Cruciferae can readily cold acclimate and the acclimation process is accompanied by expression of a specific set of low temperature-responsive genes. Structural analysis of such Iti (low temperature induced) genes has demonstrated that many of them code for polypeptides related to members of the RAB/LEA/DHN family of water stress responsive proteins. This structural similarity is indicative of common function for these proteins and suggests overlapping responses to freezing and desiccation stress. The other Iti genes characterized seem to code for novel, often very hydrophilic proteins. Enhanced freezing tolerance of A. thaliana can be induced by exposure to low temperature, mild desiccation or exogenous abscisic acid (ABA). Mutant studies have suggested that ABA-controlled processes appear to be required for a normal acclimation response. In accordance with the observed pattern of freezing tolerance induction, expression of the lti genes is responsive to the same three stimuli. However, there are marked differences in the expression patterns of the lti genes during the different modes of induction. Furthermore, the genes seem to fall into three different categories with respect to the signal pathways employed for their expression. Existence of separate response pathways to the different stimuli appears to be the most common mechanism for induction of these genes. Accordingly, stimulus specific DNA elements appear to be present in the lti promoters.