Richard G. Jensen

Adaptations to Environmental Stresses.

Environmental stresses come in many forms, yet the most prevalent stresses have in common their effect on plant water status. The availability of water for its biological roles as solvent and transport medium, as electron donor in the Hill reaction, and as evaporative coolant is often impaired by environmental conditions. Although plant species vary in their sensitivity and response to the decrease in water potential caused by drought, low temperature, or high salinity, it may be assumed that all plants have encoded capability for stress perception, signaling, and response. First, most cultivated species have wild relatives that exhibit excellent tolerance to abiotic stresses. Second, biochemical studies have revealed similarities in processes induced by stress that lead to accumulated metabolites in vascular and nonvascular plants, algae, fungi, and bacteria (Csonka, 1989; Galinski, 1993; Potts, 1994). These metabolites include nitrogen-containing compounds (proline, other amino acids, quaternary amino compounds, and polyamines) and hydroxyl compounds (sucrose, polyols, and oligosaccharides) (McCue and Hanson, 1990). Accumulation of any single metabolite is not restricted to taxonomic groupings, indicating that these are evolutionarily old traits. Third, molecular studies have revealed that a wide variety of species express a common set of genes and similar proteins (for example, Rab-related proteins and dehydrins) when stressed (Skriver and Mundy, 1990; Vilardell et al., 1994). Although functions for many of these genes have not yet been unequivocally assigned, it is likely, based on their characteristics, that these proteins play active roles in the response to stress. Learning about the biochemical and molecular mechanisms by which plants tolerate environmentat stresses is necessary for genetic engineering approaches to improving crop performance under stress. By investigating plants under stress, we can learn about the plasticity of metabolic pathways and the limits to their functioning. Also, questions of an ecological and evolutionary nature need investigation. Are the genes that confer salt tolerance on halophytes and/or drought tolerance on xerophytes evolutionarily ancient genes that have been selected against in saltand drought-sensitive plants (glycophytes) for the sake of productivity? Or have some species obtained nove1 genes in their evolutionary history that have enabled them to occupy stressful environments? How will the

Strategies for engineering water-stress tolerance in plants

Water deficit is the commonest environmental stress factor limiting plant productivity. The ability of plants to tolerate water deficit is determined by multiple biochemical pathways that facilitate retention and/or acquisition of water, protect chloroplast functions, and maintain ion homeostasis. Essential pathways include those that lead to synthesis of osmotically active metabolites and specific proteins that control ion and water flux, support scavenging of oxygen radicals, or may act as chaperones. The ability of plants to detoxify radicals under conditions of water deficit is probably the most critical requirement. Many stress-tolerant species accumulate methylated metabolites, which play a crucial dual role as osmoprotectants, and as radical scavengers. Their synthesis is correlated with stress-induced enhancement of photorespiration. However, transfer of individual genes from tolerant plants only confers marginally increased water-stress tolerance to stress-sensitive species: tolerance engineering will probably require the transfer of multiple genes.

Stress protection of transgenic tobacco by production of the osmolyte mannitol

The accumulation of sugar alcohols and other low molecular weight metabolites such as proline and glycine-betaine is a widespread response that may protect against environmental stress that occurs in a diverse range of organisms. Transgenic tobacco plants that synthesize and accumulate the sugar alcohol mannitol were engineered by introduction of a bacterial gene that encodes mannitol 1 -phosphate dehydrogenase. Growth of plants from control and mannitol-containing lines in the absence and presence of added sodium chloride was analyzed. Plants containing mannitol had an increased ability to tolerate high salinity.

Increased Resistance to Oxidative Stress in Transgenic Plants by Targeting Mannitol Biosynthesis to Chloroplasts

To investigate the potential role of a polyol, mannitol, in oxidative stress protection, a bacterial mannitol-1-phosphate dehydrogenase gene was targeted to chloroplasts by the addition of an amino-terminal transit peptide. Transgenic tobacco (Nicotiana tabacum) lines accumulate mannitol at concentrations ranging from 2.5 to 7 [mu]mol/g fresh weight. Line BS1-31 accumulated approximately 100 mM mannitol in chloroplasts and was identical to the wild type in phenotype and photosynthetic performance. The presence of mannitol in chloroplasts resulted in an increased resistance to methyl viologen (MV)-induced oxidative stress, documented by the increased retention of chlorophyll in transgenic leaf tissue following MV treatment. In the presence of MV, isolated mesophyll cells of BS1-31 exhibited higher CO2 fixation than the wild type. When the hydroxyl radical probe dimethyl sulfoxide was introduced into cells, the initial formation rate of methane sulfinic acid was significantly lower in cells containing mannitol in the chloroplast compartment than in wild-type cells, indicating an increased hydroxyl radical-scavenging capacity in BS1-31 tobacco. We suggest that the chloroplast location of mannitol can supplement endogenous radical-scavenging mechanisms and reduce oxidative damage of cells by hydroxyl radicals.

Mannitol Protects against Oxidation by Hydroxyl Radicals

Hydroxyl radicals may be responsible for oxidative damage during drought or chilling stress. We have shown that the presence of mannitol in chloroplasts can protect plants against oxidative damage by hydroxyl radicals (B. Shen, R.G. Jensen, H.J. Bohnert [1997] Plant Physiol 113: 1177–1183). Here we identify one of the target enzymes that may be protected by mannitol. Isolated thylakoids in the presence of physiological concentrations of Fe2+ generated hydroxyl radicals that were detected by the conversion of phenylalanine into tyrosine. The activity of phosphoribulokinase (PRK), a thiol-regulated enzyme of the Calvin cycle, was reduced by 65% in illuminated thylakoids producing hydroxyl radicals. Mannitol (125 mM) and sodium formate (15 mM), both hydroxyl radical scavengers, and catalase (3000 units mL-1) prevented loss of PRK activity. In contrast, superoxide dismutase (300 units mL-1) and glycine betaine (125 mM) were not effective in protecting PRK against oxidative inactivation. Ribulose-1,5-bisphosphate carboxylase/oxygenase activity was not affected by hydroxyl radicals. We suggest that the stress-protective role of mannitol may be to shield susceptible thiol-regulated enzymes like PRK plus thioredoxin, ferredoxin, and glutathione from inactivation by hydroxyl radicals in plants.

Increased Salt and Drought Tolerance by D-Ononitol Production in Transgenic Nicotiana tabacum L

A cDNA encoding myo-inositol O-methyltransferase (IMT1) has been transferred into Nicotiana tabacum cultivar SR1. During drought and salt stress, transformants (I5A) accumulated the methylated inositol D-ononitol in amounts exceeding 35 [mu]mol g-1 fresh weight In I5A, photosynthetic CO2 fixation was inhibited less during salt stress and drought, and the plants recovered faster than wild type. One day after rewatering drought-stressed plants, I5A photosynthesis had recovered 75% versus 57% recovery with cultivar SR1 plants. After 2.5 weeks of 250 mM NaCl in hydroponic solution, I5A fixed 4.9 [plus or minus] 1.4 [mu]mol CO2 m-2 s-1, whereas SR1 fixed 2.5 [plus or minus] 0.6 [mu]mol CO2 m-2 s-1. myo-Inositol, the substrate for IMT1, increases in tobacco under stress. Preconditioning of I5A plants in 50 mM NaCl increased D-ononitol amounts and resulted in increased protection when the plants were stressed subsequently with 150 mM NaCl. Pro, Suc, Fru, and Glc showed substantial diurnal fluctuations in amounts, but D-ononitol did not. Plant transformation resulting in stress-inducible, stable solute accumulation appears to provide better protection under drought and salt-stress conditions than strategies using osmotic adjustment by metabolites that are constitutively present.

Activation of ribulose bisphosphate car☐ylase in intact chloroplasts by CO2 and light

Abstract The activity of ribulose 1,5-bisphosphate (RuBP) car☐ylase in intact spinach chloroplasts is shown to depend on light and CO2. This activity was measured upon lysis of chloroplasts and assay of the initial activity using nonlimiting substrate concentrations. Incubation of chloroplasts at 25 °C in the absence of CO2 results in a gradual inactivation of the RuBP car☐ylase. In the presence of CO2 the initial activity is preserved or increased. CO2 is also able to reactivate the chloroplast car☐ylase previously inactivated in the absence of CO2. Upon illumination of the chloroplasts, additional activation was observed. This light activation results from an increased affinity for CO2 of the chloroplast car☐ylase. At pH 7.8, the enzyme in dark-adapted chloroplasts required 112 μ m CO2 for half activation, while in the light it required 24 μ m CO2. The light activation was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, carbonylcyanide 3-chlorophenylhydrazone, or dl -glyceraldehyde. Part of the light activation is most likely due to increased Mg2+ in the stroma. dl -Glyceraldehyde inhibition also suggests that some intermediate of the photosynthetic carbon cycle is involved. These results suggest that photosynthetic CO2 assimilation in the chloroplast depends upon the amount of activation of the RuBP car☐ylase. This activation is regulated by CO2 and light-induced changes in the chloroplast stroma such as pH, Mg2+, and intermediates of the photosynthetic carbon cycle.

Regulation of ribulose-1,5-bisphosphate carboxylase from tobacco: Changes in pH response and affinity for CO2 and Mg2+ induced by chloroplast intermediates☆

Abstract Ribulose-1,5-bisphosphate caryboxylase-oxygenase is activated by CO2 and Mg2+ in a process distinct from catalysis. The effect of chloroplast metabolites as they separately influenced either activation or catalysis of tobacco carboxylase was examined. Of the 28 metabolites examined, 13 effected activation of the carboxylase. The strongest positive effectors were NADPH, gluconate-6-P, glycerate-2-P, and glycerate-3-P. Negative effectors included ribose-5-P, fructose-6-P, glucose-6-P, and pyrophosphate. The concentration of CO2 or Mg2+ necessary to produce half-maximal activation is defined as Kact. NADPH and gluconate-6-P decreased the Kact(CO2) from 43 to 7.4 and 3.5 μ m , respectively (pH 8.0, 5 m m MgCl2). They also decreased the Kact(M.g2+), but had little affect on the affinity of the enzyme for CO2 during the catalytic process. Increasing Mg2+ concentration decreased the Kact(CO2) and increasing CO2 concentration decreased the Kact-(Mg2+). NADP+ and gluconate-6-P also affected the pH profile of activation, shifting it toward lower pH values. Changes in activation had no effect on the pH profile for catalysis of CO2 fixation. Effectors influenced ribulose-1,5-bisphosphate oxygenase in a manner analogous to the carboxylase. At air levels of O2 and CO2, the ratio of carboxylase to oxygenase activity was not changed by the presence of effectors, including hydroxylamine.

Ribulose bisphosphate oxygenase activity from freshly ruptured spinach chloroplasts

Abstract Suspensions of freshly lysed spinach chloroplasts, in which ribulose bisphosphate carboxylase displays an in vivo K m [CO], exhibited a ribulose bisphosphate-dependent uptake of oxygen. The kinetic properties of this oxygenase activity were examined at air levels of CO 2 (10 μ m ) and O 2 (240 μ m ). The pH optimum was 8.6–8.8 and the K M [ribulose bisphosphate] was 45 μ m . At 240 μ m O 2 , the oxygenase activity is inhibited one-half by 25 μ m CO 2 . The apparent K m (O 2 ) is large, somewhere between 1 and 2 atm. The phosphoglycolate phosphatase activity of the chloroplasts was in great excess, suggesting that phosphoglycolate formed by the oxygenase would be quickly hydrolyzed to glycolate for possible metabolism by photorespiration. A comparison of the pH dependence of both the carboxylase and oxygenase activities at air levels of CO 2 and O 2 suggests that the pH of the chloroplast stroma could regulate their relative activities and that the oxygenase activity is sufficient to account for glycolate production during photosynthesis. It is predicted that at pH 7.8, about 40% of the carbon assimilated by the Calvin cycle would go through glycolate.

Mutations in the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Increase the Formation of the Misfire Product Xylulose-1,5-Bisphosphate

The small subunit (S) increases the catalytic efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) by stabilizing the active sites generated by four large subunit (L) dimers. This stabilization appears to be due to an influence of S on the reaction intermediate 2,3-enediol, which is formed after the abstraction of a proton from the substrate ribulose-1,5-bisphosphate. We tested the functional significance of residues that are conserved among most species in the carboxy-terminal part of S and analyzed their influence on the kinetic parameters of Synecho-coccus holoenzymes. The replacements in S (F92S, Q99G, and P108L) resulted in catalytic activities ranging from 95 to 43% of wild type. The specificity factors for the three mutant enzymes were little affected (90–96% of wild type), but Km(CO2) values increased 0.5- to 2-fold. Mutant enzymes with replacements Q99G and P108L showed increased mis-protonation, relative to carboxylation, of the 2,3-enediol intermediate, forming 2 to 3 times more xylulose-1,5-bisphosphate per ribulose-1,5-bisphosphate utilized than wild-type or F92S enzymes. The results suggest that specific alterations of the L/S interfaces and of the hydrophobic core of S are transmitted to the active site by long-range interactions. S interactions with L may restrict the flexibility of active-site residues in L.