Biosynthesis of bioactive O-methylated flavonoids in Escherichia coli

Two bioactive O-methylflavonoids, sakuranetin (7-O-methylnaringenin) and ponciretin (7-O-methylnaringenin), were synthesized in Escherichia coli. Sakuranetin inhibits germination of Magnaporthe grisea, and ponciretin is a potential inhibitor of Helicobacter pylori. To achieve this, we reconstructed the naringenin biosynthesis pathway in E. coli. First, the shikimic acid pathway, which leads to the biosynthesis of tyrosine, was engineered in E. coli to increase the amount of available tyrosine. Second, several genes for the biosynthesis of ponciretin and sakuranetin such as tyrosine ammonia lyase (TAL), 4-coumaroyl CoA ligase (4CL), chalcone synthase (CHS), and O-methyltransferase (OMT) were overexpressed. In order to increase the supply the Coenzyme A (CoA), one gene (icdA, isocitrate dehydrogenase) was deleted. Using these strategies, we synthesized ponciretin and sakuranetin from glucose in E. coli at the concentration of 42.5 mg/L and 40.1 mg/L, respectively.     

Characterization of Chrysanthemum ClSOC1-1 and ClSOC1-2, homologous genes of SOC1

The MADS-box gene SOC1/TM3 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1/ Tomato MADS-box gene 3) is a main integrator in the Arabidopsis flowering pathway; its structure and function are highly conserved in many plant species. SOC1-like genes have been isolated in chrysanthemum, one of the most well-known ornamental plants, but it has not been well characterized thus far. We isolated and characterized ClSOC1-1 and ClSOC1-2, two putative orthologs of Arabidopsis SOC1, from the wild diploid chrysanthemum, Chrysanthemum lavandulifolium, to investigate the regulatory mechanisms of flowering time control in chrysanthemum. Expression analysis indicated that ClSOC1-1 and ClSOC1-2 were expressed in all examined organs/tissues (leaves, shoot apices, petioles, stems and roots) with different expression levels, and with high expression in the shoot apices and leaves during the early stage of floral transition. The expression levels of ClSOC1-1 and ClSOC1-2 in the shoot apices increased at different developmental stages with the highest expression levels after 7 days of short-day treatment. Overexpression of ClSOC1-1 and ClSOC1-2 in wild-type Arabidopsis resulted in early flowering, which was coupled with the upregulation of one of the flowering promoter genes LEAFY. Our results suggested that the ClSOC1-1 and ClSOC1-2 genes play an evolutionarily conserved role in promoting flowering in Chrysanthemum lavandulifolium and could serve as a vital target for the genetic manipulation of flowering time in the chrysanthemum.     

Development of specific and degenerate primers for CesA genes encoding cellulose synthase in flax (Linum usitatissimum L.)

Representatives of the CesA multigene family that control the synthesis of the catalytic subunits of the cellulose synthase complex were described for a number of higher plants. It has been established that the HVR2 region of these genes is class-specific and determines the involvement of the gene product in the synthesis of either the primary or secondary cell wall. The purpose of the current research was to develop degenerate and specific primers for parts of the CesA genes to allow the construction of molecular markers for the class-specific HVR2 region. Two pairs of specific primers for the CesA-1 and CesA-6 genes as well as a pair of degenerate primers for the HVR2 region of all flax CesA genes were developed, based on analysis of the CesA ESTs as well as the full-length cDNA sequences of the CesA genes in Arabidopsis, poplar, maize, and cotton that are available in the GenBank. Fragments of the expected size were amplified using flax cDNA as a template (201 bp for CesA-1, 300 bp for CesA-6, and 600 bp for HVR2). The markers developed in this research can be used for CesA gene cloning and sequencing, analysis of gene copy numbers as well as characterization of tissue- and development specific gene expression.     

Molecular evolution and sequence divergence of plant chalcone synthase and chalcone synthase-Like genes

Plant chalcone synthase (CHS) and CHS-Like (CHSL) proteins are polyketide synthases. In this study, we evaluated the molecular evolution of this gene family using representative types of CHSL genes, including stilbene synthase (STS), 2-pyrone synthase (2-PS), bibenzyl synthase (BBS), acridone synthase (ACS), biphenyl synthase (BIS), benzalacetone synthase, coumaroyl triacetic acid synthase (CTAS), and benzophenone synthase (BPS), along with their CHS homologs from the same species of both angiosperms and gymnosperms. A cDNA-based phylogeny indicated that CHSLs had diverse evolutionary patterns. STS, ACS, and 2-PS clustered with CHSs from the same species (late diverged pattern), while CTAS, BBS, BPS, and BIS were distant from their CHS homologs (early diverged pattern). The amino-acid phylogeny suggested that CHS and CHSL proteins formed clades according to enzyme function. The CHSs and CHSLs from Polygonaceae and Arachis had unique evolutionary histories. Synonymous mutation rates were lower in late diverged CHSLs than in early diverged ones, indicating that gene duplications occurred more recently in late diverged CHSLs than in early diverged ones. Relative rate tests proved that late diverged CHSLs had unequal rates to CHSs from the same species when using fatty acid synthase, which evolved from the common ancestor with the CHS superfamily, as the outgroup, while the early diverged lineages had equal rates. This indicated that late diverged CHSLs experienced more frequent mutation than early diverged CHSLs after gene duplication, allowing obtaining new functions in relatively short period of time.     

Identification of the cellulose synthase genes from the Oomycete Saprolegnia monoica and effect of cellulose synthesis inhibitors on gene expression and enzyme activity

Cellulose biosynthesis is a vital but yet poorly understood biochemical process in Oomycetes. Here, we report the identification and characterization of the cellulose synthase genes (CesA) from Saprolegnia monoica. Southern blot experiments revealed the occurrence of three CesA homologues in this species and phylogenetic analyses confirmed that Oomycete CesAs form a clade of their own. All gene products contained the D,D,D,QXXRW signature of most processive glycosyltransferases, including cellulose synthases. However, their N-terminal ends exhibited Oomycete-specific domains, i.e. Pleckstrin Homology domains, or conserved domains of an unknown function together with additional putative transmembrane domains. Mycelial growth was inhibited in the presence of the cellulose biosynthesis inhibitors 2,6-dichlorobenzonitrile or Congo Red. This inhibition was accompanied by a higher expression of all CesA genes in the mycelium and increased in vitro glucan synthase activities. Altogether, our data strongly suggest a direct involvement of the identified CesA genes in cellulose biosynthesis.     

Single-nucleotide polymorphisms in PtoCesA7 and their association with growth and wood properties in Populus tomentosa

Cellulose synthase (CesA) genes encode the enzymes that synthesize cellulose; therefore, CesAs play central roles in plant development and affect the yield and quality of wood, essential properties for industrial applications of plant biomass. To effectively manipulate wood biosynthesis in trees and improve wood quality, we thus require a better understanding of the natural variation in CesAs. Association studies have emerged as a powerful tool for identification of variation associated with quantitative traits. Here, we used a candidate gene-based association mapping approach to identify PtoCesA7 allelic variants that associate with growth and wood quality traits in Populus tomentosa. We isolated a full-length PtoCesA7 cDNA and observed high PtoCesA7 expression in xylem, consistent with the xylem-specific expression of CesA7. Nucleotide diversity and linkage disequilibrium (LD) in PtoCesA7, sampled from the P. tomentosa natural distribution, revealed that PtoCesA7 harbors high nucleotide diversity (π _T = 0.0091) and low LD (r ² ≥ 0.1, within 800 bp). By association analysis, we identified seven single-nucleotide polymorphisms (SNPs) (false discovery rate Q < 0.10) and 12 haplotypes (Q < 0.10) that associated with growth and wood properties, explaining 3.62–10.59 % of the phenotypic variance. We also validated 9 of the 10 significant marker–trait associations in at least one of three smaller subsets (climatic regions) or in a linkage-mapping population. Thus, our study identified functional PtoCesA7 allelic variants associated with growth and wood quality traits, giving new insights into genes affecting wood quality and quantity. From an applied perspective, the SNPs revealed in this study have potential applications in marker-assisted breeding.     

Ectopic expression of CaRLK1 enhances hypoxia tolerance with increasing alanine production in Nicotiana spp.

In a previous report, the pepper receptor-like kinase 1 (CaRLK1) gene was shown to be responsible for negatively regulating plant cell death caused by pathogens via accumulation of superoxide anions. Here, we examined whether this gene also plays a role in regulating cell death under abiotic stress. The total concentrations of free amino acids in CaRLK1-overexpressed cells (RLKox) increased by twofold compared with those of the wild-type Nicotiana tabacum BY-2 cells. Additionally, alanine and pyruvate concentrations increased by approximately threefold. These accumulations were associated with both the expression levels of the isocitrate lyase (ICL) and malate synthase genes and their specific activities, which were preferentially up-regulated in the RLKox cells. The expression levels of ethylene biosynthetic genes (ACC synthase and ACC oxidase) were suppressed, but those of both the metallothionein and lesion simulating disease 1 genes increased in the RLKox cells during submergence-induced hypoxia. The specific activity of catalase, which is involved in protecting ICL from reactive oxygen species, was also induced threefold in the RLKox cells. The primary roots of the transgenic plants that were exposed to hypoxic conditions grew at similar rates to those in normal conditions. We propose that CaRLK1 maintains a persistent hypoxia-resistant phenotype.     

Overexpression of <Emphasis Type="Italic">PSP1</Emphasis> enhances growth of transgenic Arabidopsis plants under ambient air conditions

Cellulose biosynthesis is mediated by cellulose synthases (CesAs), which constitute into rosette-like cellulose synthase complexe (CSC) on the plasma membrane. Two types of CSCs in Arabidopsis are believed to be involved in cellulose synthesis in the primary cell wall and secondary cell walls, respectively. In this work, we found that the two type CSCs participated cellulose biosynthesis in differentiating xylem cells undergoing secondary cell wall thickening in Populus. During the cell wall thickening process, expression of one type CSC genes increased while expression of the other type CSC genes decreased. Suppression of different type CSC genes both affected the wall-thickening and disrupted the multilaminar structure of the secondary cell walls. When CesA7A was suppressed, crystalline cellulose content was reduced, which, however, showed an increase when CesA3D was suppressed. The CesA suppression also affected cellulose digestibility of the wood cell walls. The results suggest that two type CSCs are involved in coordinating the cellulose biosynthesis in formation of the multilaminar structure in Populus wood secondary cell walls.     

Microspore embryogenesis in wheat: new marker genes for early, middle and late stages of embryo development

Microspore embryogenesis involves reprogramming of the pollen immature cell towards embryogenesis. We have identified and characterized a collection of 14 genes induced along different morphological phases of microspore-derived embryo development in wheat (Triticum aestivum L.) anther culture. SERKs and FLAs genes previously associated with somatic embryogenesis and reproductive tissues, respectively, were also included in this analysis. Genes involved in signalling mechanisms such as TaTPD1-like and TAA1b, and two glutathione S-transferase (GSTF2 and GSTA2) were induced when microspores had acquired a ‘star-like’ morphology or had undergone the first divisions. Genes associated with control of plant development and stress response (TaNF-YA, TaAGL14, TaFLA26, CHI3, XIP-R; Tad1 and WALI6) were activated before exine rupture. When the multicellular structures have been released from the exine, TaEXPB4, TaAGP31-like and an unknown embryo-specific gene TaME1 were induced. Comparison of gene expression, between two wheat cultivars with different response to anther culture, showed that the profile of genes activated before exine rupture was shifted to earlier stages in the low responding cultivar. This collection of genes constitutes a value resource for study mechanism of intra-embryo communication, early pattern formation, cell wall modification and embryo differentiation.