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

Abstract

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.

Fig 1. The enzymes of general phenylpropanoid metabolism, which are connected by black arrows, consist of PAL, C4H, and 4CL.

Dashed arrows indicate a branch pathway for lignin biosynthesis emanating from the general phenylpropanoid metabolic pathway, which is also catalyzed by 4CL. The gray arrows indicate the pathways for chalcone and resveratrol biosynthesis.

Fig 2. Schematic diagram representing the co-expression of the Ma4CL3 and MaSTS proteins.

4-S was carried by plasmid pETDuet-1/pACYCDuet-1; S4 and 4S were carried by plasmid pET28a(+); 4-T-S was also carried by plasmid pET28a(+), but with an independent T7 promoter and lac operator.

Fig 3. Gene structure of Morus notabilis 4CL genes.

Schematic diagrams with exons (gray boxes) and introns (dashed lines between exons). Gene structures were displayed by Fancy Gene (http://bio.ieo.eu/fancygene/).

Fig 4. The phylogenetic tree analysis of Mn4CL proteins.

The results of the phylogenetic analysis divided the sequences into two groups, suggesting that Mn4CL3 belongs to the class II group; whereas Mn4CL1, Mn4CL2, and Mn4CL4 belong to the class I group.

Fig 5. Sequence comparison of the deduced amino acid sequences.

Box I represents the putative AMP-binding domain, and box II represents the conserved GEICIGR putative catalytic site.

Fig 6. Expression of Ma4CL genes in different tissues and different response patterns under diverse stresses.

All of the Ma4CL genes were constitutively expressed in all of the selected organs (a), but exhibited significant differences in their magnitudes of expression. Ma4CL3 was expressed at higher levels than the other genes in the same tissues, and Ma4CL3 was strongly expressed in root bark, stem bark, and old leaves. The Ma4CL genes showed different response patterns under diverse stresses, as the induction of Ma4CL3 expression was greater that of the other genes after wounding (b), SA (c), and UV (d) treatments. Error bars on each column indicate SDs from three replicates.

Fig 7. Expression of Ma4CL genes and total flavonoid contents during mulberry fruit development.

a, b, c, and d represent Ma4CL1, Ma4CL2, Ma4CL3, and Ma4CL4 expression levels, respectively, e and f indicate total flavonoid contents during fruit development from stage S1 to S7. Error bars on each column indicate SDs from three replicates.

Fig 8. The relative expresa sion levels of Ma4CL genes under NaCl treatment.

Ma4CL1 and Ma4CL3 expression in stems peaked at 1 h, Ma4CL2 and Ma4CL4 exhibited similar double-peaked expression patterns (a–d). In roots, Ma4CL1 and Ma4CL2 expression peaked at 6 and 3 h, respectively. However, the expression of Ma4CL3 and Ma4CL4 was down-regulated at 1 and 12 h (e–h). Error bars on each column indicate SDs from three replicates. Significant differences (p < 0.05) among treatment are marked with “*”.

Fig 9. Relative expression levels of Ma4CL gene under ABA treatment.

Ma4CL genes in stems (a–d) and roots (e–h) showed different responses. Ma4CL1 and Ma4CL2 expression in stems peaked at 1 and 12 h, respectively, while Ma4CL3 and Ma4CL4 expression did not change significantly. However, Ma4CL3 expression in roots reached its maximum after 3 h, while the expression levels of the other genes increased gradually and reached their maxima after 24 h of treatment. Significant differences (p < 0.05) among treatment are marked with “*”. After 4 d of ABA treatment (i), although all of the RERS were significantly up-regulated, Ma4CL3 expression was only up-regulated 3.46-fold, while Ma4CL1, Ma4CL2, and Ma4CL4 expression was up-regulated approximately 5.23-, 16.50-, and 9.47-, respectively, compared with that of the control (“*” indicates p < 0.01). (j) The ratio of root length and stem length changed significantly under ABA treatment, and taproots became sturdier than control, and the number of root hairs increased significantly. Error bars on each column indicate SDs from three replicates.

Fig 10. Subcellular localization of the Ma4CL3 protein.

(a) EGFP, Ma4CL3::EGFP, and the EGFP::Ma4CL3 fusion strategies. (b) The positive control was a transfected plasmid containing EGFP alone. DAPI was used to indicate the nucleus. It was observed that Ma4CL3 localized to the cytomembrane mainly.

Fig 11. Fermentation test and HPLC detected.

(a) Biosynthesis routes of resveratrol constructed in recombinant E. coli.. Standard chromatograms of resveratrol (b) and the ethyl acetate extract (c). Peak 1 represents 4-cinnamate acid, and peak 2 represents resveratrol.