Plant responses to environmental stress
Elizabeth Vierling and Janice A. Kimpel
University of Arizona, Tucson, Arizona and University of Georgia, Athens, Georgia, USA
Considerable progress is being made in identifying genes that are important for tolerance to abiotic stress and in defining stress-responsive gene promoters and signal-transduction pathways. Although genetically engineered crop plants with greater resistance to environmental stress have not yet been produced, research is at a turning point where correlative changes can now
be tested for effectiveness in conferring stress tolerance.
Current Opinion in Biotechnology 1992, 3:164-1 70
It is well recognized that agricultural losses resulting from environmental stress are significant. The chal- lenge for plant breeders and biotechnologists contin- ues to be production of stress-resistant plants while maintaining acceptable yields. Recent molecular stud- ies of plant stress responses and their relevance to en- gineering stress-resistant plants are the subject of this review.
Several major areas of progress can be identified. First, many stress-induced genes have been cloned and characterized and their roles in the stress re- sponse are now being clarified. Genes involved in small molecule biosynthetic pathways important for stress tolerance, including hormones, osmolytes and phytochelatins, are being identified. Second, mecha- nisms by which plants sense stress, thus leading to adaptive responses, are being defined. This includes elucidation of signal-transduction pathways and defi- nition of transcriptional and post-transcriptional regu- latory mechanisms. Finally, restriction fragment length polymorphism (RFLP) mapping technology is now available in several plant species, providing a means of marking and tracking genetic loci associated with stress resistance. It is likely that response to stress is mediated by several genes; RFLP maps can be used to estimate additive effects and dominance of each locus associated with the phenotype.
Although the following discussion considers different stresses individually, it is important to recognize that many of the responses overlap because of similar- ities in the physiological changes that occur. For ex- ample, drought stress, salt stress and cold stress all in- volve problems of water availability. Because of such overlaps, exposure"'to one type of stress may induce a degree of tolerance to other stresses. Also, many
stress-induced genes are also developmentally regu- lated, which indicates that physiological changes at different stages of development have similarities with those experienced during stress.
Osmotic stress can be caused by several different en- vironmental factors including drought, desiccation, salt and cold. As a step toward understanding and engi- neering tolerance to these stresses, researchers have identified genes related to desiccation , genes in- duced by low turgor (encoding a putative ion chan- nel, a thiol protease and aldehyde dehydrogenase ), genes involved in the biosynthesis of compatible so- lutes (proline, pyrroline-5-carboxylate reductase, be- taine, betaine aldehyde dehydrogenase) [1,3"], genes involved in ion transport [1,3"], and genes induced by salinity (e.g. osmotin) . As the hormone abscisic acid (ABA) is involved in plant responses to osmotic stress, it is not surprising that many, though not all, of these genes are also regulated by ABA . ABA- regulated gene expression is under intense investiga- tion and both cis-acting sequences and trans-acting factors important for ABA-mediated gene expression have been identified. Further advances in the areas of drought stress, salt stress and cold stress are discussed below.
Some of the best characterized genes expressed in response to osmotic stress are the Lea (late embryo-
Abbreviations AB~abscisic acid; bZl~leucine zipper DNA binding; CHS--chalcone synthase; 4CL--4-coumarate-coenzyme A ligase;
CPRF--common plant regulatory factor; GUS--[8-glucuronidase; HSE--heat-shock promoter element; HSP--heat-shock protein; LMW--Iow molecular weight; PI--proteinase inhibitor; RAB--responsive to ABA; RFLP--restriction fragment length polymorphism;
SOD--superoxide dismutase; UV--ultraviolet.
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Plant responses to environmental stress Vierling and Kimpet 165
genesis abundant) genes. These were first identified as being expressed late in seed development and their expression is correlated with increased ABA levels and tolerance of embryos to desiccation. There are three major groups of Lea genes and homologs of each group that are regulated by osmotic stress have been found . Certain LEA-like genes/proteins have also been called RAB (responsive to ABA) or dehy- drins. This is a major example of how normal phy- siological processes such as embryo desiccation and stress can overlap. The LEA proteins are extremely hydrophilic and it has been suggested that they pro- tect other proteins from the effects of water loss. Ad- ditional support for this model has now been gained from studies of Ceratostigma plantagineum (resurrec- tion plant), which is adapted to tolerate extreme des- iccation. Piatkowski et al.  cloned ABA-responsive genes from this plant and found three that are related to previously described genes encoding LEA proteins (or dehydrins). Two genes not previously identified in other plant species were also found.
It is important to recognize that different plant organs or even different growth regions of the same organ re- spond differently to osmotic stress and also to ABA. Plant et al.  identified a protein that is expressed in drought-stressed aerial parts of tomato, but not in roots, and shows homology to a phospholipid transfer protein. The same group has linked ABA to the induc- tion of this gene by showing that it is not induced in the ABA-deficient mutant of tomato, flacca. Examining several genes, including [3-tubulin, actin, cell wall pro- teins (glycine-rich and hydroxyproline-rich proteins) and two unidentified water-deficit-induced sequences, Creelman and Mullet  documented changes in ex- pression specific to the growth-inhibited elongating region of soybean hypocotyls. The authors speculate that the decrease of 13-tubulin and actin reflects the decreased growth rate and that changes in cell wall proteins may control wall extensibility in the elongat- ing region.
Differential inhibition of shoot versus root growth is a well recognized response to drought stress. It ap- pears that although ABA accumulates in both leaves and roots, the hormone only inhibits the growth of leaves. Sharp's group has extensively studied this phys- iological phenomenon. Their recent results  indicate that proline deposition is responsible for osmotic ad- justment in root tips and allows continued growth of roots experiencing water deficit. These data not only indicate that osmotic adjustment is highly regulated, but also provide insight into the involvement of pro- line in drought tolerance.
Plants experiencing salt stress suffer from both reduced water availability and the accumulation of toxic ions, in particular Na +. McCue and Hanson [3"'] have written an excellent review of the problems and progress in
engineering plants for salt tolerance, particularly with regard to solute accumulation. Now that several genes involved in the biosynthesis of compatible solutes have been identified, understanding the regulation of so- lute production in different plant systems becomes the next challenge. Another important regulatory as- pect is the control of ion pumps. Genes have been identified that encode subunits of the tonoplast AT- Pase, which is thought to be essential for the energy requirements of increased Na + pumping. However, the extent to which transcriptional versus post-transcrip- tional processes modulate the activity of these pumps is unclear .
Metabolically engineering plants to have traits that may confer stress tolerance has recently been ac- complished by Tarczynski et al. [9"']. They intro- duced the Escherichia coli gene encoding mannitol- 1-phosphate dehydrogenase (mtlD) into tobacco, and demonstrated that the transgenic plants had elevated levels of mannitol in leaf and root tissues (exceed- ing 6 btmol gram-1 fresh weight). In E. coli, mtlD is normally involved in catabolism of mannitol-l-phos- phate to fructose-6-phosphate. In plants, it appears that excess fructose-6-phosphate drives the reaction in reverse, with a non-specific phosphatase rapidly and irreversibly converting the mannitol- l-phosphate to mannitol. These transgenic plants are excellent ma- terial for studies of the contribution of sugar alcohols to tolerance of salt or other osmotic stresses.
Several genes induced in response to salt stress have been identified, including RAB genes, salT and that encoding osmotin, of which some show tissue-specific expression . The function of these genes remains unclear beyond the proposed function of the Lea-like RAB genes. They do not appear to be strictly salt-spe- cific, but also respond to other osmotic stresses and in many cases to ABA. Gene induction is also involved in the adaptive change in photosynthesis in the salt-toler- ant plant Mesembryanthemum crystallinum. Bohnert and colleagues  have shown that the switch from C 3 to Crassulacean acid metabolism that occurs during salt stress in this plant is accompanied by new gene expression, including the induction of specific phos- phoenolpyruvate carboxylase genes.
Increased freezing tolerance following a period of cold acclimation or cold hardening is a dramatic example of how many plant species can adapt to extremes in temperature. Biochemical changes associated with low-temperature tolerance include increases in sugars, organic acids and soluble protein, the appearance of new proteins and alterations of lipids .
Changes in gene expression that correlate with cold ac- climation have been described in several species and cDNAs encoding cor (cold regulated) genes have been isolated . In Arabidopsis, the major cor genes en-
166 Plant biotechnology
code 140, 47, 24, 15 and 6.6 kD polypeptides. Interest- ingly, Lin et al. [12"] have shown that the COR polypep- tides remain soluble following boiling, which is an in- dication of their hydrophilicity. The LEA proteins have similar properties and some of the cor genes are also regulated by osmotic stress . As survival of both drought and cold stress requires tolerance to dehy- dration, it is likely that the COR proteins counteract dehydration stress. Similar cold-induced, hydrophilic proteins have been identified in several plant species [12.].
Understanding the regulation of cor gene expression is only beginning. ABA has long been associated with cold acclimation and plant responses to osmotic stress, but its role in these responses is complex. Although ABA induces cor genes in wild-type plants at room temperature, studies using Arabidopsis ABA-deficient and ABA-insensitive mutants have shown that both types of mutants still induce cor gene expression in re- sponse to cold treatment [13",14"]. Thus, ABA and cold must act through separate, but convergent, induction pathways.
Changes in membrane lipids required to maintain membrane fluidity at cold temperatures have been demonstrated to have a positive effect on cold tol- erance . A dramatic demonstration of the abil- ity to alter lipids, thereby increasing cold tolerance, has been accomplished by Wada et al. [15"]. Intro- duction of desA, a gene for fatty-acid desaturation from the chilling-resistant cyanobacterium Synechocys- tis, into chilling-sensitive Anacyst is n idulans changed the fatty acid composition of the membranes and en- abled photosynthesis to proceed uninhibited at 5C. As progress continues in the identification of enzymes in- volved in lipid metabolism in higher plants , the effects of over- and under-expression of the genes encoding these enzymes should be studied.
Another possible approach to increasing freezing toler- ance has been taken by Hightower et al. , who have introduced fish antifreeze proteins into tobacco. Anti- freeze proteins, which have not been found in plants, are composed of multiple repeats of an alanine-rich 11- amino-acid unit and act to lower the freezing point by a non-colligative mechanism. The transgenic tobacco plants showed expression of the antifreeze proteins, but increased freezing tolerance of whole plants was not measured. The ultimate goal of these workers is to test these proteins for their effectiveness in preventing ice-crystallization damage in fruits and vegetables.
Plants and other eukaryotes, as well as prokaryotes, produce a specific set of 'heat shock proteins' (HSPs) when tissue temperatures are increased, either grad- ually or abruptly, 5-10C above optimal growth tem- peratures. Regulation of HSP expression and charac-
terization of the major HSPs in plants has progressed considerably [18,19"]. Because the heat-shock response is highly conserved evolutionarily, studies of HSP func- tion in other organisms have also contributed to under- standing the response in plants.
A major regulatory point in HSP expression is the transcriptional activation of the HSP genes. The pro- moter elements required for induction of these genes are well characterized in plants and are similar to those in other eukaryotes . The heat-shock promoter element (HSE) has been successfully used to drive heat-induced expression of several different genes in transgenic plants. Scharf et al. [20.] have now cloned genes for the transcription factors from tomato (and Arabidopsis, L Nover, personal communication) which bind the HSE. These factors contain a DNA-binding do- main similar to other eukaryotic HSE-binding factors. Surprisingly, in contrast to other eukaryotes, tomato contains at least three distinct factors, all of which have the same HSE-binding domain but are highly divergent over the rest of the protein. Determining the regulatory significance of this complexity will be important for manipulating HSP gene expression.
ierling [19"] has recently reviewed molecular and functional data on HSPs in plants. Four classes of HSPs common to all eukaryotes, HSP90, HSP70, HSP60 and low molecular weight (LMW) HSPs, have been char- acterized. Proteins from the first three groups are be- lieved to function as 'molecular chaperones'  by binding to other proteins and maintaining them in a conformation necessary for correct folding, inter- action with other cellular components, or transport across membranes. For example, HSP60, also known as the ribulose bisphosphate (RuBP) carboxylase binding protein, functions in the assembly of RuBP carboxy- lase . BiP, a homolog of HSP70 that is found in the endoplasmic reticulum, has re...