Bioinformatics analysis and function prediction of NBS-LRR gene family in Broussonetia papyrifera.

Most of the currently available disease resistance (R) genes have NBS (nucleotide-binding site) and LRR (leucine-rich-repeat) domain which belongs to the NBS-LRR gene family. The whole genome sequencing of Broussonetia papyrifera provides an important bioinformatics database for the study of the NBS-LRR gene family. In this study, 328 NBS-LRR family genes were identified and classified in B. papyrifera according to different classification schemes, where there are 92 N types, 47 CN type, 54 CNL type, 29 NL types, 55 TN type, and 51 TNL type. Subsequently, we conducted bioinformatics analysis of the NBS-LRR gene family. Classification, motif analysis of protein sequences, and phylogenetic tree studies of the NBS-LRR genes in B. papyrifera provide important basis for the functional study of NBS-LRR family genes. Additionally, we performed structural analysis of the chromosomal location, physicochemical properties, and sequences identified by genetic characterization. In addition, through the analysis of GO enrichment, it was found that NBS-LRR genes were involved in defense responses and were significantly enriched in biological stimulation, immune response, and abiotic stress. In addition, we found that Bp06g0955 was the most sensitive to low temperature and encoded the RPM1 protein by analyzing the low temperature transcriptome data of B. papyrifera. Quantitative results of gene expression after 48 h of Fusarium infection showed that Bp01g3293 increased 14 times after infection, which encodes RPM1 protein. The potential of NBS-LRR gene responsive to biotic and abiotic stresses can be exploited to improve the resistance of B. papyrifera.

Identification and Investigation of the Genetic Variations and Candidate Genes Responsible for Seed Weight via GWAS in Paper Mulberry.

Seeds directly determine the survival and population size of woody plants, but the genetic basis of seed weight in woody plants remain poorly explored. To identify genetic variations and candidate genes responsible for seed weight in natural woody populations, we investigated the hundred-seed weight of 198 paper mulberry individuals from different areas. Our results showed that the hundred-seed weight of paper mulberry was significantly associated with the bioclimatic variables of sampling sites, which increased from south to north along the latitudinal-temperature gradient. Using 2,414,978 high-quality SNPs from re-sequencing data, the genome-wide association analysis of the hundred-seed weight was performed under three models, which identified 148, 19 and 12 associated genes, respectively. Among them, 25 candidate genes were directly hit by the significant SNPs, including the WRKY transcription factor, fatty acid desaturase, F-box protein, etc. Most importantly, we identified three crucial genetic variations in the coding regions of candidate genes (Bp02g2123, Bp01g3291 and Bp10g1642), and significant differences in the hundred-seed weight were detected among the individuals carrying different genotypes. Further analysis revealed that Bp02g2123 encoding a fatty acid desaturase (FAD) might be a key factor affecting the seed weight and local climate adaptation of woody plants. Furthermore, the genome-wide investigation and expression analysis of FAD genes were performed, and the results suggested that BpFADs widely expressed in various tissues and responded to multiple phytohormone and stress treatments. Overall, our study identifies valuable genetic variations and candidate genes, and provides a better understanding of the genetic basis of seed weight in woody plants.

Genome-Wide Analysis of BpYABs and Function Identification Involving in the Leaf and Silique Development in Transgenic Arabidopsis.

YABs play an important role in the leaf development of the paper mulberry (Broussonetia papyrifera) and of the heterophylly. Thus, we investigated the function of BpYABs. Gene cloning, phylogenetic analysis, motif identification, subcellular localization, transactivation activity assay, qRT-PCR, in situ hybridization, and ectopic expression were used in our study. Six BpYABs were isolated, and four of them had transcriptional activity. BpYAB1, BpYAB3, BpYAB4, and BpYAB5 were localized to the nucleus. BpYAB1 was only expressed in the flower, while BpYAB6 was not expressed in any detected tissues; the four remaining BpYABs were expressed in the bud, leaf and flower, and their expression level decreased with leaf development. Further in situ hybridization showed that BpYAB3 and BpYAB5 were expressed in the vascular tissues and lamina, but neither showed the adaxial-abaxial polarity distribution pattern in the mature leaf lamina. Ectopic expression of BpYAB2, BpYAB3, BpYAB4 and BpYAB5 induced increased expression of AtWOX1 and caused the leaf of Arabidopsis to become smaller and curl downwards. Ectopic expression also led to shorter siliques and smaller seeds, but not for BpYAB5. These results suggest that BpYABs have functional divergency and redundancy in regulating leaf and silique development.

Natural population re-sequencing detects the genetic basis of local adaptation to low temperature in a woody plant.

KEY MESSAGE: Total of 14 SNPs associated with overwintering-related traits and 75 selective regions were detected. Important candidate genes were identified and a possible network of cold-stress responses in woody plants was proposed. Local adaptation to low temperature is essential for woody plants to against changeable climate and safely survive the winter. To uncover the specific molecular mechanism of low temperature adaptation in woody plants, we sequenced 134 core individuals selected from 494 paper mulberry (Broussonetia papyrifera), which naturally distributed in different climate zones and latitudes. The population structure analysis, PCA analysis and neighbor-joining tree analysis indicated that the individuals were classified into three clusters, which showed forceful geographic distribution patterns because of the adaptation to local climate. Using two overwintering phenotypic data collected at high latitudes of 40°N and one bioclimatic variable, genome-phenotype and genome-environment associations, and genome-wide scans were performed. We detected 75 selective regions which possibly undergone temperature selection and identified 14 trait-associated SNPs that corresponded to 16 candidate genes (including LRR-RLK, PP2A, BCS1, etc.). Meanwhile, low temperature adaptation was also supported by other three trait-associated SNPs which exhibiting significant differences in overwintering traits between alleles within three geographic groups. To sum up, a possible network of cold signal perception and responses in woody plants were proposed, including important genes that have been confirmed in previous studies while others could be key potential candidates of woody plants. Overall, our results highlighted the specific and complex molecular mechanism of low temperature adaptation and overwintering of woody plants.

Identification of the PP2C gene family in paper mulberry (Broussonetia papyrifera) and its roles in the regulation mechanism of the response to cold stress.

OBJECTIVES: To study the possible roles of type-2C protein phosphatases (PP2Cs) which have been confirmed to play roles in the response to diverse abiotic stresses in paper mulberry, we launched a series of genomic and functional studies of BpPP2Cs. RESULTS: Sixty-three PP2C proteins in paper mulberry (Broussonetia papyrifera) were classified into 13 clades. Four BpPP2Cs with kinase domains were verified to be highly conserved in organisms ranging from algae to dicots. Seven pairs of BpPP2C genes were found to be expanding, and 18 BpPP2C genes had orthologues in Arabidopsis. BpPP2Cs showed broad expression in different tissues; the expression levels of 18 BpPP2Cs were changed and the phosphorylation levels of seven BpPP2C proteins increased at low temperature. Cold-response elements were found in the promoter region of 31 BpPP2Cs. Finally, Bp01g0320 was found to act as a hub protein and Bp01g0512 and Bp09g1278 played key roles in the ABA-signaling pathway and MAPK cascades, respectively. CONCLUSION: These results suggest that the PP2C gene family of paper mulberry is evolutionarily conserved and participates the regulation of the response to cold stress, which will play a vital role in further research on phosphatases in paper mulberry.

iTRAQ-Based Quantitative Proteomics Analysis Reveals the Mechanism of Golden-Yellow Leaf Mutant in Hybrid Paper Mulberry.

Plant growth and development relies on the conversion of light energy into chemical energy, which takes place in the leaves. Chlorophyll mutant variations are important for studying certain physiological processes, including chlorophyll metabolism, chloroplast biogenesis, and photosynthesis. To uncover the mechanisms of the golden-yellow phenotype of the hybrid paper mulberry plant, this study used physiological, cytological, and iTRAQ-based proteomic analyses to compare the green and golden-yellow leaves of hybrid paper mulberry. Physiological results showed that the mutants of hybrid paper mulberry showed golden-yellow leaves, reduced chlorophyll, and carotenoid content, and increased flavonoid content compared with wild-type plants. Cytological observations revealed defective chloroplasts in the mesophyll cells of the mutants. Results demonstrated that 4766 proteins were identified from the hybrid paper mulberry leaves, of which 168 proteins displayed differential accumulations between the green and mutant leaves. The differentially accumulated proteins were primarily involved in chlorophyll synthesis, carotenoid metabolism, and photosynthesis. In addition, differentially accumulated proteins are associated with ribosome pathways and could enable plants to adapt to environmental conditions by regulating the proteome to reduce the impact of chlorophyll reduction on growth and survival. Altogether, this study provides a better understanding of the formation mechanism of the golden-yellow leaf phenotype by combining proteomic approaches.

Genome-Wide Analysis of the UGT Gene Family and Identification of Flavonoids in Broussonetia papyrifera.

Broussonetia papyrifera is a multifunctional deciduous tree that is both a food and a source of traditional Chinese medicine for both humans and animals. Further analysis of the UGT gene family is of great significance to the utilization of B. papyrifera. The substrates of plant UGT genes include highly diverse and complex chemicals, such as flavonoids and terpenes. In order to deepen our understanding of this family, a comprehensive analysis was performed. Phylogenetic analysis showed that 155 BpUGTs were divided into 15 subgroups. A conserved motif analysis showed that BpUGT proteins in the same subgroups possessed similar motif structures. Tandem duplication was the primary driving force for the expansion of the BpUGT gene family. The global promoter analysis indicated that they were associated with complex hormone regulatory networks and the stress response, as well as the synthesis of secondary metabolites. The expression pattern analysis showed that the expression level of BpUGTs in leaves and roots was higher than that in fruits and stems. Next, we determined the composition and content of flavonoids, the main products of the BpUGT reaction. A total of 19 compounds were isolated and analyzed by UPLC-ESI-MS/MS in 3 species of Broussonetia including B. kazinoki, B. papyrifera, and B. kazinoki × B. papyrifera, and the number of compounds was different in these 3 species. The total flavonoid content and antioxidant capacities of the three species were analyzed respectively. All assays exhibited the same trend: the hybrid paper mulberry showed a higher total flavonoid content, a higher total phenol content and higher antioxidant activity than the other two species. Overall, our study provides valuable information for understanding the function of BpUGTs in the biosynthesis of flavonoids.

Cooperation between Broussonetia papyrifera and Its Symbiotic Fungal Community To Improve Local Adaptation of the Host.

The genetic basis of plant local adaptation has been extensively studied, yet the interplay between local adaptation, plant genetic divergence, and the microbial community remains unclear. Our study used the restriction-site associated DNA sequencing (RAD-seq) approach to explore genetic divergence in Broussonetia papyrifera and used internal transcribed spacers (ITS) to characterize fungal community. RAD-seq results show that B. papyrifera individuals could be divided into three genotypes; this genotyping result was consistent with the classification of climate type at the sample site. Most of the 101 highly differentiated genes were related to stress resistance and the microbiome. Moreover, ß-diversity results indicated that genetic divergence had a significant effect on fungal community across all compartments (P < 0.01). At genus and operational taxonomic unit (OTU) level, Mortierella, Hannaella oryzae, OTU81578 (Mortierella), and OTU1665209 (H. oryzae) were found to be the major OTUs that contribute to differences in fungal community. The properties of cooccurrence networks vary greatly among three genotypes. The results of redundancy analysis (RDA) indicated that B. papyrifera-associated fungal community was significantly related to its local adaptability. Our findings suggest that genetic divergence of B. papyrifera is closely related to local adaptation, with significant effects on the associated fungal community, which in turn would enhance host local adaptability. This improves present understanding about the coevolution of microbial communities and the host plant.IMPORTANCE The coevolution of plants with the associated fungal community and its effect on plant adaptability are not clear, especially for native trees. This study focuses on the genetic basis of local adaptation in plants and the effect of genetic divergence of Broussonetia papyrifera on the associated fungal community. We identified genes related to the microbiome that are important for local adaptation of the host. Our results show that genetic divergence in B. papyrifera significantly affects the fungal community, which has a close connection with local adaptation. This helps us to understand the relationship between local adaptation, genetic divergence, and associated fungal communities. This study highlights the effect of plant genetic divergence on associated fungal community for native trees and establishes a close connection between this effect and local adaptability in the host. In addition, these observations lay a foundation for the research of coevolution of plants and their symbiotic microbiome through genome-wide association study (GWAS).

The effect of plant compartments on the Broussonetia papyrifera-associated fungal and bacterial communities.

Plants associate with numerous microbes, but little is known about how microbiome components, especially fungi, adapt to specific plant compartments. The adaptability of microbial function to the plant compartment is also not clear especially for woody species. Here, we characterized the bacterial and fungal communities in root endosphere, stems, and rhizospheres of 33 Broussonetia papyrifera seedlings, based on amplification of 16S and ITS rRNA. Results showed that the α-diversity indexes of the bacterial community were significantly different in different plant compartments and they significantly increased from stem to root endosphere to the rhizosphere, whereas those of the fungal community were similar (p > 0.05). However, the result of constrained PCoA (CPCoA) and analysis of similarity (ANOSIM) showed that both bacterial and fungal compositions were significantly affected by plant compartments (p < 0.01). In detail, the operational taxonomic units (OTUs) distribution of the bacterial community was significantly different, but 249 of 252 fungal OTUs were shared in different plant compartments. Both the bacterial and fungal compositions were significantly influenced by plant compartments, based on the result on phyla, core OTUs, and indicator OTUs level. Further, 40 of 42 enriched KEGG pathways involving the bacteria also differed significantly among plant compartments (p < 0.01). This study provides an understanding of the influence of plant compartments on the microbiome and confirms that the disperse limitation of fungal OTUs across different plant compartments is smaller. This study sheds light on how the microbial community adapts to and thrives in different plant compartments.

The effect of environment on the microbiome associated with the roots of a native woody plant under different climate types in China.

Few studies have investigated the effect of environment on the root-associated microbiome, especially for woody plants in their native environment. The roots and rhizosphere soils of a native woody species (Broussonetia papyrifera) sampled across four different climate types in China were used to elucidate the influence of environment on the root-associated microbiome. Our results showed that the B. papyrifera root-associated microbiome contained abundant Proteobacteria and Basidiomycota, especially Pseudomonas and Rhizobium. The root-associated microbiomes were found to be significantly different under different climate types except for the bacterial community in the rhizosphere, and the proportion of bacterial operational taxonomic units (OTUs) shared among different climate types was lower than that of fungi. More than 50% of the total variance between microbiomes could be explained by 15 environmental factors, six of which, especially soil concentration phosphate and nitrate, had a significant effect. This study provided a comprehensive understanding of the root-associated microbiome of B. papyrifera and further confirmed the effect of environment on the root-associated microbiome of B. papyrifera under different climate types, with some exceptions in the rhizobacterial community and fungal OTUs. Our findings advanced knowledge of the effect of environment through an exploration of environmental factors and found that the nitrogen and phosphorus content represented the key factors.

Optical properties of silica microspheres with different functional groups.

Silica microspheres with different functional groups can be used as diagnostic material. Here, differently functionalized group (NH2-, COOH-, SiOH-, and epoxy-) silica microspheres of approximately 5 µm in diameter are prepared on garnet substrates by a self-assembly method. The surface morphologies of the silica microspheres are observed by an optical microscope. The pressure generated from the glass/silica microsphere/graphite interface is measured by a resistance strain sensor as a function of an external direct current (DC) voltage. A PAX5710 IR3 polarimeter and slit beam profiler give information on the optical polarization and optical spot distance of the polarized laser beams reflected from the differently functionalized group silica microspheres. The results reveal that the relationships between the optical polarization properties, the optical spot position of the polarized laser beam, the reflectivity of the differently functionalized group silica microspheres using circularly polarized and linearly polarized laser beams, and the external pressure show different trends. The optical properties of the differently functionalized group silica microspheres are consistent with the experimental data pressure.

Global Phosphoproteomic Analysis Reveals the Defense and Response Mechanisms of Jatropha Curcas Seedling under Chilling Stress.

As a promising energy plant for biodiesel, Jatropha curcas is a tropical and subtropical shrub and its growth is affected by one of major abiotic stress, chilling. Therefore, we adopt the phosphoproteomic analysis, physiological measurement and ultrastructure observation to illustrate the responsive mechanism of J. curcas seedling under chilling (4 °C) stress. After chilling for 6 h, 308 significantly changed phosphoproteins were detected. Prolonged the chilling treatment for 24 h, obvious physiological injury can be observed and a total of 332 phosphoproteins were examined to be significantly changed. After recovery (28 °C) for 24 h, 291 phosphoproteins were varied at the phosphorylation level. GO analysis showed that significantly changed phosphoproteins were mainly responsible for cellular protein modification process, transport, cellular component organization and signal transduction at the chilling and recovery periods. On the basis of protein-protein interaction network analysis, phosphorylation of several protein kinases, such as SnRK2, MEKK1, EDR1, CDPK, EIN2, EIN4, PI4K and 14-3-3 were possibly responsible for cross-talk between ABA, Ca2+, ethylene and phosphoinositide mediated signaling pathways. We also highlighted the phosphorylation of HOS1, APX and PIP2 might be associated with response to chilling stress in J. curcas seedling. These results will be valuable for further study from the molecular breeding perspective.

Rhizobia promote the growth of rice shoots by targeting cell signaling, division and expansion.

KEY MESSAGE: The growth-promotion of rice seedling following inoculation with Sinorhizobium meliloti 1021 was a cumulative outcome of elevated expression of genes that function in accelerating cell division and enhancing cell expansion. Various endophytic rhizobacteria promote the growth of cereal crops. To achieve a better understanding of the cellular and molecular bases of beneficial cereal-rhizobia interactions, we performed computer-assisted microscopy and transcriptomic analyses of rice seedling shoots (Oryza sativa) during early stages of endophytic colonization by the plant growth-promoting Sinorhizobium meliloti 1021. Phenotypic analyses revealed that plants inoculated with live rhizobia had increased shoot height and dry weight compared to control plants inoculated with heat-killed cells of the same microbe. At 6 days after inoculation (DAI) with live cells, the fourth-leaf sheaths showed significant cytological differences including their enlargement of parenchyma cells and reduction in shape complexity. Transcriptomic analysis of shoots identified 2,414 differentially-expressed genes (DEGs) at 1, 2, 5 and 8 DAI: 195, 1390, 1025 and 533, respectively. Among these, 46 DEGs encoding cell-cycle functions were up-regulated at least 3 days before the rhizobia ascended from the roots to the shoots, suggesting that rhizobia are engaged in long-distance signaling events during early stages of this plant-microbe interaction. DEGs involved in phytohormone production, photosynthetic efficiency, carbohydrate metabolism, cell division and wall expansion were significantly elevated at 5 and 8 DAI, consistent with the observed phenotypic changes in rice cell morphology and shoot growth-promotion. Correlation analysis identified 104 height-related DEGs and 120 dry-weight-related DEGs that represent known quantitative-trait loci for seedling vigor and increased plant height. These findings provide multiple evidences of plant-microbe interplay that give insight into the growth-promotion processes associated with this rhizobia-rice beneficial association.

Phosphoproteomic Analysis of Paper Mulberry Reveals Phosphorylation Functions in Chilling Tolerance.

Paper mulberry is a valuable woody species with a good chilling tolerance. In this study, phosphoproteomic analysis, physiological measurement, and mRNA quantification were employed to explore the molecular mechanism of chilling (4 °C) tolerance in paper mulberry. After chilling for 6 h, 427 significantly changed phosphoproteins were detected in paper mulberry seedlings without obvious physiological injury. When obvious physiological injury occurred after chilling for 48 h, a total of 611 phosphoproteins were found to be significantly changed at the phosphorylation level. Several protein kinases, especially CKII, were possibly responsible for these changes according to conserved sequence analysis. The results of Gene Ontology analysis showed that phosphoproteins were mainly responsible for signal transduction, protein modification, and translation during chilling. Additionally, transport and cellular component organization were enriched after chilling for 6 and 48 h, respectively. On the basis of the protein-protein interaction network analysis, a protein kinase and phosphatases hub protein (P1959) were found to be involved in cross-talk between Ca2+, BR, ABA, and ethylene-mediated signaling pathways. We also highlighted the phosphorylation of BpSIZ1 and BpICE1 possibly impacted on the CBF/DREB-responsive pathway. From these results, we developed a schematic for the chilling tolerance mechanism at phosphorylation level.

Identification and Functional Divergence Analysis of WOX Gene Family in Paper Mulberry.

The WOX (WUSCHEL-related homeobox) is a plant-specific transcription factor involved in plant development and stress response. However, few studies have been reported on the WOX gene in woody plants. In this study, 10 BpWOX genes were isolated from paper mulberry by RACE-PCR and categorized into three clades through phylogenetic analysis, ancient, intermediate and WUS clade. Among them, five members had the transcriptional activity detected by yeast one-hybrid and seven were uniquely localized to the nucleus through green fluorescent protein (GFP) observation. The expression patterns of BpWOX genes in different tissues and under diverse treatments were quantified by the qRT-PCR method. Results showed that BpWUS was expressed in the apical bud, stem and root, BpWOX5 and BpWOX7 functioned only in the root tip, and three BpWOXs regulated leaf development redundantly. BpWOX9 and BpWOX10 were induced by indole-3-acetic acid (IAA) or jasmonic acid (JA), while BpWOX2 was repressed by five phytohormones. Interestingly, most BpWOX genes were responsive to the abiotic stress stimuli of drought, salt, cold, and cadmium (CdCl2). Together, our study revealed that BpWOXs were functionally divergent during paper mulberry development and environmental adaptation, which might be related to their evolutionary relationships. Our work will benefit the systematic understanding of the precise function of WOX in plant development and environmental stress responses.

Genome-wide identification of the Jatropha curcas MYB family and functional analysis of the abiotic stress responsive gene JcMYB2.

BACKGROUND: The MYB family is one of the most abundant transcription factor families in plants. MYB proteins are involved in plant development, abiotic stress tolerance, hormone signal transduction and disease resistance. Here we perform genome-wide identification of MYB family transcription factors in an energy plant J. curcas, including determining family composition, phylogenetic evolution and functional prediction analysis. In addition, we further elucidate the function of the JcMYB2 gene. METHODS: The phylogenetic trees were constructed by using the neighbor-joining method in MEGA 5.2. The biological functions of some JcMYBs were predicted according to orthology. The full length cDNA of JcMYB2 was cloned by using the RACE method. GUS histochemical staining was used to test the activity of the JcMYB2 promoter. Expression patterns of JcMYB2 were detected by using qPCR Transcriptional activity JcMYB2 were confirmed through yeast one hybrid. Subcellular Localization of JcMYB2 Protein were demonstrated by transient expression in the tobacco leaf. The function of JcMYB2 in salt and freezing tolerance were detected in transgenic plants. RESULTS: A genome-wide analysis identified 128 MYB genes, including 123 R2R3-MYBs, 4 R1R2R3-MYBs and 1 4R-MYB. All of the R2R3-MYBs are further classified into 19 groups which indicated functional conservation among previously identified groups of R2R3-MYB proteins. Among of these newly identified MYBs, the JcMYB2 belongs to group G11 and its expression is induced obviously by cold, salt and MeJA (Methyl Jasmonate) and slightly by ABA (abscisic acid). JcMYB2 is localized to the nucleus and has transcriptional activity. JcMYB2 overexpressing plants are more tolerant to salt and cold stress than wild type plants. Tissue specific expression profiles showed that the JcMYB2 gene was expressed ubiquitously throughout the plant, with higher expression levels observed in the root. CONCLUSION: A comprehensive genome-wide analysis and phylogenetic relationship of R2R3-MYB subfamily in J. curcas present the global identification and functional prediction of JcR2R3-MYBs. Additionally, JcMYB2 regulates the stress response signaling networks by interacting with MeJA and ABA signaling pathway and functions in the root development of J. curcas.

The cold responsive mechanism of the paper mulberry: decreased photosynthesis capacity and increased starch accumulation.

BACKGROUND: Most studies on the paper mulberry are mainly focused on the medicated and pharmacology, fiber quality, leaves feed development, little is known about its mechanism of adaptability to abiotic stress. Physiological measurement, transcriptomics and proteomic analysis were employed to understand its response to cold stress in this study. METHODS: The second to fourth fully expanded leaves from up to down were harvested at different stress time points forthe transmission electron microscope (TEM) observation. Physiological characteristics measurement included the relative electrolyte leakage (REL), SOD activity assay, soluble sugar content, and Chlorophyll fluorescence parameter measurement. For screening of differentially expressed genes, the expression level of every transcript in each sample was calculated by quantifying the number of Illumina reads. To identify the differentially expressed protein, leaves of plants under 0, 6, 12, 24, 48 and 72 h cold stress wereharvested for proteomic analysis. Finally, real time PCR was used to verify the DEG results of the RNA-seq and the proteomics data. RESULTS: Results showed that at the beginning of cold stress, respiratory metabolism was decreased and the transportation and hydrolysis of photosynthetic products was inhibited, leading to an accumulation of starch in the chloroplasts. Total of 5800 unigenes and 38 proteins were affected, including the repressed expression of photosynthesis and the enhanced expression in signal transduction, stress defense pathway as well as secondary metabolism. Although the transcriptional level of a large number of genes has been restored after 12 h, sustained cold stress brought more serious injury to the leaf cells, including the sharp rise of the relative electrolyte leakage, the declined Fv/Fm value, swelled chloroplast and the disintegrated membrane system. CONCLUSION: The starch accumulation and the photoinhibition might be the main adaptive mechanism of the paper mulberry responded to cold stress. Most of important, enhancing the transport and hydrolysis of photosynthetic products could be the potential targets for improving the cold tolerance of the paper mulberry.

Transcriptional regulation of the paper mulberry under cold stress as revealed by a comprehensive analysis of transcription factors.

BACKGROUND: Several studies have focused on cold tolerance in multiple regulated levels. However, a genome-scale molecular analysis of the regulated network under the control of transcription factors (TFs) is still lacking, especially for trees. To comprehensively identify the TFs that regulate cold stress response in the paper mulberry and understand their regulatory interactions, transcriptomic data was used to assess changes in gene expression induced by exposure to cold. RESULTS: Results indicated that 794 TFs, belonging to 47 families and comprising more than 59% of the total TFs of this plant, were involved in the cold stress response. They were clustered into three groups, namely early, intermediate and late responsive groups which contained 95, 550 and 149 TFs, respectively. Among of these differentially expressed TFs, one bHLH, two ERFs and three CAMTAs were considered to be the key TFs functioning in the primary signal transduction. After that, at the intermediate stage of cold stress, there were mainly two biological processes that were regulated by TFs, namely cold stress resistance (including 5 bHLH, 14 ERFs, one HSF, 4 MYBs, 3 NACs, 11 WRKYs and so on) and growth and development of lateral organ or apical meristem (including ARR-B, B3, 5 bHLHs, 2 C2H2, 4 CO-like, 2 ERF, 3 HD-ZIP, 3 YABBYs, G2-like, GATA, GRAS and TCP). In late responsive group, 3 ARR-B, C3H, 6 CO-like, 2 G2-like, 2 HSFs, 2 NACs and TCP. Most of them presented the up-regulated expression at 12 or 24 hours after cold stress implied their important roles for the new growth homeostasis under cold stress. CONCLUSIONS: Our study identified the key TFs that function in the regulatory cascades mediating the activation of downstream genes during cold tress tolerance in the paper mulberry. Based on the analysis, we found that the AP2/ERF, bHLH, MYB, NAC and WRKY families might play the central and significant roles during cold stress response in the paper mulberry just as in other species. Meanwhile, many other TF families previously reported as involving in regulation of growth and development, including ARF, DBB, G2-like, GRF, GRAS, LBD, WOX and YAABY exhibited their important potential function in growth regulation under cold stress.

Proteome analysis for antifungal effects of Bacillus subtilis KB-1122 on Magnaporthe grisea P131.

Rice blast, caused by Magnaporthe grisea threatens rice production worldwide. It is important to develop novel and environment-safe strategies to control the fungus. Here we reported that Bacillus subtilis KB-1122 could strikingly inhibit the growth of M. grisea P131 in agar diffusion assays. To further understand the molecular mechanism on the suppressive role of B. subtilis on M. grisea, the antagonist-pathogen interaction of the two strains was studied by using comparative proteome analysis in this report. The cellular and culture supernatant (CSN) proteins were prepared from co-culture and subjected to two-dimensional polyacrylamide gel electrophoresis. Proteome analysis revealed 33 cellular and 18 CSN proteins showing changes upon co-culture respectively. Importantly, down-regulated cellular proteins came from M. grisea, whereas up-regulated proteins derived from B. subtilis. Results suggested that glyceraldehyde-3-phosphate dehydrogenase and serine protein kinase might contribute to antifungal activity of B. subtilis KB-1122. Of CSN proteins identified, the endo-1,4-beta-glucanase (involved in degradation of polysaccharides) was up-regulated consistently at different times of incubation. This suggests that this enzyme plays an important role in the interaction between B. subtilis KB-1122 with M. grisea P131.

Effective protein extraction protocol for proteomics studies of Jerusalem artichoke leaves.

Protein extraction is a crucial step for proteomics studies. To establish an effective protein extraction protocol suitable for two-dimensional electrophoresis (2DE) analysis in Jerusalem artichoke (Helianthus tuberosus L.), three different protein extraction methods-trichloroacetic acid/acetone, Mg/NP-40, and phenol/ammonium acetate-were evaluated using Jerusalem artichoke leaves as source materials. Of the three methods, trichloroacetic acid/acetone yielded the best protein separation pattern and highest number of protein spots in 2DE analysis. Proteins highly abundant in leaves, such as Rubisco, are typically problematic during leaf 2DE analysis, however, and this disadvantage was evident using trichloroacetic acid/acetone. To reduce the influence of abundant proteins on the detection of low-abundance proteins, we optimized the trichloroacetic acid/acetone method by incorporating a PEG fractionation approach. After optimization, 363 additional (36.2%) protein spots were detected on the 2DE gel. Our results suggest that trichloroacetic acid/acetone method is a better protein extraction technique than Mg/NP-40 and phenol/ammonium acetate in Jerusalem artichoke leaf 2DE analysis, and that trichloroacetic acid/acetone method combined with PEG fractionation procedure is the most effective approach for leaf 2DE analysis of Jerusalem artichoke.