Antonella Furini

High level transcription of a member of a repeated gene family confers dehydration tolerance to callus tissue of Craterostigma plantagineum

An experimental system has been developed which allows the identification of intermediates in the abscisic acid (ABA) signal transduction pathway leading to desiccation tolerance in plants. Desiccation tolerance in callus of the resurrection plant Craterostigma plantagineum is mediated via the plant hormone ABA, which induces the expression of gene products related to desiccation tolerance. Based on T‐DNA activation tagging, a gene (CDT‐1) was isolated which encodes a signalling molecule in the ABA transduction pathway. Constitutive overexpression of CDT‐1 leads to desiccation tolerance in the absence of ABA and to the constitutive expression of characteristic transcripts. CDT‐1 represents a novel gene with unusual features in its primary sequence.The CDT‐1 gene resembles in several features SINE retrotransposons. Mechanisms by which CDT‐1 activates the pathway could be via a regulatory RNA or via a short polypeptide.

Structure and regulation of an ABA- and desiccation-responsive gene from the resurrection plant Craterostigma plantagineum

A gene from the resurrection plant Craterostigma plantagineum (CDeT6-19) encoding a protein with sequence similarity to a major group of late embryogenesis-abundant proteins (termed rab17, dehydrin or Lea D11) is regulated by abscisic acid (ABA) and desiccation. The corresponding transcript and protein is highly inducible in vegetative and callus tissue. To analyse the mechanism of CDeT6-19 regulation its promoter was fused to the β-glucuronidase reporter gene (GUS) and introduced by PEG (polyethylene glycol) into protoplasts of Craterostigma or tobacco. With 889 bp of promoter sequence the GUS expression was significantly stimulated by ABA treatment in transient expression assays. ABA responsiveness was still observed with shorter promoter fragments, although they gave rise to lower GUS activities. Sequence comparisons with promoters from related genes of other species identified the conservation of potential ABA-responsive elements. In tobacco and Craterostigma plants stably transformed with CDeT6-19 promoter constructs a basal GUS activity is observed. However, GUS expression is enhanced by ABA or drying treatment of leaf tissues. In tobacco high promoter activity was observed in mature seeds (embryos) and in pollen.

Responses of plants to dehydration stress: a molecular analysis

Over more than a decade molecular techniques have been applied to analyse the response of plants to drought with the objective to identify genes which contribute to drought tolerance. The studies have used a variety of experimental strategies, and they have resulted in the characterization of a large number of genes which are expressed upon dehydration. A very prominent group among these genes are the so-called Lea (=late embryogenesis abundant) genes which appear to occur ubiquitously in most higher plants. A challenge for future research is still to identify the role of the gene products in dehydration stress; it is particularly necessary to distinguish gene products with a potential in osmoprotection and those which are only involved in secondary reactions. Another area of research activities has been to elucidate the dehydration stress-triggered signal transduction and the role of ABA in this process. For this part transgenic plants have been used to evaluate promoter sequences and to characterize cis-acting regulatory promoter elements crucial for a distinct expression pattern.

Agrobacterium-mediated transformation of the desiccation-tolerant plant Craterostigma plantagineum

SummaryAn efficient procedure for Agrobacterium tumefaciens- mediated transformation of the desiccation-tolerant plant Craterostigma plantagineum has been developed. Leaf explants were inoculated with A. tumefaciens strain GV3101 carrying the gene for kanamycin- or hygromycin-resistance and the ßglucuronidase reporter gene. Parameters which affected the transformation efficiency were the age of the explant, the degree of wounding and the presence of an antioxidant in the medium. Under optimal conditions, calli originated in more than 80% of leaf explants. Transformed plants were obtained from more than 50% of the cultured calli during regeneration in the presence of a suitable antibiotic. The stable integration of T-DNA was confirmed by Southern blot analysis and its expression by assays for ß-glucuronidase activity.

DIFFERENTIAL REGULATION OF TWO ABA-INDUCIBLE GENES FROM CRATEROSTIGMA PLANTAGINEUM IN TRANSGENIC ARABIDOPSIS PLANTS

In Craterostigma plantagineum the CDeT-6-19 and CDeT-27-45 genes are expressed following desiccation and/or ABA treatment. Their promoters were fused to the β-glucuronidase reporter gene (GUS) and tested in transgenic Arabidopsis. GUS activity was measured in mature Arabidopsis seeds, and the responsiveness to ABA in vegetative tissue was found to be limited to the early developmental stages. When transgenic plants were crossed with plants over-expressing the ABI3 gene, it was observed that ABI3 is not required for ABA induction of the CDeT-6-19 promoter, whereas it is crucial for expression of the CDeT-27-45 promoter.

Gene expression during dehydration stress in the resurrection plant Craterostigma plantagineum

The resurrection plant Craterostigma plantagineum is being used as an experimental model system to dissect pathways leading to desiccation tolerance. This plant can recover from severe desiccation within 24 h of contact with water. During drying or ABA treatment novel gene products accumulate rapidly in leaves and other tissues. It is proposed that these gene products contribute to the protection of cellular structures during dehydration. It has been a major goal to isolate genes and determine parameters which regulate the expression of the responsive genes. One approach was to characterize promoters. Three promoters from different lea type genes and three promoters from genes encoding inducible enzymes of the carbohydrate metabolism have been analyzed for their responsiveness to drought and ABA in transient assays and in transgenic plants. The promoter activities in transgenic plants suggest differences in the mode of the regulatory pathways and point to at least two transduction pathways. Evidence has been accumulated that the activation of at least one promoter in vegetative tissues depends on a gene product related to the Arabidopsis abi3-gene. In gel shift mobility assays it was shown that nuclear proteins from ABA treated callus and leaves specifically bind sequence elements in the promoter region. Other environmental factors such as light interact with desiccation stress and modulate the expression level of the proteins involving post-transcriptional and post-translational mechanisms. To dissect events in this signal transduction chain, two other experimental approaches have been initiated. A T-DNA based tagging experiment was carried out in order to obtain positive mutations over- expressing gene products downstream of the ABA signal. One such mutant has been obtained and has been molecularly characterized. A subtractive and differential screening is being used to isolate cDNA clones which encode transcripts expressed during the very early events of dehydration.

T-DNA Tagging of a Gene Inducing Desiccation Tolerance in Craterostigma plantagineum

Drought tolerance in the resurrection plant Craterostigma plantagineum is restricted to fully developed plants. In callus this phenomenon can be induced by treatment with abscisic acid. We have used a recently established protocol for Craterostigma transformation by Agrobacterium tumefaciens (Furini et al., 1994) for the stable integration of a T-DNA carrying a transcriptional enhancer element. By this method a gene was tagged whose induced expression resulted in the formation of a desiccation-tolerant callus in the absence of exogenously applied abscisic acid. Craterostigma genes normally induced by desiccation are constitutively induced in the transgenic callus. The tagged gene was cloned from a genomic library and used for the isolation of the corresponding cDNA clone.

Gene Expression during Water Stress

The severity of the water loss, the developmental stage and the physiological condition determine the response of plants to water stress of affected plants. We initiated research with the objective to identify gene products which may contribute to water stress tolerance using a resurrection plant and barley embryos as model systems. The unique ability of resurrection plants to withstand severe water loss greater than 90% makes them a suitable system to study water stress tolerance: upon rewatering these plants recover quickly from the stress (Gaff 1971, Bartels et al. 1990). An attractive feature of the resurrection plant Craterostigma plantagineum is that we can analyse desiccation tolerance in undifferentiated callus tissue, too. Callus withstands rapid dehydration if it is treated with the plant hormone abscisic acid (ABA) prior to the drying treatment (Bartels et al. 1990). This makes two experimental systems available from the same plant for the isolation of molecular components relevant to desiccation. Initially we isolated a large number of cDNA clones which are induced upon dehydration and/or ABA treatment (Bartels et al. 1990, Piatkowski et al. 1990, Bartels et al. 1992).

Analysis and regulation of gene expression in the resurrection plant Craterostigma plantagineum

Plant responses to water loss are very complex and depend on the severity of the water loss as well as on the developmental stage and the physiological condition of the affected plants. To study responses to protoplastic dehydration at the molecular level we initiated research with the objective to identify gene products which may contribute to water stress tolerance. The unique ability of resurrection plants to withstand severe water loss greater than 90% make them a suitable system to study water stress tolerance: upon rewatering these plants recover quickly from the stress (Gaff 1971, Bartels et al. 1990). We have chosen Craterostigma plantagineum as a representative of the resurrection plants for our studies. An attractive feature of this resurrection plant is that we can analyse desiccation tolerance in undifferentiated callus tissue, too. Callus withstands rapid dehydration if it is treated with the plant hormone abscisic acid (ABA) prior to the drying treatment (Bartels et al. 1990). This makes two experimental systems available from the same plant for the isolation of molecular components relevant to desiccation. Initially we isolated a large number of cDNA clones which are induced upon dehydration and/or ABA treatment (Bartels et al. 1990, Piatkowski et al. 1990, Bartels et al. 1992). The purpose of this chapter is to summarize some characteristic structural and regulatory features of the expressed genes.