Seasonal variations of iminosugars in mulberry leaves detected by hydrophilic interaction chromatography coupled with tandem mass spectrometry.[Pubmed: 29426416]

A rapid, sensitive, and validated method was developed for the simultaneous determination of four iminosugars, 1-deoxynojirimycin (DNJ), d-fagomine (FAG), 2-O-a-d-galactopyranosyl-DNJ (Gal-DNJ), and 4-O-β-d-glucopyranosyl-fagomine (Glu-FAG), in mulberry leaves. The method used hydrophilic interaction chromatography coupled with tandem mass spectrometry. Based on this method, the seasonal variations of iminosugars in the leaves of different mulberry species (Morus alba, Morus multicaulis Perr, Morus atropurpurea Roxb. and Morus wittiorum Hand-Mazz.) collected in Guangzhou, China, during 10 consecutive months in 2013 were investigated. The results indicated that the maximum content of DNJ in the leaves of all four species occurred in summer (June or July). The highest FAG level was found in spring (April or May). The highest levels of Gal-DNJ and Glu-FAG were recorded in autumn (September or October).

The influence of ripening stage and region on the chemical compounds in mulberry fruits (Morus atropurpurea Roxb.) based on UPLC-QTOF-MS.[Pubmed: 28888436]

Mulberries (Morus atropurpurea Roxb.) are rich in beneficial nutrients and secondary metabolites. Dramatic climate differences between western and eastern China lead to differences among the fruiting habits of mulberries grown in these regions. In this study, Xinjiang and Jiangsu, two regions in western and eastern China, respectively, were selected as sites where mulberry fruits (MFs) at different ripening stages were sampled. Their individual components, including both targeted and non-targeted chemical compounds, were detected by rapid ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Multivariate statistical analyses, including principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to compare MFs during ripening from these two regions. Potential biomarkers, which significantly contributed to the differentiation of the samples, were further identified or tentatively identified to determine the effects of ripening stages and regions on the chemical compounds in MFs. The results show that 43 compounds classified into nine different groups were identified in the MF samples from both the Xinjiang and Jiangsu regions. Among the compounds, all anthocyanins, carbohydrates and dihydroflavonols increased while phenolic acids and hydroxycoumarins decreased during ripening. Caffeoylquinic acids and some of anthocyanins could be regarded as important markers for MF ripening, and the accumulation of organic acids differentiated the samples from the two regions. Together, UPLC-QTOF-MS coupled with multivariate statistical analyses may be effective for metabolite profiling and identification of ripening degrees and cultivation regions.

Isolation and characterization of a novel chalcone synthase gene family from mulberry.[Pubmed: 28355585]

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.

Effect of Different Solvents on the Measurement of Phenolics and the Antioxidant Activity of Mulberry (Morus atropurpurea Roxb.) with Accelerated Solvent Extraction.[Pubmed: 28226182]

The effects of 9 different solvents on the measurement of the total phenolics and antioxidant activities of mulberry fruits were studied using accelerated solvent extraction (ASE). Sixteen to 22 types of phenolics (flavonols, flavan-3-ols, flavanol, hydroxycinnamic acids, hydroxybenzoic acids, and stilbenes) from different mulberry extracts were characterized and quantified using HPLC-MS/MS. The principal component analysis (PCA) was used to determine the suitable solvents to distinguish between different classes of phenolics. Additionally, the phenolic extraction abilities of ASE and ultrasound-assisted extraction (UAE) were compared. The highest extraction efficiency could be achieved by using 50% acidified methanol (50MA) as ASE solvents with 15.14 mg/gallic acid equivalents g dry weight of mulberry fruit. The PCA results revealed that the 50MA followed by 50% acidified acetone (50AA) was the most efficient solvent for the extraction of phenolics, particularly flavonols (627.12 and 510.31 μg/g dry weight, respectively), while water (W) was not beneficial to the extraction of all categories of phenolics. Besides, the results of 3 antioxidant capability assays (DPPH, ABTS free radical-scavenging assay, and ferric-reducing antioxidant power assay) showed that water-based organic solvents increased the antioxidant capabilities of the extracts compared with water or pure organic solvents. ASE was more suitable for the extraction of phenolics than UAE.

Characterization of Stilbene Synthase Genes in Mulberry (Morus atropurpurea) and Metabolic Engineering for the Production of Resveratrol in Escherichia coli.[Pubmed: 28168876]

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.

Effect of meteorological parameters and regions on accumulation pattern of phenolic compounds in different mulberry cultivars grown in China.[Pubmed: 28068840]

The compositions of phenolic compounds in 10 cultivars of two mulberry species (Morus atropurpurea Roxb. and Morus alba Linn.) grown in four regions of China were analysed using HPLC-TOF-MS. Results showed that a total of 27 phenolic compounds were identified in these mulberry cultivars. The Taiwan cultivar from the Jiangsu region showed the highest concentration of hydroxycinnamic acids, quercetin derivatives, and anthocyanins. Multivariate statistical analysis revealed that six mulberry cultivars grown in the region of Jiangsu, Ningxia, and Guangdong were differentiated regarding their species feature and regional characteristics. Cyanidin rhamnosylglucoside, 4-caffeoylquinic acid, dihydroquercetin, and quercetin were further screened out to be the key compounds for the differentiation of these cultivars. The correlation between the regional climate feature (including rainfall, humidity, and temperature) and the accumulation of phenolic compounds in these mulberry cultivars was established.

Complete chloroplast genome sequence of cultivated Morus L. species.[Pubmed: 27813600]

The complete chloroplast genome (cpDNA) sequences of two cultivated species of Morus L. (Morus atropurpurea and Morus multicaulis) are reported and reconstructed in this study, and were compared with that of wild Morus mongolica. In M. atropurpurea, the circular genome is 159,113 bp in size and comprises two identical inverted repeat (IR) regions of 25,707 bp each, separated by a large single-copy (LSC) region of 87,824 bp and a small single-copy (SSC) region of 19,875 bp. The cpDNA sequence of M. multicaulis is longer than that of M. atropurpurea (159,154 bp), and consists of two IRs (25,678 bp), a LSC region (87,763 bp), and a SSC region (20,035 bp). Each cpDNA contains 112 unique genes including 78 protein-coding genes, 30 transfer RNA genes, and 4 ribosomal RNA genes, with a GC content of 36.2%. There were 83 simple sequence repeats (SSRs) with mononucleotides being the most common (60) and di-, tri-, tetra-, and hexanucleotides appearing less frequently in M. atropurpurea. M. multicaulis contains 81 SSRs containing 63 mononucleotide repeats. The genes and SSRs identified in this study may enhance understanding of cpDNA evolution at both intra- and interspecific levels. MEGA 6.0 was used to construct a phylogenetic tree of 27 species, which revealed that M. atropurpurea and M. multicaulis are more related to their congeners than to others. The cpDNA of M. atropurpurea and M. multicaulis and its structural analysis are important for the chloroplast genome project, development of molecular markers for Morus species, and breeding of varieties.

Phenolic Profiles, Antioxidant Activities, and Neuroprotective Properties of Mulberry (Morus atropurpurea Roxb.) Fruit Extracts from Different Ripening Stages.[Pubmed: 27588828]

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Molecular characterization and expression analysis of the mulberry Na(+)/H(+) exchanger gene family.[Pubmed: 26730882]

Na(+)/H(+) exchangers (NHXs) have important roles in cellular pH, and Na(+) and K(+) homeostasis in plants. Mulberry is not only an important traditional economic woody plant known for its leaves, which are the exclusive food source of the silkworm Bombyx mori, but it can also adapt to many different adverse conditions, including saline environments. However, little is known about the NHXs in this important perennial tree. In this study, we identified and cloned seven putative NHX gene family members from Morus atropurpurea based on a genome-wide analysis of the Morus genome database. A phylogenetic analysis and genomic organization of mulberry NHXs suggested that the mulberry NHX family forms three distinct subgroups. Transcriptome data and real-time PCR of different mulberry varieties under normal culture conditions revealed that the mulberry NHX family has a different tissue-specific pattern in the two mulberry species. The MaNHX genes' expression analyses under different stresses (salt and drought) and signal molecules (abscisic acid, salicylic acid, hydrogen peroxide and methyl jasmonate) revealed that MaNHXs not only could be induced by salt, drought and abscisic acid as describe in the literature, but were also induced by other signal molecules, which indicated that MaNHX members exhibited diverse and complicated expression patterns in different mulberry tissues under various abiotic stresses, phytohormones and plant signaling molecules. Our results provide some insights into new and emerging cellular and physiological functions of this group of H(+)-coupled cation exchangers, beyond their function in salt tolerance, and also provide the basis for further characterizations of MaNHXs' physiological functions.