Fusant Trichoderma HF9 with enhanced extracellular chitinase and protein content

Strain improvement was carried out to obtain higher chitinase and protein by inter-specific protoplast fusion between Trichoderma harzianum and Trichoderma viride. Fusant HF9 and parental strains of Trichoderma were compared for chitinase and protein production. 1% of glucose, sucrose and fungal cell wall (Rhizoctonia solani), were used as carbon source for cultivation of Trichoderma and fungal cell wall was the best to induce chitinase and protein. Usage of 0.5% colloidal chitin for the fungal growth under aerated conditions at pH 6.5 and 28°C led to higher chitinase and protein production. In these conditions fusant Trichoderma HF9 in comparison with parent strains had 3-, 2.5- and 1.5-fold increase of total chitinase, specific chitinase and protein, respectively. SDS-PAGE analysis revealed that it had 9 major protein bands with up-regulation compared to parent strains. Amino acid analysis showed that protein of culture filtrate of T. harzianum, T. viride and fusant Trichoderma HF9 had 8, 6 and 10 amino acids, respectively. The results obtained suggested that fusant HF9 could be an integration of T. harzianum and T. viride through protoplast fusion.     

Screening of Chromium-Resistant Bacteria for Plant Growth-Promoting Activities

Chromium-resistant plant growth-promoting bacteria (PGPB) were isolated from the electroplating industry waste disposal site soils at Coimbatore, India, using LB medium. The strain tolerated chromium concentrations up to 500 mg Cr⁶⁺L^?1 on LB medium. The strain was identified as Pseudomonas sp. VRK3 based on its morphological, physiological, and biochemical characteristics following Bergey's manual of determinative bacteriology. Evaluation of plant growth-promoting parameters revealed the intrinsic ability of the strain for the production of indole-3-acetic acid (IAA), siderophore, and solubilization of insoluble phosphate. Pseudomonas sp. VRK3 utilized tryptophan as a precursor for the growth and production of IAA (105.77 μg mL^?1) and also exhibited the production of siderophore. The strain utilized tricalcium phosphate as the sole source of phosphate exhibiting a high rate of phosphate solubilization (0.49 μg mL^?1). Pseudomonas sp. VRK3 showed a significant extent of chromium uptake and accumulation in their cell walls. Furthermore, Pseudomonas sp. VRK3 was demonstrated to possess mobilization of chromium from the soil that would enhance chromium availability to the plant. Potential use of this Pseudomonas sp. VRK3 as PGPB needs further testing in enhancing the growth and chromium uptake by the plants in pots under nursery conditions.     

Effect of Eugenia Jambolana seed kernel on antioxidant defense system in streptozotocin-induced diabetes in rats

Increased oxidative stress has been suggested to be involved in the pathogenesis and progression of diabetic tissue damage. The aim of this study was to investigate the effect of ethanolic extract of Eugenia jambolana seed kernel on antioxidant defense systems of plasma and pancreas in streptozotocin-induced diabetes in rats. The levels of glucose, vitamin-C, vitamin-E, ceruloplasmin, reduced glutathione and lipidperoxides were estimated in plasma of control and experimental groups of rats. The levels of lipidperoxides, reduced glutathione and activities of superoxide dismutase, catalase and glutathione peroxidase were assayed in pancreatic tissue of control and experimental groups of rats. A significant increase in the levels of plasma glucose, vitamin-E, ceruloplasmin, lipid peroxides and a concomitant decrease in the levels of vitamin-C, reduced glutathione were observed in diabetic rats. The activities of pancreatic antioxidant enzymes were altered in diabetic rats. These alterations were reverted back to near normal level after the treatment with Eugenia jambolana seed kernel and glibenclamide. Histopathological studies also revealed that the protective effect of Eugenia jambolana seed kernel on pancreatic beta-cells. The present study shows that Eugenia jambolana seed kernel decreased oxidative stress in diabetic rats, which inturn may be due to its hypoglycemic property.     

Enhanced enzymatic hydrolysis of mild alkali pre-treated rice straw at high-solid loadings using in-house cellulases in a bench scale system

In the present study, scale-up systems for cellulase production and enzymatic hydrolysis of pre-treated rice straw at high-solid loadings were designed, fabricated and tested in the laboratory. Cellulase production was carried out using tray fermentation at 45 °C by Aspergillus terreus in a temperature-controlled humidity chamber. Enzymatic hydrolysis studies were performed in a horizontal rotary drum reactor at 50 °C with 25 % (w/v) solid loading and 9 FPU g^?1 substrate enzyme load using in-house as well commercial cellulases. Highly concentrated fermentable sugars up to 20 % were obtained at 40 h with an increased saccharification efficiency of 76 % compared to laboratory findings (69.2 %). These findings demonstrate that we developed a simple and less energy intensive bench scale system for efficient high-solid saccharification. External supplementation of commercial β-glucosidase and hemicellulase ensured better hydrolysis and further increased the saccharification efficiency by 14.5 and 20 %, respectively. An attempt was also made to recover cellulolytic enzymes using ultrafiltration module and nearly 79–84 % of the cellulases and more than 90 % of the sugars were recovered from the saccharification mixture.     

Natural variation involving deletion alleles of FRIGIDA modulate temperature‐sensitive flowering responses in Arabidopsis thaliana

Ambient temperature is one of the major environmental factors that modulate plant growth and development. There is extensive natural genetic variation in thermal responses of plants exemplified by the variation exhibited by the accessions of Arabidopsis thaliana. In this work we have studied the enhanced temperature response in hypocotyl elongation and flowering shown by the Tsu‐0 accession in long days. Genetic mapping in the Col‐0 × Tsu‐0 recombinant inbred line (RIL) population identified several QTLs for thermal response including three major effect loci encompassing candidate genes FRIGIDA (FRI), FLOWERING LOCUS C (FLC) and FLOWERING LOCUS T (FT). We confirm and validate these QTLs. We show that the Tsu‐0 FRI allele, which is the same as FRI‐Ler is associated with late flowering but only at lower temperatures in long days. Using transgenic lines and accessions, we show that the FRI‐Ler allele confers temperature‐sensitive late flowering confirming a role for FRI in photoperiod‐dependent thermal response. Through quantitative complementation with heterogeneous inbred families, we further show that cis‐regulatory variation at FT contributes to the observed hypersensitivity of Tsu‐0 to ambient temperature. Overall our results suggest that multiple loci that interact epistatically govern photoperiod‐dependent thermal responses of A. thaliana.     

Genome‐wide association analyses reveal complex genetic architecture underlying natural variation for flowering time in canola

Optimum flowering time is the key to maximize canola production in order to meet global demand of vegetable oil, biodiesel and canola‐meal. We reveal extensive variation in flowering time across diverse genotypes of canola under field, glasshouse and controlled environmental conditions. We conduct a genome‐wide association study and identify 69 single nucleotide polymorphism (SNP) markers associated with flowering time, which are repeatedly detected across experiments. Several associated SNPs occur in clusters across the canola genome; seven of them were detected within 20 Kb regions of a priori candidate genes; FLOWERING LOCUS T, FRUITFUL, FLOWERING LOCUS C, CONSTANS, FRIGIDA, PHYTOCHROME B and an additional five SNPs were localized within 14 Kb of a previously identified quantitative trait loci for flowering time. Expression analyses showed that among FLC paralogs, BnFLC.A2 accounts for ~23% of natural variation in diverse accessions. Genome‐wide association analysis for FLC expression levels mapped not only BnFLC.C2 but also other loci that contribute to variation in FLC expression. In addition to revealing the complex genetic architecture of flowering time variation, we demonstrate that the identified SNPs can be modelled to predict flowering time in diverse canola germplasm accurately and hence are suitable for genomic selection of adaptative traits in canola improvement programmes.     

Microbial Inoculants with Multifaceted Traits Suppress Rhizoctonia Populations and Promote Plant Growth in Cotton

The suppressive effects of microbial inoculants on cotton seedling mortality were assessed in Rhizoctonia solani‐infested soil. Per cent mortality ranged from 16 to 32 (60–120 days after sowing, DAS) and significant differences were recorded at 120 DAS, especially after drenching with compost tea of Azotobacter sp. and Anabaena torulosa—Trichoderma viride‐biofilmed formulations. The activity of hydrolytic enzymes was reduced in diseased root tissues due to a majority of the microbially inoculated treatments, compared with healthy root tissues. Per cent changes in the amounts of glomalin‐related soil proteins (GRSPs) were 2 to 85% greater than those of the uninoculated experimental controls. These microbial inoculants altered the rhizosphere bacterial communities as evident from the Denaturing gradient gel electrophoresis (DGGE) banding patterns and, also reduced the population of R. solani. While the copy numbers of the internal transcribed spacer (ITS) gene of R. solani in the uninoculated (infested soil) were approximately 1.47 × 10¹¹ per g soil, they were 1.34–1.42 × 10⁵ per g soil after the application of A. torulosa, Anabaena laxa and A. torulosa–Bacillus sp. Increases in yield (ranging from 3 to 23%) due to various microbial inoculants relative to uninoculated controls illustrated their promise as plant growth‐promoting and disease‐suppressing agents. This study illustrates the modulation of rhizosphere ecology through microbial inoculants as a mechanism of disease suppression and sustaining plant growth.     

ORMDL/serine palmitoyltransferase stoichiometry determines effects of ORMDL3 expression on sphingolipid biosynthesis

The ORM1 (Saccharomyces cerevisiae)-like proteins (ORMDLs) and their yeast orthologs, the Orms, are negative homeostatic regulators of the initiating enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT). Genome-wide association studies have established a strong correlation between elevated expression of the endoplasmic reticulum protein ORMDL3 and risk for childhood asthma. Here we test the notion that elevated levels of ORMDL3 decrease sphingolipid biosynthesis. This was tested in cultured human bronchial epithelial cells (HBECs) (an immortalized, but untransformed, airway epithelial cell line) and in HeLa cells (a cervical adenocarcinoma cell line). Surprisingly, elevated ORMDL3 expression did not suppress de novo biosynthesis of sphingolipids. We determined that ORMDL is expressed in functional excess relative to SPT at normal levels of expression. ORMDLs and SPT form stable complexes that are not increased by elevated ORMDL3 expression. Although sphingolipid biosynthesis was not decreased by elevated ORMDL3 expression, the steady state mass levels of all major sphingolipids were marginally decreased by low level ORMDL3 over-expression in HBECs. These data indicate that the contribution of ORMDL3 to asthma risk may involve changes in sphingolipid metabolism, but that the connection is complex.     

Populus GT43 family members group into distinct sets required for primary and secondary wall xylan biosynthesis and include useful promoters for wood modification

The plant GT43 protein family includes xylosyltransferases that are known to be required for xylan backbone biosynthesis, but have incompletely understood specificities. RT‐qPCR and histochemical (GUS) analyses of expression patterns of GT43 members in hybrid aspen, reported here, revealed that three clades of the family have markedly differing specificity towards secondary wall‐forming cells (wood and extraxylary fibres). Intriguingly, GT43A and B genes (corresponding to the Arabidopsis IRX9 clade) showed higher specificity for secondary‐walled cells than GT43C and D genes (IRX14 clade), although both IRX9 and IRX14 are required for xylosyltransferase activity. The remaining genes, GT43E, F and G (IRX9‐L clade), showed broad expression patterns. Transient transactivation analyses of GT43A and B reporters demonstrated that they are activated by PtxtMYB021 and PNAC085 (master secondary wall switches), mediated in PtxtMYB021 activation by an AC element. The high observed secondary cell wall specificity of GT43B expression prompted tests of the efficiency of its promoter (pGT43B), relative to the CaMV 35S (35S) promoter, for overexpressing a xylan acetyl esterase (CE5) or downregulating REDUCED WALL ACETYLATION (RWA) family genes and thus engineering wood acetylation. CE5 expression was weaker when driven by pGT43B, but it reduced wood acetyl content substantially more efficiently than the 35S promoter. RNAi silencing of the RWA family, which was ineffective using 35S, was achieved when using GT43B promoter. These results show the utility of the GT43B promoter for genetically engineering properties of wood and fibres.