Changying Liu

Characterization and Functional Analysis of 4-Coumarate:CoA Ligase Genes in Mulberry

A small, multigene family encodes 4-coumarate:CoA ligases (4CLs) that catalyze the ligation of CoA to hydroxycinnamic acids, a branch point directing metabolites to flavonoid or monolignol pathways. In this study, we characterized four 4CL genes from M. notabilis Genome Database, and cloned four Ma4CL genes from M. atropurpurea cv. Jialing No.40. A tissue-specific expression analysis indicated that Ma4CL3 was expressed at higher levels than the other genes, and that Ma4CL3 was strongly expressed in root bark, stem bark, and old leaves. Additionally, the expression pattern of Ma4CL3 was similar to the trend of the total flavonoid content throughout fruit development. A phylogenetic analysis suggested that Mn4CL1, Mn4CL2, and Mn4CL4 belong to class I 4CLs, and Mn4CL3 belongs to class II 4CLs. Ma4CL genes responded differently to a series of stresses. Ma4CL3 expression was higher than that of the other Ma4CL genes following wounding, salicylic acid, and ultraviolet treatments. An in vitro enzyme assay indicated that 4-coumarate acid was the best substrate among cinnamic acid, 4-coumarate acid, and caffeate acid, but no catalytic activity to sinapate acid and ferulate acid. The results of subcellular localization experiments showed that Ma4CL3 localized to the cytomembrane, where it activated transcription. We used different vectors and strategies to fuse Ma4CL3 with stilbene synthase (STS) to construct four Ma4CL-MaSTS co-expression systems to generate resveratrol. The results indicated that only a transcriptional fusion vector, pET-Ma4CL3-T-MaSTS, which utilized a T7 promoter and lac operator for the expression of MaSTS, could synthesize resveratrol.

Mulberry Transcription Factor MnDREB4A Confers Tolerance to Multiple Abiotic Stresses in Transgenic Tobacco

The dehydration responsive element binding (DREB) transcription factors have been reported to be involved in stress responses. Most studies have focused on DREB genes in subgroups A-1 and A-2 in herbaceous plants, but there have been few reports on the functions of DREBs from the A-3–A-6 subgroups and in woody plants. Moreover, mulberry trees are ecologically and economically important perennial woody plants, but there has been little research on its stress physiology, biochemistry and molecular biology. In this study, a DREB gene from the mulberry tree, designated as MnDREB4A, classified into the A-4 subgroup by our previous study, was selected for further characterization. Our results showed that the MnDREB4A protein was localized to the nucleus where it activated transcription. The promoter of MnDREB4A can direct prominent expression downstream of the β-glucuronidase (GUS) gene under heat, cold, drought and salt stress, and GUS staining was deepest after 12 h of stress treatment. The MnDREB4A-overexpression transgenic tobacco showed the improved growth phenotype under untreated conditions, such as greener leaves, longer roots, and lower water loss and senescence rates. Overexpression of MnDREB4A in tobacco can significantly enhance tolerance to heat, cold, drought, and salt stresses in transgenic plants. The leaf discs and seedlings of transgenic plants reduced leaf wilting and senescence rates compared to the wild type plants under the different stress conditions. Further investigation showed that transgenic plants also had higher water contents and proline contents, and lower malondialdehyde contents under untreated condition and stress conditions. Our results indicate that the MnDREB4A protein plays an important role in plant stress tolerance.

Isolation and expression analysis of anthocyanin biosynthetic genes in Morus alba L.

Anthocyanins from mulberry fruits are used in medicine. However, little anthocyanin can be detected in other tissues and sometimes also mulberry fruits are colorless. The aim of this study was to investigate which gene or genes have the strongest correlation with the anthocyanin biosynthesis. The expression of several anthocyanin synthesis genes were determined in different tissues of two white and two purple fruit cultivars. Genes encoding dihydroflavonol reductase (MaDFR) and anthocyanidin synthase (MaANS) showed a high expression only in fruit tissue of purple-fruit cultivars. During the development of mulberry fruits, the anthocyanin content was well correlated with the transcripts abundance of MaDFR, MaANS, and MaCHS (encoding chalcone synthase). The skin of female mulberry flowers turns red under irradiance because of up-regulated expressions of MaCHS, MaDFR, and MaANS. These three genes may control the anthocyanin biosynthesis in mulberry and up-regulation of them may greatly increase the anthocyanin content.

Characterization and expression of genes involved in the ethylene biosynthesis and signal transduction during ripening of mulberry fruit.

Although ethylene is well known as an essential regulator of fruit development, little work has examined the role ethylene plays in the development and maturation of mulberry (Morus L.) fruit. To study the mechanism of ethylene action during fruit development in this species, we measured the ethylene production, fruit firmness, and soluble solids content (SSC) during fruit development and harvest. By comparing the results with those from other climacteric fruit, we concluded that Morus fruit are probably climacteric. Genes associated with the ethylene signal transduction pathway of Morus were characterized from M. notabilis Genome Database, including four ethylene receptor genes, a EIN2-like gene, a CTR1-like gene, four EIN3-like genes, and a RTE1-like gene. The expression patterns of these genes were analyzed in the fruit of M. atropurpurea cv. Jialing No.40. During fruit development, transcript levels of MaETR2, MaERS, MaEIN4, MaRTE, and MaCTR1 were lower at the early stages and higher after 26 days after full bloom (DAF), while MaETR1, MaEIL1, MaEIL2, and MaEIL3 remained constant. In ripening fruit, the transcripts of MaACO1 and MaACS3 increased, while MaACS1 and MaACO2 decreased after harvest. The transcripts of MaACO1, MaACO2, and MaACS3 were inhibited by ethylene, and 1-MCP (1–methylcyclopropene) upregulated MaACS3. The transcripts of the MaETR-like genes, MaRTE, and MaCTR1 were inhibited by ethylene and 1-MCP, suggesting that ethylene may accelerate the decline of MaETRs transcripts. No significant changes in the expression of MaEIN2, MaEIL1, and MaEIL3 were observed during ripening or in response to ethylene, while the expressions of MaEIL2 and MaEIL4 increased rapidly after 24 h after harvest (HAH) and were upregulated by ethylene. The present study provides insights into ethylene biosynthesis and signal transduction in Morus plants and lays a foundation for the further understanding of the mechanisms underlying Morus fruit development and ripening.

Characterization of Stilbene Synthase Genes in Mulberry (Morus atropurpurea) and Metabolic Engineering for the Production of Resveratrol in Escherichia coli

Stilbenes have been recognized for their beneficial physiological effects on human health. Stilbene synthase (STS) is the key enzyme of resveratrol biosynthesis and has been studied in numerous plants. Here, four MaSTS genes were isolated and identified in mulberry (Morus atropurpurea Roxb.). The expression levels of MaSTS genes and the accumulation of trans-resveratrol, trans-oxyresveratrol, and trans-mulberroside A were investigated in different plant organs. A novel coexpression system that harbored 4-coumarate:CoA ligase gene (Ma4CL) and MaSTS was established. Stress tests suggested that MaSTS genes participate in responses to salicylic acid, abscisic acid, wounding, and NaCl stresses. Additionally, overexpressed MaSTS in transgenic tobacco elevated the trans-resveratrol level and increased tolerance to drought and salinity stresses. These results revealed the major MaSTS gene, and we evaluated its function in mulberry, laying the foundation for future research on stilbene metabolic pathways in mulberry.

De novo assembly of mulberry (Morus alba L.) transcriptome and identification of candidate unigenes related to salt stress responses

Mulberry (Morus alba L.) is a kind of plant with strong adaptation to drought, salt stress, water logging, and other environmental stresses. However, there is little knowledge on the molecular mechanism involved in its response and resistance to environmental stresses, including salt stress. In this study, a total of 101589 unigenes were obtained from 24 Morus salinity subtranscriptomes using Illumina RNA-sequencing technology, and led to 34.72% of the assembled reads being matched to known transcripts. The number of down-regulated DEGs (differentially expressed genes) under salt stress is more than that of up-regulated DEGs, and these down-regulated DEGs enriched in the process related to stress response by GO and KEGG enrichment analysis. It is notable that some genes showed diverse response patterns against salt stress in genotype- and tissue-dependent manners. The DEGs involved in signal transduction and transcription regulation were found to be more enriched in low-salt-tolerant genotypes and the majority of these responsive genes showed decreased transcript abundance, which may result in low tolerance of low-salt-tolerant genotypes. The results of this study will advance our understanding of the salt response in Morus and provide the basis for further genetic improvement of salt tolerance in Morus and other plants.

Isolation and characterization of a novel chalcone synthase gene family from mulberry

Chalcone synthase (CHS) is the pivotal enzyme that catalyzes the first committed step of the phenylpropanoid pathway leading to flavonoids. Here, five CHS genes were determined in mulberry (Morus atropurpurea Roxb.). Interestingly, phylogenetic analysis tended to group three MaCHSs in the stilbene synthase (STS) family and initially annotated these as MaSTSs. A co-expression system that harbored a 4-coumarate:CoA ligase gene and one of the candidate genes was established to determine the functions of this novel gene family. The fermentation result demonstrated that MaSTS in fact encoded a CHS enzyme, and was consequently retermed MaCHS. Tissue-specific expression analysis indicated that MaCHS1/MaCHS2 was highly abundant in fruit, and MaCHS4 had significant expression in root bark, stem bark and old leaves, while MaCHS3 and MaCHS5 were more expressed in old leaves. Subcellular localization experiments showed that MaCHS was localized to the cytoplasm. Transcription levels suggested MaCHS genes were involved in a series of defense responses. Over-expression of MaCHS in transgenic tobacco modified the metabolite profile, and resulted in elevated tolerance to a series of environmental stresses. This study comprehensively evaluated the function of MaCHS genes and laid the foundation for future research on MaCHS in mulberry.

Characterization and expression of abscisic acid signal transduction genes during mulberry fruit ripening

The plant hormone abscisic acid (ABA) plays an important role in fruit development. To analyze the transcriptional regulation of ABA signaling pathway-related genes during mulberry (Morus alba L.) fruit development and ripening, 17 genes participating in ABA signal transduction were isolated from the Morus notabilis genome database, including five MnPYL, six MnPP2C, and six MnSnRK2 genes. The result of multiple sequence alignment revealed that their functional amino acid residues and domains are conserved within each gene family. The expression profiles of ABA signaling pathway-related genes in the fruit of Morus atropurpurea cv. Jialing No. 40 were measured by quantitative real-time polymerase chain reaction. The transcripts of MaPYL4, MaPP2C5, and MaSnRK2.6 were expressed at a relatively higher level during the entire development process. Moreover, the transcript levels of MaPYL1/3/5, MaPP2C2/4/5/6, and MaSnRK2.3/2.6 were lower during the early-maturation stage and higher during the post-maturation stage, which suggests that they may play a vital role in regulating mulberry fruit ripening. In addition, these genes showed strong responses to exogenous ABA, fluridone, glucose, and sucrose. This study also indicated that exogenous ABA, sucrose, and glucose may promote fruit maturity, while fluridone significantly inhibits fruit ripening.

Screening, cloning and expression analysis of a cellulase derived from the causative agent of hypertrophy sorosis scleroteniosis, Ciboria shiraiana.

A cellulase gene (KJ700939, CsCelA) from Ciboria shiraiana that is highly expressed during the infection of mulberry fruit was screened by quantitative real-time PCR (qRT-PCR). Using cDNA isolated from infected mulberry fruits as template, the full-length 1170-bp sequence of CsCelA was obtained, which encodes a 390-amino acid protein with a putative signal peptide of 24 amino acids. The 998-bp fragment encoding the mature peptide of CsCelA was cloned into the multiple cloning site of the pPIC9K vector and overexpressed as an active protein of 55.3kDa in the methylotrophic yeast Pichia pastoris. The specific activity of induced supernatants of the recombinant cellulase (CsCelA) was 17.44U/ml and 135U/g for freeze-dried powder. The Kmax and Vmax of CsCelA for sodium carboxymethylcellulose (CMC) were 4.6mg/ml and 107.2U/mg, respectively. The supernatant and freeze-dried powder of the recombinant cellulase exhibited stable activity from pH4.0 to 9.0, and at temperatures ranging from 30°C to 55°C. Finally, the activity of the recombinant cellulase was assessed by enzymatic hydrolysis of the cell walls of mulberry leaves. CsCelA showed an endo-cellulase mode of cleavage, as assessed by thin layer chromatography (TLC).

Characterization and expression profiles of MaACS and MaACO genes from mulberry (Morus alba L.)

Abstract1-Aminocyclopropane-1-carboxylic acid synthase (ACS) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) are encoded by multigene families and are involved in fruit ripening by catalyzing the production of ethylene throughout the development of fruit. However, there are no reports on ACSor ACO genes in mulberry, partly because of the limited molecular research background. In this study, we have obtained five ACS gene sequences and two ACO gene sequences from Morus Genome Database. Sequence alignment and phylogenetic analysis of MaACO1 and MaACO2 showed that their amino acids are conserved compared with ACO proteins from other species. MaACS1 and MaACS2 are type I, MaACS3 and MaACS4 are type II, and MaACS5 is type III, with different C-terminal sequences. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) expression analysis showed that the transcripts of MaACS genes were strongly expressed in fruit, and more weakly in other tissues. The expression of MaACO1 and MaACO2 showed different patterns in various mulberry tissues. MaACS and MaACO genes demonstrated two patterns throughout the development of mulberry fruit, and bothof them were strongly up-regulated by abscisic acid (ABA) and ethephon.