Showing papers in "Plant Physiology in 1995"

Root Architecture and Plant Productivity.

Abstract: Water and nutrient availability limit plant growth in a11 but a very few natural ecosystems. They limit yield in most agricultural ecosystems, and in the United States and other industrialized nations, intensive irrigation and fertilization have generated serious environmental problems. The acquisition of soil resources by plant root systems is therefore a subject of considerable interest in agriculture and ecology, as well as a complex and challenging problem in basic plant biology. Symbioses between roots and otlier organisms (notably mycorrhizas and N-fixing bacteria), modification of the rhizosphere through root exudates, and the uptake and transport characteristics of root axes are a11 important dimensions of this problem that are being actively researched by plant biologists. Another aspect of this problem that has received less attention, despite its probable importance, is root architecture. Recent methodological innovations present opportunities for improved under.standing of the functional importance of root architecture in the efficient acquisition of soil resources and plant adaptation to suboptimal soil conditions. The purpose of this Update is to briefly summarize conceptual issues and recent developments in the study of root architecture and to propose a framework for understanding its physiological basis.

Overexpression of [delta]-Pyrroline-5-Carboxylate Synthetase Increases Proline Production and Confers Osmotolerance in Transgenic Plants

Abstract: Proline (Pro) accumulation has been correlated with tolerance to drought and salinity stresses in plants. Therefore, overproduction of Pro in plants may lead to increased tolerance against these abiotic stresses. To test this possibility, we overexpressed in tobacco the mothbean [delta]-pyrroline-5-carboxylate synthetase, a bifunctional enzyme able to catalyze the conversion of glutamate to [delta]-pyrroline-5-carboxylate, which is then reduced to Pro. The transgenic plants produced a high level of the enzyme and synthesized 10- to 18-fold more Pro than control plants. These results suggest that activity of the first enzyme of the pathway is the rate-limiting factor in Pro synthesis. Exogenous supply of nitrogen further enhanced Pro production. The osmotic potentials of leaf sap from transgenic plants were less decreased under water-stress conditions compared to those of control plants. Overproduction of Pro also enhanced root biomass and flower development in transgenic plants under drought-stress conditions. These data demonstrated that Pro acts as an osmoprotectant and that overproduction of Pro results in the increased tolerance to osmotic stress in plants.

Aluminum Toxicity and Tolerance in Plants

Abstract: Aluminum (Al) is the most abundant metal in the earth's crust, comprising about 7% of its mass. Since many plant species are sensitive to micromolar concentrations of Al, the potential for soils to be Al toxic is considerable. Fortunately, most of the Al is bound by ligands or occurs in other nonphytotoxic forms such as aluminosilicates and precipitates. However, solubilization of this Al is enhanced by low pH and Al toxicity is a major factor limiting plant production on acid soils. Soil acidification can develop naturally when basic cations are leached from soils, but it can be accelerated by some farming practices and by acid rain (Kennedy, 1986). Strategies to maintain production on these soils include the application of lime to raise the soil pH and the use of plants that are tolerant of acid soils. Although Al toxicity has been identified as a problem of acid soils for over 70 years, our knowledge about the primary sites of toxicity and the chain of events that finally affects plant growth remains largely speculative. In this paper we review recent progress that has been made in our understanding of Al toxicity and the mechanisms of Al tolerance in plants.

Lectins as Plant Defense Proteins

Abstract: Many plant species contain carbohydrate-binding proteins, which are commonly referred to as either lectins or agglutinins. Generally speaking, lectins are proteins that bind reversibly to specific monoor oligosaccharides. Since the initial discovery of a hemagglutinating factor in castor bean extracts by Stillmark in 1888, several hundred of these proteins have been isolated and characterized in some detail with respect to their carbohydrate-binding specificity, molecular structure, and biochemical properties. Lectins from different plant species often differ with respect to their molecular structure and specificity. It is important, therefore, to realize that all plant lectins are artificially classified together solely on the basis of their ability to recognize and bind carbohydrates. Moreover, the question arises whether proteins with a completely different structure and sugar-binding specificity fulfill the same physiological role. No conclusive answer can be given to this question as yet, for the simple reason that the role of most plant lectins is not known with certainty. There is, however, growing evidence that most lectins play a role in the plant's defense against different kinds of plant-eating organisms. The idea that lectins may be involved in plant defense is not new. In an earlier review, Chrispeels and Raikhel (1991) critically assessed the defensive role of the phytohemagglutinin family and a number of chitin-binding proteins. During the last few years important progress has been made in the study of plant lectins in general and in the understanding of their effects on other organisms in particular. In this Update we summarize the recent developments that support the defensive role of plant lectins and, in addition, discuss earlier work in this field against the background of our present knowledge of this group of plant proteins.

Mechanisms of Cadmium Mobility and Accumulation in Indian Mustard

Abstract: Indian mustard (Brassica juncea L.), a high biomass crop plant, accumulated substantial amounts of cadmium, with bioaccumulation coefficients (concentration of Cd in dry plant tissue/concentration in solution) of up to 1100 in shoots and 6700 in roots at nonphytotoxic concentrations of Cd (0.1 [mu]g/mL) in solution. This was associated with a rapid accumulation of phytochelatins in the root, where the majority of the Cd was coordinated with sulfur ligands, probably as a Cd-S4 complex, as demonstrated by x-ray absorption spectroscopy. In contrast, Cd moving in the xylem sap was coordinated predominantly with oxygen or nitrogen ligands. Cd concentrations in the xylem sap and the rate of Cd accumulation in the leaves displayed similar saturation kinetics, suggesting that the process of Cd transport from solution through the root and into the xylem is mediated by a saturable transport system(s). However, Cd translocation to the shoot appeared to be driven by transpiration, since ABA dramatically reduced Cd accumulation in leaves. Within leaves, Cd was preferentially accumulated in trichomes on the leaf surface, and this may be a possible detoxification mechanism.

Ion homeostasis in NaCl stress environments

Abstract: Homeostasis can be defined as the tendency of a cell or an organism to maintain internal steady state, even in response to any environmental perturbation or stimulus tending to disturb normality, because of the coordinate responses of its constituent components. Typically, ions constantly flux in and out of cells in a controlled fashion with net flux adjusted to accommodate cellular requirements, thus creating an ionic homeostasis. When plant cells are exposed to salinity, mediated by high NaCl concentrations, kinetic steady states of ion transport for Na+ and Cland other ions, such as K+ and Ca2+, are disturbed (Binzel et al., 1988). High apoplastic levels of Na+ and Clalter aqueous and ionic thermodynamic equilibria, resulting in hyperosmotic stress, ionic imbalance, and toxicity. Thus, it is vital for the plant to re-establish cellular ion homeostasis for metabolic functioning and growth, that is, to adapt to the saline environment. Comparisons of what have been interpreted to be adaptive responses among various species lead to the conclusion that some salt-tolerant plants have evolved specialized complex mechanisms that allow adaptation to saline stress conditions. In fact, these unique mechanisms, such as salt glands, exist in few plant species and cannot be presumed to be ubiquitously functional for salt adaptation of all plants. However, intrinsically cellular-based mechanisms appear to be common to all genotypes and are a requisite for salt tolerance. Of paramount importance are those mechanisms that function to regulate ion homeostasis while mediating osmotic adjustment through the accumulation and intracellular compartmentation of ions that are predominant in the external environment. In this update we will focus principally on Na+ homeostasis in sodic environments; however, we also include discussions of H+, K+, Ca2+, and Clbecause of the interrelationship of these ions with Na+ homeostasis. Ion transport processes across the plasma membrane and the tonoplast will be emphasized because these are presumed to be most essential for the control of intracellular Na+ uptake and vacuolar compartmentation.

Dissection of Oxidative Stress Tolerance Using Transgenic Plants.

Abstract: Environmental stress is the major limiting factor in plant productivity. Much of the injury to plants caused by stress exposure is associated with oxidative damage at the cellular level. Widespread losses of forests and crops due to ozone pollution provide a highly visible example of oxidative stress (see Tingey et al., 1993, for a review), but less obvious losses caused by oxidative damage associated with periods of cold or drought also take their toll in the accumulation of incremental setbacks during a growing season. The role of ROIs in plant stress damage is indicated by the increased production of ROIs and the increased oxidative damage in tissues during stress. In plants, the highly energetic reactions of photosynthesis and an abundant oxygen supply make the chloroplast a particularly rich source of ROIs. High light intensity can lead to excess reduction of PSI so that CO2 fixation cannot keep pace and NADP+ pools are reduced. Under these conditions, 02 can compete for electrons from PSI, leading to the generation of ROIs through the Mehler reaction. When CO2 fixation is limited by environmental conditions such as cold temperatures or low CO2 availability (closed stomata), excess PSI reduction and increased ROI production can occur even at moderate light intensities. Efficient removal of ROIs from chloroplasts is critical, since H202 concentrations as low as 10 ptM can inhibit photosynthesis by 50% (Kaiser, 1979). Although the toxicity of *?2and H202 themselves is relatively low, their metal-dependent conversion to the highly toxic -OH via the Haber-Weiss reaction is thought to be responsible for the majority of the biological damage associated with these molecules. Antioxidant systems of plant chloroplasts include enzymes such as SOD and APX, and nonenzymatic components such as ascorbic acid and glutathione. The proposed ROI scavenging pathway of chloroplasts is shown in Figure 1 (Asada, 1994). Superoxide radicals are produced by the reduction of molecular oxygen at PSI via the Mehler reaction. This ?2- is rapidly dismuted to H202 by SOD that is associated with the thylakoid. The H202 produced is

Plant defensins: novel antimicrobial peptides as components of the host defense system.

Abstract: Various mechanisms to fend off microbial invaders have been devised by all living organisms, including microorganisms themselves. The most sophisticated of these mechanisms relies on the synthesis of immunoglobulins directed against specific microbial targets. However, immunoglobulin-based immunity operates only in a relatively minor subset of living species, namely the higher vertebrates. A much more ancient and widespread defense strategy involves the production of small peptides that exert antimicrobial properties. As products of single genes, antimicrobial peptides can be synthesized in a swift and flexible way, and because of their small size they can be produced by the host with a minimal input of energy and biomass. Wellknown examples of antimicrobial peptides are the cecropins that accumulate in the hemolymph of many invertebrates in response to injury or infection (reviewed by Boman and Hultmark, 1987) and the magainins that are secreted by glands in the skin of amphibians (reviewed by Bevins and Zasloff, 1990). Cecropins and magainins are small (20-40 residues) basic peptides displaying an amphipathic a-helical structure that can integrate in microbial membranes to form ion channels (Duclohier, 1994). Another class of antimicrobial peptides is formed by the Cys-rich peptides, which in contrast to cecropins and magainins, have a complex cystine-stabilized three-dimensional folding pattern often involving antiparallel ,3-sheets. Defensins are one class among the numerous types of Cys-rich antimicrobial peptides, which differ in length, number of cystine, bonds, or folding pattern (reviewed by Boman, 1995). Insect defensins (34-43 residues, three disulfide bridges) are, like cecropins, produced in a pathogeninducible manner by the insect fat body and secreted in the hemolymph (reviewed by Hoffmann and Hetru, 1992). Mammalian defensins (29-34 amino acids, three disulfide bridges) are produced by various specialized cells in the mammalian body (reviewed by Lehrer et al., 1993; Ganz and Lehrer, 1994). For example, they are very abundant in granules of phagocytic blood cells. These granules fuse with phagocytosis vesicles containing microorganisms, where the defensins are thought to contribute, together with other antimicrobial proteins and active oxygen species, to killing of the engulfed microorganisms. Defensins are also secreted by epithelial cells of the intestines and airways, where they may help maintain the normal microbial flora in a steady state. In addition, the expression of defensins in the airway epithelium has been shown to be up-regulated after exposure to bacterial lipopolysaccharides (Diamond et al., 1993). The importance of defensins in innate immunity of humans is underscored by the observation that certain disorders characterized by recurrent infections are associated with a lack of defensins in blood phagocytes (Ganz et al., 1988). Moreover, transposon mutants of a pathogenic Salmonella strain known to infect and grow inside phagocytes simultaneously lost their resistance to defensins (and other antimicrobial peptides) and their virulence (Groisman et al., 1992). Recently, we characterized a novel class of plant peptides whose structural and functional properties resemble those of insect and mammalian defensins. Hence, we termed this family of peptides "plant defensins" (Terras et al., 1995).

Phytochelatins and Related Peptides (Structure, Biosynthesis, and Function)

Abstract: Plants obtain micronutrients such as Cu and Zn from aquatic and terrestrial environments that may also provide the metals Cd, Pb, and Hg. These essential and nonessentia1 metals exist at low to high concentrations depending on natural and manmade disturbances. In a fluctuating environment the plants may experience shifting internal concentrations of these bioreactive metals. Therefore, it is beneficial for plants to have mechanisms that (a) maintain internal concentrations of essential metals between deficient and toxic limits and (b) keep nonessential metals below their toxicity thresholds. The proteins and peptides known as MTs sequester metals and thereby may accomplish cellular metal homeostasis and detoxification. These molecules are rich in Cys’s that provide thiols for binding the metals mentioned so far. The early work on plants was modeled after equine renal MT, a metal-induced protein with 20 Cys’s distributed uniquely within the sequence of 60 amino acids and M, of approximately 9000 (Robinson et al., 1993). Studies of yeasts, algae, and plants relied heavily on isolating the metal-induced components of M , of approximately 9000 that bound Cd or Cu. In many of these preparations Cys, Glu, and Gly accounted for 45 to 97% of the amino acids, which is inconsistent with a dose relationship to the archetypal form, equine renal MT. Two independent groups, one working with the fission yeast Schizosaccharomyces pombe (Kondo et al., 1984) and the other working with cultured cells of Rauvolfia serpentina (Grill et al., 1985), showed that the molecules binding Cd were a family of peptides with the primary structure (y-Glu-Cys),-Gly, where n = 2 to 7 depending on the organism. The peptide bond in the repeating Glu-Cys pairs is a y-carboxyamide linkage not synthesized on ribosomes. Consequently, these and related peptides are now designated class I11 MTs, which are defined as atypical, nontranslationally synthesized metal thiolate polypeptides (Robinson et al., 1993). No consensus has been reached concerning a trivial name for the peptides. The name cadystin holds priority (Kondo et al., 1984), with phytochelatin being popularly used for algae and plants (Grill et al., 1985). Recent progress on class I11 MTs

Arabidopsis Mutants Lacking Phenolic Sunscreens Exhibit Enhanced Ultraviolet-B Injury and Oxidative Damage

Abstract: We have assessed ultraviolet-B (UV-B)-induced injury in wild-type Arabidopsis thaliana and two mutants with altered aromatic secondary product biosynthesis. Arabidopsis mutants defective in the ability to synthesize UV-B-absorbing compounds (flavonoids in transparent testa 5 [tt5] and sinapate esters in ferulic acid hydroxylase 1 [fah1]) are more sensitive to UV-B than is the wild-type Landsberg erecta. Despite its ability to accumulate UV-absorptive flavonoid compounds, the ferulic acid hydroxylase mutant fah1 exhibits more physiological injury (growth inhibition and foliar lesions) than either wild type or tt5. The extreme UV-B sensitivity of fah1 demonstrates the importance of hydroxycinnamate esters as UV-B protectants. Consistent with the whole-plant response, the highest levels of lipid and protein oxidation products were seen in fah1. Ascorbate peroxidase enzyme activity was also increased in the leaves of UV-B-treated plants in a dose- and genotype-dependent manner. These results demonstrate that, in A. thaliana, hydroxycinnamates are more effective UV-B protectants than flavonoids. The data also indicate that A. thaliana responds to UV-B as an oxidative stress, and sunscreen compounds reduce the oxidative damage caused by UV-B.

Cadmium-Sensitive, cad1 Mutants of Arabidopsis thaliana Are Phytochelatin Deficient

Abstract: An allelic series of cad1, cadmium-sensitive mutants of Arabidopsis thaliana, was isolated. These mutants were sensitive to cadmium to different extents and were deficient in their ability to form cadmium-peptide complexes as detected by gel-filtration chromatography. Each mutant was deficient in its ability to accumulate phytochelatins (PCs) as detected by high-performance liquid chromatography and the amount of PCs accumulated by each mutant correlated with its degree of sensitivity to cadmium. The mutants had wild-type levels of glutathione, the substrate for PC biosynthesis, and in vitro assays demonstrated that each of the mutants was deficient in PC synthase activity. These results demonstrate conclusively the importance of PCs for cadmium tolerance in plants.

How and Why Do Plants Inactivate Homologous (Trans)genes

Abstract: Gene silencing in transgenic plants has emerged in the last 5 years as a topic of intense interest for both applied and basic plant scientists. From the applied side, gene silencing has come as an unwelcome surprise. Early reviews of the prospects for plant genetic engineering did not pinpoint this as a potential obstacle. Rather, the anticipated challenge was to identify tissueand stage-specific promoters that could be used to obtain regulated transgene expression. Yet, silencing of transgenes is turning out to be a substantial problem, as described recently in an aptly titled review, "Transgene Inactivation: Plants Fight Back!" (Finnegan and McElroy, 1994). According to this article, of 30 companies polled, nearly all reported some problems with unwanted silencing of transgenes. While companies are struggling to find ways to avoid silencing, a small cadre of basic scientists has become fascinated by the phenomenon and is analyzing a variety of silencing systems. To this latter group, the phenomenon of silencing represents more than just an unwanted response to foreign genes; rather, it has opened a door that might lead to a deeper understanding of previously unsuspected ways that plants naturally use homologous or complementary nucleic acid sequences to modify gene expression, both in the nucleus at the DNA level and in the nucleus or cytoplasm as a means to control excess production of mRNA or replication of RNA pathogens. Initial studies of transgenic plants concentrated on those showing tissue-specific and/or high levels of expression. Plants that did not exhibit the desired expression characteristics were usually discarded. It was only a matter of time, however, until such plants would begin to be treated as objects of scientific investigation in their own right. In the late 1980s, papers started to appear that were devoted solely to describing cases of silencing that involved transgene/transgene or transgene/endogenous gene interactions. A central feature of all of these studies was that silencing was associated with multiple copies of homologous DNA sequences, which could comprise protein-coding regions, promoters, or both. Although the initial impulse was to lump these first cases together as a single phenomenon, it has since become clear that several different mechanisms are probably involved. These different mechanisms are united, however, in the sense that they all involve variations on nucleic acid interactions: DNA-DNA, RNA-RNA, and DNA-RNA. A general term that encompasses all of these phenomena is "homology-dependent gene silencing." Numerous detailed reviews of this topic are available (Jorgensen, 1992; Matzke and Matzke, 1993; Flavell, 1994; Matzke et al., 1994b; Mol et al., 1994).

Salicylic acid inhibits synthesis of proteinase inhibitors in tomato leaves induced by systemin and jasmonic acid

Abstract: Salicylic acid (SA) and acetylsalicylic acid (ASA), previously shown to inhibit proteinase inhibitor synthesis induced by wounding, oligouronides (H.M. Doherty, R.R. Selvendran, D.J. Bowles [1988] Physiol Mol Plant Pathol 33: 377-384), and linolenic acid (H. Pena-Cortes, T. Albrecht, S. Prat, E.W. Weiler, L. Willmitzer [1993] Planta 191: 123-128), are shown here to be potent inhibitors of systemin-induced and jasmonic acid (JA)-induced synthesis of proteinase inhibitor mRNAs and proteins. The inhibition by SA and ASA of proteinase inhibitor synthesis induced by systemin and JA, as well as by wounding and oligosaccharide elicitors, provides further evidence that both oligosaccharide and polypeptide inducer molecules utilize the octadecanoid pathway to signal the activation of proteinase inhibitor genes. Tomato (Lycopersicon esculentum) leaves were pulse labeled with [35S]methionine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the inhibitory effects of SA are shown to be specific for the synthesis of a small number of JA-inducible proteins that includes the proteinase inhibitors. Previous results have shown that SA inhibits the conversion of 13S-hydroperoxy linolenic acid to 12-oxo-phytodienoic acid, thereby inhibiting the signaling pathway by blocking synthesis of JA. Here we report that the inhibition of synthesis of proteinase inhibitor proteins and mRNAs by SA in both light and darkness also occurs at a step in the signal transduction pathway, after JA synthesis but preceding transcription of the inhibitor genes.

Improved Performance of Transgenic Fructan-Accumulating Tobacco under Drought Stress.

Abstract: Fructans are polyfructose molecules produced by approximately 15% of the flowering plant species. It is possible that, in addition to being a storage carbohydrate, fructans have other physiological roles. Owing to their solubility they may help plants survive periods of osmotic stress induced by drought or cold. To investigate the possible functional significance of fructans, use was made of transgenic tobacco (Nicotiana tabacum) plants that accumulate bacterial fructans and hence possess an extra sink for carbohydrate. Biomass production was analyzed during drought stress with the use of lines differing only in the presence of fructans. Fructan-producing tobacco plants performed significantly better under polyethylene-glycol-mediated drought stress than wild-type tobacco. The growth rate of the transgenic plants was significantly higher (+55%), as were fresh weight (+33%) and dry weight (+59%) yields. The difference in weight was observed in all organs and was particularly pronounced in roots. Under unstressed control conditions the presence of fructans had no significant effect on growth rate and yield. Under all conditions the total nonstructural carbohydrate content was higher in the transgenic plants. We conclude that the introduction of fructans in this non-fructan-producing species mediates enhanced resistance to drought stress.

Changes in Isozyme Profiles of Catalase, Peroxidase, and Glutathione Reductase during Acclimation to Chilling in Mesocotyls of Maize Seedlings.

Abstract: The response of antioxidants to acclimation and chilling in various tissues of dark-grown maize (Zea mays L.) seedlings was examined in relation to chilling tolerance and protection from chilling-induced oxidative stress. Chilling caused an accumulation of H2O2 in both the coleoptile + leaf and the mesocotyl (but not roots), and acclimation prevented this accumulation. None of the antioxidant enzymes were significantly affected by acclimation or chilling in the coleoptile + leaf or root. However, elevated levels of glutathione in acclimated seedlings may contribute to an enhanced ability to scavenge H2O2 in the coleoptile + leaf. In the mesocotyl (visibly most susceptible to chilling), catalase3 was elevated in acclimated seedlings and may represent the first line of defense from mitochondria-generated H2O2. Nine of the most prominent peroxidase isozymes were induced by acclimation, two of which were located in the cell wall, suggesting a role in lignification. Lignin content was elevated in mesocotyls of acclimated seedlings, likely improving the mechanical strength of the mesocotyl. One cytosolic glutathione reductase isozyme was greatly decreased in acclimated seedlings, whereas two others were elevated, possibly resulting in improved effectiveness of the enzyme at low temperature. When taken together, these responses to acclimation illustrate the potential ways in which chilling tolerance may be improved in preemergent maize seedlings.

Cold Acclimation of Arabidopsis thaliana (Effect on Plasma Membrane Lipid Composition and Freeze-Induced Lesions)

Abstract: Maximum freezing tolerance of Arabidopsis thaliana L. Heyn (Columbia) was attained after 1 week of cold acclimation at 2[deg]C. During this time, there were significant changes in both the lipid composition of the plasma membrane and the freeze-induced lesions that were associated with injury. The proportion of phospholipids increased from 46.8 to 57.1 mol% of the total lipids with little change in the proportions of the phospholipid classes. Although the proportion of di-unsaturated species of phosphatidylcholine and phosphatidylethanolamine increased, mono-unsaturated species were still the preponderant species. The proportion of cerebrosides decreased from 7.3 to 4.3 mol% with only small changes in the proportions of the various molecular species. The proportion of free sterols decreased from 37.7 to 31.2 mol%, but there were only small changes in the proportions of sterylglucosides and acylated sterylglucosides. Freezing tolerance of protoplasts isolated from either nonacclimated or cold-acclimated leaves was similar to that of leaves from which the protoplasts were isolated (-3.5[deg]C for nonacclimated leaves; -10[deg]C for cold-acclimated leaves). In protoplasts isolated from nonacclimated leaves, the incidence of expansion-induced lysis was [less than or equal to]10% at any subzero temperature. Instead, freezing injury was associated with formation of the hexagonal II phase in the plasma membrane and subtending lamellae. In protoplasts isolated from cold-acclimated leaves, neither expansion-induced lysis nor freeze-induced formation of the hexagonal II phase occurred. Instead, injury was associated with the "fracture-jump lesion," which is manifested as localized deviations of the plasma membrane fracture plane to subtending lamellae. The relationship between the freeze-induced lesions and alterations in the lipid composition of the plasma membrane during cold acclimation is discussed.

Expansin Mode of Action on Cell Walls (Analysis of Wall Hydrolysis, Stress Relaxation, and Binding)

Abstract: The biochemical mechanisms underlying cell wall expansion in plants have long been a matter of conjecture. Previous work in our laboratory identified two proteins (named "expansins") that catalyze the acid-induced extension of isolated cucumber cell walls. Here we examine the mechanism of expansin action with three approaches. First, we report that expansins did not alter the molecular mass distribution or the viscosity of solutions of matrix polysaccharides. We conclude that expansins do not hydrolyze the major pectins or hemicelluloses of the cucumber wall. Second, we investigated the effects of expansins on stress relaxation of isolated walls. These studies show that expansins account for the pH-sensitive and heat-labile components of wall stress relaxation. In addition, these experiments show that expansins do not cause a progressive weakening of the walls, as might be expected from the action of a hydrolase. Third, we studied the binding of expansins to the cell wall and its components. The binding characteristics are consistent with this being the site of expansin action. We found that expansins bind weakly to crystalline cellulose but that this binding is greatly increased upon coating the cellulose with various hemicelluloses. Xyloglucan, either solubilized or as a coating on cellulose microfibrils, was not very effective as a binding substrate. Expansins were present in growing cell walls in low quantities (approximately 1 part in 5000 on a dry weight basis), suggesting that they function catalytically. We conclude that expansins bind at the interface between cellulose microfibrils and matrix polysaccharides in the wall and induce extension by reversibly disrupting noncovalent bonds within this polymeric network. Our results suggest that a minor structural component of the matrix, other than pectin and xyloglucan, plays an important role in expansin binding to the wall and, presumably, in expansin action.

The shikimate pathway as an entry to aromatic secondary metabolism.

Abstract: The shikimate pathway is often referred to as the common aromatic biosynthetic pathway, even though nature does not synthesize all aromatic compounds by this route. This metabolic sequence converts the primary metabolites PEP and erythrose-4-P to chorismate, the last common precursor for the three aromatic amino acids Phe, Tyr, and Trp and for p-amino and p-hydroxy benzoate (Fig. 1). The shikimate pathway is found in bacteria, fungi, and plants. In monogastric animals, Phe and Trp are essential amino acids that have to come with the diet and Tyr is directly derived from Phe. Since bacteria use in excess of 90% of their metabolic energy for protein biosynthesis, for most prokaryotes, the three aromatic amino acids represent nearly the entire output of aromatic biosynthesis, and regulatory mechanisms for shikimate pathway activity are triggered by the intracellular concentrations of Phe, Tyr, and Trp. This is not so in higher plants, in which the aromatic amino acids are the precursors for a large variety of secondary metabolites with aromatic ring structures that often make up a substantial amount of the total dry weight of a plant. Among the many aromatic secondary metabolites are flavonoids, many phytoalexins, indole acetate, alkaloids such as morphine, UV light protectants, and,

MgATP-Dependent Transport of Phytochelatins Across the Tonoplast of Oat Roots

Abstract: In Cd-exposed oat (Avena sativa) roots Cd was found to be associated primarily with the phytochelatin ([gamma]-glutamylcysteinyl)3-glutamic acid [([gamma]EC)3G], with a peptide to Cd ratio of 1:3 (cysteine to Cd ratio of 1:1), even though both ([gamma]EC)2G and ([gamma]EC)3G were present in the roots. Phytochelatins are known to accumulate in the vacuoles of plant cells on exposure to Cd, but the mechanism is not clear. Here we present evidence for the transport of the phytochelatins ([gamma]EC)3G and ([gamma]EC)2G as well as the Cd complex Cd-([gamma]EC)3G across the tonoplast of oat roots. Transport of ([gamma]EC)3G had a Km, for MgATP of 0.18 mM and a Vmax of 0.7 to 1 nmol mg-1 protein min-1. Transport of ([gamma]EC)3G was also energized by MgGTP and to a lesser extent MgUTP and was highly sensitive to orthovanadate, with a 50%-inhibitory concentration of 0.9 [mu]M. The Cd complex Cd-([gamma]EC)3G and ([gamma]EC)2G were also transported in a MgATP-dependent, vanadate-sensitive manner. Therefore, this process is a candidate for the transport of both phytochelatins, and Cd as its peptide complex, from the cytoplasm into the vacuole.

Overexpression of glutathione reductase but not glutathione synthetase leads to increases in antioxidant capacity and resistance to photoinhibition in poplar trees.

Abstract: A poplar hybrid, Populus tremula x Populus alba, was transformed with the bacterial genes for either glutathione reductase (GR) (gor) or glutathione synthetase (GS) (gshII). When the gor gene was targeted to the chloroplasts, leaf GR activities were up to 1000 times greater than in all other lines. In contrast, targeting to the cytosol resulted in 2 to 10 times the GR activity. GR mRNA, protein, and activity levels suggest that bacterial GR is more stable in the chloroplast. When the gshII gene was expressed in the cytosol, GS activities were up to 100 times greater than in other lines. Overexpression of GR or GS in the cytosol had no effect on glutathione levels, but chloroplastic-GR expression caused a doubling of leaf glutathione and an increase in reduction state. The high-chloroplastic-GR expressors showed increased resistance to photoinhibition. The herbicide methyl viologen inhibited CO2 assimilation in all lines, but the increased leaf levels of glutathione and ascorbate in the high-chloroplastic-GR expressors persisted despite this treatment. These results suggest that overexpression of GR in the chloroplast increases the antioxidant capacity of the leaves and that this improves the capacity to withstand oxidative stress.

Plant Protein Kinase Families and Signal Transduction

Abstract: Enzymes of the eukaryotic protein kinase superfamily catalyze the reversible transfer of the gamma-phosphate from ATP to amino acid side chains of proteins. Protein kinase function can be counteracted by the action of phosphoprotein phosphatases. Phosphorylation status of a protein can have profound effects on its activity and interaction with other proteins. An estimated 1 to 3% of functional eukaryotic genes encode protein kinases, suggesting that they are involved in many aspects of cellular regulation and metabolism. In plants, protein phosphorylation has been implicated in responses to many signals, including light, pathogen invasion, hormones, temperature stress, and nutrient deprivation. Activities of several plant metabolic and regulatory enzymes are also controlled by reversible phosphorylation. As might be expected from this diversity of function, there is a large array of different protein kinases. Purification of protein kinases and their subsequent cloning, facilitated by the PCR and advances in homology-based cloning techniques, as well as functional analyses, including complementation of conditional yeast mutants and positional cloning of mutant plant genes, has already led to identification of more than 70 plant protein kinase genes. However, the precise functional roles of specific protein kinases and phosphatases during plant growth and development have been elucidated for only a few.

Isolation and Expression of Three Gibberellin 20-Oxidase cDNA Clones from Arabidopsis

Abstract: Using degenerate oligonucleotide primers based on a pumpkin (Cucurbita maxima) gibberellin (GA) 20-oxidase sequence, six different fragments of dioxygenase genes were amplified by polymerase chain reaction from Arabidopsis thaliana genomic DNA. One of these was used to isolate two different full-length cDNA clones, At2301 and At2353, from shoots of the GA-deficient Arabidopsis mutant ga1–2. A third, related clone, YAP169, was identified in the Database of Expressed Sequence Tags. The cDNA clones were expressed in Escherichia coli as fusion proteins, each of which oxidized GA12 at C-20 to GA15, GA24, and the C19 compound GA9, a precursor of bioactive GAs; the C20 tricarboxylic acid compound GA25 was formed as a minor product. The expression products also oxidized the 13-hydroxylated substrate GA53, but less effectively than GA12. The three cDNAs hybridized to mRNA species with tissue-specific patterns of accumulation, with At2301 being expressed in stems and inflorescences, At2353 in inflorescences and developing siliques, and YAP169 in siliques only. In the floral shoots of the ga1–2 mutant, transcript levels corresponding to each cDNA decreased dramatically after GA3 application, suggesting that GA biosynthesis may be controlled, at least in part, through down-regulation of the expression of the 20-oxidase genes.

Pathogenesis-Related PR-1 Proteins Are Antifungal (Isolation and Characterization of Three 14-Kilodalton Proteins of Tomato and of a Basic PR-1 of Tobacco with Inhibitory Activity against Phytophthora infestans)

Abstract: Three distinct basic 14-kD proteins, P14a, P14b, and P14c, were isolated from tomato (Lycopersicon esculentum Mill. cv Baby) leaves infected with Phytophthora infestans. They exhibited antifungal activity against P. infestans both in vitro (inhibition of zoospore germination) and in vivo with a tomato leaf disc assay (decrease in infected leaf surface). Serological cross-reactions and amino acid sequence comparisons showed that the three proteins are members of the PR-1 group of pathogenesis-related (PR) proteins. P14a and P14b showed high similarity to a previously characterized P14, whereas P14c was found to be very similar to a putative basic-type PR-1 from tobacco predicted from isolated DNA clones. This protein, named PR-1 g, was purified from virus-infected tobacco (Nicotiana tabacum Samsun NN) leaves and characterized by amino acid microsequencing, along with the well-known acidic tobacco PR-1a, PR-1b, and PR-1c. The various tomato and tobacco PR-1 proteins were compared for their biological activity and found to display differential fungicidal activity against P. infestans in both the in vitro and in vivo assays, the most efficient being the newly characterized tomato P14c and tobacco PR-1g.

Hydrogen Peroxide Stimulates Salicylic Acid Biosynthesis in Tobacco

Abstract: Hydrogen peroxide induced the accumulation of free benzoic acid (BA) and salicylic acid (SA) in tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaves. Six hours after infiltration with 300 mM H2O2, the levels of BA and SA in leaves increased 5-fold over the levels detected in control leaves. The accumulation of BA and SA was preceded by the rapid activation of benzoic acid 2-hydroxylase (BA2H) in the H2O2-infiltrated tissues. This enzyme catalyzes the formation of SA from BA. Enzyme activation could be reproduced in vitro by addition of H2O2 or cumene hydroperoxide to the assay mixture. H2O2 was most effective in vitro when applied at 6 mM. In vitro activation of BA2H by peroxides was inhibited by the catalase inhibitor 3-amino-1,2,4-triazole. We suggest that H2O2 activates SA biosynthesis via two mechanisms. First, H2O2 stimulates BA2H activity directly or via the formation of its substrate, molecular oxygen, in a catalase-mediated reaction. Second, higher BA levels induce the accumulation of BA2H protein in the cells and provide more substrate for this enzyme.

Salicylic Acid in Rice (Biosynthesis, Conjugation, and Possible Role).

Abstract: Salicylic acid (SA) is a natural inducer of disease resistance in some dicotyledonous plants Rice seedlings (Oryza sativa L) had the highest levels of SA among all plants tested for SA content (between 001 and 3719 [mu]g/g fresh weight) The second leaf of rice seedlings had slightly lower SA levels than any younger leaves To investigate the role of SA in rice disease resistance, we examined the levels of SA in rice (cv M-201) after inoculation with bacterial and fungal pathogens SA levels did not increase after inoculation with either the avirulent pathogen Pseudomonas syringae D20 or with the rice pathogens Magnaporthe grisea, the causal agent of rice blast, and Rhizoctonia solani, the causal agent of sheath blight However, leaf SA levels in 28 rice varieties showed a correlation with generalized blast resistance, indicating that SA may play a role as a constitutive defense compound Biosynthesis and metabolism of SA in rice was studied and compared to that of tobacco Rice shoots converted [14C]cinnamic acid to SA and the lignin precursors p-coumaric and ferulic acids, whereas [14C]benzoic acid was readily converted to SA The data suggest that in rice, as in tobacco, SA is synthesized from cinnamic acid via benzoic acid In rice shoots, SA is largely present as a free acid; however, exogenously supplied SA was converted to [beta]-O-D-glucosylSA by an SA-inducible glucosyltransferase (SA-GTase) A 7-fold induction of SA-GTase activity was observed after 6 h of feeding 1 mM SA Both rice roots and shoots showed similar patterns of SA-GTase induction by SA, with maximal induction after feeding with 1 mM SA

Low Temperature Induces the Accumulation of Phenylalanine Ammonia-Lyase and Chalcone Synthase mRNAs of Arabidopsis thaliana in a Light-Dependent Manner.

Abstract: Anthocyanins, which accumulate in leaves and stems in response to low temperature and changes in light intensity, are synthesized through the phenylpropanoid pathway that is controlled by key enzymes that include phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) In this work we demonstrate that PAL and CHS mRNAs accumulate in leaves of Arabidopsis thaliana (L) Heynh upon exposure to low temperature in a light-dependent manner The regulation of the PAL1 gene expression by low temperature and light was examined by analyzing the expression of the [beta]-glucuronidase (uidA) reporter gene in transgenic Arabidopsis plants containing the uidA gene of Escherichia coli under the control of the PAL1 promoter The results indicate that the accumulation of PAL1 mRNA is transcriptionally regulated Histochemical staining for [beta]-glucuronidase activity showed that the PAL1 promoter is preferentially activated in photosynthetically active cells, paralleling anthocyanin accumulation Moreover, we show that light may also be implicated in the regulation of the CHS gene in response to bacterial infiltration Finally, using two transparent testa Arabidopsis mutants that are unable to accumulate anthocyanins, we demonstrate that these pigments are not required for successful development of freezing tolerance in this species

The Electronic Plant Gene Register

Abstract: Plant Gene Register titles for PGR 99–174 to PGR 99–187 appear below. The sequences have beendeposited in GenBank and the articles listed online through the World Wide Web.To cite an electronic Plant Gene Register article as a bibliographic reference, follow the stylegiven below:Park S, Thornburg RW (1998) Characterization of UMP kinase cDNAs from rice (accession nos.AF187062 and AF187063) (PGR 99–174). Plant Physiol

Characterization of the Cell-Wall Polysaccharides of Arabidopsis thaliana Leaves

Abstract: The cell-wall polysaccharides of Arabidopsis thaliana leaves have been isolated, purified, and characterized. The primary cell walls of all higher plants that have been studied contain cellulose, the three pectic polysaccharides homogalacturonan, rhamnogalacturonan I and rhamnogalacturonan II, the two hemicelluloses xyloglucan and glucuronoarabinoxylan, and structural glycoproteins. The cell walls of Arabidopsis leaves contain each of these components and no others that we could detect, and these cell walls are remarkable in that they are particularly rich in phosphate buffer-soluble polysaccharides (34% of the wall). The pectic polysaccharides of the purified cell walls consist of rhamnogalacturonan I (11%), rhamnogalacturonon II (8%), and homogalacturonan (23%). Xyloglucan (XG) accounts for 20% of the wall, and the oligosaccharide fragments generated from XG by endoglucanase consist of the typical subunits of other higher plant XGs. Glucuronoarabinoxylan (4%), cellulose (14%) and protein (14%) account for the remainder of the wall. Except for the phosphate buffer-soluble pectic polysaccharides, the polysaccharides of Arabidopsis leaf cell walls occur in proportions similar to those of other plants. The structure of the Arabidopsis cell-wall polysaccharides are typical of those of many other plants.

Methanol Emission from Leaves (Enzymatic Detection of Gas-Phase Methanol and Relation of Methanol Fluxes to Stomatal Conductance and Leaf Development)

Abstract: We recently reported the detection of methanol emissions from leaves (R. MacDonald, R. Fall [1993] Atmos Environ 27A: 1709–1713). This could represent a substantial flux of methanol to the atmosphere. Leaf methanol production and emission have not been investigated in detail, in part because of difficulties in sampling and analyzing methanol. In this study we used an enzymatic method to convert methanol to a fluorescent product and verified that leaves from several species emit methanol. Methanol was emitted almost exclusively from the abaxial surfaces of hypostomatous leaves but from both surfaces of amphistomatous leaves, suggesting that methanol exits leaves via stomates. The role of stomatal conductance was verified in experiments in which stomates were induced to close, resulting in reduced methanol. Free methanol was detected in bean leaf extracts, ranging from 26.8 [mu]g g-1 fresh weight in young leaves to 10.0 [mu]g g-1 fresh weight in older leaves. Methanol emission was related to leaf development, generally declining with increasing leaf age after leaf expansion; this is consistent with volatilization from a cellular pool that declines in older leaves. It is possible that leaf emission could be a major source of methanol found in the atmosphere of forests.

Is the Reaction Catalyzed by 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase a Rate-Limiting Step for Isoprenoid Biosynthesis in Plants?

Abstract: 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes the irreversible conversion of 3-hydroxy-3-methylglutaryl coenzyme A to mevalonate and is considered a key regulatory step controlling isoprenoid metabolism in mammals and fungi. The rate-limiting nature of this enzyme for isoprenoid biosynthesis in plants remains controversial. To investigate whether HMGR activity could be limiting in plants, we introduced a constitutively expressing hamster HMGR gene into tabacco (Nicotiana tabaccum L.) plants to obtain unregulated HMGR activity. The impact of the resulting enzyme activity on the biosynthesis and accumulation of particular isoprenoids was evaluated. Expression of the hamster HMGR gene led to a 3- to 6-fold increase in the total HMGR enzyme activity. Total sterol accumulation was consequently increased 3- to 10-fold, whereas end-product sterols such as sitosterol, campesterol, and stigmasterol were increased only 2-fold. The level of cycloartenol, a sterol biosynthetic intermediate, was increased more than 100-fold. Although the synthesis of total sterols appears to be limited normally by HMGR activity, these results indicate that the activity of one or more later enzyme(s) in the pathway must also be involved in determining the relative accumulation of end-product sterols. The levels of other isoprenoids such as carotenoids, phytol chain of chlorophyll, and sesquiterpene phytoalexins were relatively unaltered in the transgenic plants. It appears from these results that compartmentation, channeling, or other rate-determining enzymes operate to control the accumulation of these other isoprenoid end products.