Histone variant H2A.Z is required for plant salt response by regulating gene transcription.

As a well-conserved histone variant, H2A.Z epigenetically regulates plant growth and development as well as the interaction with environmental factors. However, the role of H2A.Z in response to salt stress remains unclear, and whether nucleosomal H2A.Z occupancy work on the gene responsiveness upon salinity is obscure. Here, we elucidate the involvement of H2A.Z in salt response by analysing H2A.Z disorder plants with impaired or overloaded H2A.Z deposition. The salt tolerance is dramatically accompanied by H2A.Z deficiency and reacquired in H2A.Z OE lines. H2A.Z disorder changes the expression profiles of large-scale of salt responsive genes, announcing that H2A.Z is required for plant salt response. Genome-wide H2A.Z mapping shows that H2A.Z level is induced by salt condition across promoter, transcriptional start site (TSS) and transcription ending sites (-1 kb to +1 kb), the peaks preferentially enrich at promoter regions near TSS. We further show that H2A.Z deposition within TSS provides a direct role on transcriptional control, which has both repressive and activating effects, while it is found generally H2A.Z enrichment negatively correlate with gene expression level response to salt stress. This study shed light on the H2A.Z function in salt tolerance, highlighting the complex regulatory mechanisms of H2A.Z on transcriptional activity for yielding appropriate responses to particularly environmental stress.

Endogenous melatonin involved in plant salt response by impacting auxin signaling.

KEY MESSAGE: The study on melatonin biosynthesis mutant snat1snat2 revealed that endogenous melatonin plays an important role in salt responsiveness by mediating auxin signaling. Melatonin is a pleiotropic signaling molecule, which, besides being involved in multiple growth and developmental processes, also mediates environmental stress responses. However, whether and how endogenous melatonin is involved in salt response has not been determined. In this study, we elucidated the involvement of endogenous melatonin in salt response by investigatiing the impact of salt stress on a double mutant of Arabidopsis (snat1snat2) defective in melatonin biosynthesis genes SNAT1 and SNAT2. This mutant was found to exhibit salt sensitivity, manifested by unhealthy growth, ion imbalance and ROS accumulation under salt stress. Transcriptomic profiles of snat1snat2 revealed that the expression of a large number of salt-responsive genes was affected by SNAT defect, and these genes were closely related to the synthesis of auxin and several signaling pathways. In addition, the salt-sensitive growth phenotype of snat1snat2 was alleviated by the application of exogenous auxin. Our results show that endogenous melatonin may be essential for plant salt tolerance, a function that could be correlated with diverse activity in mediating auxin signaling.

Arabidopsis nucleoporin NUP96 mediates plant salt tolerance by modulating the transcription of salt-responsive genes.

MAIN CONCLUSION: Physiological and molecular tests show that NUP96 plays an important role in the plant response to salt stress, resulting from the reprogramming of transcriptomic profiles, which are likely to be mediated by the influence on the nuclear/cytosol shuttling of the key regulators of salt tolerance. As a key component of the nuclear pore complex (NPC), nucleoporin 96 (NUP96) is critical for modulating plant development and interactions with environmental factors, but whether NUP96 is involved in the salt response is still unknown. Here, we analyzed the role of Arabidopsis NUP96 under salt stress. The loss-of-function mutant nup96 exhibited salt sensitivity in terms of rosette growth and root elongation, and showed attenuated capacity in maintaining ion and ROS homeostasis, which could be compensated for by the overexpression of NUP96. RNA sequencing revealed that many salt-responsive genes were misregulated after NUP96 mutation, and especially NUP96 is required for the expression of a large portion of salt-induced genes. This is likely correlated with the activity in facilitating nuclear/cytosol transport of the underlying regulators in salt tolerance such as the transcription factor ATAP2, targeted by eight downregulated genes in nup96 under salt stress. Our results illustrate that NUP96 plays an important role in the salt response, probably by regulating the nucleocytoplasmic shuttling of key mRNAs or proteins associated with plant salt responsiveness.

Redox Regulation of Salt Tolerance in Eutrema salsugineum by Proteomics.

Salt stress severely restricts plant growth and crop production, which is accompanied by accumulation of reactive oxygen species (ROS) that disturb cell redox homeostasis and oxidize redox-sensitive proteins. Eutrema salsugineum, a halophytic species closely related to Arabidopsis, shows a high level of tolerance to salinity and is increasingly used as a model plant in abiotic stress biology. To understand redox modifications and signaling pathways under salt stress, we used tandem mass tag (TMT)-based proteomics to quantify the salt-induced changes in protein redox modifications in E. salsugineum. Salt stress led to increased oxidative modification levels of 159 cysteine sites in 107 proteins, which play roles in carbohydrate and energy metabolism, transport, ROS homeostasis, cellular structure modulation, and folding and assembly. These lists of unknown redox reactive proteins in salt mustard lay the foundation for future research to understand the molecular mechanism of plant salt response. However, glutathione peroxidase (GPX) is one of the most important antioxidant enzymes in plants. Our research indicates that EsGPX may be involved in regulating ROS levels and that plants with overexpressed EsGPX have much improved salt tolerance.

Enhancement of sulfur metabolism and antioxidant machinery confers Bacillus sp. Jrh14-10-induced alkaline stress tolerance in plant.

Alkaline stress is a major environmental challenge that restricts plant growth and agricultural productivity worldwide. Plant growth-promoting rhizobacteria (PGPR) can be used to effectively enhance plant abiotic stress in an environment-friendly manner. However, PGPR that can enhance alkalinity tolerance are not well-studied and the mechanisms by which they exert beneficial effects remain elusive. In this study, we isolated Jrh14-10 from the rhizosphere soil of halophyte Halerpestes cymbalaria (Pursh) Green and found that it can produce indole-3-acetic acid (IAA) and siderophore. By 16S rRNA gene sequencing, it was classified as Bacillus licheniformis. Inoculation Arabidopsis seedlings with Jrh14-10 significantly increased the total fresh weight (by 148.1%), primary root elongation (by 1121.7%), and lateral root number (by 108.8%) under alkaline stress. RNA-Seq analysis showed that 3389 genes were up-regulated by inoculation under alkaline stress and they were associated with sulfur metabolism, photosynthetic system, and oxidative stress response. Significantly, the levels of Cys and GSH were increased by 144.3% and 48.7%, respectively, in the inoculation group compared to the control under alkaline stress. Furthermore, Jrh14-10 markedly enhanced the activities of antioxidant enzymes, resulting in lower levels of O2•-, H2O2, and MDA as well as higher levels of Fv/Fm in alkaline-treated seedlings. In summary, Jrh14-10 can improve alkaline stress resistance in seedlings which was accompanied by an increase in sulfur metabolism-mediated GSH synthesis and antioxidant enzyme activities. These results provide a mechanistic understanding of the interactions between a beneficial bacterial strain and plants under alkaline stress.

Analysis of N6-methyladenosine reveals a new important mechanism regulating the salt tolerance of sugar beet (Beta vulgaris).

Salinity is an important environmental factor in crop growth and development. N6-methyladenosine (m6A) is an essential epigenetic modification that regulates plant-environment interaction. Sugar beet is a major sugar-yielding crop that has a certain tolerance to salt, but the dynamic response elicited by the m6A modification of transcripts under salt stress remains unknown. In this study, sugar beet was exposed to 300 mM NaCl to investigate its physiological response to high salinity and transcriptome-wide m6A modification profile. After the salt treatment, 7737 significantly modified m6A sites and 4981 differentially expressed genes (DEGs) were identified. Among the 312 m6A-modified DEGs, 113 hypomethylated DEGs were up-regulated and 99 hypermethylated DEGs were down-regulated, indicating a negative correlation between m6A modification and gene expression. Well-known salt tolerance genes (e.g., sodium/hydrogen exchanger 1, choline monooxygenase, and nucleoredoxin 2) and phospholipid signaling pathway genes (phosphoinositol-specific phospholipase C, phospholipase D, diacylglycerol kinase 1, etc.) were also among the m6A-modified genes. Further analysis showed that m6A modification may regulate salt-tolerant related gene expression by controlling mRNA stability. Therefore, changes in m6A modification may negatively regulate the expression of the salt-resistant genes in sugar beet, at least in part by modulating the stability of the mRNA via demethylase BvAlkbh10B. These findings could provide a better understanding of the epigenetic mechanisms of salt tolerance in sugar beets and uncover new candidate genes for improving the production of sugar beets planted in high-salinity soil.

Unique Features of the m6A Methylome and Its Response to Salt Stress in the Roots of Sugar Beet (Beta vulgaris).

Salt is one of the most important environmental factors in crop growth and development. N6-methyladenosine (m6A) is an epigenetic modification that regulates plant-environment interaction at transcriptional and translational levels. Sugar beet is a salt-tolerant sugar-yielding crop, but how m6A modification affects its response to salt stress remains unknown. In this study, m6A-seq was used to explore the role of m6A modification in response to salt stress in sugar beet (Beta vulgaris). Transcriptome-wide m6A methylation profiles and physiological responses to high salinity were investigated in beet roots. After treatment with 300 mM NaCl, the activities of peroxidase and catalase, the root activity, and the contents of Na+, K+, and Ca2+ in the roots were significantly affected by salt stress. Compared with the control plants, 6904 differentially expressed genes (DEGs) and 566 differentially methylated peaks (DMPs) were identified. Association analysis revealed that 243 DEGs contained DMP, and 80% of these DEGs had expression patterns that were negatively correlated with the extent of m6A modification. Further analysis verified that m6A methylation may regulate the expression of some genes by controlling their mRNA stability. Functional analysis revealed that m6A modifications primarily affect the expression of genes involved in energy metabolism, transport, signal transduction, transcription factors, and cell wall organization. This study provides evidence that a post-transcriptional regulatory mechanism mediates gene expression during salt stress by affecting the stability of mRNA in the root.

Growth promotion of Sargassum fusiforme by epiphytic microbes is dependent on the extent of interspecific interactions of the microbial community.

Profound growth differences such as seedling length and biomass are often observed during the cultivation of Sargassum fusiforme despite the absence of detectable variance in abiotic factors that could have affected this process. This highlights the importance of biotic factors such as epiphytic microbiota in controlling seedling growth. Yet, how, and to what extent microbial activities can affect host growth in the presence of seawater flow and continuous erosion remains debatable. Particularly, the contribution of microbial network interactions to the growth of macroalgae remains poorly understood. This study aimed to compare the physicochemical properties of S. fusiforme seedlings via 16S rRNA gene Illumina sequencing-based profiling of the epiphytic microbial communities of seedlings with different lengths. Significantly different epiphytic bacterial communities were observed among S. fusiforme seedlings of different lengths. The result showed that community from longer seedlings maintained higher bacterial diversity with the taxa Gammaproteobacteria, Burkholderiales, Alteromonadales, Vibrionaceae, Ralstonia, Colwelliaceae, and Thalassotalea being selectively enriched. More importantly, microbial interspecific interactions, which were predominantly positive, were enhanced consistently in communities of the longer seedlings, indicative of reinforced prevalent and mutually cooperative relationships among the microorganisms associated with S. fusiforme seedlings of greater length. Furthermore, longer seedlings also displayed up-regulation of microbial functional potentials involved in N fixation and mineralization, P mineralization and transportation, and ion transportation compared with shorter ones. Lastly, stochastic processes dominated the community assembly of the epiphytic microorganisms. These findings could provide new insights into the relationship between microbial communities and growth in S. fusiforme seedlings and enable us to predict the community diversity and assembly of macroalgae-associated microbial communities. This could have important implications for linking microbial community diversity and network interactions to their host productivity.

A Salt-Tolerant Strain of Trichoderma longibrachiatum HL167 Is Effective in Alleviating Salt Stress, Promoting Plant Growth, and Managing Fusarium Wilt Disease in Cowpea.

Salt stress is a constraint factor in agricultural production and restricts crops yield and quality. In this study, a salt-tolerant strain of Trichoderma longibrachiatum HL167 was obtained from 64 isolates showing significant salt tolerance and antagonistic activity to Fusarium oxysporum. T. longibrachiatum HL167 inhibited F. oxysporum at a rate of 68.08% in 200 mM NaCl, penetrated F. oxysporum under 200 mM NaCl, and eventually induced F. oxysporum hyphae breaking, according to electron microscope observations. In the pot experiment, pretreatment of cowpea seedlings with T. longibrachiatum HL167 reduced the accumulation level of ROS in tissues and the damage caused by salt stress. Furthermore, in the field experiment, it was discovered that treating cowpea with T. longibrachiatum HL167 before root inoculation with F. oxysporum can successfully prevent and control the development of cowpea Fusarium wilt, with the best control effect reaching 61.54%. Moreover, the application of HL 167 also improved the K+/Na+ ratio of cowpea, alleviated the ion toxicity of salt stress on cowpea, and HL167 was found to effectively colonize the cowpea roots. T. longibrachiatum HL167, which normally survives in saline-alkali environments and has the functions of disease prevention and plant growth promotion capabilities, has important research implications for improving the saline-alkali soil environment and for the sustainable development of green agriculture.

Overexpression of an aquaporin gene EsPIP1;4 enhances abiotic stress tolerance and promotes flowering in Arabidopsis thaliana.

Aquaporins are water channel proteins that play an essential role in plant growth and development. Despite extensive functional characterization of aquaporins in model plants such as Arabidopsis, their contributions to abiotic stress tolerance in non-model plants are still poorly understood. As a close relative of Arabidopsis thaliana, Eutrema salsugineum is an excellent model for studying salt tolerance. Here, we identified and functionally characterized EsPIP1;4, a gene encoding a plasma membrane intrinsic protein (PIP) aquaporin in E. salsugineum. Overexpression of EsPIP1;4 in Arabidopsis improved seed germination and root growth of transgenic plants under abiotic stress, which was accompanied by an increase in proline accumulation, reduction in MDA, and decrease in the rate of ion leakage. Under abiotic stress, transgenic plants overexpressing EsPIP1;4 also showed increased antioxidant enzyme activity, and enhanced K+/Na+ ratio compared to control plants. Furthermore, overexpression of EsPIP1;4 promoted flowering by regulating genes in multiple flowering pathways. Together, our results demonstrated that an aquaporin from E. salsugineum improves abiotic stress tolerance and promotes flowering.

The halophyte gene ScVTC2 confers resistance to oxidative stress via AsA-mediated photosynthetic enhancement.

Various abiotic stresses commonly cause excessive production of reactive oxygen species (ROS) and result in oxidative stress, which challenges the physiological homeostasis of plants. Maintaining a delicate balance between ROS generation and removal is critical for plants to cope with stressful environments. Suaeda corniculata is a typical euhalophyte with strong tolerance to salt stress, but its mechanism of ROS detoxification to prevent oxidative stress is unknown. Here, a combined analysis of RNA-Seq and photosynthetic assays was performed on S. corniculata under oxidative stress to uncover the underlying mechanism that modulates oxidative tolerance. Our results showed that all genes involved in the pathway of ROS scavenging, especially the AsA-GSH pathway, were highly enriched under oxidative stress. Notably, VTC2 (GGPase), which functions in the L-galactose pathway of AsA synthesis, was significantly upregulated. Arabidopsis transgenic plants with heterologous expression of ScVTC2 showed elevated AsA and increased tolerance to oxidative stress. Furthermore, ScVTC2 also established better photosynthetic capacity in these plants upon oxidative treatment. Thus, ScVTC2 not only functioned as an effective ROS scavenger but also as a protector of the photosynthetic apparatus in S. corniculata and allowed plants to respond to and tolerate oxidative stress.

Protein complex formation in methionine chain-elongation and leucine biosynthesis.

During the past two decades, glucosinolate (GLS) metabolic pathways have been under extensive studies because of the importance of the specialized metabolites in plant defense against herbivores and pathogens. The studies have led to a nearly complete characterization of biosynthetic genes in the reference plant Arabidopsis thaliana. Before methionine incorporation into the core structure of aliphatic GLS, it undergoes chain-elongation through an iterative three-step process recruited from leucine biosynthesis. Although enzymes catalyzing each step of the reaction have been characterized, the regulatory mode is largely unknown. In this study, using three independent approaches, yeast two-hybrid (Y2H), coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC), we uncovered the presence of protein complexes consisting of isopropylmalate isomerase (IPMI) and isopropylmalate dehydrogenase (IPMDH). In addition, simultaneous decreases in both IPMI and IPMDH activities in a leuc:ipmdh1 double mutants resulted in aggregated changes of GLS profiles compared to either leuc or ipmdh1 single mutants. Although the biological importance of the formation of IPMI and IPMDH protein complexes has not been documented in any organisms, these complexes may represent a new regulatory mechanism of substrate channeling in GLS and/or leucine biosynthesis. Since genes encoding the two enzymes are widely distributed in eukaryotic and prokaryotic genomes, such complexes may have universal significance in the regulation of leucine biosynthesis.

Arabidopsis Glutathione-S-Transferases GSTF11 and GSTU20 Function in Aliphatic Glucosinolate Biosynthesis.

Glutathione (GSH) conjugation with intermediates is required for the biosynthesis of glucosinolate (GSL) by serving as a sulfur supply. Glutathione-S-transferases (GSTs) primarily work on GSH conjugation, suggesting their involvement in GSL metabolism. Although several GSTs, including GSTF11 and GSTU20, have been recently postulated to act in GSL biosynthesis, molecular evidence is lacking. Here, we demonstrated that GSTF11 and GSTU20 play non-redundant, although partially overlapping, roles in aliphatic GSL biosynthesis. In addition, GSTU20 plays a more important role than GSTF11, which is manifested by the greater loss of aliphatic GSLs associated with GSTU20 mutant and a greater number of differentially expressed genes in GSTU20 mutant compared to GSTF11 mutant. Moreover, a double mutation leads to a greater aggregate loss of aliphatic GSLs, suggesting that GSTU20 and GSTF11 may function in GSL biosynthesis in a dosage-dependent manner. Together, our results provide direct evidence that GSTU20 and GSTF11 are critically involved in aliphatic GSL biosynthesis, filling the knowledge gap that has been speculated in recent decades.

Proteomics and phosphoproteomics revealed molecular networks of stomatal immune responses.

MAIN CONCLUSION: Dynamic protein and phosphoprotein profiles uncovered the overall regulation of stomata movement against pathogen invasion and phosphorylation states of proteins involved in ABA, SA, calcium and ROS signaling, which may modulate the stomatal immune response. Stomatal openings represent a major route of pathogen entry into the plant, and plants have evolved mechanisms to regulate stomatal aperture as innate immune response against bacterial invasion. However, the mechanisms underlying stomatal immunity are not fully understood. Taking advantage of high-throughput liquid chromatography mass spectrometry (LC-MS), we performed label-free proteomic and phosphoproteomic analyses of enriched guard cells in response to a bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. In total, 495 proteins and 1229 phosphoproteins were identified as differentially regulated. These proteins are involved in a variety of signaling pathways, including abscisic acid and salicylic acid hormone signaling, calcium and reactive oxygen species signaling. We also showed that dynamic changes of phosphoprotein WRKY transcription factors may play a crucial role in regulating stomata movement in plant immunity. The identified proteins/phosphoproteins and the pathways form interactive molecular networks to regulate stomatal immunity. This study has provided new insights into the multifaceted mechanisms of stomatal immunity. The differential proteins and phosphoproteins are potential targets for engineering or breeding of crops for enhanced pathogen defense.

Nuclear pore complex components have temperature-influenced roles in plant growth and immunity.

Nuclear pore complexes (NPCs) are main channels controlling nucleocytoplasmic transport and are composed of approximately 30 nucleoporins (NUPs). Emerging evidence suggests that some NUP genes have specialized functions that challenge the traditional view of NPCs as structures of uniform composition. Here, we analysed the role of six outer-ring components of NPC at normal and warm growth temperatures by examining their loss-of-function mutants in Arabidopsis thaliana. All six NUP subunits, NUP85, NUP96, NUP 133, NUP 160, SEH1 and HOS1, have a non-redundant temperature-influenced function in one or more of the processes, including rosette growth, leaf architecture and intracellular immune receptor-mediated disease resistance. At the molecular level, NUP85 and NUP133 are required for mRNA export only at warm temperature and play a larger role in the localization of transcription factor at warm temperature. In addition, NUP96 and HOS1 are essential for the expression of high temperature-responsive genes, which is correlated with their larger activity in facilitating nuclear accumulation of the transcription factor PIF4 at warm temperature. Our results show that subunits of NPC have differential roles at different temperatures, suggesting the existence of temperature-influenced NPC complexes and activities.

Proteomic Analysis of Auricularia auricula-judae Under Freezing Treatment Revealed Proteins and Pathways Associated With Melanin Reduction.

Auricularia auricula-judae is an edible nutrient-rich mushroom, which is a traditional medicinal resource in China. It is known that environment stimuli will affect the production of melanin by A. auricula-judae, but the mechanism of the effects of freezing treatment on melanin accumulation remains unknown. In the present study, the synthesis of melanin in A. auricula-judae was analyzed by physiological assays and a proteomics approach. Our findings showed that a longer freezing treatment causes a lighter color of A. auricula-judae fruiting bodies. The proteomic analysis showed that proteins involved in glycolysis/gluconeogenesis, tyrosine metabolism, ribosome, and arginine biosynthesis might contribute to the color differences in the A. auricula-judae after freezing treatment. This work will be expected to provide valuable information on the physiological and molecular mechanisms of freezing treatment on the color quality of A. auricula-judae.

Genome-wide characterization and expression analysis of aquaporins in salt cress (Eutrema salsugineum).

Aquaporins (AQPs) serve as water channel proteins and belong to major intrinsic proteins (MIPs) family, functioning in rapidly and selectively transporting water and other small solutes across biological membranes. Importantly, AQPs have been shown to play a critical role in abiotic stress response pathways of plants. As a species closely related to Arabidopsis thaliana, Eutrema salsugineum has been proposed as a model for studying salt resistance in plants. Here we surveyed 35 full-length AQP genes in E. salsugineum, which could be grouped into four subfamilies including 12 plasma membrane intrinsic proteins (PIPs), 11 tonoplast intrinsic proteins (TIPs), nine NOD-like intrinsic proteins (NIPs), and three small basic intrinsic proteins (SIPs) by phylogenetic analysis. EsAQPs were comprised of 237-323 amino acids, with a theoretical molecular weight (MW) of 24.31-31.80 kDa and an isoelectric point (pI) value of 4.73-10.49. Functional prediction based on the NPA motif, aromatic/arginine (ar/R) selectivity filter, Froger's position and specificity-determining position suggested quite differences in substrate specificities of EsAQPs. EsAQPs exhibited global expressions in all organs as shown by gene expression profiles and should be play important roles in response to salt, cold and drought stresses. This study provides comprehensive bioinformation on AQPs in E. salsugineum, which would be helpful for gene function analysis for further studies.

Metabolite signatures of grasspea suspension-cultured cells illustrate the complexity of dehydration response.

MAIN CONCLUSION: This represents the first report deciphering the dehydration response of suspension-cultured cells of a crop species, highlighting unique and shared pathways, and adaptive mechanisms via profiling of 330 metabolites. Grasspea, being a hardy legume, is an ideal model system to study stress tolerance mechanisms in plants. In this study, we investigated the dehydration-responsive metabolome in grasspea suspension-cultured cells (SCCs) to identify the unique and shared metabolites crucial in imparting dehydration tolerance. To reveal the dehydration-induced metabolite signatures, SCCs of grasspea were exposed to 10% PEG, followed by metabolomic profiling. Chromatographic separation by HPLC coupled with MRM-MS led to the identification of 330 metabolites, designated dehydration-responsive metabolites (DRMs), which belonged to 28 varied functional classes. The metabolome was found to be constituted by carboxylic acids (17%), amino acids (13.5%), flavonoids (10.9%) and plant growth regulators (10%), among others. Pathway enrichment analysis revealed predominance of metabolites involved in phytohormone biosynthesis, secondary metabolism and osmotic adjustment. Exogenous application of DRMs, arbutin and acetylcholine, displayed improved physiological status in stress-resilient grasspea as well as hypersensitive pea, while administration of lauric acid imparted detrimental effects. This represents the first report on stress-induced metabolomic landscape of a crop species via a suspension culture system, which would provide new insights into the molecular mechanism of stress responses and adaptation in crop species.

Microbispora sitophila sp. nov., a novel actinobacterium isolated from rhizosphere soil of wheat (Triticum aestivum L.).

A novel actinobacterium, designated strain NEAU-D428T, was isolated from rhizosphere soil of wheat and characterized using a polyphasic approach. Morphological and chemotaxonomic characteristics of the strain coincided with members of the genus Microbispora. The 16S rRNA gene sequence analysis showed that the isolate was most closely related to Microbispora bryophytorum NEAU-TX2-2T (99.2 %). Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the strain clustered with Microbispora clausenae CLES2T (99.1 %), but formed a separate subclade in the phylogenomic tree within the genus Microbispora. The menaquinones were identified as MK-9(H4), MK-9(H2) and MK-9(H0). The phospholipid profile was found to consist of diphosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, ninhydrin-positive glycophospholipid, phosphatidylinositol and phosphatidylinositol mannoside. The major fatty acids were identified as iso-C16 : 0, C16 : 0, 10-methyl C17 : 0 and C18 : 0. Digital DNA-DNA hybridization and average nucleotide identity values between strain NEAU-D428T and M. bryophytorum NEAU-TX2-2T, Microbispora camponoti 2C-HV3T, M. clausenae CLES2T, 'Microbispora cellulosiformans' Gxj-6T and Microbispora fusca NEAU-HEGS1-5T were 47.6 and 92.2 %, 47.5 and 92.2 %, 55.9 and 94.0 %, 33.1 and 86.8 %, and 33.6 and 87.1 %, respectively. These results and some physiological and biochemical properties demonstrated that the strain could be distinguished from its closest relatives. Therefore, it is proposed that strain NEAU-D428T should be classified as representative of a novel species of the genus Microbispora, for which the name Microbispora sitophila sp. nov. is proposed. The type strain is NEAU-D428T (=CGMCC 4.7523T=DSM 109822T).

Metabolomics of Early Stage Plant Cell-Microbe Interaction Using Stable Isotope Labeling.

Metabolomics has been used in unraveling metabolites that play essential roles in plant-microbe (including pathogen) interactions. However, the problem of profiling a plant metabolome with potential contaminating metabolites from the coexisting microbes has been largely ignored. To address this problem, we implemented an effective stable isotope labeling approach, where the metabolome of a plant bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 was labeled with heavy isotopes. The labeled bacterial cells were incubated with Arabidopsis thaliana epidermal peels (EPs) with guard cells, and excessive bacterial cells were subsequently removed from the plant tissues by washing. The plant metabolites were characterized by liquid chromatography mass spectrometry using multiple reactions monitoring, which can differentiate plant and bacterial metabolites. Targeted metabolomic analysis suggested that Pst DC3000 infection may modulate stomatal movement by reprograming plant signaling and primary metabolic pathways. This proof-of-concept study demonstrates the utility of this strategy in differentiation of the plant and microbe metabolomes, and it has broad applications in studying metabolic interactions between microbes and other organisms.