Molecular Identification of Juglans Regia Endophyte LTL-G3, Its Antifungal Potential and Bioactive Substances.

Background: Endophyte is one of the potential biocontrol agents for inhibiting plant pathogens. However, the mechanisms and characteristics involved in the inhibition of different phytopathogenic fungi by endophytes, especially walnut endophytes, are still largely unknown. Objectives: The present study aimed to identify the walnut endophytic fungus LTL-G3 from a genetic point of view, assess the strain's antifungal activity, and determine the bioactivities of the substances it produces against plant pathogens. Materials and Methods: The homologous sequence of strain LTL-G3 was examined, and typical strains of the Trichoderma virens group were used to build NJ phylogenetic trees and analyze the taxonomic position of the strain. The biocontrol agent's antagonistic potential for many plant pathogenic fungi. By using silica gel G chromatography, the active components of the strain were separated and purified. The active components were identified using GC-MS and NMR. Results: The strain LTL-G3 was identified as Trichoderma virens. Its fermentation and secondary metabolite extracts had a broad spectrum and strong inhibitory effect on the spread of six plant pathogens (Botrytis cinerea, Fusarium graminearum, Gloeosporium fructigenum, Phytophthora capsici, Rhizoctonia solani, and Valsa mali) evaluated, of which, its inhibition rate against Valsa mali reached 76.6% (fermentation extract) and 100% (ethyl acetate and n-butanol extracts). On silica gel G chromatography, bioactive compounds were divided into 6 fractions and 7 sub-fractions. Fr.2-2 was the sub-fraction that showed the greatest inhibitory against V. mali, as an inhibition percentage of 89.36% in 1 mg. mL-1. Fifteen key inhibitory chemicals identified using GC-MS. By examining the NMR data, the chemical make-up of the precipitated white solid was identified. The inhibition rate against V. mali increased by over 95% at a dosage of 1 mg. mL-1, indicating a significant linear association between compound A and that rate. Conclusions: The strain LTL-G3 can be applied as an efficient biological control agent against V. mali, and its highly inhibitive secondary metabolites provide the mechanism for this action.

Multiple transcriptional regulation of walnut JrGSTTau1 gene in response to osmotic stress.

Glutathione S-transferases (GSTs) are important plant proteins involved in biotic and abiotic stress responses. A gene from Juglans regia, JrGSTTau1 was previously cloned and functionally characterized as an enzyme involved in improving cold tolerance in plants. To clarify the functional mechanism of JrGSTTau1 and its role in stress response, here, the JrGSTTau1 promoter including the up-stream regulators was examined using yeast one-hybrid together with transient expression assays, and the osmotic stress response ability was confirmed by comparing with wild-type plants. The 1500 bp JrGSTTau1 promoter displayed high GUS expression activity and was enhanced by mannitol stress. The promoter is composed of abundant cis-elements, some of which were osmotic stress response-related motifs, such as ABRE, DRE and MYB, indicating that the expression of JrGSTTau1 is regulated by potential up-stream regulators under abiotic stress. The transcription factors (TFs) of JrDREB2A, JrMYC2, JrMYB44, JrDof1 and JrWRKY7 were identified, which shared a similar response with JrGSTTau1 when exposed to PEG6000 in walnut leaf and root. These results implied that JrDREB2A, JrMYC2, JrMYB44, JrDof1 and JrWRKY7 may act as up-stream regulators of JrGSTTau1 to regulate or combine functionality with JrGSTTau1 in osmotic stress response. Furthermore, compared with the WT plants, the transgenic tobacco plants that overexpress JrGSTTau1 showed improved tolerance to drought induced by osmotic stress, in which antioxidant enzymes, proline and reactive oxygen species (ROS) are involved. Our results demonstrated the positive role played by JrGSTTau1 in osmotic tolerance, which is regulated by multiple up-stream regulators.

The walnut transcription factor JrGRAS2 contributes to high temperature stress tolerance involving in Dof transcriptional regulation and HSP protein expression.

BACKGROUND: GRAS transcription factor (TF) family is unique and numerous in higher plants with diverse functions that involving in plant growth and development processes, such as gibberellin (GA) signal transduction, root development, root nodule formation, and mycorrhiza formation. Walnut tree is exposed to various environmental stimulus that causing concern about its resistance mechanism. In order to understand the molecular mechanism of walnut to adversity response, a GRAS TF (JrGRAS2) was cloned and characterized from Juglans regia in this study. RESULTS: A 1500 bp promoter fragment of JrGRAS2 was identified from the genome of J. regia, in which the cis-elements were screened. This JrGRAS2 promoter displayed expression activity that was enhanced significantly by high temperature (HT) stress. Yeast one-hybrid assay, transient expression and chromatin immunoprecipitation (Chip)-PCR analysis revealed that JrDof3 could specifically bind to the DOFCOREZM motif and share similar expression patterns with JrGRAS2 under HT stress. The transcription of JrGRAS2 was induced by HT stress and up-regulated to 6.73-~11.96-fold in the leaf and 2.53-~4.50-fold in the root to control, respectively. JrGRAS2 was overexpressed in Arabidopsis, three lines with much high expression level of JrGRAS2 (S3, S7, and S8) were selected for HT stress tolerance analysis. Compared to the wild type (WT) Arabidopsis, S3, S7, and S8 exhibited enhanced seed germination rate, fresh weight accumulation, and activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and glutathione-S-transferase (GST) under HT stress. In contrast, the Evans blue staining, electrolyte leakage (EL) rates, hydrogen dioxide (H2O2) and malondialdehyde (MDA) content of transgenic seedlings were all lower than those of WT exposed to HT stress. Furthermore, the expression of heat shock proteins (HSPs) in S3, S7, and S8 was significant higher than those in WT plants. The similar results were obtained in JrGRAS2 transient overexpression walnut lines under normal and HT stress conditions. CONCLUSIONS: Our results suggested that JrDof3 TF contributes to improve the HT stress response of JrGRAS2, which could effectively control the expression of HSPs to enhance HT stress tolerance. JrGRAS2 is an useful candidate gene for heat response in plant molecular breeding.

The complete chloroplast genome of the Juglans regia (Juglandales: Julandaceae).

The complete nucleotide sequence of Juglans regia chloroplast genome (cpDNA) was carried out using Illumina Hiseq 2500. The genome is 160 537 bp in length, and the overall base composition of H-strand is 31.59% for A, 18.35% for C, 17.76% for G and 32.30% for T. Similar to the other higher plants, cpDNA have a large single copy (LSC) region of 90 059 bp and a small single copy(SSC) of 18 412 bp, is separated by a pair of inverted repeats (IRa and IRb) of 26 033 bp. There are 113 genes in the cpDNA, including 80 protein-coding genes, 4 ribosomal RNA genes (4 rRNA species), 36 transfer RNA genes (19 tRNA species). To further investigate the evolution status of J. regia in the level of chloroplast genome, tree constructed using MEGA 6.0 with NJ and ML methord demonstrated J. regia was clustered in Fagales, which was identical with APG III.

In planta characterization of a tau class glutathione S-transferase gene from Juglans regia (JrGSTTau1) involved in chilling tolerance.

KEY MESSAGE: JrGSTTau1 is an important candidate gene for plant chilling tolerance regulation. A tau subfamily glutathione S-transferase (GST) gene from Juglans regia (JrGSTTau1, GeneBank No.: KT351091) was cloned and functionally characterized. JrGSTTau1 was induced by 16, 12, 10, 8, and 6 °C stresses. The transiently transformed J. regia showed much greater GST, glutathione peroxidase (GPX), superoxide dismutase (SOD), and peroxidase (POD) activities and lower H2O2, malondialdehyde (MDA), reactive oxygen species (ROS), and electrolyte leakage (EL) rate than prokII (empty vector control) and RNAi::JrGSTTau1 under cold stress, indicating that JrGSTTau1 may be involved in chilling tolerance. To further confirm the role of JrGSTTau1, JrGSTTau1 was heterologously expressed in tobacco, transgenic Line5, Line9, and Line12 were chosen for analysis. The germinations of WT, Line5, Line9, and Line12 were similar, but the fresh weight, primary root length, and total chlorophyll content (tcc) of the transgenic lines were significantly higher than those of WT under cold stress. When cultivated in soil, the GST and SOD activities of transgenic tobacco were significantly higher than those of WT; however, the MDA and H2O2 contents of WT were on average 1.47- and 1.96-fold higher than those of Line5, Line9, and Line12 under 16 °C. The DAB, Evans blue, and PI staining further confirmed these results. Furthermore, the abundances of NtGST, MnSOD, NtMAPK9, and CDPK15 were elevated in 35S::JrGSTTau1 tobacco compared with WT. These results suggested that JrGSTTau1 improves the plant chilling tolerance involved in protecting enzymes, ROS scavenging, and stress-related genes, indicating that JrGSTTau1 is a candidate gene for the potential application in molecular breeding to enhance plant abiotic stress tolerance.

Synthesis and characterization of carboxymethyl xylan-g-poly(propylene oxide) and its application in films.

Carboxymethyl xylan-g-poly(propylene oxide) (CMX-g-PPO) was successfully synthesized by grafting poly(propylene oxide) chains onto xylan from bamboo using the Al(Oi-Pr)3 initiated ring-opening polymerization of propylene oxides, followed by carboxymethylation with sodium chloroacetate under microwave irradiation. The synthesized CMX-g-PPO was well characterized by FT-IR, (13)C NMR, and AFM. The AFM imaging showed that the average sizes of xylan were 422.1×67.4×1.2nm, while the average sizes of grafting branches PPO were 128.0×38.5×5.1nm, which firstly provided an irrefutable and visual evidence for the structure of grafted xylan at single molecular level. Subsequently, a serial of CMX-g-PPO/CS films were prepared without addition of any plasticizers. The surface morphologies, wettability, water vapor barrier properties, mechanical properties, and thermal stabilities of the obtained films were characterized and compared with those of the control films by AFM, contact angle, WVP, tensile testing, and TGA, respectively.