Isolation and characterisation of aerobic endospore forming Bacilli from sugarcane rhizosphere for the selection of strains with agriculture potentialities

Eighteen aerobic endospore forming strains were isolated from sugarcane rhizosphere in N-free medium. A phenotypic description and analysis of the 5' end hypervariable region sequences of 16S rRNA revealed a high diversity of Bacillus and related genera. Isolates were identified, and four genera were obtained: seven strains belonged to Bacillus (Bacillaceae family), four belonged to Paenibacillus, six belonged to Brevibacillus and one strain was identified as Cohnella (Paenibacillaceae family). Four Brevibacillus strains showed in vitro inhibitory activity against plant pathogens fungi Curvularia and Fusarium. Seventy-four percent of the isolated bacteria grew on pectin as the only carbon source, showing polygalacturonase activity. Pectate lyase activity was detected for the first time in a Brevibacillus genus strain. All isolates showed endoglucanase activity. Calcium phosphate solubilisation was positive in 83.3% of the isolates, with higher values than those reported for Bacillus inorganic phosphate solubilising strains. High ethylene plant hormone secretion in the culture medium was detected in 22% of the bacteria. This is the first report of ethylene secretion in Paenibacillaceae isolates. Indole-3-acetic acid production was found in a Brevibacillus genus isolate. It was reported for the first time the presence of Cohnella genus strain on sugarcane rhizosphere bearing plant growth promoting traits. The sugarcane isolate Brevibacillus B65 was identified as a plant growth inoculant because it showed wider spectra of plant stimulation capabilities, including an antifungal effect, extracellular hydrolases secretion, inorganic phosphate solubilisation and plant hormone liberation. In this work, sugarcane was shown to be a suitable niche for finding aerobic endospore forming 'Bacilli' with agriculture biotechnological purposes.

Untersuchungen zur Genexpression in Wurzeln der reblausresistenten Unterlagsrebsorte ’Börner’

Die reblausresistente Unterlagsrebsorte ’Börner’ reagiert auf einen Reblausbefall mit einer Hypersensitivitätsreaktion (HR), die sich in Form von Nekrosen an Blättern und Wurzeln zeigt. Im Rahmen dieser Dissertation wurde der Resistenzmechanismus mittels differenzieller Genexpressionsanalysen untersucht. Unter Anwendung der suppressiven subtraktiven Hybridisierung, der DNA-Microarraytechnik sowie der GeneFishingTM-Methode erfolgte ein Vergleich zwischen der Genexpression in hypersensitivem Wurzelgewebe und Normalgewebe der Unterlagsrebe ’Börner’. Neben der Reblaus induzierten HR wurde insbesondere auf die experimentelle Induktion durch das Pflanzenhormon Indol-3-Essigsäure (IES) zurückgegriffen. Damit sollten Kenntnisse über die Rolle der IES als auslösender Faktor der Resistenzreaktion gewonnen werden. Die Ergebnisse bestätigen die Annahme, dass die IES Pathogenabwehrreaktionen in ’Börner’ induziert. So konnten Hinweise auf die transkriptionelle Aktivierung Resistenz und HR assoziierter Proteine gefunden werden, wie z.B. Phytoalexine und pathogen-related (PR)-Proteine sowie Vertreter aus der hypersensitive-induced response-Familie. Es konnten weiterhin wertvolle Informationen im Hinblick auf die Transduktion des IES-Signals im Zusammenhang mit der Aktivierung von Resistenzreaktionen gewonnen werden. So wurden Hinweise auf die Beteiligung der Signalsubstanzen Ethylen, Salicylsäure, Jasmonsäure, Calcium sowie reaktiver Sauerstoffspezies gefunden. Es konnten zudem Anhaltspunkte für die Aktivierung des Auxin induzierten Ubiquitin/26S-Proteolyseweges und weiterer Signalkomponenten, wie z.B. Kinasen und Transkriptionsfaktoren, ermittelt werden. Auch auf die Beteiligung von Auxinrezeptoren konnte aufgrund der Resultate geschlossen werden. Damit war es im Rahmen der Dissertation möglich, potenzielle Signaltransduktionswege zu erarbeiten, die für weiterführende Untersuchungen des Reblausresistenzmechanismus von entscheidender Bedeutung sind.

Isolation of plant growth promoting bacteria from torch lake sediments and their role in phytoremediation of metal contaminated soil

In this study, we isolated eight copper-resistant bacteria from Torch Lake sediment contaminated by copper mine tailings (stamp sand). Sequence analysis of gyrB and rpoD genes revealed that these organisms are closer to various Pseudomonas species. These eight bacterial isolates were also resistant to zinc, cesium, lead, arsenate and mercury. Further characterization showed that all the strains produced plant growth promoting indole-3-acetic acid (IAA), iron chelating siderophore and solubilized mineral phosphate and metals. The effect of bacterial inoculation on plant growth and copper uptake by maize (Zea mays) and sunflower (Helianthus annuus) was investigated using one of the isolates (Pseudomonas sp. TLC 6-6.5-4) with higher IAA production and phosphate and metal soubilization, which resulted in a significant increase in copper accumulation in maize and sunflower, and an increase in the total biomass of maize. Genes involved in copper resistance of Pseudomonas sp. TLC 6-6.5-4 was analyzed by transposon mutational analysis. Two copper sensitive mutants with significant reduction in copper resistance were identified: CSM1, a mutant disrupted in trp A gene (tryptophan synthase alpha subunit); CSM2, a mutant disrupted in clpA gene (ATP-dependent Clp protease). Proteomic and metabolomic analysis were performed to identify biochemical and molecular mechanisms involved in copper resistance using CSM2 due to its lower minimum inhibitory concentration compared with CSM1 and the wild type. The effect of different bacterial inoculation methods on plant growth, copper uptake and soil enzyme activities was investigated. Four different delivery methods were used including soil inoculation (before or after plant emergence), seed coating and root dipping. Soil inoculation before sowing seeds and coating seeds with PGPB led to better growth of maize, higher copper uptake and an increase in soil invertase and dehydrogenase activities. Proteomic and metabolomic analyses were performed to investigate the effect of bacterial inoculation on maize grown in normal soil and stamp sand. Our results revealed that bacterial inoculation led to environment-dependent effects on maize proteome and metabolome.

The auxin influx carrier is essential for correct leaf positioning

Auxin is of vital importance in virtually every aspect of plant growth and development, yet, even after almost a century of intense study, major gaps in our knowledge of its synthesis, distribution, perception, and signal transduction remain. One unique property of auxin is its polar transport, which in many well-documented cases is a critical part of its mode of action. Auxin is actively transported through the action of both influx and efflux carriers. Inhibition of polar transport by the efflux inhibitor N-1-naphthylphthalamic acid (NPA) causes a complete cessation of leaf initiation, a defect that can be reversed by local application of the auxin, indole-3-acetic acid (IAA), to the responsive zone of the shoot apical meristem. In this study, we address the role of the auxin influx carrier in the positioning and outgrowth of leaf primordia at the shoot apical meristem of tomato. By using a combination of transport inhibitors and synthetic auxins, we demonstrate that interference with auxin influx has little effect on organ formation as such, but prevents proper localization of leaf primordia. These results suggest the existence of functional auxin concentration gradients in the shoot apical meristem that are actively set up and maintained by the action of efflux and influx carriers. We propose a model in which efflux carriers control auxin delivery to the shoot apical meristem, whereas influx and efflux carriers regulate auxin distribution within the meristem.

Auxin Regulates the Initiation and Radial Position of Plant Lateral Organs

Leaves originate from the shoot apical meristem, a small mound of undifferentiated tissue at the tip of the stem. Leaf formation begins with the selection of a group of founder cells in the so-called peripheral zone at the flank of the meristem, followed by the initiation of local growth and finally morphogenesis of the resulting bulge into a differentiated leaf. Whereas the mechanisms controlling the switch between meristem propagation and leaf initiation are being identified by genetic and molecular analyses, the radial positioning of leaves, known as phyllotaxis, remains poorly understood. Hormones, especially auxin and gibberellin, are known to influence phyllotaxis, but their specific role in the determination of organ position is not clear. We show that inhibition of polar auxin transport blocks leaf formation at the vegetative tomato meristem, resulting in pinlike naked stems with an intact meristem at the tip. Microapplication of the natural auxin indole-3-acetic acid (IAA) to the apex of such pins restores leaf formation. Similarly, exogenous IAA induces flower formation on Arabidopsis pin-formed1-1 inflorescence apices, which are blocked in flower formation because of a mutation in a putative auxin transport protein. Our results show that auxin is required for and sufficient to induce organogenesis both in the vegetative tomato meristem and in the Arabidopsis inflorescence meristem. In this study, organogenesis always strictly coincided with the site of IAA application in the radial dimension, whereas in the apical–basal dimension, organ formation always occurred at a fixed distance from the summit of the meristem. We propose that auxin determines the radial position and the size of lateral organs but not the apical–basal position or the identity of the induced structures.

Increased glutamine in leaves of poplar transgenic with pine GS1a caused greater anthranilate synthetase a-subunit (ASA1) transcript and protein abundances: an auxin-related mechanism for enhanced growth in GS transgenics?

The initial reaction in the pathway leading to the production of indole-3-acetic acid (IAA) in plants is the reaction between chorismate and glutamine to produce anthranilate, catalysed by the enzyme anthranilate synthase (ASA; EC 4.1.3.27). Compared with non-transgenic controls, leaves of transgenic poplar with ectopic expression of the pine cytosolic glutamine synthetase (GS1a; EC 6.3.1.2) produced significantly greater glutamine and significantly enhanced ASA a-subunit (ASA1) transcript and protein (approximately 130% and 120% higher than in the untransformed controls, respectively). Similarly, tobacco leaves fed with 30 mM glutamine and 2 mM chorismate showed enhanced ASA1 transcript and protein (175% and 90% higher than controls, respectively). Furthermore, free IAA was significantly elevated both in leaves of GS1a transgenic poplar and in tobacco leaves fed with 30 mM glutamine and 2 mM chorismate. These results indicated that enhanced cellular glutamine may account for the enhanced growth in GS transgenic poplars through the regulation of auxin biosynthesis

The Agrobacterium vitis T-6b oncoprotein induces auxin-independent cell expansion in tobacco

Among the Agrobacterium T-DNA genes, rolB, rolC, orf13, orf8, lso, 6b and several other genes encode weakly homologous proteins with remarkable effects on plant growth. The 6b oncogene induces tumors and enations. In order to study its properties we have used transgenic tobacco plants that carry a dexamethasone-inducible 6b gene, dex-T-6b. Upon induction, dex-T-6b plants develop a large array of morphological modifications, some of which involve abnormal cell expansion. In the present investigation, dex-T-6b-induced expansion was studied in intact leaves and an in vitro leaf disc system. Although T-6b and indole-3-acetic acid (IAA) both induced expansion and were non-additive, T-6b expression did not increase IAA levels, nor did it induce an IAA-responsive gene. Fusicoccin (FC) is known to stimulate expansion by increasing cell wall plasticity. T-6b- and FC-induced expansion were additive at saturating FC concentrations, indicating that T-6b does not act by a similar mechanism to FC. T-6b expression led to higher leaf osmolality values, in contrast to FC, suggesting that the T-6b gene induces expansion by increasing osmolyte concentrations. Metabolite profiling showed that glucose and fructose played a major role in this increase. We infer that T-6b disrupts the osmoregulatory controls that govern cell expansion during development and wound healing.

Subcellular localization and tissue specific expression of amidase 1 from Arabidopsis thaliana

Amidase 1 (AMI1) from Arabidopsis thaliana converts indole-3-acetamide (IAM), into indole-3-acetic acid (IAA). AMI1 is part of a small isogene family comprising seven members in A. thaliana encoding proteins which share a conserved glycine- and serine-rich amidase-signature. One member of this family has been characterized as an N-acylethanolamine-cleaving fatty acid amidohydrolase (FAAH) and two other members are part of the preprotein translocon of the outer envelope of chloroplasts (Toc complex) or mitochondria (Tom complex) and presumably lack enzymatic activity. Among the hitherto characterized proteins of this family, AMI1 is the only member with indole-3-acetamide hydrolase activity, and IAM is the preferred substrate while N-acylethanolamines and oleamide are not hydrolyzed significantly, thus suggesting a role of AMI1 in auxin biosynthesis. Whereas the enzymatic function of AMI1 has been determined in vitro, the subcellular localization of the enzyme remained unclear. By using different GFP-fusion constructs and an A. thaliana transient expression system, we show a cytoplasmic localization of AMI1. In addition, RT-PCR and anti-amidase antisera were used to examine tissue specific expression of AMI1 at the transcriptional and translational level, respectively. AMI1-expression is strongest in places of highest IAA content in the plant. Thus, it is concluded that AMI1 may be involved in de novo IAA synthesis in A. thaliana.

Characterization of four bifunctional plant IAM/PAM-amidohydrolases capable of contributing to auxin biosynthesis.

Amidases [EC 3.5.1.4] capable of converting indole-3-acetamide (IAM) into the major plant growth hormone indole-3-acetic acid (IAA) are assumed to be involved in auxin de novo biosynthesis. With the emerging amount of genomics data, it was possible to identify over forty proteins with substantial homology to the already characterized amidases from Arabidopsis and tobacco. The observed high conservation of amidase-like proteins throughout the plant kingdom may suggest an important role of theses enzymes in plant development. Here, we report cloning and functional analysis of four, thus far, uncharacterized plant amidases from Oryza sativa, Sorghum bicolor, Medicago truncatula, and Populus trichocarpa. Intriguingly, we were able to demonstrate that the examined amidases are also capable of converting phenyl-2-acetamide (PAM) into phenyl-2-acetic acid (PAA), an auxin endogenous to several plant species including Arabidopsis. Furthermore, we compared the subcellular localization of the enzymes to that of Arabidopsis AMI1, providing further evidence for similar enzymatic functions. Our results point to the presence of a presumably conserved pathway of auxin biosynthesis via IAM, as amidases, both of monocot, and dicot origins, were analyzed.

YUCCA8 and YUCCA9 overexpression reveals a link between auxin signaling and lignification through the induction of ethylene biosynthesis.

Auxin is associated with the regulation of virtually every aspect of plant growth and development. Many previous genetic and biochemical studies revealed that, among the proposed routes for the production of auxin, the so-called indole-3-pyruvic acid (IPA) pathway is the main source for indole-3-acetic acid (IAA) in plants. The IPA pathway involves the action of 2 classes of enzymes, tryptophan-pyruvate aminotransferases (TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1(TAA1)/TRYPTOPHAN AMINOTRANSFERASE RELATED (TAR)) and flavin monooxygenases (YUCCA). Both enzyme classes appear to be encoded by small gene families in Arabidopsis consisting of 5 and 11 members, respectively. We recently showed that it is possible to induce transcript accumulation of 2 YUCCA genes, YUC8 and YUC9, by methyl jasmonate treatment. Both gene products were demonstrated to contribute to auxin biosynthesis in planta.1 Here we report that the overexpression of YUC8 as well as YUC9 led to strong lignification of plant aerial tissues. Furthermore, new evidence indicates that this abnormally strong secondary growth is linked to increased levels of ethylene production.

High temperature promotes auxin-mediated hypocotyl elongation in Arabidopsis

Physiological studies with excised stem segments have implicated the plant hormone indole-3-acetic acid (IAA or auxin) in the regulation of cell elongation. Supporting evidence from intact plants has been somewhat more difficult to obtain, however. Here, we report the identification and characterization of an auxin-mediated cell elongation growth response in Arabidopsis thaliana. When grown in the light at high temperature (29°C), Arabidopsis seedlings exhibit dramatic hypocotyl elongation compared with seedlings grown at 20°C. This temperature-dependent growth response is sharply reduced by mutations in the auxin response or transport pathways and in seedlings containing reduced levels of free IAA. In contrast, mutants deficient in gibberellin and abscisic acid biosynthesis or in ethylene response are unaffected. Furthermore, we detect a corresponding increase in the level of free IAA in seedlings grown at high temperature, suggesting that temperature regulates auxin synthesis or catabolism to mediate this growth response. Consistent with this possibility, high temperature also stimulates other auxin-mediated processes including auxin-inducible gene expression. Based on these results, we propose that growth at high temperature promotes an increase in auxin levels resulting in increased hypocotyl elongation. These results strongly support the contention that endogenous auxin promotes cell elongation in intact plants.

Auxin-Growth Relationships in Maize Coleoptiles and Pea Internodes and Control by Auxin of the Tissue Sensitivity to Auxin

Growth of a zone of maize (Zea mays L.) coleoptiles and pea (Pisum sativum L.) internodes was greatly suppressed when the organ was decapitated or ringed at an upper position with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) mixed with lanolin. The transport of apically applied 3H-labeled indole-3-acetic acid (IAA) was similarly inhibited by NPA. The growth suppressed by NPA or decapitation was restored by the IAA mixed with lanolin and applied directly to the zone, and the maximal capacity to respond to IAA did not change after NPA treatment, although it declined slightly after decapitation. The growth rate at IAA saturation was greater than the rate in intact, nontreated plants. It was concluded that growth is limited and controlled by auxin supplied from the apical region. In maize coleoptiles the sensitivity to IAA increased more than 3 times when the auxin level was reduced over a few hours with NPA treatment. This result, together with our previous result that the maximal capacity to respond to IAA declines in pea internodes when the IAA level is enhanced for a few hours, indicates that the IAA concentration-response relationship is subject to relatively slow adaptive regulation by IAA itself. The spontaneous growth recovery observed in decapitated maize coleoptiles was prevented by an NPA ring placed at an upper position of the stump, supporting the view that recovery is due to regenerated auxin-producing activity. The sensitivity increase also appeared to participate in an early recovery phase, causing a growth rate greater than in intact plants.

Dim-Red-Light-Induced Increase in Polar Auxin Transport in Cucumber Seedlings1 : I. Development of Altered Capacity, Velocity, and Response to Inhibitors

We have developed and characterized a system to analyze light effects on auxin transport independent of photosynthetic effects. Polar transport of [3H]indole-3-acetic acid through hypocotyl segments from etiolated cucumber (Cucumis sativus L.) seedlings was increased in seedlings grown in dim-red light (DRL) (0.5 μmol m−2 s−1) relative to seedlings grown in darkness. Both transport velocity and transport intensity (export rate) were increased by at least a factor of 2. Tissue formed in DRL completely acquired the higher transport capacity within 50 h, but tissue already differentiated in darkness acquired only a partial increase in transport capacity within 50 h of DRL, indicating a developmental window for light induction of commitment to changes in auxin transport. This light-induced change probably manifests itself by alteration of function of the auxin efflux carrier, as revealed using specific transport inhibitors. Relative to dark controls, DRL-grown seedlings were differentially less sensitive to two inhibitors of polar auxin transport, N-(naphth-1-yl) phthalamic acid and 2,3,5-triiodobenzoic acid. On the basis of these data, we propose that the auxin efflux carrier is a key target of light regulation during photomorphogenesis.

Alteration of Hormone Levels in Transgenic Tobacco Plants Overexpressing the Rice Homeobox Gene OSH1

The rice (Oryza sativa L.) homeobox gene OSH1 causes morphological alterations when ectopically expressed in transgenic rice, Arabidopsis thaliana, and tobacco (Nicotiana tabacum L.) and is therefore believed to function as a morphological regulator gene. To determine the relationship between OSH1 expression and morphological alterations, we analyzed the changes in hormone levels in transgenic tobacco plants exhibiting abnormal morphology. Levels of the plant hormones indole-3-acetic acid, abscisic acid, gibberellin (GA), and cytokinin (zeatin and trans-zeatin [Z]) were measured in leaves of OSH1-transformed and wild-type tobacco. Altered plant morphology was found to correlate with changes in hormone levels. The more severe the alteration in phenotype of transgenic tobacco, the greater were the changes in endogenous hormone levels. Overall, GA1 and GA4 levels decreased and abscisic acid levels increased compared with wild-type plants. Moreover, in the transformants, Z (active form of cytokinin) levels were higher and the ratio of Z to Z riboside (inactive form) also increased. When GA3 was supplied to the shoot apex of transformants, internode extension was restored and normal leaf morphology was also partially restored. However, such GA3-treated plants still exhibited some morphological abnormalities compared with wild-type plants. Based on these data, we propose the hypothesis that OSH1 affects plant hormone metabolism either directly or indirectly and thereby causes changes in plant development.

Higher Activity of an Aldehyde Oxidase in the Auxin-Overproducing superroot1 Mutant of Arabidopsis thaliana1

Aldehyde oxidase (AO; EC 1.2.3.1) activity was measured in seedlings of wild type or an auxin-overproducing mutant, superroot1 (sur1), of Arabidopsis thaliana. Activity staining for AO after native polyacrylamide gel electrophoresis separation of seedling extracts revealed that there were three major bands with AO activity (AO1–3) in wild-type and mutant seedlings. One of them (AO1) had a higher substrate preference for indole-3-aldehyde. This AO activity was significantly higher in sur1 mutant seedlings than in the wild type. The difference in activity was most apparent 7 d after germination, the same time required for the appearance of the remarkable sur1 phenotype, which includes epinastic cotyledons, elongated hypocotyls, and enhanced root development. Higher activity was observed in the root and hypocotyl region of the mutant seedlings. We also assayed the indole-3-acetaldehyde oxidase activity in extracts by high-performance liquid chromatography detection of indole-3-acetic acid (IAA). The activity was about 5 times higher in the extract of the sur1 seedlings, indicating that AO1 also has a substrate preference for abscisic aldehyde. Treatment of the wild-type seedlings with picloram or IAA caused no significant increase in AO1 activity. This result suggested that the higher activity of AO1 in sur1 mutant seedlings was not induced by IAA accumulation and, thus, strongly supports the possible role of AO1 in IAA biosynthesis in Arabidopsis seedlings.