Light Moderates the Induction of Phosphoenolpyruvate Carboxylase by NaCl and Abscisic Acid in Mesembryanthemum crystallinum

In Mesembryanthemum crystallinum, phosphoenolpyruvate carboxylase is synthesized de novo in response to osmotic stress, as part of the switch from C₃-photosynthesis to Crassulacean acid metabolism. To better understand the environmental signals involved in this pathway, we have investigated the effects of light on the induced expression of phosphoenolpyruvate carboxylase mRNA and protein in response to stress by 400 millimolar NaCl or 10 micromolar abscisic acid in hydroponically grown plants. When plants were grown in high-intensity fluorescent or incandescent light (850 microeinsteins per square meter per second), NaCl and abscisic acid induced approximately an eightfold accumulation of phosphoenolpyruvate carboxylase mRNA when compared to untreated controls. Levels of phosphoenolpyruvate carboxylase protein were high in these abscisic acid- and NaCl-treated plants, and detectable in the unstressed control. Growth in high-intensity incandescent (red) light resulted in approximately twofold higher levels of phosphoenolpyruvate carboxylase mRNA in the untreated plants when compared to control plants grown in high-intensity fluorescent light. In low light (300 microeinsteins per square meter per second fluorescent), only NaCl induced mRNA levels significantly above the untreated controls. Low light grown abscisic acid- and NaCl-treated plants contained a small amount of phosphoenolpyruvate carboxylase protein, whereas the (untreated) control plants did not contain detectable amounts of phosphoenolpyruvate carboxylase. Environmental stimuli, such as light and osmotic stress, exert a combined effect on gene expression in this facultative halophyte.     

Removal of feedback inhibition of delta(1)-pyrroline-5-carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic stress

The Delta(1)-pyrroline-5-carboxylate synthetase (P5CS; EC not assigned) is the rate-limiting enzyme in proline (Pro) biosynthesis in plants and is subject to feedback inhibition by Pro. It has been suggested that the feedback regulation of P5CS is lost in plants under stress conditions. We compared Pro levels in transgenic tobacco (Nicotiana tabacum) plants expressing a wild-type form of Vigna aconitifolia P5CS and a mutated form of the enzyme (P5CSF129A) whose feedback inhibition by Pro was removed by site-directed mutagenesis. Transgenic plants expressing P5CSF129A accumulated about 2-fold more Pro than the plants expressing V. aconitifolia wild-type P5CS. This difference was further increased in plants treated with 200 mM NaCl. These results demonstrated that the feedback regulation of P5CS plays a role in controlling the level of Pro in plants under both normal and stress conditions. The elevated Pro also reduced free radical levels in response to osmotic stress, as measured by malondialdehyde production, and significantly improved the ability of the transgenic seedlings to grow in medium containing up to 200 mM NaCl. These findings shed new light on the regulation of Pro biosynthesis in plants and the role of Pro in reducing oxidative stress induced by osmotic stress, in addition to its accepted role as an osmolyte.     

Osmoregulation in Rhizobium meliloti: Production of Glutamic Acid in Response to Osmotic Stress

Rhizobium meliloti, like many other bacteria, accumulates high levels of glutamic acid when osmotically stressed. The effect was found to be proportional to the osmolarity of the growth medium. NaCl, KCI, sucrose, and polyethylene glycol elicited this response. The intracellular levels of glutamate and K⁺ began to increase immediately when cells were shifted to high-osmolarity medium. Antibiotics that inhibit protein synthesis did not affect this increase in glutamate production. Cells growing in conventional media at any stage in the growth cycle could be suspended in medium causing osmotic stress and excess glutamate accumulated. The excess glutamate did not appear to be excreted, and the intracellular level eventually returned to normal when osmotically stressed cells were suspended in low-osmolarity medium. A glt mutant lacking glutamate synthase and auxotrophic for glutamate accumulated excess glutamate in response to osmotic stress. Addition of isoleucine, glutamine, proline, or arginine stimulated glutamate accumulation to wild-type levels when the mutant cells were suspended in minimal medium with NaCl to cause osmotic stress. In both wild-type and mutant cells, inhibitors of transaminase activity, including azaserine and aminooxyacetate, reduced glutamate levels. The results suggest that the excess glutamate made in response to osmotic stress is derived from degradation of amino acids and transamination of 2-ketoglutarate.     

The relationship between proline content, the expression level of P5CS (Δ1-pyrroline-5-carboxylate synthetase), and drought tolerance in Tibetan hulless barley (Hordeum vulgare var. nudum)

Many plants accumulate proline (Pro) when suffered from drought; thus, the relationship between Pro accumulation and plant drought tolerance becomes an increasing concern. Pro is synthesized from either glutamine or ornithine, and the former pathway dominates under osmotic stress conditions. In this study, the dynamic accumulation of free Pro under drought stress in 10 genotypes of Tibetan hulless barley (Hordeum vulgare var. nudum) with water lose rate (WLR) of 0.3304 to 0.5839 g/(h g dry wt) was investigated. However, no correlation between Pro accumulation and drought tolerance was found. Furthermore, the barley stripe mosaic virus establisheding virus-induced gene silencing was employed to suppress the expression of the encoding gene Δ¹-Pyrroline-5-Carboxylate Synthetase (P5CS), which catalyzes the ratelimiting step of Glu pathway in Pro biosynthesis. By the quantitative real-time polymerase chain reaction, the decrease of the P5CS expression was found, and a consequent Pro degradation was also detected in P5CS-silenced plants. However, neither increased WLR of detached leaves nor decreased survival rate under drought stress was found compared with control plants. These results suggested that the repressed expression of P5CS and decreased content of free Pro may not interfere with the drought tolerance of Tibetan hulless barley.     

Overexpression of the Malus hupehensis MhNPR1 gene increased tolerance to salt and osmotic stress in transgenic tobacco

Earlier, we have reported that overexpression of Malus hupehensis Non-expressor of pathogenesis related gene 1 (MhNPR1) gene in tobacco could induce the expression of pathogenesis-related genes and enhance resistance to fungus Botrytis cinerea. In this study, we showed that MhNPR1 can be induced by NaCl, PEG6000, low temperature (4 °C), abscisic acid and apple aphids’ treatments in M. hupehensis. Heterogonous expression of MhNPR1 gene in tobacco conferred enhanced resistance to NaCl at the stage of seed germination, and conferred resistance to mannitol at the stage of seed germination and to PEG6000 at the stage of seedlings. Furthermore, overexpression of MhNPR1 in transgenic tobacco led to higher expression levels of osmotic-stress related genes compared with wild-type plants. This was the first report of a novel function of NPR1 that overexpression of MhNPR1 gene has a positive effect on salt and osmotic stress in tobacco, which differs from the function that overexpressing of AtNPR1 gene has a negative effect on dehydration and salt stress in rice.     

Effect of thermal,oxidative, acidic,osmotic, or nutritional stresses on subsequent culturability of Escherichia coli in seawater

Survival of stressed Escherichia coli with or without the rpoS gene was assessed after 2 and 6 days in sterile seawater. Cells were submitted to thermal (48°C), acidic (pH 5.1), oxidative (H₂O₂ 1mm), nutritional (C, N, P starvation), or osmotic (NaCl 0.5m) stresses for periods ranging from 0 to 4 h. We found a stress-mediated cross protection against seawater relative to controls. Viability was higher when cells were acid, oxidatively, nutritionally or osmotically stressed. Survival increased in cells stressed at 37°C as compared with 20°C. With the exception of osmotic stress, we found that this stress-induced cross protection was rpoS dependent.Correspondence to: P.M. Munro.     

Isolation and characterization of a Δ1-pyrroline-5-carboxylate synthetase (NtP5CS) from Nitraria tangutorum Bobr. and functional comparison with its Arabidopsis homologue

Several functional and regulatory proteins play important roles in controlling plant stress tolerance. Proline (Pro) is one of the most accumulated osmolytes correlated with tolerance to stresses. Δ¹-Pyrroline-5-carboxylate synthetase (P5CS) is a rate-limiting enzyme in Pro biosynthesis. In the present study, we isolated the cDNA for a P5CS gene (NtP5CS) from the halophyte Nitraria tangutorum. Phylogenetic analysis and subcellular localization analysis of NtP5CS-GFP protein in onion cells showed that NtP5CS was a new P5CS gene and was involved in Pro synthesis in N. tangutorum. Expression of the NtP5CS gene was induced by salt stress, dehydration, and high and low temperatures. Escherichia coli overexpressing AtP5CS or NtP5CS exhibited better growth in all treatments, including high salinity, high alkalinity, dehydration, osmotic, heat and cold stresses. Additionally, NtP5CS recombinant E. coli cells grew better than did AtP5CS recombinant cells in response to abiotic stresses. Our data demonstrate that the P5CS from a halophytic species functions more efficiently than its homologue from a glycophytic species in improving the stress tolerance of E. coli.     

Dehydration-Stress-Regulated Transgene Expression in Stably Transformed Rice Plants

To confer abscisic acid (ABA) and/or stress-inducible gene expression, an ABA-response complex (ABRC1) from the barley (Hordeum vulgare L.) HVA22 gene was fused to four different lengths of the 5′ region from the rice (Oryza sativa L.) Act1 gene. Transient assay of β-glucuronidase (GUS) activity in barley aleurone cells shows that, coupled with ABRC1, the shortest minimal promoter (Act1–100P) gives both the greatest induction and the highest level of absolute activity following ABA treatment. Two plasmids with one or four copies of ABRC1 combined with the same Act1–100P and HVA22(I) of barley HVA22 were constructed and used for stable expression of uidA in transgenic rice plants. Three Southern blot-positive lines with the correct hybridization pattern for each construct were obtained. Northern analysis indicated that uidA expression is induced by ABA, water-deficit, and NaCl treatments. GUS activity assays in the transgenic plants confirmed that the induction of GUS activity varies from 3- to 8-fold with different treatments or in different rice tissues, and that transgenic rice plants harboring four copies of ABRC1 show 50% to 200% higher absolute GUS activity both before and after treatments than those with one copy of ABRC1.     

Proline Accumulation in Developing Grapevine Fruit Occurs Independently of Changes in the Levels of Δ1-Pyrroline-5-Carboxylate Synthetase mRNA or Protein

Mature fruit of grapevine (Vitis vinifera) contains unusually high levels of free proline (Pro; up to 24 μmol or 2.8 mg/g fresh weight). Pro accumulation does not occur uniformly throughout berry development but only during the last 4 to 6 weeks of ripening when both berry growth and net protein accumulation have ceased. In contrast, the steady-state levels of both the mRNA encoding V. vinifera Δ¹-pyrroline-5-carboxylate synthetase (VVP5CS), a key regulatory enzyme in Pro biosynthesis, and its protein product remain relatively uniform throughout fruit development. In addition, the steady-state protein levels of Pro dehydrogenase, the first enzyme in Pro degradation, increased throughout early fruit development but thereafter remained relatively constant. The developmental accumulation of free Pro late in grape berry ripening is thus clearly distinct from the osmotic stress-induced accumulation of Pro in plants. It is not associated with either sustained increases in steady-state levels of P5CS mRNA or protein or a decrease in steady-state levels of Pro dehydrogenase protein, suggesting that other physiological factors are important for its regulation.     

Effects of Osmoprotectants upon NaCl Stress in Rice

Plants accumulate a number of osmoprotective substances in response to NaCl stress, one of them being proline (Pro). While characterizing some of the changes in solute accumulation in NaCl-stressed rice (Oryza sativa L.), we identified several other potential osmoprotectants. One such substance, trehalose, begins to accumulate in small amounts in roots after 3 d. We performed a series of experiments to compare the effects of Pro and trehalose on ion accumulation to determine whether the two chemicals protect the same physiological processes. We found that Pro either has no effect or, in some cases, exasperates the effect of NaCl on growth inhibition, chlorophyll loss, and induction of a highly sensitive marker for plant stress, the osmotically regulated salT gene. By contrast, low to moderate concentrations of trehalose reduce Na+ accumulation, salT expression, and growth inhibition. Somewhat higher concentrations (10 mM) prevent NaCl-induced loss of chlorophyll in blades, preserve root integrity, and enhance growth. The results of this study indicate that during osmotic stress trehalose or carbohydrates might be more important for rice than Pro.     

Molecular dissection of the response of a rice leucine-rich repeat receptor-like kinase (LRR-RLK) gene to abiotic stresses

Leucine-rich repeat (LRR) receptor-like kinase (RLK) proteins play key roles in a variety of biological pathways. In a previous study, we analyzed the members of the rice LRR-RLK gene family using in silico analysis. A total of 23 LRR-RLK genes were selected based on the expression patterns of a genome-wide dataset of microarrays. The Oryza sativa gamma-ray induced LRR-RLK1 (OsGIRL1) gene was highly induced by gamma irradiation. Therefore, we studied its expression pattern in response to various different abiotic and phytohormone treatments. OsGIRL1 was induced on exposure to abiotic stresses such as salt, osmotic, and heat, salicylic acid (SA), and abscisic acid (ABA), but exhibited downregulation in response to jasmonic acid (JA) treatment. The OsGIRL1 protein was clearly localized at the plasma membrane. The truncated proteins harboring juxtamembrane and kinase domains (or only harboring a kinase domain) exhibited strong autophosphorylation. The biological function of OsGIRL1 was investigated via heterologous overexpression of this gene in Arabidopsis plants subjected to gamma-ray irradiation, salt stress, osmotic stress, and heat stress. A hypersensitive response was observed in response to salt stress and heat stress, whereas a hyposensitive response was observed in response to gamma-ray treatment and osmotic stress. These results provide critical insights into the molecular functions of the rice LRR-RLK genes as receptors of external signals.     

Expression of a carbonic anhydrase gene is induced by environmental stresses in Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Expression of the gene (OsCA1) coding for carbonic anhydrase (CA) in leaves and roots of rice was induced by environmental stresses from salts (NaCl, NaHCO₃ and Na₂CO₃), and osmotic stress (10%, w/v, PEG 6000). CA activity of rice seedlings more than doubled under some of these stresses. Transgenic Arabidopsis over-expressing OsCA1 had a greater salt tolerance at the seedling stage than wild-type plants in 1/2 MS medium with 5 mM NaHCO₃, 50 mM NaCl, on 100 mM NaCl. Thus CA expression responds to environmental stresses and is related to stress tolerance in rice.     

Exogenous ABA induces salt tolerance in indica rice (Oryza sativa L.): The role of OsP5CS1 and OsP5CR gene expression during salt stress

Abscisic acid (ABA) applied exogenously at 100 μM prior to and during the salt-stress period induced salt tolerance in both the salt-susceptible (LPT123) and the genetically related salt-resistant (LPT123-TC171) rice lines, enhanced the survival rate by 20%, and triggered proline (Pro) accumulation earlier than that by salt-stress alone, supporting a role for Pro as an osmoprotectant. In both rice lines, salt-stress induced OsP5CS1 gene expression, suggesting that proline accumulation occurs via OsP5CS1 gene expression during salt stress. An increase in the endogenous ABA level was required for the induction of OsP5CS1 gene expression by salt stress. Under salt stress, topical ABA application-induced OsP5CS1 gene expression only in the salt-resistant line but up-regulated OsP5CR gene expression in both rice lines, suggesting that the increased proline accumulation and salt resistance induced by topical ABA application may result from the up-regulation of OsP5CR and not, directly at least, from OsP5CS1. Moreover, exogenous ABA application up-regulates OsCam1-1 (the salt-stress-responsive calmodulin) gene expression, and calmodulin was shown to play a role in the signal transduction cascade in proline accumulation during salt stress. These data suggest the role of the calmodulin signaling cascade and the induction of OsP5CR gene expression in proline accumulation by exogenous ABA application.