Rice EIL1 interacts with OsIAAs to regulate auxin biosynthesis mediated by the tryptophan aminotransferase MHZ10/OsTAR2 during root ethylene responses.

Ethylene plays essential roles in adaptive growth of rice (Oryza sativa). Understanding of the crosstalk between ethylene and auxin (Aux) is limited in rice. Here, from an analysis of the root-specific ethylene-insensitive rice mutant mao hu zi 10 (mhz10), we identified the tryptophan aminotransferase (TAR) MHZ10/OsTAR2, which catalyzes the key step in indole-3-pyruvic acid-dependent Aux biosynthesis. Genetically, OsTAR2 acts downstream of ethylene signaling in root ethylene responses. ETHYLENE INSENSITIVE3 like1 (OsEIL1) directly activated OsTAR2 expression. Surprisingly, ethylene induction of OsTAR2 expression still required the Aux pathway. We also show that Os indole-3-acetic acid (IAA)1/9 and OsIAA21/31 physically interact with OsEIL1 and show promotive and repressive effects on OsEIL1-activated OsTAR2 promoter activity, respectively. These effects likely depend on their EAR motif-mediated histone acetylation/deacetylation modification. The special promoting activity of OsIAA1/9 on OsEIL1 may require both the EAR motifs and the flanking sequences for recruitment of histone acetyltransferase. The repressors OsIAA21/31 exhibit earlier degradation upon ethylene treatment than the activators OsIAA1/9 in a TIR1/AFB-dependent manner, allowing OsEIL1 activation by activators OsIAA1/9 for OsTAR2 expression and signal amplification. This study reveals a positive feedback regulation of ethylene signaling by Aux biosynthesis and highlights the crosstalk between ethylene and Aux pathways at a previously underappreciated level for root growth regulation in rice.

An RRM domain protein SOE suppresses transgene silencing in rice.

Gene expression is regulated at multiple levels, including RNA processing and DNA methylation/demethylation. How these regulations are controlled remains unclear. Here, through analysis of a suppressor for the OsEIN2 over-expressor, we identified an RNA recognition motif protein SUPPRESSOR OF EIN2 (SOE). SOE is localized in nuclear speckles and interacts with several components of the spliceosome. We find SOE associates with hundreds of targets and directly binds to a DNA glycosylase gene DNG701 pre-mRNA for efficient splicing and stabilization, allowing for subsequent DNG701-mediated DNA demethylation of the transgene promoter for proper gene expression. The V81M substitution in the suppressor mutant protein mSOE impaired its protein stability and binding activity to DNG701 pre-mRNA, leading to transgene silencing. SOE mutation enhances grain size and yield. Haplotype analysis in c. 3000 rice accessions reveals that the haplotype 1 (Hap 1) promoter is associated with high 1000-grain weight, and most of the japonica accessions, but not indica ones, have the Hap 1 elite allele. Our study discovers a novel mechanism for the regulation of gene expression and provides an elite allele for the promotion of yield potentials in rice.

Global analysis of seed transcriptomes reveals a novel PLATZ regulator for seed size and weight control in soybean.

Seed size and weight are important factors that influence soybean yield. Combining the weighted gene co-expression network analysis (WGCNA) of 45 soybean accessions and gene dynamic changes in seeds at seven developmental stages, we identified candidate genes that may control the seed size/weight. Among these, a PLATZ-type regulator overlapping with 10 seed weight QTLs was further investigated. This zinc-finger transcriptional regulator, named as GmPLATZ, is required for the promotion of seed size and weight in soybean. The GmPLATZ may exert its functions through direct binding to the promoters and activation of the expression of cyclin genes and GmGA20OX for cell proliferation. Overexpression of the GmGA20OX enhanced seed size/weight in soybean. We further found that the GmPLATZ binds to a 32-bp sequence containing a core palindromic element AATGCGCATT. Spacing of the flanking sequences beyond the core element facilitated GmPLATZ binding. An elite haplotype Hap3 was also identified to have higher promoter activity and correlated with higher gene expression and higher seed weight. Orthologues of the GmPLATZ from rice and Arabidopsis play similar roles in seeds. Our study reveals a novel module of GmPLATZ-GmGA20OX/cyclins in regulating seed size and weight and provides valuable targets for breeding of crops with desirable agronomic traits.

The GDSL Lipase MHZ11 Modulates Ethylene Signaling in Rice Roots.

Ethylene plays important roles in plant growth and development, but the regulation of ethylene signaling is largely unclear, especially in crops such as rice (Oryza sativa). Here, by analysis of the ethylene-insensitive mutant mao huzi 11 (mhz11), we identified the GDSL lipase MHZ11, which modulates ethylene signaling in rice roots. MHZ11 localized to the endoplasmic reticulum membrane and has acyl-hydrolyzing activity. This activity affects the homeostasis of sterols in rice roots and is required for root ethylene response. MHZ11 overexpression caused constitutive ethylene response in roots. Genetically, MHZ11 acts with the ethylene receptor ETHYLENE RESPONSE SENSOR2 (OsERS2) upstream of CONSTITUTIVE TRIPLE RESPONSE2 (OsCTR2) and ETHYLENE INSENSITIVE2 (OsEIN2). The mhz11 mutant maintains more OsCTR2 in the phosphorylated form whereas MHZ11 overexpression promotes ethylene-mediated inhibition of OsCTR2 phosphorylation. MHZ11 colocalized with the ethylene receptor OsERS2, and its effect on OsCTR2 phosphorylation requires ethylene perception and initiation of ethylene signaling. The mhz11 mutant overaccumulated sterols and blocking sterol biosynthesis partially rescued the mhz11 ethylene response, likely by reducing receptor-OsCTR2 interaction and OsCTR2 phosphorylation. We propose that MHZ11 reduces sterol levels to impair receptor-OsCTR2 interactions and OsCTR2 phosphorylation for triggering ethylene signaling. Our study reveals a mechanism by which MHZ11 participates in ethylene signaling for regulation of root growth in rice.

Zinc-finger protein GmZF351 improves both salt and drought stress tolerance in soybean.

Abiotic stress is one of the most important factors reducing soybean yield. It is essential to identify regulatory factors contributing to stress responses. A previous study found that the tandem CCCH zinc-finger protein GmZF351 is an oil level regulator. In this study, we discovered that the GmZF351 gene is induced by stress and that the overexpression of GmZF351 confers stress tolerance to transgenic soybean. GmZF351 directly regulates the expression of GmCIPK9 and GmSnRK, leading to stomata closing, by binding to their promoter regions, which carry two CT(G/C)(T/A)AA elements. Stress induction of GmZF351 is mediated through reduction in the H3K27me3 level at the GmZF351 locus. Two JMJ30-demethylase-like genes, GmJMJ30-1 and GmJMJ30-2, are involved in this demethylation process. Overexpression of GmJMJ30-1/2 in transgenic hairy roots enhances GmZF351 expression mediated by histone demethylation and confers stress tolerance to soybean. Yield-related agronomic traits were evaluated in stable GmZF351-transgenic plants under mild drought stress conditions. Our study reveals a new mode of GmJMJ30-GmZF351 action in stress tolerance, in addition to that of GmZF351 in oil accumulation. Manipulation of the components in this pathway is expected to improve soybean traits and adaptation under unfavorable environments.

GmJAZ3 interacts with GmRR18a and GmMYC2a to regulate seed traits in soybean.

Seed weight is usually associated with seed size and is one of the important agronomic traits that determine yield. Understanding of seed weight control is limited, especially in soybean plants. Here we show that Glycine max JASMONATE-ZIM DOMAIN 3 (GmJAZ3), a gene identified through gene co-expression network analysis, regulates seed-related traits in soybean. Overexpression of GmJAZ3 promotes seed size/weight and other organ sizes in stable transgenic soybean plants likely by increasing cell proliferation. GmJAZ3 interacted with both G. max RESPONSE REGULATOR 18a (GmRR18a) and GmMYC2a to inhibit their transcriptional activation of cytokinin oxidase gene G. max CYTOKININ OXIDASE 3-4 (GmCKX3-4), which usually affects seed traits. Meanwhile, the GmRR18a binds to the promoter of GmMYC2a and activates GmMYC2a gene expression. In GmJAZ3-overexpressing soybean seeds, the protein contents were increased while the fatty acid contents were reduced compared to those in the control seeds, indicating that the GmJAZ3 affects seed size/weight and compositions. Natural variation in JAZ3 promoter region was further analyzed and Hap3 promoter correlates with higher promoter activity, higher gene expression and higher seed weight. The Hap3 promoter may be selected and fixed during soybean domestication. JAZ3 orthologs from other plants/crops may also control seed size and weight. Taken together, our study reveals a novel molecular module GmJAZ3-GmRR18a/GmMYC2a-GmCKXs for seed size and weight control, providing promising targets during soybean molecular breeding for better seed traits.

Nuclear factor Y subunit GmNFYA competes with GmHDA13 for interaction with GmFVE to positively regulate salt tolerance in soybean.

Soybean is an important crop worldwide, but its production is severely affected by salt stress. Understanding the regulatory mechanism of salt response is crucial for improving the salt tolerance of soybean. Here, we reveal a role for nuclear factor Y subunit GmNFYA in salt tolerance of soybean likely through the regulation of histone acetylation. GmNFYA is induced by salt stress. Overexpression of GmNFYA significantly enhances salt tolerance in stable transgenic soybean plants by inducing salt-responsive genes. Analysis in soybean plants with transgenic hairy roots also supports the conclusion. GmNFYA interacts with GmFVE, which functions with putative histone deacetylase GmHDA13 in a complex for transcriptional repression possibly by reducing H3K9 acetylation at target loci. Under salt stress, GmNFYA likely accumulates and competes with GmHDA13 for interaction with GmFVE, leading to the derepression and maintenance of histone acetylation for activation of salt-responsive genes and finally conferring salt tolerance in soybean plants. In addition, a haplotype I GmNFYA promoter is identified with the highest self-activated promoter activity and may be selected during future breeding for salt-tolerant cultivars. Our study uncovers the epigenetic regulatory mechanism of GmNFYA in salt-stress response, and all the factors/elements identified may be potential targets for genetic manipulation of salt tolerance in soybean and other crops.

A transcriptional regulatory module controls lipid accumulation in soybean.

Soybean (Glycine max) is one of the most important oilseed crops. However, the regulatory mechanism that governs the process of oil accumulation in soybean remains poorly understood. In this study, GmZF392, a tandem CCCH zinc finger (TZF) protein which was identified in our previous RNA-seq analysis of seed-preferred transcription factors, was found to function as a positive regulator of lipid production. GmZF392 promotes seed oil accumulation in both transgenic Arabidopsis and stable transgenic soybean plants by binding to a bipartite cis-element, containing TG- and TA-rich sequences, in promoter regions, activating the expression of genes in the lipid biosynthesis pathway. GmZF392 physically interacts with GmZF351, our previously identified transcriptional regulator of lipid biosynthesis, to synergistically promote downstream gene expression. Both GmZF392 and GmZF351 are further upregulated by GmNFYA, another transcription factor involved in lipid biosynthesis, directly (in the former case) and indirectly (in the latter case). Promoter sequence diversity analysis showed that the GmZF392 promoter may have been selected at the origin of the Glycine genus and further mildly selected during domestication from wild soybeans to cultivated soybeans. Our study reveals a regulatory module containing three transcription factors in the lipid biosynthesis pathway, and manipulation of the module may improve oil production in soybean and other oilseed crops.

A class B heat shock factor selected for during soybean domestication contributes to salt tolerance by promoting flavonoid biosynthesis.

Soybean (Glycine max) production is severely affected in unfavorable environments. Identification of the regulatory factors conferring stress tolerance would facilitate soybean breeding. In this study, through coexpression network analysis of salt-tolerant wild soybeans, together with molecular and genetic approaches, we revealed a previously unidentified function of a class B heat shock factor, HSFB2b, in soybean salt stress response. We showed that HSFB2b improves salt tolerance through the promotion of flavonoid accumulation by activating one subset of flavonoid biosynthesis-related genes and by inhibiting the repressor gene GmNAC2 to release another subset of genes in the flavonoid biosynthesis pathway. Moreover, four promoter haplotypes of HSFB2b were identified from wild and cultivated soybeans. Promoter haplotype II from salt-tolerant wild soybean Y20, with high promoter activity under salt stress, is probably selected for during domestication. Another promoter haplotype, III, from salt-tolerant wild soybean Y55, had the highest promoter activity under salt stress, had a low distribution frequency and may be subjected to the next wave of selection. Together, our results revealed the mechanism of HSFB2b in soybean salt stress tolerance. Its promoter variations were identified, and the haplotype with high activity may be adopted for breeding better soybean cultivars that are adapted to stress conditions.

A Histone Code Reader and a Transcriptional Activator Interact to Regulate Genes for Salt Tolerance.

Plant homeodomain (PHD) finger proteins are involved in various developmental processes and stress responses. They recognize and bind to epigenetically modified histone H3 tail and function as histone code readers. Here we report that GmPHD6 reads low methylated histone H3K4me0/1/2 but not H3K4me3 with its N-terminal domain instead of the PHD finger. GmPHD6 does not possess transcriptional regulatory ability but has DNA-binding ability. Through the PHD finger, GmPHD6 interacts with its coactivator, LHP1-1/2, to form a transcriptional activation complex. Using a transgenic hairy root system, we demonstrate that overexpression of GmPHD6 improves stress tolerance in soybean (Glycinemax) plants. Knocking down the LHP1 expression disrupts this role of GmPHD6, indicating that GmPHD6 requires LHP1 functions during stress response. GmPHD6 influences expression of dozens of stress-related genes. Among these, we identified three targets of GmPHD6, including ABA-stress-ripening-induced CYP75B1 and CYP82C4 Overexpression of each gene confers stress tolerance in soybean plants. GmPHD6 is recruited to H3K4me0/1/2 marks and recognizes the G-rich elements in target gene promoters, whereas LHP1 activates expression of these targets. Our study reveals a mechanism involving two partners in a complex. Manipulation of the genes in this pathway should improve stress tolerance in soybean or other legumes/crops.

Selection for a Zinc-Finger Protein Contributes to Seed Oil Increase during Soybean Domestication.

Seed oil is a momentous agronomical trait of soybean (Glycine max) targeted by domestication in breeding. Although multiple oil-related genes have been uncovered, knowledge of the regulatory mechanism of seed oil biosynthesis is currently limited. We demonstrate that the seed-preferred gene GmZF351, encoding a tandem CCCH zinc finger protein, is selected during domestication. Further analysis shows that GmZF351 facilitates oil accumulation by directly activating WRINKLED1, BIOTIN CARBOXYL CARRIER PROTEIN2, 3-KETOACYL-ACYL CARRIER PROTEIN SYNTHASE III, DIACYLGLYCEROL O-ACYLTRANSFERASE1, and OLEOSIN2 in transgenic Arabidopsis (Arabidopsis thaliana) seeds. Overexpression of GmZF351 in transgenic soybean also activates lipid biosynthesis genes, thereby accelerating seed oil accumulation. The ZF351 haplotype from the cultivated soybean group and the wild soybean (Glycine soja) subgroup III correlates well with high gene expression level, seed oil contents and promoter activity, suggesting that selection of GmZF351 expression leads to increased seed oil content in cultivated soybean. Our study provides novel insights into the regulatory mechanism for seed oil accumulation, and the manipulation of GmZF351 may have great potential in the improvement of oil production in soybean and other related crops.

Soybean NIMA-Related Kinase1 Promotes Plant Growth and Improves Salt and Cold Tolerance.

NEK (NIMA-related kinase) is known as a family of serine/threonine kinases which mainly participate in microtubule-related mitotic events in fungi, mammals and other eukaryotes. Our previous studies found that Arabidopsis NEK6 plays an important role in plant response to abiotic stress. We further investigated roles of the NEK family in soybean and found that at least eight members can respond to abiotic stresses. Among them, only GmNEK1, a novel NEK member which is distantly related to Arabidopsis NEK6, enhanced plant growth and promoted salt and cold tolerance in transgenic Arabidopsis plants. The growth of soybean plants harboring GmNEK1-overexpressing hairy roots under saline condition was also improved. A series of stress-related genes including RH3, CORI3 and ALDH10A8 were found to be up-regulated in GmNEK1-overexpressing Arabidopsis plants and soybean hairy roots. Moreover, soybean plants with GmRH3-overexpressing hairy roots exhibited increased salt tolerance, while soybean plants with GmRH3-RNAi (RNA interference) roots were more sensitive to salt stress than the wild-type plants. Our study uncovers a novel role for GmNEK1 in promoting plant adaptive growth under adverse conditions at least partially through up-regulation of GmRH3. Manipulation of these genes in soybean or other crops may improve growth and production under stress conditions.

Lower Serum Vitamin D Level Was Associated with Risk of Dry Eye Syndrome.

BACKGROUND To determine the association between serum 25(OH)D and dry eye syndrome (DES) incidence. This study was also designed to determine whether serum 25(OH)D levels were associated with ocular parameter of DES patients. MATERIAL AND METHODS This is a case-control study with 70 DES cases and 70 healthy controls. Clinical data included body mass index (BMI, kg/m²), smoking history, diabetes, and blood pressure. Serum 25(OH)D was chosen as the main parameter and reflected the level of vitamin D. The DES parameters included ocular surface disease index (OSDI) scales, tear film breakup time (TBUT) and Schirmer test I. The differences in each parameter between case and control groups were detected and the association of serum 25(OH)D and DES parameter were detected. RESULTS It was shown that 25(OH)D levels were lower in patients with DES than in healthy controls. When the 25(OH)D levels was stratified, vitamin D deficiency was more common in the DES cases. In advanced studies, it was found that there were statistically significant associations between serum 25(OH) D levels and the Schimer test, TBUT, and OSDI scales. CONCLUSIONS A significant association between serum 25(OH)D level and DES incidence was detected in this study. Considering the relatively small sample size of this study, larger studies are needed in the future.

The Alfin-like homeodomain finger protein AL5 suppresses multiple negative factors to confer abiotic stress tolerance in Arabidopsis.

Plant homeodomain (PHD) finger proteins affect processes of growth and development by changing transcription and reading epigenetic histone modifications, but their functions in abiotic stress responses remain largely unclear. Here we characterized seven Arabidopsis thaliana Alfin1-like PHD finger proteins (ALs) in terms of the responses to abiotic stresses. ALs localized to the nucleus and repressed transcription. Except AL6, all the ALs bound to G-rich elements. Mutations of the amino acids at positions 34 and 35 in AL6 caused loss of ability to bind to G-rich elements. Expression of the AL genes responded differentially to osmotic stress, salt, cold and abscisic acid treatments. AL5-over-expressing plants showed higher tolerance to salt, drought and freezing stress than Col-0. Consistently, al5 mutants showed reduced stress tolerance. We used ChIP-Seq assays to identify eight direct targets of AL5, and found that AL5 binds to the promoter regions of these genes. Knockout mutants of five of these target genes exhibited varying tolerances to stresses. These results indicate that AL5 inhibits multiple signaling pathways to confer stress tolerance. Our study sheds light on mechanisms of AL5-mediated signaling in abiotic stress responses, and provides tools for improvement of stress tolerance in crop plants.

Tobacco Translationally Controlled Tumor Protein Interacts with Ethylene Receptor Tobacco Histidine Kinase1 and Enhances Plant Growth through Promotion of Cell Proliferation.

Ethylene is an important phytohormone in the regulation of plant growth, development, and stress response throughout the lifecycle. Previously, we discovered that a subfamily II ethylene receptor tobacco (Nicotiana tabacum) Histidine Kinase1 (NTHK1) promotes seedling growth. Here, we identified an NTHK1-interacting protein translationally controlled tumor protein (NtTCTP) by the yeast (Saccharomyces cerevisiae) two-hybrid assay and further characterized its roles in plant growth. The interaction was further confirmed by in vitro glutathione S-transferase pull down and in vivo coimmunoprecipitation and bimolecular fluorescence complementation assays, and the kinase domain of NTHK1 mediates the interaction with NtTCTP. The NtTCTP protein is induced by ethylene treatment and colocalizes with NTHK1 at the endoplasmic reticulum. Overexpression of NtTCTP or NTHK1 reduces plant response to ethylene and promotes seedling growth, mainly through acceleration of cell proliferation. Genetic analysis suggests that NtTCTP is required for the function of NTHK1. Furthermore, association of NtTCTP prevents NTHK1 from proteasome-mediated protein degradation. Our data suggest that plant growth inhibition triggered by ethylene is regulated by a unique feedback mechanism, in which ethylene-induced NtTCTP associates with and stabilizes ethylene receptor NTHK1 to reduce plant response to ethylene and promote plant growth through acceleration of cell proliferation.

Tobacco ankyrin protein NEIP2 interacts with ethylene receptor NTHK1 and regulates plant growth and stress responses.

Ethylene is a gaseous hormone that regulates many processes involved in plant growth, development and stress responses. Previously, we found that the tobacco ethylene receptor NTHK1 (Nicotiana tabacum histidine kinase 1) promotes seedling growth and affects plant salt stress responses. In this study, NTHK1 ethylene receptor-interacting protein 2 (NEIP2) was identified and further characterized in relation to these processes. NEIP2 contains three ankyrin repeats that mediate an interaction with NTHK1 as demonstrated by yeast two-hybrid, glutathione S-transferase (GST) pull-down and co-immunoprecipitation assays. NTHK1 phosphorylates NEIP2 in vitro. Salt stress and ethylene treatment induce NEIP2 accumulation in the first few hours and then the NEIP2 can be phosphorylated in planta. The overexpression of NTHK1 enhances NEIP2 accumulation in the presence of ethylene and salt stress. NEIP2 overexpression promotes plant growth but reduces ethylene responses, which is consistent with the functions of NTHK1. Additionally, NEIP2 improves plant performance under salt and oxidative stress. These results suggest that ethylene-induced NEIP2 probably acts as a brake to reduce ethylene response but resumes growth through interaction with NTHK1. Manipulation of NEIP2 may be beneficial for crop improvement.

pts promoter influences antibiotic resistance via proton motive force and ROS in Escherichia coli.

Introduction: Glucose level is related to antibiotic resistance. However, underlying mechanisms are largely unknown. Methods: Since glucose transport is performed by phosphotransferase system (PTS) in bacteria, pts promoter-deleted K12 (Δpts-P) was used as a model to investigate effect of glucose metabolism on antibiotic resistance. Gas chromatography-mass spectrometry based metabolomics was employed to identify a differential metabolome in Δpts-P compared with K12, and with glucose as controls. Results: Δpts-P exhibits the resistance to ß-lactams and aminoglycosides but not to quinolones, tetracyclines, and macrolide antibiotics. Inactivated pyruvate cycle was determined as the most characteristic feature in Δpts-P, which may influence proton motive force (PMF), reactive oxygen species (ROS), and nitric oxide (NO) that are related to antibiotic resistance. Thus, they were regarded as three ways for the following study. Glucose promoted PMF and ß-lactams-, aminoglycosides-, quinolones-mediated killing in K12, which was inhibited by carbonyl cyanide 3-chlorophenylhydrazone. Exogenous glucose did not elevated ROS in K12 and Δpts-P, but the loss of pts promoter reduced ROS by approximately 1/5, which was related to antibiotic resistance. However, NO was neither changed nor related to antibiotic resistance. Discussion: These results reveal that pts promoter regulation confers antibiotic resistance via PMF and ROS in Escherichia coli.

A translational regulator MHZ9 modulates ethylene signaling in rice.

Ethylene plays essential roles in rice growth, development and stress adaptation. Translational control of ethylene signaling remains unclear in rice. Here, through analysis of an ethylene-response mutant mhz9, we identified a glycine-tyrosine-phenylalanine (GYF) domain protein MHZ9, which positively regulates ethylene signaling at translational level in rice. MHZ9 is localized in RNA processing bodies. The C-terminal domain of MHZ9 interacts with OsEIN2, a central regulator of rice ethylene signaling, and the N-terminal domain directly binds to the OsEBF1/2 mRNAs for translational inhibition, allowing accumulation of transcription factor OsEIL1 to activate the downstream signaling. RNA-IP seq and CLIP-seq analyses reveal that MHZ9 associates with hundreds of RNAs. Ribo-seq analysis indicates that MHZ9 is required for the regulation of ~ 90% of genes translationally affected by ethylene. Our study identifies a translational regulator MHZ9, which mediates translational regulation of genes in response to ethylene, facilitating stress adaptation and trait improvement in rice.

A Microtitre Plate Dilution Method for Minimum Killing Concentration Is Developed to Evaluate Metabolites-Enabled Killing of Bacteria by ß-lactam Antibiotics.

Because, as of yet, there are few new antibiotics active against multidrug-resistant bacteria are being explored, compounds including metabolites that might help us tide over this crisis are greatly expected. A recently adopted method to evaluate the potentiation of metabolites is the plate-counting test. However, the method is time-consuming, strenuous, and unfeasible for a large scale of screening. A minimum inhibitory concentration (MIC) test by using a microtitre plate dilution method is convenient and economic for a large scale of identification, but it cannot be used to detect the potentiation. Here, the microtitre plate dilution method was modified to develop a novel test for evaluating metabolites that enable the killing of bacterial pathogens by antibiotics, designed as minimum killing concentration (MKC). To do this, bacterial number, incubation time, ionic strength of M9 medium, and inosine concentration are optimized using Escherichia coli. Different from the MIC test, which uses 5 × 104 CFU cells and performed in LB medium, the MKC test needed 1 × 107 CFU - 2 × 107 CFU cells and was carried out in M9 medium. Moreover, MKC test was suitable for bactericidal antibiotics such as cephalosporins, penicillins and carbapenems and was proportional to the plate-counting test. The developed MKC test was feasible for different metabolites and clinically multidrug-resistant pathogens, and measurement of minimum bactericidal concentration (MBC). Therefore, the MKC test was developed to accelerate the identification of compounds that promote antibiotic-mediated killing efficacy.

Melatonin regulates gene expressions through activating auxin synthesis and signaling pathways.

Background: Both melatonin and indole-3-acetic acid (IAA) are derived from tryptophan. And the most interesting and unsolved puzzle in melatonin research is that what is the relationship between melatonin and auxin? Methods: In this study, we performed transcriptome analysis with a time series method to disclose the connection of the two metabolites in soybean. Results: Our results reveal that melatonin and IAA treatments cause substantial overlaps in gene expression changes. Common genes of melatonin and IAA treatments could be sorted into clusters with very similar expression tendency. A KEGG assay showed that exogenous applied melatonin enriched differentially expressed genes in auxin biosynthesis and signaling pathways. For details, melatonin up-regulates several YUCCA genes which participate in auxin biosynthesis; melatonin also enhances expression levels of auxin receptor coding genes, such as TIR1, AFB3 and AFB5; dozens of genes involved in auxin transport, such as AUXI and PIN, are regulated by melatonin similarly as by auxin; auxin-responsive genes, such as IAA, ARF, GH3 and SAUR-like genes, intensively respond to melatonin as well as to auxin. A DR5 promoter mediated GUS staining assay showed that low concentration of melatonin could induce auxin biosynthesis in a dosage manner, whereas high concentration of melatonin would eliminate such effect. At last, gene ontology (GO) analysis suggests that melatonin treatment has similar characteristics as auxin treatment in many processes. However, the two molecules still keep their own features respectively. For example, melatonin takes part in stress responses, while IAA treatment enriches the GO terms that related to cell growth. Conclusion: Taken together, exogenous applied melatonin, if not exceeds the appropriate concentration, could promote auxin responses range from biosynthesis to signaling transduction. Thus, our research is a key part to explain the auxin-like roles of melatonin in regulating plant growth.