Dorothea Bartels

Drought and Salt Tolerance in Plants

Agricultural productivity worldwide is subject to increasing environmental constraints, particularly to drought and salinity due to their high magnitude of impact and wide distribution. Traditional breeding programs trying to improve abiotic stress tolerance have had some success, but are limited by the multigenic nature of the trait. Tolerant plants such as Craterostigma plantagenium, Mesembryanthemum crystallinum, Thellungiella halophila and other hardy plants could be valuable tools to dissect the extreme tolerance nature. In the last decade, Arabidopsis thaliana, a genetic model plant, has been extensively used for unravelling the molecular basis of stress tolerance. Arabidopsis also proved to be extremely important for assessing functions for individual stress-associated genes due to the availability of knock-out mutants and its amenability for genetic transformation. In this review, the responses of plants to salt and water stress are described, the regulatory circuits which allow plants to cope with stress are presented, and how the present knowledge can be applied to obtain tolerant plants is discussed.

Molecular cloning of abscisic acid-modulated genes which are induced during desiccation of the resurrection plant Craterostigma plantagineum.

Leaves of the resurrection plant Craterostigma plantagineum Hochst, can be desiccated up to 1% relative water content and are still viable after rehydration. To clone genes related to this extreme desiccation tolerance, an in-vitro system was first developed which allows the induction of the same resurrection response in callus tissue upon treatment with abscisic acid (ABA). Several proteins and in-vitro-synthesized polypeptides were then identified which can be induced both in desiccation-tolerant, naturally dried leaves and in ABA-treated calli surviving after rehydration. Complementary-DNA clones corresponding to mRNAs expressed only in desiccation-tolerant tissues were obtained and classified into several gene families. In hybrid-selected translation experiments, representative cDNA clones were associated with water stress and ABA-inducible polypeptides abundantly expressed in dried leaves and ABA-treated calli. The expression pattern of several of these abundant transcripts was analyzed in RNA-hybridization experiments. Upon stress or ABA treatment the transcription levels increased rapidly, but they declined after relief from the stress state. This, together with data on genomic copy numbers indicated that a set of abundantly expressed genes are involved in the desiccation process of resurrection plants. Data on endogenous ABA contents before and after stress applications and on the physiological effects of exogenous ABA treatments indicate that in Craterostigma plantagineum the induction of an extreme desiccation tolerance is mediated by this plant hormone.

Acquisition of Desiccation Tolerance and Longevity in Seeds of Arabidopsis thaliana (A Comparative Study Using Abscisic Acid-Insensitive abi3 Mutants)

Two new abscisic acid (ABA)-insensitive mutants of Arabidopsis thaliana affected in the abi3 locus are described. These new mutants are severely ABA insensitive. Like the earlier described abi3–1 and the ABA-deficient and -insensitive double mutant aba,abi3, these new mutants vary in the extent of ABA-correlated physiological responses. Mutant seeds fail to degrade chlorophyll during maturation and show no dormancy, and desiccation tolerance and longevity are poorly developed. Carbohydrate accumulation as well as synthesis of LEA or RAB proteins are often suggested to be essential for acquisition of desiccation tolerance. In this work two points are demonstrated. (a) Accumulation of carbohydrates as such does not correlate with acquisition of desiccation tolerance or longevity. It is suggested that a low ratio of mono- to oligosac-charides rather than the absolute amount of carbohydrates controls seed longevity or stability to desiccation tolerance. (b) Synthesis of a few assorted proteins, which is responsive to ABA in the later part of seed maturation, is not correlated with desiccation tolerance or longevity.

Water Deficit Triggers Phospholipase D Activity in the Resurrection Plant Craterostigma plantagineum

Phospholipids play an important role in many signaling pathways in animal cells. Signaling cascades are triggered by the activation of phospholipid cleaving enzymes such as phospholipases C, D (PLD), and A2. Their activities result in the formation of second messengers and amplification of the initial signal. In this study, we provide experimental evidence that PLD is involved in the early events of dehydration in the resurrection plant Craterostigma plantagineum. The enzymatic activity of the PLD protein was activated within minutes after the onset of dehydration, and although it was not inducible by abscisic acid, PLD activity did increase in response to mastoparan, which suggests a role for heterotrimeric G proteins in PLD regulation. Two cDNA clones encoding PLDs, CpPLD-1 and CpPLD-2, were isolated. The CpPLD-1 transcript was constitutively expressed, whereas CpPLD-2 was induced by dehydration and abscisic acid. Immunological studies revealed changes in the subcellular localization of the PLD protein in response to dehydration. Taken together, the data on enzymatic activity as well as transcript and protein distributions allowed us to propose a role for PLD in the events leading to desiccation tolerance in C. plantagineum.

The role of small RNAs in abiotic stress

It was recently discovered that plants respond to environmental stress not only with a specific gene expression programme at the mRNA and protein level but also small RNAs as response modulators play an important role. The small RNAs lead to cleavage or translational inhibition of mRNAs via complementary target sites. Different examples are described where small RNAs have been shown to be involved in stress responses. A link between hormonal action and small RNA activities has frequently been observed thus coupling exogenous factors with endogenous transmitters. Using the CDT‐1 gene from the desiccation tolerant plant Craterostigma plantagineum as an example, it is discussed that generation of novel small RNAs could be an evolutionary pathway in plants to adapt to extreme environments.

Desiccation leads to the rapid accumulation of both cytosolic and chloroplastic proteins in the resurrection plant Craterostigma plantagineum Hochst

A number of desiccation-related and abscisic-acid (ABA)-inducible transcripts have been isolated from the resurrection plant Craterostigma plantagineum (Scrophulariaceae). They have been analysed at the transcriptional level (D. Bartels et al., 1990, Planta 181, 27–34) and their nucleotide sequences determined (D. Piatkowski et al., 1990, Plant Physiol. 94, 1682–1688). Three such genes encoded polypeptides with substantial homologies to proteins abundantly expressed during late embryogenesis in many higher plants; two other genes encoded novel transcripts. The temporal expression patterns of these gene products and their distribution in different organs of the plant and in callus tissues have now been analysed immunologically. For this, in-situ RNA hybridizations and immunocytochemical studies using tissue sections were carried out at both the light and electron microscope level. All of the products were found to be present in leaf tissue, and some were also found in roots and in seeds. Three desiccation-related proteins were localized in the cytosol, while two others, one associated with the thylakoid membranes, the other soluble in the stroma, were detected in the chloroplast. In C. plantagineum the severe ultrastructural changes observed during the desiccation-rehydration process indicate the need for protectants: the gene products characterized in this publication may be good candidates for this role.

Targeting detoxification pathways: an efficient approach to obtain plants with multiple stress tolerance?

A serious factor limiting the engineering of stress tolerance has been our ignorance about the function of stress-induced genes. A stress-activated novel aldose-aldehyde reductase was cloned from alfalfa. The ectopic expression of this gene in tobacco resulted in tolerance to oxidative stress and dehydration. Physiological analysis suggested that aldose reductase probably functions by reducing the level of reactive aldehydes. This provides a promising perspective for the development of crop plants with improved stress tolerance.

A desiccation-related Elip-like gene from the resurrection plant Craterostigma plantagineum is regulated by light and ABA.

The resurrection plant Craterostigma plantagineum tolerates an extreme loss of cellular water. Therefore this plant is being studied as model system to analyse desiccation tolerance at the molecular level. Upon dehydration, new transcripts are abundantly expressed in different tissues of the plant. One such desiccation‐related nuclear gene (dsp‐22 for desiccation stress protein) encodes a mature 21 kDa protein which accumulates in the chloroplasts. Sequence analysis indicates that dsp‐22 is closely related to early light inducible genes (Elip) of higher plants and to a carotene biosynthesis related gene (cbr) isolated from the green alga Dunaliella bardawil. In contrast to other desiccation‐related genes, light is an essential positive factor regulating the expression of dsp‐22: ABA‐mediated gene activation leads to the accumulation of the transcript only in the presence of light. During the desiccation process, light acts at the transcriptional and post‐transcriptional levels. The implications of these different controls and the possible role of the dsp‐22 protein in the desiccation/rehydration process are discussed.

An ABA and GA modulated gene expressed in the barley embryo encodes an aldose reductase related protein.

In most higher plants a period of desiccation is the terminal event in embryogenesis. Excised barley embryos acquire desiccation tolerance at a precise developmental stage and cDNA clones have been isolated which are temporally linked with desiccation tolerance. One such clone (pG22–69) with a putative gene product of 34 kd displays high structural homology to mammalian genes encoding an NADPH dependent aldose reductase involved in the synthesis of sorbitol. This first aldose reductase gene of plants is expressed constitutively during embryo maturation and is modulated by the plant hormones abscisic acid (ABA) and gibberellic acid (GA). Immunohistochemistry showed that the protein is preferentially expressed in tissues formed at early stages in embryogenesis. Measurements of enzymatic activity indicate that pG22–69 encodes an active aldose reductase. The finding of this reductase activity and the cloning of the corresponding gene supports the existence of a metabolic pathway in plants playing a role in the synthesis of osmolytes like sorbitol. The significance of this work is that genes of related structure and functions are being used in diverse organisms to fulfil stress related biological requirements.

Multiple PLDs Required for High Salinity and Water Deficit Tolerance in Plants

High salinity and drought have received much attention because they severely affect crop production worldwide. Analysis and comprehension of the plants response to excessive salt and dehydration will aid in the development of stress-tolerant crop varieties. Signal transduction lies at the basis of the response to these stresses, and numerous signaling pathways have been implicated. Here, we provide further evidence for the involvement of phospholipase D (PLD) in the plants response to high salinity and dehydration. A tomato (Lycopersicon esculentum) α-class PLD, LePLDα1, is transcriptionally up-regulated and activated in cell suspension cultures treated with salt. Gene silencing revealed that this PLD is indeed involved in the salt-induced phosphatidic acid production, but not exclusively. Genetically modified tomato plants with reduced LePLDα1 protein levels did not reveal altered salt tolerance. In Arabidopsis (Arabidopsis thaliana), both AtPLDα1 and AtPLDδ were found to be activated in response to salt stress. Moreover, pldα1 and pldδ single and double knock-out mutants exhibited enhanced sensitivity to high salinity stress in a plate assay. Furthermore, we show that both PLDs are activated upon dehydration and the knock-out mutants are hypersensitive to hyperosmotic stress, displaying strongly reduced growth.