Strain-specific salt tolerance and osmoregulatory mechanisms in Azospirillum brasilense

Salinity stress inhibits the growth and nitrogen fixation ability of the plant growth-promoting rhizobacterium Azospirillum brasilense. Five strains of A. brasilense were isolated from the rhizosphere of Indian cereals and grasses and identified on the basis of their phenotypic features and 16S rRNA gene sequence. The five Indian isolates and two standard strains of A. brasilense, Sp7 and Cd, showed notable differences in growth, acetylene-reducing activity under salt stress, and ability to take up and use glycine betaine for the restoration of growth and acetylene-reducing activity under salt stress. Salt stress also enhanced the production of exopolysaccharides and cell aggregates, the extent of which varied in different strains of A. brasilense at different carbon to nitrogen ratios in the culture medium. It can be concluded that the production of exopolysaccharides and cell aggregates is a more consistent physiological response of A. brasilense to salt stress than is the uptake and osmoprotection by glycine betaine.     

Effect of root exudates on the exopolysaccharide composition and the lipopolysaccharide profile of Azospirillum brasilense Cd under saline stress

The effect of wheat root exudates on the exopolysaccharide (EPS) composition and the lipopolysaccharide (LPS) profile of Azospirillum brasilense Cd under saline stress was studied. EPS of A. brasilense Cd was composed of glucose (47%), mannose (3%), xylose (4%), fucose (28%), rhamnose (6%), arabinose (1%) and galactose (11%). Under saline stress, A. brasilense produced a totally different EPS, composed mainly of galactose. Root exudates induced changes in A. brasilense EPS composition only under normal conditions, consisting of higher amounts of arabinose and xylose compared with EPS of bacteria grown without root exudates. No changes were induced by root exudates when A. brasilense was grown under saline stress. Additionally, root exudates induced changes in the LPS profile, both under normal and stress conditions.     

Isolation, fractionation and some properties of polysaccharides produced in a bound form by Azospirillum brasilense and their possible involvement in Azospirillum-wheat root interactions

Abstract It was shown that Azospirillum brasilense strains Sp7, Sp107, Sp245, and S17 when cultivated in a liquid synthetic malate medium to the end of the exponential phase of growth, produced at least two complex polysaccharide-containing components. The components were arbitrarily called lipopolysaccharide-protein complex and polysaccharide-lipid complex. These complexes were shown to interact with a wheat germ agglutinin. From polysaccharide-lipid complexes, acidic polysaccharides were isolated and their specific rotation, molecular masses, affinity for wheat germ agglutinin, and monosaccharide composition were determined. The polysaccharides of all strains contained rhamnose, galacturonic acid, and glucosamine, while the polysaccharides of strains Sp7 and S17 included additional fucose and mannose, respectively, and both had galactose. It is suggested that lipopolysaccharide-protein complexes, polysaccharide-lipid complexes, and polysaccharides may be involved in the process of interaction of azospirilla with wheat root surfaces.     

Poly-β-hydroxybutyrate metabolism in Azospirillum brasilense and the ecological role of PHB in the rhizosphere

Abstract Azospirillum brasilense is a rhizosphere microorganism which has potential use for promoting plant growth in economically important crops. Its ability to survive the adverse conditions imposed by nutrient starvation and competition in the rhizosphere is of great importance. A. brasilense accumulates up to 70% of its cell dry weight with poly-β-hydroxybutyrate (PHB). In the presence of stress factors such as ultraviolet radiation, desiccation and osmotic stress, PHB-rich cells survived better than PHB-poor cells. Polymer-rich cells of Azospirillum fixed N₂ in the absence of exogenous carbon and combined nitrogen. The enzymes of the PHB cycle in both the synthesis and degradation processes as well as during starvation were more active in PHB-rich cells. After 24 h of starvation there was a peak of activity of d (−)β-hydroxybutyrate dehydrogenase, β-ketothiolase and thiophorase due to PHB degradation. Additionally, acetoacetyl-CoA reductase dropped to a minimum level because PHB could not be synthesized. The possible utilization of PHB as a sole carbon and energy source by A. brasilense and other bacteria during establishment, proliferation and survival in the rhizosphere will be discussed.     

Cloning of rat olfactory bulb tubulin tyrosine ligase cDNA: a dominant negative mutant and an antisense cDNA increase the proliferation rate of cells in culture

In this paper we describe the cloning of rat olfactory bulb tubulin tyrosine ligase (TTL) cDNA, and investigate the physiological role of TTL in cultured CHO-K1 cells. Comparison of the deduced amino acid sequence of rat TTL cDNA with those of bovine and pig showed approximately 90% of identity. Transient transfection of CHO-K1 cells with a dominant negative mutant of TTL that contains the binding site to the substrate (tubulin) but not the catalytic domain, significantly decreased the endogenous TTL activity as determined in vitro. Similar results were obtained using a construction encoding for the antisense sequence of TTL. The reduction in TTL activity is not accompanied by a decrease in the tyrosination levels of microtubules, as judged by immunofluorescence analysis. Strikingly, the number of cells in the plates transfected with the mutant TTL or the antisense TTL cDNA was, after 72 h of culture, two and three times higher, respectively, than the number of cells in the control plates. These results support the hypothesis that TTL may play a role in the regulation of the cell cycle in living cells.     

Function of a novel GDSL-type pepper lipase gene, <Emphasis Type="Italic">CaGLIP1</Emphasis>, in disease susceptibility and abiotic stress tolerance

GDSL-type lipase is a hydrolytic enzyme whose amino acid sequence contains a pentapeptide motif (Gly-X-Ser-X-Gly) with active serine (Ser). Pepper GDSL-type lipase (CaGLIP1) gene was isolated and functionally characterized from pepper leaf tissues infected by Xanthomonas campestris pv. vesicatoria (Xcv). The CaGLIP1 protein was located in the vascular tissues of Arabidopsis root. The CaGLIP1 gene was preferentially expressed in pepper leaves during the compatible interaction with Xcv. Treatment with salicylic acid, ethylene and methyl jasmonate induced CaGLIP1 gene expression in pepper leaves. Sodium nitroprusside, methyl viologen, high salt, mannitol-mediated dehydration and wounding also induced early and transient CaGLIP1 expression in pepper leaf tissues. Virus-induced gene silencing of CaGLIP1 in pepper conferred enhanced resistance to Xcv, accompanied by the suppressed expression of basic PR1 (CaBPR1) and defensin (CaDEF1) genes. The CaGLIP1 lipase produced in Escherichia coli hydrolyzed the substrates of short and long chain nitrophenyl esters. The CaGLIP1-overexpressing Arabidopsis exhibited enhanced hydrolytic activity toward short and long chain nitrophenyl ester, as well as enhanced susceptibility to the bacterial pathogen Pseudomonas syringae pv. tomato and the biotrophic oomycete Hyaloperonospora parasitica. SA-induced expression of AtPR1 and AtGST1, also was delayed in CaGLIP1-overexpressing plants by SA application. During seed germination and plant growth, the CaGLIP1 transgenic plants showed drought tolerance and differential expression of drought- and abscisic acid (ABA)-inducible genes AtRD29A, AtADH and AtRab18. ABA treatment differentially regulated seed germination and gene expression in wild-type and CaGLIP1 transgenic Arabidopsis. Overexpression of CaGLIP1 also regulated glucose- and oxidative stress signaling. Together, these results indicate that CaGLIP1 modulates disease susceptibility and abiotic stress tolerance. The nucleotide sequence data reported here has been deposited in the GenBank database under the accession number AY775336.     

Development of oxidative stress tolerance resulted in reduced ability to undergo morphologic transitions and decreased pathogenicity in a t-butylhydroperoxide-tolerant mutant of Candida albicans

We tested the hypothesis that adaptation of Candida albicans to chronic oxidative stress inhibits the formation of hyphae and reduces pathogenicity. Candida albicans cells were exposed to increasing concentrations of t-butylhydroperoxide (tBOOH), a lipid peroxidation-accelerating agent, and mutants with heritable tBOOH tolerance were isolated. Hypha formation by the mutants was negligible on Spider agar, indicating that the development of oxidative stress tolerance prevented Candida cells from undergoing dimorphic switches. One of the mutants, C. albicans AF06, was five times less pathogenic in mice than its parental strain, due to its reduced germ tube-, pseudohypha- and hypha-forming capability, and decreased phospholipase secretion. An increased oxidative stress tolerance may therefore be disadvantageous when this pathogen leaves blood vessels and invades deep organs. The AF06 mutant was characterized by high intracellular concentrations of endogenous oxidants, reduced monounsaturated and polyunsaturated fatty acid contents, the continuous induction of the antioxidative defense system, decreased cytochrome c-dependent respiration, and increased alternative respiration. The mutation did not influence growth rate, cell size, cell surface, cellular ultrastructures, including mitochondria, or recognition by human polymorphonuclear leukocytes. The selection of oxidative stress-tolerant respiratory Candida mutants may also occur in vivo, when reduced respiration helps the fungus to cope with antimycotic agents.     

Comparative analysis of transcriptional responses to the cryoprotectants, dimethyl sulfoxide and trehalose, which confer tolerance to freeze–thaw stress in Saccharomyces cerevisiae

We have used microarray analysis to monitor the gene expression profile of Saccharomyces cerevisiae BY4743 in the presence of the cryoprotectants, dimethyl sulfoxide (Me₂SO) and trehalose. Analysis of these profiles suggests that both cryoprotectants increased the expression of genes involved in protein synthesis, ribosomal biogenesis, fatty acid biosynthesis, ergosterol biosynthesis, cell wall biosynthesis, and cellular accumulation of low molecular compounds such as glycerol, arginine and proline. Cryoprotectant treatment reduced the expression of genes involved in the β-oxidation of fatty acids. In addition, Me₂SO increased the expression of genes involved in protein refolding and trehalose increased the expression of genes involved in spore formation. This study supported that exposure to cryoprotectants prior to freezing not only reduce the freeze–thaw damage but also provide various process to the recovery from freeze–thaw damage.     

Increased tolerance to salt- and water-deficit stress in transgenic lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L.) by constitutive expression of LEA

A late embryogenesis abundant (LEA) protein gene, ME-leaN4, from rape (Brassica napus) was successfully introduced into lettuce (Lactuca sativa L.) using Agrobacterium tumefaciens-mediated transformation. Infection by Agrobacterium strain EHA101 containing the binary vector pIG121-LEA was applied. Six independent transgenic lettuce plants were generated as a result. Transgenic lettuce demonstrated enhanced growth ability as compared to non-transformed control plants under salt-stress and water-deficit stress conditions. After 10-day growth under 100 mM NaCl condition in the hydroponics, average plant length and fresh weight of transgenic lettuce were 2.8 cm and 2.5 g plant^–1, while control plants were only 0.2 cm and 0.3 g plant^–1, respectively. The increased tolerance was also reflected by delayed wilting of leaves caused by water-deficit stress. These results suggest that growth characteristics were improved in transgenic lettuce plants constitutively expressing the rape LEA gene in response to salt- and water-deficit stress. Byong-Jin Park, Zaochang Liu These two authors contributed equally to this work.     

Overexpression of <Emphasis Type="Italic">OsiSAP8</Emphasis>, a member of stress associated protein (SAP) gene family of rice confers tolerance to salt,drought and cold stress in transgenic tobacco and rice

We describe here the isolation and characterization of OsiSAP8, a member of stress Associated protein (SAP) gene family from rice characterized by the presence of A20 and AN1 type Zinc finger domains. OsiSAP8 is a multiple stress inducible gene, induced by various stresses, namely heat, cold, salt, desiccation, submergence, wounding, heavy metals as well as stress hormone Abscisic acid. OsiSAP8 protein fused to GFP was localized towards the periphery of the cells in the epidermal cells of infiltrated Nicotiana benthamiana leaves. Yeast two hybrid analysis revealed that A20 and AN1 type zinc-finger domains of OsiSAP8 interact with each other. Overexpression of the gene in both transgenic tobacco and rice conferred tolerance to salt, drought and cold stress at seed germination/seedling stage as reflected by percentage of germination and gain in fresh weight after stress recovery. Transgenic rice plants were tolerant to salt and drought during anthesis stage without any yield penalty as compared to unstressed transgenic plants. OsiSAP8 is deposited in the Genbank with the Accession number AY345599.     

Ectopic expression of <Emphasis Type="Italic">ThCYP1</Emphasis>, a stress-responsive cyclophilin gene from <Emphasis Type="Italic">Thellungiella halophila</Emphasis>, confers salt tolerance in fission yeast and tobacco cells

The halophyte Thellungiella halophila (salt cress) is an ideal model system for studying the molecular mechanisms of salinity tolerance in plants. Herein, we report the identification of a stress-responsive cyclophilin gene (ThCYP1) from T. halophila, using fission yeast as a functional system. The expression of ThCYP1 is highly inducible by salt, abscisic acid (ABA), H₂O₂ and heat shock. Ectopic overexpression of the ThCYP1 gene enhance the salt tolerance capacity of fission yeast and tobacco (Nicotiana tabacum L.) cv. Bright Yellow 2 (BY-2) cells significantly. ThCYP1 is expressed constitutively in roots, stems, leaves and flowers, with higher expression occurring in the roots and flowers. The ThCYP1 proteins are distributed widely within the cell, but are enriched significantly in the nucleus. The present results suggest that ThCYP1 may participate in response to stresses in the salt cress, perhaps by regulating appropriate folding of certain stress-related proteins, or in the signal transduction processes.     

Aggregates formed as a result of the expression of yeast <Emphasis Type="Italic">Met2</Emphasis> gene in transgenic tobacco plants,stimulate the production of stress-protective metabolites and increased the plants tolerance to heat stress

Methionine biosynthesis has taken different evolutionary pathways in bacteria, fungi and plants. To gain insight into these differences and to search for new ways of manipulating methionine biosynthesis in plants, the yeast (Saccharomyces cerevisiae) Met2 gene and the bacteria (Leptospira meyeri) MetX gene, both encoding homoserine O-acetyltransferase, were expressed in tobacco plants. We found protein aggregates in extracts of these transgenic plants, whose levels were much higher in plants grown at 35 °C than at 25 °C. It appears that the yeast and the bacterial proteins are heat labile and tend to change their intracellular conformation. These conformational changes of the transgenic proteins were more prominent at high temperature and most probably triggered aggregation of the yeast and the bacterial proteins. Moreover, plants expressing the yeast gene that grew at 35 °C over-accumulated stress-associated metabolites, such as phenolic compounds, including tannins, as well as the amino acid arginine. In addition, the transgenic plants expressing high levels of the foreign genes show growth retardation, which further suggests that, these plants suffer from internal stress. The changes in protein conformation and the consequent triggering of stress response may account for the ability of these transgenic plants to tolerate more extreme heat stress (60 °C) than the wild-type plants.