Molecular and Phenotypic Investigation on Antibacterial Activities of Limonene Isomers and Its Oxidation Derivative against Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes a devastating bacterial leaf blight in rice. Here, the antimicrobial effects of D-limonene, L-limonene, and its oxidative derivative carveol against Xoo were investigated. We revealed that carveol treatment at ≥ 0.1 mM in liquid culture resulted in significant decrease in Xoo growth rate (> 40%) in a concentration-dependent manner, and over 1 mM, no growth was observed. The treatment with D-limonene and L-limonene also inhibited the Xoo growth but to a lesser extent compared to carveol. These results were further elaborated with the assays of motility, biofilm formation and xanthomonadin production. The carveol treatment over 1 mM caused no motilities, basal level of biofilm formation (< 10%), and significantly reduced xanthomonadin production. The biofilm formation after the treatment with two limonene isomers was decreased in a concentration-dependent manner, but the degree of the effect was not comparable to carveol. In addition, there was negligible effect on the xanthomonadin production mediated by the treatment of two limonene isomers. Field emission-scanning electron microscope (FE-SEM) unveiled that all three compounds used in this study cause severe ultrastructural morphological changes in Xoo cells, showing shrinking, shriveling, and holes on their surface. Moreover, quantitative real-time PCR revealed that carveol and D-limonene treatment significantly down-regulated the expression levels of genes involved in virulence and biofilm formation of Xoo, but not with L-limonene. Together, we suggest that limonenes and carveol will be the candidates of interest in the development of biological pesticides.

The complete chloroplast genome of Chrysanthemum zawadskii Herbich (Asteraceae) isolated in Korea.

We have determined the complete chloroplast genome of Chrysanthemum zawadskii Herbich isolated in Korea. The circular chloroplast genome of C. zawadskii is 151,137 bp long and has four subregions: 83,041 bp of large single copy and 18,350 bp of small single copy regions are separated by 24,873 bp of inverted repeat regions including 133 genes (87 protein-coding genes, eight rRNA genes, 37 tRNAs, and one pseudogene). There are 65 to 152 single nucleotide polymorphisms and 33 to 64 insertion and deletion regions (178 bp to 372 bp in length) identified against three available chloroplast genomes of C. zawadskii. The phylogenetic tree shows that C. zawadskii is clustered as a paraphyletic group with C. zawadskii subsp. coreanum, displaying incongruency between species and clades.

Electrospray patterning of yeast cells for applications in alcoholic fermentation.

Yeast cells patterned by pulsed jet electrospray showed a high alcoholic fermentation rate. Multi-dimensional patterns of individual yeast cells were produced by varying the experimental parameters of the electrospray system. The electrospray process, which employed a vibrational electric field, could control patterns of viable yeast cells at a cellular resolution. This novel system for electrospraying viable cells can be applied to biological process engineering including whole cell biochip techniques and micro fermentation processes for biochemical studies.

Functional and genomic characterization of a wound- and methyl jasmonate-inducible chalcone isomerase in Eremochloa ophiuroides [Munro] Hack.

Eremochloa ophiuroides, a perennial warm-season lawn grass, has a characteristic phenotype of red pigmentation in tissues during maturation. The putative gene families associated with the red coloration were previously identified in E. ophiuroides. These genes encode chalcone synthases, flavonol 3-hydroxylases, and flavonol 3'-hydroxylases, acting on the early flavonoid-biosynthesis pathway. Here, a type-I chalcone isomerase (CHI) gene was isolated from E. ophiuroides based on leaf-transcriptome data, and the corresponding enzyme was functionally characterized in vitro and in planta. Complementation of Arabidopsis tt5 mutants by overexpressing EoCHI recapitulated the wild-type seed coat color. Wounding and methyl jasmonate treatments significantly elevated the transcript level of EoCHI and total anthocyanin content in shoots. Confocal microscopy indicated the localization of EoCHI to the endoplasmic reticulum. The genomic EoCHI sequence contained two introns with a novel pattern of exon‒intron organization. Further examinations on genomic structures of CHI family from ancient to advanced plant lineages should be of interests to decipher evolutionary pathways of extant plant CHI genes.

Transcriptome-guided identification and functional characterization of key terpene synthases involved in constitutive and methyl jasmonate-inducible volatile terpene formation in Eremochloa ophiuroides (Munro) Hack.

Centipedegrass (Eremochloa ophiuroides [Munro] Hack.) is a warm-season turfgrass, widely planted in residential lawns and recreational fields. Here, we uncovered three major terpenes released from the shoots of Eo: (E)-ß-ocimene (6%), α-muurolene (87.8%), and eremophilene (6.2%). Methyl jasmonate (MeJA) treatment increased the emission of monoterpenes, including (E)- and (Z)-ß-ocimene, limonene, and myrcene, as well as sesquiterpene blends of (E)-caryophyllene, α-copaene, (+)-cyclosativene, and α-farnesene. RNA sequencing analysis predicted 14 putative Eo terpene synthase (EoTPS) genes, and two full-length EoTPS were successfully amplified: Eo7816 (1722 bp) and Eo6039 (1701 bp). Phylogenetic analysis revealed that Eo7816 and Eo6039 belonged to the clades of TPS-b and TPS-a, respectively. The Arabidopsis transgenic plants overexpressing Eo7816 exclusively released (E)-ß-ocimene (96%) with (Z)-ß-ocimene and myrcene. In contrast, Eo6039-overexpressing Arabidopsis plants emitted significant amounts of α-muurolene (69.4%) and eremophilene (21.8%). Together, we demonstrated that the two TPSs play roles in producing major volatile terpenes in Eo.

Novel functions of peroxiredoxin Q from Deinococcus radiodurans R1 as a peroxidase and a molecular chaperone.

Deinococcus radiodurans R1 is extremely resistant to ionizing radiation and oxidative stress. In this study, we characterized DR0846, a candidate peroxiredoxin in D. radiodurans. DR0846 is a peroxiredoxin Q containing two conserved cysteine residues. DR0846 exists mainly in monomeric form with an intramolecular disulfide bond between the two cysteine residues. We found that DR0846 functions as a molecular chaperone as well as a peroxidase. A mutational analysis indicates that the two cysteine residues are essential for enzymatic activity. A double-deletion mutant lacking DR0846 and catalase DR1998 exhibits decreased oxidative and heat shock stress tolerance with respect to the single mutants or the wild-type cells. These results suggest that DR0846 contributes to resistance against oxidative and heat stresses in D. radiodurans.

Inhibitory effect of gamma irradiation and its application for control of postharvest green mold decay of Satsuma mandarins.

Gamma irradiation has been shown to be effective for the control of postharvest fungi in vitro, but little is known regarding antifungal action, responses to gamma irradiation, and its application to fresh produce. Gamma irradiation was evaluated for its in vitro and in vivo antifungal activity against Penicillium digitatum on Satsuma mandarin fruits. Green mold was inhibited in a dose-dependent manner. Gamma irradiation showed a complete inhibition of spore germination, germ tube elongation, and mycelial growth of P. digitatum, particularly at 1.0kGy. To further investigate the mechanisms by which gamma irradiation inhibits fungal growth, the membrane integrity and cellular leakage of conidia were tested, indicating that gamma irradiation results in the loss of plasma membrane integrity, causing the release of intracellular contents such as soluble proteins. In vivo assays demonstrated that established doses can completely inhibit the growth of fungal pathogens, but such high doses cause severe fruit damage. Thus, to eliminate the negative impact on fruit quality, gamma irradiation at lower doses was evaluated for inhibition of P. digitatum, in combination with a chlorine donor, sodium dichloro-s-triazinetrione (NaDCC). Interestingly, only a combined treatment with 0.4kGy of gamma irradiation and 10ppm of NaDCC exhibited significant synergistic antifungal activity against green mold decay. The mechanisms by which the combined treatment decreased the green mold decay of mandarin fruits can be directly associated with the disruption of cell membrane of the fungal pathogen, which resulted in a loss of cytoplasmic material from the hyphae. These findings suggest that a synergistic effect of combining treatment with gamma irradiation with NaDCC has potential as an antifungal approach to reduce the severity of green mold in mandarin fruits.

Direct suppression of a rice bacterial blight (Xanthomonas oryzae pv. oryzae) by monoterpene (S)-limonene.

Rice bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a severe disease of rice plants. Upon pathogen infection, rice biosynthesizes phytoalexins, including diterpenoids such as momilactones, phytocassanes, and oryzalexins. However, information on headspace volatiles in response to Xoo infection is limited. We have examined headspace volatile terpenes, induced by the infection of Xoo, and investigated their biological roles in the rice plant. Monoterpenes α-thujene, α-pinene, sabinene, myrcene, α-terpene, and (S)-limonene and sesquiterpenes cyclosativene, α-copaene, and ß-elemene were detected from 1-week-old Xoo-infected rice seedlings, by solid-phase microextraction-gas chromatography-mass spectrometry. All monoterpenes were constitutively released from rice seedlings before Xoo infection. However, (S)-limonene emission was further elicited after exposure of the seedlings to Xoo in coincidence with upregulation of limonene synthase gene (OsTPS20) transcripts. Only the stereospecific (S)-limonene [and not (R)-limonene or other monoterpenes] severely inhibited Xoo growth, as confirmed by disc diffusion and liquid culture assays. Rice seedlings showed suppressed pathogenic symptoms suggestive of resistance to Xoo infection after foliar treatment with (S)-limonene. Collectively, our findings suggest that (S)-limonene is a volatile phytoanticipin, which plays a significant role in suppressing Xoo growth in rice seedlings.

Herbaspirillum sp. strain GW103 alleviates salt stress in Brassica rapa L. ssp. pekinensis.

Mutual interactions between plant and rhizosphere bacteria facilitate plant growth and reduce risks of biotic and abiotic stresses. The present study demonstrates alleviation of salt stress in Brassica rapa L. ssp. perkinensis (Chinese cabbage) by Herbaspirillum sp. strain GW103 isolated from rhizosphere soil of Phragmites australis. The strain was capable of producing plant beneficial factors, such as auxin, siderophore, and 1-aminocylopropane-1-carboxylic acid deaminase. Treatment of strain GW103 on Chinese cabbage under salt stress increased K(+)/Na(+) ratio in roots generating balance in the ratio of ion homeostasis and consequently contributed to the increase of biomass. In addition, root colonization potential of the strain was observed by green fluorescent protein (GFP)-tagging approach. These results strongly suggest the beneficial impact of strain GW103 by inducing the alleviation of salt stress and development of stress tolerance in Chinese cabbage via plant-microbe interaction.

Paenibacillus gelatinilyticus sp. nov. a psychrotolerant bacterium isolated from a reclaimed soil and amended description of Paenibacillus shenyangensis.

A rod shaped, Gram-stain positive, non-motile, facultative anaerobic and gelatin hydrolysing bacterium, strain PG1(T), was isolated from reclaimed land soil in Kyehwa-do, Republic of Korea. Strain PG1(T) showed highest 16S rRNA gene sequence similarity (97.4 and 96.5%, respectively) to Paenibacillus shenyangensis A9(T) and Paenibacillus hunanensis FeL05(T), and clustered closely with the members of the family Paenibacillaceae. DNA-DNA hybridization studies revealed a genomic relatedness of 47 ± 9% with P. shenyangensis A9(T). The predominant fatty acids of strain PG1(T) were identified to be anteiso-C(15:0) (46.7%) and C(16:0) (22.7%). Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified phospholipid were found to be the major polar lipids. The genomic DNA G+C content was found to be 47.7 mol%. This polyphasic characterisation of the newly isolated strain PG1(T) justifies its description as representative of a novel species in the genus Paenibacillus, for which the name Paenibacillus gelatinilyticus sp. nov., (type strain = PG1(T) = KCTC 33642(T) = JCM 30624(T)) is proposed.

Rice terpene synthase 20 (OsTPS20) plays an important role in producing terpene volatiles in response to abiotic stresses.

This study examined the volatile terpenes produced by rice seedlings in response to oxidative stress induced by various abiotic factors. Solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) analyses revealed that when exposed to UV-B radiation, rice seedlings emitted a bouquet of monoterpene mixtures in a time-dependent manner. The mixtures comprised limonene, sabinene, myrcene, α-terpinene, ß-ocimene, γ-terpinene, and α-terpinolene. Among them, (S)-limonene was the most abundant volatile, discriminated by chiral SPME-GC-MS. The volatile profiles collected from rice plants treated with both γ-irradiation and H2O2 were identical to those observed in the UV-B irradiated plants, thus indicating that the volatile mixtures were specifically produced in response to oxidative stress, particularly in the presence of H2O2. Using a reverse genetics approach, we isolated full-length rice terpene synthase 20 (OsTPS20, 599 amino acids, 69.39 kDa), which was further characterized as an (S)-limonene synthase by removing the N-terminal signal peptide (63 amino acids) of the protein. The recombinant OsTPS20 protein catalyzed the conversion of geranyl diphosphate to (S)-limonene and other minor monoterpenes, essentially covering all of the volatile compounds detected from the plant. Moreover, qRT-PCR revealed that the transcript levels of OsTPS20 were significantly induced in response to oxidative stress, thereby suggesting that OsTPS20 plays a major role in producing terpene volatiles during abiotic stress. Detailed biochemical analyses and the unusual domain characteristics of OsTPS20 are also discussed in this report.

Bioleaching characteristics, influencing factors of Cu solubilization and survival of Herbaspirillum sp. GW103 in Cu contaminated mine soil.

This study was aimed at assess the potential of diazotrophic bacteria, Herbaspirillum sp. GW103, for bioleaching of Cu in mine soil. The strain exhibited resistance to As (550mgL(-1)), Cu (350mgL(-1)), Zn (300mgL(-1)) and Pb (200mgL(-1)). The copper resistance was further confirmed by locating copA and copB genes. The survival of the isolate GW103 during bioleaching was analyzed using green fluorescent protein tagged GW103. Response surface methodology based Box-Behnken design was used to optimize the physical and chemical conditions for Cu bioleaching. Five significant variables (temperature, incubation time, CaCO3, coconut oil cake (COC), agitation rate) were selected for the optimization. Second-order polynomials were established to identify the relationship between Cu bioleaching and variables. The optimal conditions for maximum Cu bioleaching (66%) were 30°C, 60h of incubation with 1.75% of CaCO3 and 3% COC at 140rpm. The results of Cu sequential extraction studies indicated that the isolate GW103 leached Cu from ion-exchangeable, reducible, strong organic and residual fractions. Obtained results point out that the isolate GW103 could be used for bioleaching of Cu from mine soils.

1-aminocyclopropane-1-carboxylate deaminase from Pseudomonas fluorescens promoting the growth of Chinese cabbage and its polyclonal antibody.

Bacterial 1-aminocyclopropane-1-carboxlyate (ACC) deaminase (AcdS) is an enzyme that cleaves ACC, a precursor of the plant hormone ethylene, into α-ketobutyrate and ammonia. The acdS gene was cloned from Pseudomonas fluorescens, which was capable of improving the seedling of Chinese cabbage under salinity condition. The recombinant AcdS (rAcdS) exhibited optimal activity at pH 8.5 and 30°C. Strong activity was sustained at up to 100 mM NaCl. The polyclonal anti-P. fluorescens AcdS antibody was produced in a rabbit that had been immunized with the purified rAcdS. This antibody successfully recognized the homologous antigens derived from the total proteins of isolated plant growth-promoting microorganisms. A statistically significant correlation was observed between the intensity of hybridization signal and AcdS activity measured by a biochemical method, suggesting its application as a useful indicator for active deaminases.

Inhibition of corneal neovascularization by subconjunctival and topical bevacizumab and sunitinib in a rabbit model.

PURPOSE: To compare the effects of subconjunctival injection and topical application of bevacizumab and sunitinib on experimentally induced corneal neovascularization (CNV). METHODS: CNV was induced by sutures in the right eyes of 36 rabbits. After suture removal, the rabbits were divided into 6 groups with 6 rabbits in each group. In groups 1, 2, and 3, the eyes received a subconjunctival injection of 0.1 mL of normal saline, 2.5 mg/0.1 mL of bevacizumab, and 0.25 mg/0.1 mL of sunitinib, respectively, immediately after suture removal. A booster injection of the same agent was repeated 1 week later in each group. In groups 4, 5, and 6, the eyes received topical applications of saline, bevacizumab (5 mg/mL), and sunitinib (0.5 mg/mL), respectively. These solutions were applied twice a day for 2 weeks, starting immediately after suture removal. CNV was analyzed through biomicroscopy and through histological examination using hematoxylin and eosin and CD31 immunohistochemical staining. RESULTS: On day 14, the mean percentages of areas of CNV in sunitinib-treated eyes were smaller compared with saline-treated or bevacizumab-treated eyes in both the subconjunctival (P = 0.003 and 0.032, respectively) and topical groups (P < 0.001 in both). The topical administration of sunitinib was significantly more effective than the subconjunctival injection of the same drug at 1 week (P = 0.011). Upon histological examination of samples from the topical group, sunitinib-treated eyes showed lower vascularity than saline-treated and bevacizumab-treated eyes (P = 0.036 and 0.046, respectively). CONCLUSIONS: These results suggest that sunitinib is more effective than bevacizumab for the inhibition of CNV. Furthermore, topical administration of sunitinib yields better results than a subconjunctival injection of the same medication.

Genome sequence of Herbaspirillum sp. strain GW103, a plant growth-promoting bacterium.

Herbaspirillum sp. strain GW103 was isolated from rhizosphere soil of the reed Phragmites australis on reclaimed land. Here we report the 5.05-Mb draft genome sequence of the strain, providing bioinformation about the agronomic benefits of this strain, such as multiple traits relevant to plant root colonization and plant growth promotion.