Cloning of A Full-Length cDNA Encoding 4-Coumarate:CoA Ligase of Amorpha fruticosa by PCR-Based Methods

An Amorpha fruticosa cDNA encoding 4 coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism related to lignin forming, was cloned by degenerating oligo primed polymerase chain reaction (PCR) and rapid amplification of cDNA end (RACE) PCR. We designed 5′RACE primers based on 4CLA1 fragment which obtained from degenerate PCR. Inverse PCR and nested PCR enabled cloning of the remainder fragments of the gene included 5′ and 3′ end sequence. The ORF encodes a polypeptide of 540 amino acids. The predicted amino acid sequence exhibits significant homology with those of other cloned 4CL genes, contain domains typical of predicted 4CL proteins, in particular a postulated AMP binding site, catalytic domain, and conserved Cys residues.

Allelic variation in the coumarate 3-hydroxylase gene associated with wood properties of Catalpa fargesii Bur.

Coumarate 3-hydroxylase(C3h)genes participate in the synthesis of lignin and may affect the properties of wood that are important for its commercial value.A better understanding of the natural variation in C3h genes and their associations to wood properties is required to effectively improve wood quality.We used a candidate gene-based association mapping approach to identify CfC3h allelic variants associated with traits that affect the wood properties of Catalpa fargesii.We first isolated the full-length CfC3h cDNA(1825 bp),which was expressed at relatively high levels in xylem according to real time-polymerase chain reaction.In totally,17 common single-nucleotide polymorphisms(minor allele frequency>5%)were identified through cloning and sequencing the CfC3h locus from a mapping population(including 88 unrelated natural C.fargesii individuals collected from main distribution area).Nucleotide diversity and linkage disequilibrium(LD)in CfC3h indicate that CfC3h has low nucleotide diversity(π_(t)=0.0031 andθ_(w)=0.0103)and relatively low LD(within 1800 bp;r^(2)≥0.1).An association analysis identified eight common single-nucleotide polymorphisms(SNPs)(false discovery rate,Q<0.10)and ten haplotypes(Q<0.10)associated with wood properties,explaining 4.92-12.09%of the phenotypic variance in an association population consisted of 125 unrelated natural individuals(The 88 individuals from the mapping population were comprised in the association population).Our study would provide new insight into C3h gene affecting wood quality,and the SNP markers identified would have potential applications in marker-assisted breeding in the future.

Process for the Obtention of Coumaric Acid from Coumarin: Analysis of the Reaction Conditions

Coumaric acid can be obtained from basic hydrolysis of coumarin, through a reaction process consisting on opening the lactone ring and cis-trans isomerization. Parameters such as reaction time, temperature, NaOH concentration, solvent and reaction atmosphere, have been thoroughly studied and analyzed, in order to determine the appropriate conditions for the maximum conversion efficiency of coumarin into coumaric acid. Experimental results show that the best conditions are a 1 hour reaction time, at 160℃, with a 20% sodium hydroxide aqueous solution, and in an inert reaction atmosphere.

ART1 and putrescine contribute to rice aluminum resistance via OsMYB30 in cell wall modification

Cell wall is the first physical barrier to aluminum(Al)toxicity.Modification of cell wall properties to change its binding capacity to Al is one of the major strategies for plant Al resistance;nevertheless,how it is regulated in rice remains largely unknown.In this study,we show that exogenous application of putrescines(Put)could significantly restore the Al resistance of art1,a rice mutant lacking the central regulator Al RESISTANCE TRANSCRIPTION FACTOR 1(ART1),and reduce its Al accumulation particularly in the cell wall of root tips.Based on RNA-sequencing,yeast-onehybrid and electrophoresis mobility shift assays,we identified an R2R3 MYB transcription factor OsMYB30 as the novel target in both ART1-dependent and Put-promoted Al resistance.Furthermore,transient dual-luciferase assay showed that ART1 directly inhibited the expression of OsMYB30,and in turn repressed Os4CL5-dependent 4-coumaric acid accumulation,hence reducing the Al-binding capacity of cell wall and enhancing Al resistance.Additionally,Put repressed OsMYB30 expression by eliminating Alinduced H2O2accumulation,while exogenous H2O2promoted OsMYB30 expression.We concluded that ART1 confers Put-promoted Al resistance via repression of OsMYB30-regulated modification of cell wall properties in rice.