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

Introduction It is well recognized that agricultural losses resulting from environmental stress are significant. The challenge for plant breeders and biotechnologists continues to b e production of stress-resistant plants while maintaining acceptable yields. Recent molecular studies of plant stress responses and their relevance to engineering stress-resistant plants are the subject of this review. Several major areas of progress can be identified. First, m a n y stress-induced genes have been cloned and characterized and their roles in the stress response are n o w being clarified. G e n e s involved in small molecule biosynthetic pathways important for stress tolerance, including hormones, osmolytes and phytochelatins, are being identified. Second, mechanisms b y which plants sense stress, thus leading to adaptive responses, are being defined. This includes elucidation of signal-transduction pathways and definition of transcriptional and post-transcriptional regulatory mechanisms. Finally, restriction fragment length p o l y m o r p h i s m (RFLP) m a p p i n g technology is n o w available in several plant species, providing a m e a n s of marking and tracking genetic loci associated with stress resistance. It is likely that response to stress is mediated b y several genes; RFLP m a p s 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 m a n y of the responses overlap because of similarities in the physiological changes that occur. For example, drought stress, salt stress and cold stress all involve problems of water availability. Because of such overlaps, exposure"'to o n e type of stress may induce a degree of tolerance to other stresses. Also, m a n y

stress-induced genes are also developmentally regulated, which indicates that physiological changes at different stages of development have similarities with those experienced during stress.

Osmotic stress

Osmotic stress can be caused by several different environmental factors including drought, desiccation, salt and cold. As a step toward understanding and engineering tolerance to these stresses, researchers have identified genes related to desiccation [1], genes induced b y low turgor (encoding a putative ion channel, a thiol protease and aldehyde dehydrogenase [2]), genes involved in the biosynthesis of compatible solutes (proline, pyrroline-5-carboxylate reductase, betaine, betaine aldehyde dehydrogenase) [1,3"], genes involved in ion transport [1,3"], and genes induced by salinity (e.g. osmotin) [1]. As the h o r m o n e 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 b y ABA [1]. ABAregulated gene expression is under intense investigation and both cis-acting sequences and trans-acting factors important for ABA-mediated gene expression have b e e n identified. Further advances in the areas of drought stress, salt stress and cold stress are discussed below.

Drought stress 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. © Current Biology Ltd ISSN 0958-1669

Plant responses to environmental stress Vierling and Kimpet

genesis abundant) genes. These were first identified as being expressed late in seed d e v e l o p m e n t 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 [1]. Certain LEA-like genes/proteins have also b e e n called RAB (responsive to ABA) or dehydrins. This is a major example of h o w normal physiological processes such as embryo desiccation and stress can overlap. The LEA proteins are extremely hydrophilic and it has b e e n suggested that they protect other proteins from the effects of water loss. Additional support for this model has n o w b e e n gained from studies of Ceratostigma p l a n t a g i n e u m (resurrection plant), which is adapted to tolerate extreme desiccation. Piatkowski et al. [4] cloned ABA-responsive genes from this plant and found three that are related to previously described genes encoding LEA proteins (or dehydrins). T w o 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 respond differently to osmotic stress and also to ABA. Plant et al. [5] 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 induction 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 proteins (glycine-rich and hydroxyproline-rich proteins) and two unidentified water-deficit-induced sequences, Creelman and Mullet [6] documented changes in expression 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 m a y control wall extensibility in the elongating region. Differential inhibition of shoot versus root growth is a well recognized response to drought stress. It appears that although ABA accumulates in both leaves and roots, the h o r m o n e only inhibits the growth of leaves. Sharp's group has extensively studied this physiological p h e n o m e n o n . Their recent results [7] indicate that proline deposition is responsible for osmotic adjustment 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 proline in drought tolerance.

Salt stress 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. N o w that several genes involved in the biosynthesis of compatible solutes have b e e n identified, understanding the regulation of solute production in different plant systems b e c o m e s the next challenge. Another important regulatory aspect is the control of ion pumps. Genes have b e e n identified that encode subunits of the tonoplast ATPase, which is thought to b e essential for the energy requirements of increased Na + pumping. However, the extent to which transcriptional versus post-transcriptional processes modulate the activity of these p u m p s is unclear [8]. Metabolically engineering plants to have traits that m a y confer stress tolerance has recently been accomplished b y Tarczynski et al. [9"']. They introduced the Escherichia coli gene encoding mannitol1-phosphate dehydrogenase ( m t l D ) into tobacco, and demonstrated that the transgenic plants had elevated levels of mannitol in leaf and root tissues (exceeding 6 btmol gram-1 fresh weight). In E. coli, m t l D is normally involved in catabolism of mannitol-l-phosphate 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 material 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 b e e n identified, including RAB genes, salT and that encoding osmotin, of which some show tissue-specific expression [1]. The function of these genes remains unclear b e y o n d the p r o p o s e d function of the Lea-like RAB genes. They do not a p p e a r to be strictly salt-specific, 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-tolerant plant M e s e m b r y a n t h e m u m crystallinum. Bohnert and colleagues [10] have s h o w n that the switch from C3 to Crassulacean acid metabolism that occurs during salt stress in this plant is accompanied b y new gene expression, including the induction of specific phosphoenolpyruvate carboxylase genes.

Cold stress/acclimation

Increased freezing tolerance following a period of cold acclimation or cold hardening is a dramatic example of h o w m a n y 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 n e w proteins and alterations of lipids [11]. Changes in gene expression that correlate with cold acclimation have been described in several species and cDNAs encoding cor (cold regulated) genes have b e e n isolated [11]. In Arabidopsis, the major cor genes en-

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Plant biotechnology code 140, 47, 24, 15 and 6.6 kD polypeptides. Interestingly, Lin et al. [12"] have shown that the COR polypeptides remain soluble following boiling, which is an indication of their hydrophilicity. The LEA proteins have similar properties and some of the c o r genes are also regulated by osmotic stress [11]. As survival of both drought and cold stress requires tolerance to dehydration, it is likely that the COR proteins counteract dehydration stress. Similar cold-induced, hydrophilic proteins have b e e n identified in several plant species [12.]. Understanding the regulation of c o r gene expression is only beginning. ABA has long b e e n 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 r o o m temperature, studies using Arabidopsis ABA-deficient and ABA-insensitive mutants have s h o w n that both types of mutants still induce cor gene expression in response to cold treatment [13",14"]. Thus, ABA and cold must act through separate, but convergent, induction pathways. Changes in m e m b r a n e lipids required to maintain m e m b r a n e fluidity at cold temperatures have b e e n demonstrated to have a positive effect on cold tolerance [11]. A dramatic demonstration of the ability to alter lipids, thereby increasing cold tolerance, has b e e n accomplished by Wada et al. [15"]. Introduction of desA, a gene for fatty-acid desaturation from the chilling-resistant cyanobacterium Synechocystis, into chilling-sensitive A n a c y s t i s n i d u l a n s changed the fatty acid composition of the m e m b r a n e s and enabled photosynthesis to proceed uninhibited at 5°C. As progress continues in the identification of enzymes involved in lipid metabolism in higher plants [16], the effects of over- and under-expression of the genes encoding these enzymes should be studied. Another possible approach to increasing freezing tolerance has b e e n taken b y Hightower et al. [17], w h o have introduced fish antifreeze proteins into tobacco. Antifreeze proteins, which have not b e e n found in plants, are c o m p o s e d of multiple repeats of an alanine-rich 11amino-acid unit and act to lower the freezing point b y a non-colligative mechanism. The transgenic tobacco plants s h o w e d expression of the antifreeze proteins, but increased freezing tolerance of w h o l e plants was not measured. The ultimate goal of these workers is to test these proteins for their effectiveness in preventing ice-crystallization d a m a g e in fruits and vegetables.

Heat stress Plants and other eukaryotes, as well as prokaryotes, produce a specific set of 'heat shock proteins' (HSPs) w h e n tissue temperatures are increased, either gradually or abruptly, 5-10°C above optimal growth temperatures. 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 function in other organisms have also contributed to understanding the response in plants. A major regulatory point in HSP expression is the transcriptional activation of the HSP genes. The promoter elements required for induction of these genes are well characterized in plants and are similar to those in other eukaryotes [18]. The heat-shock p r o m o t e r element (HSE) has b e e n successfully used to drive heat-induced expression of several different genes in transgenic plants. Scharf et al. [20.] have n o w cloned genes for the transcription factors from tomato (and Arabidopsis, L Nover, personal communication) which bind the HSE. These factors contain a DNA-binding domain 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 b e important for manipulating HSP gene expression. ¥ierling [19"] has recently reviewed molecular and functional data on HSPs in plants. Four classes of HSPs c o m m o n to all eukaryotes, HSP90, HSP70, HSP60 and low molecular weight (LMW) HSPs, have b e e n characterized. Proteins from the first three groups are believed to function as 'molecular chaperones' [21] b y binding to other proteins and maintaining them in a conformation necessary for correct folding, interaction with other cellular components, or transport across membranes. For example, HSP60, also k n o w n as the ribulose bisphosphate (RuBP) carboxylase binding protein, functions in the assembly of RuBP carboxylase [21]. BiP, a h o m o l o g of HSP70 that is found in the endoplasmic reticulum, has recently b e e n identified as participating in seed storage protein deposition [22,23]-- another example of molecular chaperone activity. Exciting data relating to the mechanism of HSP70 action has b e e n gained from crystallization of the amino-terminal portion of the protein, which exhibits ATPase activity essential for function [24"']. The LMW I-fSPs may also act as molecular chaperones, although direct evidence is lacking. Plants appear to be unique a m o n g eukaryotes in having LMW HSP homologs not only in the cytoplasm, but also in the endoplasmic reticulum [19"] and chloroplast [25]. This is an interesting parallel to HSP70, homologs of which are found in these three compartments as well as in mitochondria. It is assumed that HSP expression is necessary for surviving heat stress, presumably to provide molecular chaperones, although most of the supporting data are derived from studies of HSPs or h o m o l o g o u s proteins that are present constitutively. There are no published studies in which HSP expression has b e e n enhanced or reduced b y gene manipulations in transgenic plants.

Plant responses to environmental stress Vierling and Kimpel Oxidative stress Oxidative stress occurs w h e n the concentrations of reactive oxygen species, such as the superoxide radical (O2'-), hydrogen peroxide, or the hydroxyl radical ( O H ) , increase in cells [26"]. Plants have evolved several mechanisms to protect against these potentially damaging molecules, including the synthesis of scavenger sulphydryl compounds and enzymes such as peroxidases, catalases and superoxide dismutases (SODs). Increases in the activity of these enzymes are strongly correlated with imposition of stress (e.g. infection, ultraviolet (UV) irradiation, herbicide application, elevated o z o n e and sulphur dioxide in the air, and chilling) [27]. In yeast, E. coli and Drosophila, these enzymes are critical for aerobic g r o w t h - - d o e s it follow that constitutive or over-expression of these genes will improve plant resistance to stress? Initial work b y T e p p e r m a n and Dunsmuir [28] indicated that overexpression of a chloroplast Cu/Zn-SOD did not improve resistance to superoxide toxicity. Resuits from h u m a n and animal studies also suggest that overproduction of SOD can cause detrimental effects. However, there are at least three k n o w n forms of SOD, suggesting that other forms might provide some level of protection. Bowler et al. [29"] have overexpressed a Mn-SOD e n z y m e targeted to both mitochondria and chloroplasts of tobacco. In tissues of the transgenic plants with elevated chloroplast Mn-SOD activity, protection against light-dependent paraquat damage was significantly increased. In the dark, however, tissues that only moderately overproduced Mn-SOD were actually more sensitive to herbicide damage. The phenotype is p r o b a b l y determined by the overall ratio of the oxygen radical and hydrogen peroxide, which react non-enzymatically to form the extremely toxic hydroxyl radical. The oxygen radical is used b y SODs as a substrate in a reaction that releases hydrogen peroxide. ThUs, SODs can dramatically affect the ratio of these reactive oxygen species. The authors suggest that the best a p p r o a c h may be to engineer plants with both a SOD and a peroxidase as the formation of hydroxyl radicals should b e quite rare in tissues producing both enzymes.

Ultraviolet light stress Some plants achieve protection from the damaging effects of UV irradiation by synthesizing flavonoid pigments. The first enzyme in the committed pathway to flavanoid pigment production is chalcone synthase (CHS). Previous w o r k [30] identified a cis-acting DNA sequence in the parsley CHS promoter, unit 1, that contains two separate regions (box I and b o x II) necessary for UV light inducibili W of CHS. Weisshaur et al. [31"] determined that unit 1 is both necessary and sufficient for UV light inducibility, and they isolated three cDNAs encoding trans-acting factors that bind to the b o x II core sequence (ACGTGGC). Preliminary anal-

ysis indicates that all three cDNAs encode a leucine zipper DNA-binding (bZIP) motif. These bZIP motifs are similar to all other identified bZIP regions of higher plants. The sequences recognized b y all these plant bZIP domains contain the core ACGT sequence present in b o x II. Consequently, the parsley trans-factors have b e e n n a m e d c o m m o n plant regulatory factors (CPRF)-I, CPRF-2, and CPRF-3. H o w all these plant trans-acting factors regulate very different promoters despite recognizing the same four-base-pair sequence remains to be resolved. These authors hypothesize that at least two cis elements are required for activation in response to a stimulus, one of which is typically an ACGT type. Perhaps most intriguing, CPRF-1 is itself induced by UV light. Maximal accumulation of CPRF-1 occurs at the same time as the maximal increase in CHS mRNA levels, consistent with a causal relationship between CPRF-1 synthesis and CHS mRNA accumulation. In addition to UV-light inducibility, CHS is induced u p o n pathogen challenge and is also developmentally regulated. Studies b y Wingender et al. [32] with soybean and b y Fritze et al. [331 with snapdragon define the CHS promoter as a linear array of separable cis-acting elements, subsets of which are responsive to the various stimuli. The binding affinities and distribution of the trans-acting factors that interact with these elements are highly conserved, as p r o m o t e r function may be correctly induced in heterologous expression systems (soybean in parsley, snapdragon in tobacco). Despite this strong functional conservation a m o n g the k n o w n trans-acting factors, there is no strong sequence conservation in the identified cis-acting elements of the CHS promoters.

Arabidopsis thaliana offers an opportunity to dissect the mechanism of induction of these genes b y the use of mutants. Feinbaume et al. [34] have d e v e l o p e d a set of transformed plants carrying either the full length CHS promoter fused to a [3-glucuronidase (GUS) reporter gene or any one of a set of 5' deletions of the promoter fused to GUS. Seeds from these plants are being mutagenized and the p r o g e n y screened for unusual (e.g. high, low or non-inducible) levels of GUS activity. The first screen revealed one mutant with much reduced expression of both the e n d o g e n o u s CHS gene and the CHS-GUS fusion gene. A similar approach is being used to dissect the molecular mechanisms by which other stresses are perceived. The enzyme 4 - c o u m a r a t e - c o e n z y m e A ligase (4CL), which acts at the b r a n c h p o i n t where the phenylpropanoid b a c k b o n e is directed into several endproduct-specific pathways, is also induced by stress. In parsley, expression of the gene encoding 4CL is induced by UV irradiation, wounding and pathogen infection, and is also u n d e r developmental regulation. The cis-acting element in the parsley 4CL-1 promoter that controls stress inducibility is distinct from that controlling developmental regulation [35]. As seen for the CHS promoter, factors interacting with the parsley 4CLq promoter appear highly conserved, as the developmental and stress-inducible regulation can be

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168 Plantbiotechnology expressed in transgenic tobacco. In a surprising observation [35], stress responsiveness of this gene was found to require the presence of cis-acting elements not only in the promoter region of the gene but also in the coding region.

Wounding Genes coding for repair or protective functions are induced in response to wounding. These include cell-wall structural proteins [36] and enzymes, lignin, suberin, flavonoid [37] and isoprenoid synthetic enzymes [38], fructosidases (for energy mobilization) [39] and proteinase inhibitors (PIs). The induction of tomato PIs, which are effective against m a n y chewing insects, has been studied by Ryan's laboratory (Washington State) for the past twenty years. Last year they reported the discovery of a previously u n k n o w n type of h o r m o n e in plants, a small, 18-amino-acid peptide [40"]. The peptide, n a m e d systemin, is responsible for the systemic induction of the PI genes that occurs following wounding of a single leaf. This landmark discovery, coupled with the finding that methyl jasmonate also systemically induces the PI genes [41"], led Ryan to propose a signal transduction pathway for w o u n d i n g (International Society for Plant Molecular Biology Meeting October 1991, Tucson, Arizona, USA). In his model, systemin is released at the w o u n d site u p o n cell disruption and m o v e s throughout the plant. It binds to receptors o n the plasma m e m b r a n e , causing the release of linolenic acid which is further metabolized to jasmonic acid (the soluble f o r m of methyl jasmonate). Jasmonic acid then acts directly or indirectly to trigger activation of the wound-responsive genes. Pena-Cortes et al. [42] s h o w e d that wound-inducibility of PI genes in potato and tomato is mediated by ABA. Using wild-type and ABA-deficient lines transformed with PI p r o m o t e r - G U S coding region constructs, they demonstrated that activation of the P1 promoter b y wounding, both locally and systemically, requires ABA. ABA must therefore b e included in the signal transduction scheme, perhaps with the role of mediating the jasmonic acid signalling. The PI genes are also developmentally regulated, but this regulation involves cis-acting elements that are separable from the wound-responsive elements, and their developmental expression is not affected b y ABA [42,43].

problem because of the complexi W and n u m b e r of changes that occur under stress conditions. Instances where transfer of a single gene will positively affect tolerance are likely to b e limited, complicating the job of plant improvement. For the biotechnologist, problems in metabolic engineering are also significant, as very clearly outlined by McCue and Hanson [3"'] and demonstrated b y the transgenic-plant w o r k manipulating SODs [28,29"]. However, in addition to the identification of m a n y genes that are clearly involved in stress tolerance, characterization of more gene promoters has e n h a n c e d our ability to direct gene expression both temporally and spatially in order to effect very specific changes in transgenic plants. Current plant transformation technology should enable relatively rapid hypothesis testing in model species and final testing in different crop species. A promising n e w approach involves using RFLPs to m a p quantitative trait loci [44"] associated with stress tolerance. This is an important technique for the future, both in terms of manipulating such traits in a breeding program and understanding the genetic nature of stress tolerance. The use of wild species to introgress desirable traits not present in cultivated species remains a goal of plant breeders. For traits that are controlled by m a n y genes, RFLP m a p s m a y accelerate the introgression process. Both transgenic plant studies and RFLP-based studies aimed at understanding and improving plant stress tolerance are n o w underway in many laboratories.

Acknowledgements EV thanks Drs J Cushman, H Bohnert a n d M T h o m a s h o w for helpful discussions.

References and recommended reading Papers of particular interest, p u b l i s h e d within the annual period of review, have b e e n highlighted as: of special interest •. of outstanding interest 1.

SKRIVERK, MUNDY J: G e n e E x p r e s s i o n i n R e s p o n s e to AbsCetsic Acid and O s m o t i c Stress. Plant Cell 1990, 2:503-512.

2.

GUERREROFD, JONES JT, MULLET JE: T u r g o r - r e s p o n s i v e G e n e Transcription a n d RNA Levels i n c r e a s e R a p i d l y w h e n Pea Shoots are Wilted. Sequence and E x p r e s s i o n o f T h r e e I n d u c i b l e G e n e s . Plant Mol Biol 1990, 15:11-26.

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Conclusions By manipulating gene expression and metabolic pathways in transgenic plants, it is n o w possible to determine h o w m a n y of the changes correlated with stress are important to stress tolerance. Nonetheless, ability to manipulate stress tolerance remains a long-term

MCCUEKF, HANSON AD: D r o u g h t a n d Salt T o l e r a n c e : Tow a r d s Understanding a n d A p p l i c a t i o n . Trends Biotechnol 1990, 8:358-361. An excellent s u m m a r y of a p p r o a c h e s and problems to be considered in engineering plant stress tolerance. A g o o d source of basic information o n osmolyte accumulation during osmotic stress. 4.

PIATKOWSKID, SCHNEIDER K, SALAMINI F, BARTELS D: C h a r -

a c t e r i z a t i o n o f Five Abscisic Acid-responsive cDNA C l o n e s Isolated f r o m t h e D e s i c c a t i o n - t o l e r a n t P l a n t Ceratostigma plantagineum and T h e i r R e l a t i o n s h i p to Other Water S t r e s s G e n e s . Plant Physiol 1990, 94:1682-1688.

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PLANTAL, COHEN A, MOSES MS, BRAY EA: N u c l e o t i d e Seq u e n c e a n d Spatial E x p r e s s i o n P a t t e r n o f a D r o u g h t a n d A b s c i s i c - a c i d - i n d u c e d G e n e o f T o m a t o . Plant Physiol 1991, 97:900-906.

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CREELMANRA, MULLETJE: W a t e r Deficit M o d u l a t e s G e n e Expression in Growing Zones of Soybean Seedlings. A n a l y s i s o f D i f f e r e n t i a l l y E x p r e s s e d cDNAs, a N e w ~t u b u l i n G e n e , a n d E x p r e s s i o n o f G e n e s E n c o d i n g Cell Wall P r o t e i n s . Plant Mol Biol 1991, 17:591-608.

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VOETBERG GS, SHARP RE: G r o w t h o f t h e M a i z e Prim a r y R o o t at Low W a t e r P o t e n t i a l s . Plant Physiol 1991, 96:1125-1130.

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NARASIMHANML, BINZEL ML, PEREZ-PRAT E, CHEN Z, NELSON DE, SINGH NK, BRESSAN RA, HASEGAWAPM: NaC1 Regulat i o n o f T o n o p l a s t ATPase 7 0 - k i l o d a l t o n S u b u n i t m R N A i n T o b a c c o Cells. Plant Physiol 1991, 97:562-568.

TA~CZYNSmMC, JENSEN RC, BOHNERT HJ: E x p r e s s i o n o f a Bacterial m t l D Gene in Transgenic Tobacco Leads to t h e P r o d u c t i o n a n d t h e A c c u m u l a t i o n o f t h e S u g a r A l c o h o l , M a n n i t o L Proc Natl Acad Sci USA 1992, in press. The first example of increasing osmolyte production by genetic engineering. An e n z y m e responsible for mannitol catabolism causes mannitol accumulation in transgenic tobacco. Tobacco does not normally accumulate mannitol so a n e w metabolic pathway has b e e n introduced.

Discusses the relevance of HSPs to plant thermotolerance and identifies areas of future research. 20.

SCHARFKD, ROSE S, ZOTF W, SCH{SFFL F, NOVER L: T h r e e T o m a t o G e n e s C o d e f o r H e a t S t r e s s T r a n s c r i p t i o n Fact o r s w i t h a R e g i o n o f R e m a r k a b l e H o m o l o g y to t h e D N A - b t n d i n g D o m a i n o f t h e Y e a s t HSP. EMBO J 1990, 9:4495-4502. The s e q u e n c e of the conserved DNA-binding d o m a i n of the heatshock transcription factors is presented. Unusual features of the plant heat-shock factors, including the existence of three distinct factors and induction of two of these by heat are described. Demonstrates the conservation of the c/s-sequences a n d trans-factors involved in the heat shock response a m o n g divergent eukaryotes. 21.

ELLISRJ: Molecttlar C h a p e r o n e s : T h e P l a n t C o n n e c t i o n . Science 1990, 250:954-958.

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BOSTONRS, EONTES EBP, SHANK BB, WROBEL RL: I n c r e a s e d E x p r e s s i o n o f t h e Maize I m m u n o g l o b u l i n B i n d i n g Prot e i n H o m o l o g b-70 i n T h r e e Z e i n R e g u l a t o r y M u t a n t s . Plant Cell 1991, 3:497-505.

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MAROCCOA, SANTUCCIA, CERIOLI S, MOTTO M, DIFONZO N, THOMPSON R, SALAMINI F: T h r e e H i g h - l y s i n e M u t a t i o n s C o n t r o l t h e Level o f A T P - b i n d i n g HSP70-1ike P r o t e i n s i n t h e Maize E n d o s p e r m . Plant Cell 1991, 3:507-515.

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CUSHMANJC, DEROCHER EJ, BOHNERT HT: G e n e E x p r e s s i o n D u r i n g A d a p t a t i o n t o Salt Stress. In Environmental Inj u r y to Plants. Edited by Katteman FJ. San Diego: Academic Press, 1990:173-203.

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THOMASHOWMF: M o l e c u l a r G e n e t i c s o f C o l d A c c l i m a t i o n i n H i g h e r P l a n t s . Adv Genetics 1990, 28:99-131.

LIN C, G u o W'W, EVERSON E, THOMASHOW ME: C o l d Accli•n a t i o n i n A r a b i d o p s i s and W h e a t . Plant Physiol 1990, 94:1078-1083. The 'boiling soluble' property of the COR polypeptides is described. All the major in vitro translation products of mRNAs, w h i c h are induced by cold acclimation, remain soluble after boiling.

24. •.

FLAHERTYKM, McKAY DB, KABSCH W, HOLMES KC: Sinlilarity of the Three-dimensional Structures of Actin and t h e ATPase F r a g t n e n t s o f a 7 0 - k D a H e a t S h o c k C o g n a t e P r o t e i n . Proc Natl Acad Sci USA 1991, 88:5041-5045. X-ray crystal structure data s h o w i n g that the amino-terminal half of HSP70 is structurally similar to actin. Continued progress in structural analysis of HSP70 is essential to defining the m e c h a n i s m of molecular chaperone action. 25.

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NORDINK, HEINO P, PALVA ET: S e p a r a t e S i g n a l P a t h w a y s Regulate the Expression of a Low-temperature-induced G e n e i n A r a b i d o p s i s t h a l i a n a (L.) H e y n h . Plant Mol Biol 1991, 16:1061-1071. A g o o d demonstration of h o w h o r m o n e deficiem and insensitive mutams (in this'case ABA) can be u s e d to probe regulatory pathways. GILMOURS, THOMASHOWMF: C o l d A c c l i m a t i o n a n d Coldr e g u l a t e d G e n e E x p r e s s i o n i n ABA M u t a n t s o f Arab i d o p s i s thaliana. Plant Mol Biol 1991, 17:1233-1240. Further support for separate but convergent pathways for ABA and cold regulation of cor genes.

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SCANDALIOSJG: R e s p o n s e o f P l a n t A n t i o x i d a n t D e f e n s e G e n e s to E n v i r o n m e n t a l S t r e s s . In Genomtc Responses to Environmental Stress. Edited by Scandalios JG. San Diego: Academic Press, Inc; Adv Genetics 1990, 28:1-41. A thorough review of the biochemistry of oxidative stress and the response of plants to it. Currem k n o w l e d g e of enzymology, especially of catalase a n d superoxide dlsmutase, and molecular biology in plants is summarized. 27.

TSANG EWT, BOWLER C, HEROUART D, VAN CAMp W, VILLARROEL R, GENETELLO C, VAN MONTAGU M, INZE D: Differential Regulation of Superoxide Dismutases in P l a n t s E x p o s e d to E n v i r o a x m e n t a l S t r e s s . Plant Cell 1991, 3:783-792.

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TEPPERMANJM, DUNSMUIRP: T r a n s f o r m e d P l a n t s w i t h Elev a t e d Levels o f C h l o r o p l a s t S u p e r o x i d e D i s m u t a s e a r e n o t M o r e R e s i s t a n t to S u p e r o x i d e T o x i c i t y . Plant Mol Biol 1991, 14:501-511.

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W A D AH, GOMBOS Z, MURATA N: E n h a n c e m e n t o f c h i l l i n g T o l e r a n c e o f a C y a n o b a c t e r i u m b y G e n e t i c Man i p u l a t i o n o f Fatty Acid D e s a t u r a t i o n . Nature 1990, 347:200-203. A demonstration that lipid composition can be genetically engineered in cyanobacteria. Alterations in lipid composition conferred increased cold tolerance on photosynthetic activity.

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SOMERVILLEC, BROWSE J: P l a n t Lipids: M e t a b o l i s m , Mut a n t s a n d M e m b r a n e s . Science 1991, 252:80-87.

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HIGHTOWERR, BADEN C, PENZES E, LUND P, DUNSMUIRP: Expression of Antifreeze Proteins in Transgenic Plants. Plant Mol Biol 1991, 17:1013-1021.

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GURLEYWB, KEY JL: T r a n s c r i p t i o n a l R e g u l a t i o n o f t h e H e a t S h o c k R e s p o n s e - - A P l a n t P e r s p e c t i v e . Biochemistry 1991, 30:1-12.

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VIERL1NGE: R o l e s o f H e a t S h o c k P r o t e i n s i n P l a n t s . A n n u Rev Plant Physiol Plant Mol Biol 1991, 42:579-620. Summarizes the characteristics of the major classes of HSPs a n d the progress in molecular biology of the corresponding g e n e s in plants.

CHEN Q, VIERLING E: A n a l y s i s o f C o n s e r v e d D o m a i n s Identifxes a U n i q u e S t r u c t u r a l F e a t u r e o f a C h l o r o p l a s t H e a t S h o c k P r o t e i n . Mol Gen Genet 1991, 226:425-431.

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BOWLER C, SLOOTEN L, VANDENBRANDEN S, DE RYCKE R, BOTrERMAN J, SYBESMA C, VAN MONTAGU M, INZE D: M a n g a n e s e S u p e r o x i d e D i s m u t a s e C a n R e d u c e Cellular D a m a g e M e d i a t e d b y O x y g e n Radicals i n T r a n s g e n i c P l a n t s . EMBO J 1991, 10:1723-1732. A good demonstration of the progress in plant transformation: MnSOD g e n e s are targeted to either mitochondria or chloroplasts. 30.

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SCHULZE-LEFERTP, DANGL JL, BECKER-ANDR£ M, I-IAHLBROCK K, SCI-IULZ W: I n d u c i b l e In Vivo F o o t p r i n t s Deft_tie Seq u e n c e s N e c e s s a r y f o r UV L i g h t A c t i v a t i o n o f t h e Parsl e y C h a l c o n e S y n t h a s e G e n e . EMBO J 1989, 8:651-656.

WEISSHAARB, ARMSTRONG GA, BLOCK A, DA COSTA E SILVA O, HAHLBROCKK: L i g h t - i n d u c i b l e a n d C o n s t i t u t i v e l y Expressed DNA-binding Proteins Recognizing a Plant P r o m o t e r E l e m e n t w i t h F u n c t i o n a l R e l e v a n c e i n Light R e s p o n s i v e n e s s . EMBO J 1991, 10:1777-1786. Interesting comparison of the DNA-binding proteins of higher plains that recognize a similar core s e q u e n c e (ACGT).

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WINGENDER R, ROHRIG H, HORICKE C, SCHELLJ: Cis-regulat o r y E l e m e n t s I n v o l v e d i n Ultraviolet Light Regulation a n d Plant Defense. Plant Cell 1990, 2:1019-1026.

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FRITZEK, STAIGERD, CZAJAI, WALDENR, SCHELLJ, WING D: D e v e l o p m e n t a l a n d UV Light R e g u l a t i o n o f t h e Snapd r a g o n C h a l c o n e S y n t h a s e P r o m o t e r . Plant Cell 1991, 3:893-905.

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FEINBAUMEILL, STORZ G, AUSUBELFM: H i g h I n t e n s i t y a n d Blue Light Regulated E x p r e s s i o n o f C h i m e r i c C h a i c o n e S y n t h a s e Genes i n T r a n s g e n i c A r a b i d o p s i s t h a l i a n a Plants. Mol Gen Genet 1991, 226:449-456.

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DOUGLAS CJ, HAUFEE KD, ITES-MORALES M-E, ELLARD M, PASZKOWSKI U, HAHLBROCK K, DANGL JL: E x o n i c Seq u e n c e s are R e q u i r e d f o r Elicitor a n d Light Activation o f a P l a n t D e f e n s e Gene, b u t P r o m o t e r S e q u e n c e s a r e Sufficient for Tissue Specific E x p r e s s i o n . EMBO J 1991, 10:1767-1775.

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SHOWALTERAM, ZHOU J, RUMEAUD, WORST SG, VARNERJE: T o m a t o E x t e n s i n a n d E x t e n s i n - l i k e cDNAs: Structure a n d E x p r e s s i o n i n R e s p o n s e to W o u n d i n g . Plant Mol Biol 1991, 16:547-565.

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KEITH B, DONG X, AUSUBEL FM, FINK GR: Differential I n d u c t i o n o f 3 - d e o x y - n - a r a b i n o - h e p t u l o s o n a t e 7p h o s p h a t e synthase genes in Arabidopsis thaliana b y W o u n d i n g a n d P a t h o g e n i c Attack. Proc Natl A c a d Sci USA 1991, 88:8821-8825.

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g A N GZ, PARKH, LACY GH, CRAMERCL: Differential Activ a t i o n o f Potato 3-hydrox-y-3-methylglutaryl C o e n z y m e A R e d u c t a s e G e n e s b y W o u n d i n g a n d P a t h o g e n Challenge. Plant Cell 1991, 3:397-405.

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STURMA, CHRISPEELS MJ: cDNA C l o n i n g o f C a r r o t Extracellular ~-fructosidase a n d its E x p r e s s i o n i n R e s p o n s e to W o u n d i n g a n d Bacterial Infection. Plant Cell 1990, 2:1107-1119.

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PEARCEG, STRYDOMD, JOHNSON S, RYANCA: A P o l y p e p t i d e F r o m T o m a t o Leaves I n d u c e s W o u n d d n d u c i b l e Prot e i n a s e I n h i b i t o r P r o t e i n s . Science 1991, 253:895-898. A landmark paper that introduces a class of hormonal molecules never previously described for plants. 41.

FARMEREE, RYAN CA: I n t e r p l a n t C o m m u n i c a t i o n : Airb o r n e Methyl J a s m o n a t e I n d u c e s S y n t h e s i s o f Prot e i n a s e I n h i b i t o r s i n P l a n t Leaves. Proc Natl A c a d Sci USA 1990, 87:7713-7717. Identifies a second volatile compound (ethylene being the first) that mediates interplant communication. Methyl jasmonate released from sagebrush can induce PI synthesis in nearby tomato plants. 42.

PENA-CORTESH, WILLMITZER L, SANCHEZ-SERRANOJJ: Abscisic Acid Mediates W o u n d I n d u c t i o n b u t n o t Developmental-specific Expression of the Proteinase I n h i b i t o r H Gene Family. Plant Cell 1991, 3:963-972.

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WINGATEVPM, RYAN CA: U n i q u e l y Regulated P r o t e i n a s e I n h i b i t o r I Gene i n a Wild Tomato Species. Plant Physiol 1991, 97:496-501.

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PATERSONAH, DAMON S, HEWITrJD, ZAMIRD, RABINOWITCH HD, LINCOLN SE, LANDER ES, TANKSLEY SD: M e n d e l i a n Factors U n d e r l y i n g Quantitative Traits i n Tomato: C o m p a r i s o n Across Species, G e n e r a t i o n s a n d E n v i r o n merits. Genetics 1991, 127:181-197. Shows feasibility of breeding for quantitative traits using quantitative trait loci identified by RFLP mapping. Suggests that variation in a polygenic trait (e.g. soluble solids in fruits) may be the result of allelic variation in orthologous genes.

E Vierling, Department of Biochemistry, Life Sciences South, University of Arizona, Tucson, Arizona 85721, USA. JA Kimpel, Department of Agronomy, Miller Plant Sciences Building, University of Georgia, Athens, Georgia 30602, USA.