SilkMeta: a comprehensive platform for sharing and exploiting pan-genomic and multi-omic silkworm data.

The silkworm Bombyx mori is a domesticated insect that serves as an animal model for research and agriculture. The silkworm super-pan-genome dataset, which we published last year, is a unique resource for the study of global genomic diversity and phenotype-genotype association. Here we present SilkMeta (http://silkmeta.org.cn), a comprehensive database covering the available silkworm pan-genome and multi-omics data. The database contains 1082 short-read genomes, 546 long-read assembled genomes, 1168 transcriptomes, 294 phenotype characterizations (phenome), tens of millions of variations (variome), 7253 long non-coding RNAs (lncRNAs), 18 717 full length transcripts and a set of population statistics. We have compiled publications on functional genomics research and genetic stock deciphering (mutant map). A range of bioinformatics tools is also provided for data visualization and retrieval. The large batch of omics data and tools were integrated in twelve functional modules that provide useful strategies and data for comparative and functional genomics research. The interactive bioinformatics platform SilkMeta will benefit not only the silkworm but also the insect biology communities.

Dehydrogenase MnGutB1 catalyzes 1-deoxynojirimycin biosynthesis in mulberry.

As the prevalence of diabetes continues to increase, the number of individuals living with diabetes complications will reach an unprecedented magnitude. Continuous use of some synthetic agents to reduce blood glucose levels causes severe side effects, and thus, the demand for nontoxic, affordable drugs persists. Naturally occurring compounds, such as iminosugars derived from the mulberry (Morus spp.), have been shown to reduce blood glucose levels. In mulberry, 1-deoxynojirimycin (DNJ) is the predominant iminosugar. However, the mechanism underlying DNJ biosynthesis is not completely understood. Here, we showed that DNJ in mulberry is derived from sugar and catalyzed through 2-amino-2-deoxy-D-mannitol (ADM) dehydrogenase MnGutB1. Combining both targeted and nontargeted metabolite profiling methods, DNJ and its precursors ADM and nojirimycin (NJ) were quantified in mulberry samples from different tissues. Purified His-tagged MnGutB1 oxidized the hexose derivative ADM to form the 6-oxo compound DNJ. The mutant MnGutB1 D283N lost this remarkable capability. Furthermore, in contrast to virus-induced gene silencing of MnGutB1 in mulberry leaves that disrupted the biosynthesis of DNJ, overexpression of MnGutB1 in hairy roots and light-induced upregulation of MnGutB1 enhanced DNJ accumulation. Our results demonstrated that hexose derivative ADM, rather than lysine derivatives, is the precursor in DNJ biosynthesis, and it is catalyzed by MnGutB1 to form the 6-oxo compound. These results represent a breakthrough in producing DNJ and its analogs for medical use by metabolic engineering or synthetic biology.

Metabolic and Transcriptional Analysis Reveals Flavonoid Involvement in the Drought Stress Response of Mulberry Leaves.

Abiotic stress, especially drought stress, poses a significant threat to terrestrial plant growth, development, and productivity. Although mulberry has great genetic diversity and extensive stress-tolerant traits in agroforestry systems, only a few reports offer preliminary insight into the biochemical responses of mulberry leaves under drought conditions. In this study, we performed a comparative metabolomic and transcriptomic analysis on the "drooping mulberry" (Morus alba var. pendula Dippel) under PEG-6000-simulated drought stress. Our research revealed that drought stress significantly enhanced flavonoid accumulation and upregulated the expression of phenylpropanoid biosynthetic genes. Furthermore, the activities of superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) content were elevated. In vitro enzyme assays and fermentation tests indicated the involvement of flavonol synthase/flavanone 3-hydroxylase (XM_010098126.2) and anthocyanidin 3-O-glucosyltransferase 5 (XM_010101521.2) in the biosynthesis of flavonol aglycones and glycosides, respectively. The recombinant MaF3GT5 protein was found to recognize kaempferol, quercetin, and UDP-glucose as substrates but not 3-/7-O-glucosylated flavonols and UDP-rhamnose. MaF3GT5 is capable of forming 3-O- and 7-O-monoglucoside, but not di-O-glucosides, from kaempferol. This implies its role as a flavonol 3, 7-O-glucosyltransferase. The findings from this study provided insights into the biosynthesis of flavonoids and could have substantial implications for the future diversified utilization of mulberry.

The Mulberry SPL Gene Family and the Response of MnSPL7 to Silkworm Herbivory through Activating the Transcription of MnTT2L2 in the Catechin Biosynthesis Pathway.

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, as unique plant transcription factors, play important roles in plant developmental regulation and stress response adaptation. Although mulberry is a commercially valuable tree species, there have been few systematic studies on SPL genes. In this work, we identified 15 full-length SPL genes in the mulberry genome, which were distributed on 4 Morus notabilis chromosomes. Phylogenetic analysis clustered the SPL genes from five plants (Malus × domestica Borkh, Populus trichocarpa, M. notabilis, Arabidopsis thaliana, and Oryza sativa) into five groups. Two zinc fingers (Zn1 and Zn2) were found in the conserved SBP domain in all of the MnSPLs. Comparative analyses of gene structures and conserved motifs revealed the conservation of MnSPLs within a group, whereas there were significant structure differences among groups. Gene quantitative analysis showed that the expression of MnSPLs had tissue specificity, and MnSPLs had much higher expression levels in older mulberry leaves. Furthermore, transcriptome data showed that the expression levels of MnSPL7 and MnSPL14 were significantly increased under silkworm herbivory. Molecular experiments revealed that MnSPL7 responded to herbivory treatment through promoting the transcription of MnTT2L2 and further upregulating the expression levels of catechin synthesis genes (F3'H, DFR, and LAR).

Hierarchical Action of Mulberry miR156 in the Vegetative Phase Transition.

The vegetative phase transition is a prerequisite for flowering in angiosperm plants. Mulberry miR156 has been confirmed to be a crucial factor in the vegetative phase transition in Arabidopsis thaliana. The over-expression of miR156 in transgenic Populus × canadensis dramatically prolongs the juvenile phase. Here, we find that the expression of mno-miR156 decreases with age in all tissues in mulberry, which led us to study the hierarchical action of miR156 in mulberry. Utilizing degradome sequencing and dual-luciferase reporter assays, nine MnSPLs were shown to be directly regulated by miR156. The results of yeast one-hybrid and dual-luciferase reporter assays also revealed that six MnSPLs could recognize the promoter sequences of mno-miR172 and activate its expression. Our results demonstrate that mno-miR156 performs its role by repressing MnSPL/mno-miR172 pathway expression in mulberry. This work uncovered a miR156/SPLs/miR172 regulation pathway in the development of mulberry and fills a gap in our knowledge about the molecular mechanism of vegetative phase transition in perennial woody plants.

Origin and functional differentiation of (E)-ß-ocimene synthases reflect the expansion of monoterpenes in angiosperms.

The acquisition of new metabolic activities is a major force driving evolution. We explored, from the perspectives of gene family expansion and the evolutionary adaptability of proteins, how new functions have arisen in which terpene synthases diverged. Monoterpenoids are diverse natural compounds that can be divided into cyclic and acyclic skeleton forms according to their chemical structure. We demonstrate, through phylogenetic reconstructions and genome synteny analyses, that the (E)-ß-ocimene synthases, which are acyclic monoterpene synthases (mTPSs), appear to have arisen several times in independent lineages during plant evolution. Bioinformatics analyses and classical mutation experiments identified four sites (I388, F420, S446, and F485) playing important roles in the neofunctionalization of mTPSs. Incubation of neryl diphosphate with Salvia officinalis 1,8-cineole synthase (SCS) and mutated proteins show that these four sites obstruct the isomerization of geranyl diphosphate. Quantum mechanical/molecular mechanical molecular dynamics simulations of models of SCS, SCSY420F/I446S, and SCSN338I/Y420F/I446S/L485F with (3R)-linalyl diphosphate suggest that mutations changed the configuration of the intermediate to obtain new activities. These results provide new perspectives on the evolution of mTPSs, explain the convergent evolution of (E)-ß-ocimene synthases at the molecular level, and identify key residues to control the specificity of engineered mTPSs.

Horizontal transfers of LTR retrotransposons in seven species of Rosales.

Horizontal transposable element transfer (HTT) events have occurred among a large number of species and play important roles in the composition and evolution of eukaryotic genomes. HTTs are also regarded as effective forces in promoting genomic variation and biological innovation. In the present study, HTT events were identified and analyzed in seven sequenced species of Rosales using bioinformatics methods by comparing sequence conservation and Ka/Ks value of reverse transcriptase (RT) with 20 conserved genes, estimating the dating of HTTs, and analyzing the phylogenetic relationships. Seven HTT events involving long terminal repeat (LTR) retrotransposons, two HTTs between Morus notabilis and Ziziphus jujuba, and five between Malus domestica and Pyrus bretschneideri were identified. Further analysis revealed that these LTR retrotransposons had functional structures, and the copy insertion times were lower than the dating of HTTs, particularly in Mn.Zj.1 and Md.Pb.3. Altogether, the results demonstrate that LTR retrotransposons still have potential transposition activity in host genomes. These results indicate that HTT events are another strategy for exchanging genetic material among species and are important for the evolution of genomes.

LysM1 in MmLYK2 is a motif required for the interaction of MmLYP1 and MmLYK2 in the chitin signaling.

KEY MESSAGE: Two LysM-containing proteins, namely, MmLYP1 and MmLYK2, were identified in mulberry. These proteins might be involved in chitin signaling. The LysM1 of MmLYK2 is critical for their interactions. Chitin is a major component of fungal cell walls and acts as an elicitor in plant innate immunity. Lysin motif (LysM)-containing proteins are essential for chitin recognition. However, related studies have been rarely reported in woody plants. In this study, in mulberry, the expression of a LysM-containing protein, MmLYP1, was significantly up-regulated after treatment with chitin and pathogenic fungi. In addition, MmLYP1 has an affinity for insoluble chitin polymers. Thus, MmLYP1 might function in chitin signaling. Since MmLYP1 lacks an intracellular domain, additional protein kinases are required for this signaling. An LysM-containing kinase, MmLYK2, was then identified. Expression of the MmLYK2 did not change significantly after chitin treatment, and the affinity of MmLYK2 for insoluble chitin was not high. The structure of MmLYP1 is similar to that of the chitin elicitor-binding proteins in rice and Arabidopsis. However, MmLYK2 has two LysM motifs, while the chitin elicitor receptor kinase 1 proteins in rice and Arabidopsis have one and three LysM motifs, respectively. The LysM1 of MmLYK2 interacted with all four LysM motifs in MmLYP1 and MmLYK2 in yeast. The chimera lacking the LysM1 of MmLYK2 did not interact with MmLYP1 and MmLYK2 in yeast and Nicotiana benthamiana cells. The LysM1 in MmLYK2 is the key motif in the interaction between MmLYP1 and MmLYK2, which may be involved in chitin signaling.

Evolutionary and functional analysis of mulberry type III polyketide synthases.

BACKGROUND: Type III polyketide synthases are important for the biosynthesis of flavonoids and various plant polyphenols. Mulberry plants have abundant polyphenols, but very little is known about the mulberry type III polyketide synthase genes. An analysis of these genes may provide new targets for genetic improvement to increase relevant secondary metabolites and enhance the plant tolerance to biotic and abiotic stresses. RESULTS: Eighteen genes encoding type III polyketide synthases were identified, including six chalcone synthases (CHS), ten stilbene synthases (STS), and two polyketide synthases (PKS). Functional characterization of four genes representing most of the MnCHS and MnSTS genes by coexpression with 4-Coumaroyl-CoA ligase in Escherichia coli indicated that their products were able to catalyze p-coumaroyl-CoA and malonyl-CoA to generate naringenin and resveratrol, respectively. Microsynteny analysis within mulberry indicated that segmental and tandem duplication events contributed to the expansion of the MnCHS family, while tandem duplications were mainly responsible for the generation of the MnSTS genes. Combining the evolution and expression analysis results of the mulberry type III PKS genes indicated that MnCHS and MnSTS genes evolved mainly under purifying selection to maintain their original functions, but transcriptional subfunctionalization occurred during long-term species evolution. Moreover, mulberry leaves can rapidly accumulated oxyresveratrol after UV-C irradiation, suggesting that resveratrol was converted to oxyresveratrol. CONCLUSIONS: Characterizing the functions and evolution of mulberry type III PKS genes is crucial for advancing our understanding of these genes and providing the basis for further studies on the biosynthesis of relevant secondary metabolites in mulberry plants.

Identification of the mulberry genes involved in ethylene biosynthesis and signaling pathways and the expression of MaERF-B2-1 and MaERF-B2-2 in the response to flooding stress.

The phytohormone ethylene is essential to plant growth and development. It plays crucial roles in responses to biotic and abiotic stress. The mulberry tree is an important crop plant in countries in which people rear silkworms for silk production. The availability of the mulberry genome has made it possible to identify mulberry genes involved in ethylene biosynthesis and signal pathways. A total of 145 mulberry genes were identified by both homology-based and hidden Markov model (HMM) search, including 29 genes associated with ethylene biosynthesis and 116 genes in the AP2/ERF family. Studies on gene structure have provided a genetic basis for understanding the functions of these genes. The differences in gene expression were also observed in different tissues. The expression of two mulberry genes in the AP2/ERF family, MaERF-B2-1 and MaERF-B2-2, was found to be associated with the response to flooding stress.

Identification and characterization of genes involved in the jasmonate biosynthetic and signaling pathways in mulberry (Morus notabilis).

Jasmonate (JA) is an important phytohormone regulating growth, development, and environmental response in plants, particularly defense response against herbivorous insects. Recently, completion of the draft genome of the mulberry (Morus notabilis) in conjunction with genome sequencing of silkworm (Bombyx mori) provides an opportunity to study this unique plant-herbivore interaction. Here, we identified genes involved in JA biosynthetic and signaling pathways in the genome of mulberry for the first time, with the majority of samples showing a tissue-biased expression pattern. The analysis of the representative genes 12-oxophytodienoic acid reductase (OPRs) and jasmonate ZIM-domain (JAZs) was performed and the results indicated that the mulberry genome contains a relatively small number of JA biosynthetic and signaling pathway genes. A gene encoding an important repressor, MnNINJA, was identified as an alternative splicing variant lacking an ethylene-responsive element binding factor-associated amphiphilic repression motif. Having this fundamental information will facilitate future functional study of JA-related genes pertaining to mulberry-silkworm interactions.

Genome-wide identification and expression analyses of cytochrome P450 genes in mulberry (Morus notabilis).

Cytochrome P450s play critical roles in the biosynthesis of physiologically important compounds in plants. These compounds often act as defense toxins to prevent herbivory. In the present study, a total of 174 P450 genes of mulberry (Morus notabilis C.K.Schn) were identified based on bioinformatics analyses. These mulberry P450 genes were divided into nine clans and 47 families and were found to be expressed in a tissue-preferential manner. These genes were compared to the P450 genes in Arabidopsis thaliana. Families CYP80, CYP92, CYP728, CYP733, CYP736, and CYP749 were found to exist in mulberry, and they may play important roles in the biosynthesis of mulberry secondary metabolites. Analyses of the functional and metabolic pathways of these genes indicated that mulberry P450 genes may participate in the metabolism of lipids, other secondary metabolites, xenobiotics, amino acids, cofactors, vitamins, terpenoids, and polyketides. These results provide a foundation for understanding of the structures and biological functions of mulberry P450 genes.

[The proteasome inhibitor MG132 attenuates skeletal muscle atrophy in a rat model of chronic obstructive pulmonary disease].

OBJECTIVE: To investigate the effect of the proteasome inhibitor MG-132 on skeletal muscle atrophy in a rat model of chronic obstructive pulmonary disease (COPD) and its potential mechanisms. METHODS: The COPD rat model was established by instillation of LPS and exposure to the cigarette smoke. Then the COPD rats were randomly divided into 3 groups (each group n = 12): COPD model control group, MG-132 high dose group (MG-132 0.1 mg·kg(-1)·d(-1)) and low dose group (MG-132 0.05 mg·kg(-1)·d(-1)), and normal control group. After 1 week and 4 week, 6 rats of each group were sacrificed, and then the following parameters were determined: the weight of the diaphragm muscle, the concentration of TNF-α in the serum and diaphragm via enzyme-linked immunosorbent assay (ELISA). Muscle atrophy F-box protein (MAFbx), NF-κBp65, and IκB-α mRNA levels were determined by RT-PCR. The protein levels of MAFbx, NF-κBp65 and IκB-α in diaphragm were measured by Western blot. The single factor analysis of variance was used for statistical analysis among the groups, while t test was used for comparison between 2 groups, and Pearson linear correlation analysis was also performed. RESULTS: The weight of diaphragm muscle from 1 week and 4 week normal control group [(0.99 ± 0.06) mg and (1.20 ± 0.04) mg] were reduced as compared to those of COPD model control group [(0.83 ± 0.09) mg and (1.01 ± 0.06) mg], high dose group [(0.85 ± 0.02) mg and (1.11 ± 0.06) mg], and low dose group [(0.83 ± 0.03) mg and (1.04 ± 0.02) mg]. The reduction of diaphragm muscle weight in the high dose group and the low dose group was significantly less than that in the COPD model control group, with a more marked difference as compared with the 4 week high dose group. The TNF-α levels in diaphragm from 4 week high dose group [(106 ± 8) ng/L] and low dose group [(122 ± 7) ng/L] were decreased as compared to that of the COPD model control group [(143 ± 24) ng/L]. The levels of NF-κBp65 and MAFbx mRNA from the 4 week high dose group (2.17 ± 0.42) and low dose group (1.74 ± 0.14) and the protein expression (1.13 ± 0.04 and 1.27 ± 0.05) were also decreased as compared to those of the COPD model control group (mRNA 2.81 ± 0.31 and 4.87 ± 0.34, protein expression 1.32 ± 0.04 and 1.44 ± 0.07). The levels of IκB-α mRNA and protein expression (0.96 ± 0.08 and 0.83 ± 0.06) were higher than those of the COPD model control group (0.25 ± 0.02 and 0.58 ± 0.06), (t = 1.57-24.9, P < 0.05). The levels of the TNF-α levels in serum and diaphragm were correlated positively with the levels of MAFbx and NF-κBp65 mRNA and protein expression (r = 0.672-0.875, P < 0.01), but negatively with the levels of IκB-α mRNA and protein expression (r = -0.656--0.927, P < 0.01). CONCLUSIONS: The proteasome inhibitor MG-132 significantly inhibited IκB-α degradation thus preventing NF-κB activation. This effect resulted in preventing skeletal muscle atrophy in the COPD rats.

The gap-free genome of mulberry elucidates the architecture and evolution of polycentric chromosomes.

Mulberry is a fundamental component of the global sericulture industry, and its positive impact on our health and the environment cannot be overstated. However, the mulberry reference genomes reported previously remained unassembled or unplaced sequences. Here, we report the assembly and analysis of the telomere-to-telomere gap-free reference genome of the mulberry species, Morus notabilis, which has emerged as an important reference in mulberry gene function research and genetic improvement. The mulberry gap-free reference genome produced here provides an unprecedented opportunity for us to study the structure and function of centromeres. Our results revealed that all mulberry centromeric regions share conserved centromeric satellite repeats with different copies. Strikingly, we found that M. notabilis is a species with polycentric chromosomes and the only reported polycentric chromosome species up to now. We propose a compelling model that explains the formation mechanism of new centromeres and addresses the unsolved scientific question of the chromosome fusion-fission cycle in mulberry species. Our study sheds light on the functional genomics, chromosome evolution, and genetic improvement of mulberry species.

Chromosome restructuring and number change during the evolution of Morus notabilis and Morus alba.

Mulberry (Morus spp.) is an economically important plant as the main food plant used for rearing domesticated silkworm and it has multiple uses in traditional Chinese medicine. Two basic chromosome numbers (Morus notabilis, n = 7, and Morus alba, n = 14) have been reported in the genus Morus, but the evolutionary history and relationship between them remain unclear. In the present study, a 335-Mb high-quality chromosome-scale genome was assembled for the wild mulberry species M. notabilis. Comparative genomic analyses indicated high chromosomal synteny between the 14 chromosomes of cultivated M. alba and the six chromosomes of wild M. notabilis. These results were successfully verified by fluorescence in situ hybridization. Chromosomal fission/fusion events played crucial roles in the chromosome restructuring process between M. notabilis and M. alba. The activity of the centromere was another key factor that ensured the stable inheritance of chromosomes. Our results also revealed that long terminal repeat retrotransposons were a major driver of the genome divergence and evolution of the mulberry genomes after they diverged from each other. This study provides important insights and a solid foundation for studying the evolution of mulberry, allowing the accelerated genetic improvement of cultivated mulberry species.

[Effect of qufeng zhidong simplified recipe on the behavior of the tic disorder rats].

OBJECTIVE: To study the effects qufeng zhidong Simplified Recipe (QZSR) on the behavior of the tic disorder (TD) rats. METHODS: Fifty male SD rats were randomly divided to the normal group, the model group, the QZSR-1 group, the QZSR-2 group, and the QZSR group, 10 in each group. Two mg/kg apomorphine (APO) was intraperitoneally injected to rats in the model group, the QZSR-1 group, the QZSR-2 group, and the QZSR group, while equal volume of normal saline was intraperitoneally injected to rats in the normal group, both once daily for 7 successive weeks. At the 4th week equal volume of normal saline was intraperitoneally injected to rats in the model group and the normal group, while corresponding medicinal liquid was intraperitoneally injected to those in the rest groups, both once daily for 7 successive weeks. At the 2nd and 4th week of intervention, rats' improvement degrees of stereotyped behavior and the open-field test were monitored, and their experimental results were analyzed. RESULTS: At the 2nd and 4th week of intervention, when compared with those of the model group, the score of stereotyped behavior decreased, the numbers of passing-panel, straightening, and dejecta pill were reduced, and the number of grooming increased in the QZSR-1 group, the QZSR-2 group, and the QZSR group. But there was no difference among the three groups (P>0.05). CONCLUSION: QZSR could significantly reduce APO induced stereotyped behavior scores of TD rats, improve their locomotor activities, and reinforce their adaptive faculty.

Dynamic changes in transposable element and gene methylation in mulberry (Morus notabilis) in response to Botrytis cinerea.

DNA methylation has been proposed to regulate plant stress resistance. However, the dynamic changes in DNA methylation in woody plants and their correlations with pathogenic responses are not fully understood. Here, we present single-base maps of the DNA methylomes of mulberry (Morus notabilis) leaves that were subjected to a mock treatment or inoculation with Botrytis cinerea. Compared with the former, the latter showed decreased mCG and mCHG levels and increased mCHH levels. DNA methylation inhibitors reduced resistance gene methylation levels and enhanced mulberry resistance, suggesting that the hypomethylation of resistance genes affects mulberry resistance to B. cinerea. Virus-induced gene silencing of MnMET1 enhanced the expression of mulberry-resistance genes, thereby increasing the plant's resistance to B. cinerea. We also found that MITEs play a dominant role in controlling DNA methylation levels. MITEs appear to be the main sources of 24-nt siRNAs that regulate gene expression through the RNA-directed DNA methylation pathway.

Ciboria carunculoides Suppresses Mulberry Immune Responses Through Regulation of Salicylic Acid Signaling.

Ciboria carunculoides is the dominant causal agent of mulberry sclerotial disease, and it is a necrotrophic fungal pathogen with a narrow host range that causes devastating diseases in mulberry fruit. However, little is known about the interaction between C. carunculoides and mulberry. Here, our transcriptome sequencing results showed that the transcription of genes in the secondary metabolism and defense-related hormone pathways were significantly altered in infected mulberry fruit. Due to the antimicrobial properties of proanthocyanidins (PAs), the activation of PA biosynthetic pathways contributes to defense against pathogens. Salicylic acid (SA) and jasmonic acid (JA) are major plant defense hormones. However, SA signaling and JA signaling are antagonistic to each other. Our results showed that SA signaling was activated, while JA signaling was inhibited, in mulberry fruit infected with C. carunculoides. Yet SA mediated responses are double-edged sword against necrotrophic pathogens, as SA not only activates systemic acquired resistance (SAR) but also suppresses JA signaling. We also show here that the small secreted protein CcSSP1 of C. carunculoides activates SA signaling by targeting pathogenesis-related protein 1 (PR1). These findings reveal that the infection strategy of C. carunculoides functions by regulating SA signaling to inhibit host defense responses.

The evolution of the expansin gene family in Brassica species.

Expansin gene (EXP) family plays important roles in plant growth and crop improvement. However, it has not been well studied in the Brassica genus that includes several important agricultural and horticultural crops. To get insight to the evolution and expansion of EXP family in Brassica, Brassica EXPs which are homologues of 35 known AtEXPs of Arabidopsis were comprehensively and systematically analyzed in the present study. In total, 340 Brassica EXPs were clustered into four groups that corresponded multiple alignment to four subfamilies of AtEXPs, with divergent conserved motifs and cis-acting elements among groups. To understand the expansion of EXP family, an integrated genomic block system was constructed among Arabidopsis and Brassica species based on 24 known ancestral karyotype blocks. Obvious gene loss, segmental duplication, tandem duplication and DNA sequence repeat events were found during the expansion of Brassica EXPs, of which the segmental duplication was possibly the major driving force. The divergence time was estimated in 1109 orthologs pairs of EXPs, revealing the divergence of Brassica EXPs from AtEXPs during ~30 MYA, and the divergence of EXPs among Brassica species during 13.50-17.94 MYA. Selective mode analysis revealed that the purifying selection was the major contributor to expansion of Brassica EXPs. This study provides new insights into the evolution and expansion of the EXP family in Brassica genus.

Mulberry genes MnANR and MnLAR confer transgenic plants with resistance to Botrytis cinerea.

Proanthocyanidins (PAs) are major defense-related phenolics in mulberry, but the mechanism underlying their biosynthesis remains uncharacterized. In this study, the relationship between the expression of genes encoding anthocyanidin reductase (ANR) or leucoanthocyanidin reductase (LAR) and PA biosynthesis was investigated in white and red mulberry fruits. In ripening fruits, the MnANR and MnLAR transcription levels tended to decrease, whereas the catechin and epicatechin contents initially increased and then decreased. In contrast, the PA content exhibited a clearly different trend. The ectopic expression of MnANR and MnLAR in tobacco increased the resistance to Botrytis cinerea, as evidenced by the less extensive disease symptoms of the transgenic plants compared with the wild-type plants. In vitro experiments revealed that the transgenic tobacco crude leaf extract had an obvious inhibitory effect on B. cinerea. Moreover, the ectopic expression of MnANR and MnLAR in tobacco inhibited the expression of anthocyanin biosynthesis genes, resulting in decreased anthocyanin contents in flowers. The results of this study may be useful for elucidating the mechanism underlying PA biosynthesis. Furthermore, ANR and LAR represent potential targets for improving the resistance of mulberry and related plant species to B. cinerea.