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.     

Physiological,structural and molecular traits activated in strawberry plants after inoculation with the plant growth‐promoting bacterium Azospirillum brasilense REC3

The plant growth‐promoting strain REC3 of Azospirillum brasilense, isolated from strawberry roots, prompts growth promotion and systemic protection against anthracnose disease in this crop. Hence, we hypothesised that A. brasilense REC3 can induce different physiological, structural and molecular responses in strawberry plants. Therefore, the aim of this work was to study these traits activated in Azospirillum‐colonised strawberry plants, which have not been assessed until now. Healthy, in vitro micropropagated plants were root‐inoculated with REC3 under hydroponic conditions; root and leaf tissues were sampled at different times, and oxidative burst, phenolic compound content, malondialdehyde (MDA) concentration, callose deposition, cell wall fortification and gene expression were evaluated. Azospirillum inoculation enhanced levels of soluble phenolic compounds after 12 h post‐inoculation (hpi), while amounts of cell wall bound phenolics were similar in inoculated and control plants. Other early responses activated by REC3 (at 24 hpi) were a decline of lipid peroxidation and up‐regulation of strawberry genes involved in defence (FaPR1), bacterial recognition (FaFLS2) and H₂O₂ depuration (FaCAT and FaAPXc). The last may explain the apparent absence of oxidative burst in leaves after bacterial inoculation. Also, REC3 inoculation induced delayed structural responses such as callose deposition and cell wall fortification (at 72 hpi). Results showed that A. brasilense REC3 is capable of exerting beneficial effects on strawberry plants, reinforcing their physiological and cellular characteristics, which in turns contribute to improve plant performance.     

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.     

Heterologous expression of OsSIZ1, a rice SUMO E3 ligase,enhances broad abiotic stress tolerance in transgenic creeping bentgrass

Sumoylation is a posttranslational regulatory process in higher eukaryotes modifying substrate proteins through conjugation of small ubiquitin‐related modifiers (SUMOs). Sumoylation modulates protein stability, subcellular localization and activity; thus, it regulates most cellular functions including response to environmental stress in plants. To study the feasibility of manipulating SUMO E3 ligase, one of the important components in the sumoylation pathway in transgenic (TG) crop plants for improving overall plant performance under adverse environmental conditions, we have analysed TG creeping bentgrass (Agrostis stolonifera L.) plants constitutively expressing OsSIZ1, a rice SUMO E3 ligase. Overexpression of OsSIZ1 led to increased photosynthesis and overall plant growth. When subjected to water deficiency and heat stress, OsSIZ1 plants exhibited drastically enhanced performance associated with more robust root growth, higher water retention and cell membrane integrity than wild‐type (WT) controls. OsSIZ1 plants also displayed significantly better growth than WT controls under phosphate‐starvation conditions, which was associated with a higher uptake of phosphate (Pi) and other minerals, such as potassium and zinc. Further analysis revealed that overexpression of OsSIZ1 enhanced stress‐induced SUMO conjugation to substrate in TG plants, which was associated with modified expression of stress‐related genes. This strongly supports a role sumoylation plays in regulating multiple molecular pathways involved in plant stress response, establishing a direct link between sumoylation and plant response to environmental adversities. Our results demonstrate the great potential of genetic manipulation of sumoylation process in TG crop species for improved resistance to broad abiotic stresses.     

Arabidopsis RING E3 ubiquitin ligase JUL1 participates in ABA‐mediated microtubule depolymerization,stomatal closure,and tolerance response to drought stress

Ubiquitination is a critical post‐translational protein modification that has been implicated in diverse cellular processes, including abiotic stress responses, in plants. In the present study, we identified and characterized a T‐DNA insertion mutant in the At5g10650 locus. Compared to wild‐type Arabidopsis plants, at5g10650 progeny were hyposensitive to ABA at the germination stage. At5g10650 possessed a single C‐terminal C3HC4‐type Really Interesting New Gene (RING) motif, which was essential for ABA‐mediated germination and E3 ligase activity in vitro. At5g10650 was closely associated with microtubules and microtubule‐associated proteins in Arabidopsis and tobacco leaf cells. Localization of At5g10650 to the nucleus was frequently observed. Unexpectedly, At5g10650 was identified as JAV1‐ASSOCIATED UBIQUITIN LIGASE1 (JUL1), which was recently reported to participate in the jasmonate signaling pathway. The jul1 knockout plants exhibited impaired ABA‐promoted stomatal closure. In addition, stomatal closure could not be induced by hydrogen peroxide and calcium in jul1 plants. jul1 guard cells accumulated wild‐type levels of H₂O₂ after ABA treatment. These findings indicated that JUL1 acts downstream of H₂O₂ and calcium in the ABA‐mediated stomatal closure pathway. Typical radial arrays of microtubules were maintained in jul1 guard cells after exposure to ABA, H₂O₂, and calcium, which in turn resulted in ABA‐hyposensitive stomatal movements. Finally, jul1 plants were markedly more susceptible to drought stress than wild‐type plants. Overall, our results suggest that the Arabidopsis RING E3 ligase JUL1 plays a critical role in ABA‐mediated microtubule disorganization, stomatal closure, and tolerance to drought stress.     

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.     

Extended access of cocaine self‐administration results in tolerance to the dopamine‐elevating and locomotor‐stimulating effects of cocaine

Tolerance to the neurochemical and psychoactive effects of cocaine after repeated use is a hallmark of cocaine addiction in humans. However, comprehensive studies on tolerance to the behavioral, psychoactive, and neurochemical effects of cocaine following contingent administration in rodents are lacking. We outlined the consequences of extended access cocaine self‐administration as it related to tolerance to the psychomotor activating, dopamine (DA) elevating, and DA transporter (DAT) inhibiting effects of cocaine. Cocaine self‐administration (1.5 mg/kg/inj; 40 inj; 5 days), which resulted in escalation of first hour intake, caused reductions in evoked DA release and reduced maximal rates of uptake through the DAT as measured by slice voltammetry in the nucleus accumbens core. Furthermore, we report reductions in cocaine‐induced uptake inhibition and a corresponding increase in the dose of cocaine required for 50% inhibition of DA uptake (K_i) at the DAT. Cocaine tolerance at the DAT translated to reductions in cocaine‐induced DA overflow as measured by microdialysis. In addition, cocaine‐induced elevations in locomotor activity and stereotypy were reduced, while rearing behavior was enhanced in animals with a history of cocaine self‐administration. Here, we demonstrate both neurochemical and behavioral cocaine tolerance in an extended‐access rodent model of cocaine abuse, which allows for a better understanding of the neurochemical and psychomotor tolerance that develops to cocaine in human addicts.

     

From model to crop: functional characterization of SPL8 in M. truncatula led to genetic improvement of biomass yield and abiotic stress tolerance in alfalfa

Biomass yield, salt tolerance and drought tolerance are important targets for alfalfa (Medicago sativa L.) improvement. Medicago truncatula has been developed into a model plant for alfalfa and other legumes. By screening a Tnt1 retrotransposon‐tagged M. truncatula mutant population, we identified three mutants with enhanced branching. Branch development determines shoot architecture which affects important plant functions such as light acquisition, resource use and ultimately impacts biomass production. Molecular analyses revealed that the mutations were caused by Tnt1 insertions in the SQUAMOSA PROMOTER BINDING PROTEIN‐LIKE 8 (SPL8) gene. The M. truncatula spl8 mutants had increased biomass yield, while overexpression of SPL8 in M. truncatula suppressed branching and reduced biomass yield. Scanning electron microscopy (SEM) analysis showed that SPL8 inhibited branching by directly suppressing axillary bud formation. Based on the M. truncatula SPL8 sequence, alfalfa SPL8 (MsSPL8) was cloned and transgenic alfalfa plants were produced. MsSPL8 down‐regulated or up‐regulated alfalfa plants exhibited similar phenotypes to the M. truncatula mutants or overexpression lines, respectively. Specifically, the MsSPL8 down‐regulated alfalfa plants showed up to 43% increase in biomass yield in the first harvest. The impact was even more prominent in the second harvest, with up to 86% increase in biomass production compared to the control. Furthermore, down‐regulation of MsSPL8 led to enhanced salt and drought tolerance in transgenic alfalfa. Results from this research offer a valuable approach to simultaneously improve biomass production and abiotic stress tolerance in legumes.     

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.     

Increase in antioxidant enzyme activity,stress tolerance and biocontrol efficacy of Pichia kudriavzevii with the transition from a yeast-like to biofilm morphology

Morphological change, such as from yeast-like to biofilm, has been recently considered to be involved in the mode of action of some antagonistic yeasts used as postharvest biocontrol agents. In the present study, the biocontrol yeast, Pichia kudriavzevii, reversibly shifted from a yeast-like morphology on yeast peptone dextrose (YPD) medium with 2% agar to a biofilm morphology on YPD with 0.3% agar. The tolerance of P. kudriavzevii to heat and oxidative stresses, as well as the biocontrol efficacy against postharvest diseases on pear fruit, increased significantly from the yeast-like form to the biofilm form. The activity of antioxidant enzymes, including catalase and superoxidase dismutase, in the biofilm form was also significantly higher. The elevated activity of antioxidant enzymes was associated with less protein and lipid oxidation in the biofilm form, compared to the yeast-like form, under heat and oxidative stresses. These results suggest that activation of antioxidant system with the morphology shift contributes to the enhancement of abiotic stress tolerance and biocontrol performance of P. kudriavzevii. These findings provide new information on the biology of yeast antagonists that is essential for their potential application and development.     

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.