GENETIC AND MOLECULAR DISSECTION OF OSMOTIC AND ABA SIGNALS

Extreme environments such as high soil salinity, drought, high temperature or cold require special adaptation of plants. Moreover, environmental stress respresent serious limitation for agricultural productivity. Osmotic stress develops in plants during drought or when the soil has high salt content. Our group is studying the regulation of osmotic stress responses in the model organism, Arabidopsis thaliana. Using genetic approaches such as T-DNA insertion mutagenesis and random cDNA transfer, we have identified several Arabidopsis genes which have an influence on resposes to salt, drought and oxidative stress or ABA signalling. Proline metabolism is used to study metabolic responses to such environmental contrains.

Development of genetic tools to identify and study novel regulatory genes

T-DNA insertion mutagenesis has been developed in Arabidopsis as an important tool for the identification and functional characterization of regulatory genes which can control osmotic stress tolerance. We have established a tagged mutant collection with annotation of more than thousand insertion sites (Szabados et al., 2002). Using a promoter trap technique which employed the firefly luciferase as reporter gene, numerous in situ gene fusions have been identified where the T-DNA insert was inserted in stress-responsive genes. A stress-responsive ABC transporter has been characterized, which was activated by high salinity, osmotic pressure and ABA (Alvarado et al., 2004). Screening for altered stress sensitivity, several tagged mutants have been identified with reduced stress tolerance. In the case of the ppr40 mutant, germination and growth were hypersensitive to salt, high osmotics, abscisic acid (ABA) and oxidative agents. The insertion inactivated the PPR40 gene which codes for a previously unknown PPR domain protein, involved in the regulation of mitochondrial electron transport (Zsigmond et al., 2008). Recently we have developed an efficient way to identify and clone novel regulatory genes through the construction and employment of the Controlled Overexpression System, which is based on a transformation competent cDNA library. The utility of the system has been confirmed in three genetic screens. Stress regulatory genes have been identified which control salt tolerance, ABA sensitivity and expression of stress-induced genes (Papdi et al., 2008).

List of identified T-DNA insertion sites. Mutants are available for research purposes.

Proline metabolism and stress regulation

Proline accumulation during drought or salt stress is a well-known phenomenon in plants. In order to characterize regulation of proline accumulation, the Arabidopsis P5CS1 and P5CS2 genes, which encode the rate-limiting enzyme of proline biosynthesis, the pyrroline-5-carboxylate synthase, have been cloned and characterized in the last decade. We showed that functional divergence of the two P5CS genes is manifested in differential transcriptional regulation of these genes during plant development and in responses to biotic and abiotic stress (Ábrahám et al., 2003, Fabro et al., 2004). Moreover, differences in intracellular localization of the GFP-tagged P5CS1 and P5CS2 proteins suggested that intracellular compartmentalization is important to regulate housekeeping and stress-induced proline accumulation. The importance of proline accumulation in osmotic stress tolerance has been confirmed with loss-of-function insertion p5cs1 mutants, which showed enhanced salt sensitivity and oxidative damage. The p5cs2 mutations lead to embryo lethality, and therefore confirmed the essential housekeeping function of the P5CS2 gene (Székely et al., 2008).