Ferulate 5-hydroxylase (F5H), a cytochrome P450 monooxygenase, has been indicated to function in the monolignol biosynthesis of sinapyl alcohol, namely the precursor of syringyl lignin during phenylpropanoid metabolism. In the present study, a full-length cDNA clone encoding F5H, designated as TbF5H1, was cloned from water chestnuts (Trapa bicornis Osbeck.) using RT-PCR and RACE. Sequence analysis of TbF5H1 protein revealed the presence of highly conserved motifs shard by P450 s, such as heme binding cysteine, oxygen binding I-helix, and E-R-R triade. Subsequently, TbF5H1 was expressed in bacterial system as a recombinant protein, which was used to ascertain kinetic parameters of the protein with the Km and Vmax values of 2.40 μM and 3.35 pkat mg−1, respectively. Together with high identity with other plant F5Hs based amino acid sequences, this result suggested that TbF5H1 had a biological function similar to its homologues in S lignin synthesis. Western blotting analysis using anti-TbF5H1 serum towards total proteins extracted from roots, stems, leaves, and flowers, detected a protein with a molecular weight of about 57 kDa. Quantitative real-time PCR analysis revealed the transcript level of TbF5H1 was higher in leaves than in other tested tissues. Interestingly, TbF5H1 was differentially expressed in response to various stresses, including salt (NaCl), hydrogen peroxide (H2O2) and heavy metal (lead), as well as elicitation by methyl ester jasmonate (MeJA). The expression of TbF5H1 was not significantly altered under NaCl or lead stress conditions, whereas a significant increase in TbF5H1 expression was detected after elicitation of H2O2 and MeJA, respectively. Moreover, there existed a fine coordination between expression of TbF5H1 and contents of certain nonenzymatic antioxidants. These results indicated that integrated with some redox components, TbF5H1 gene should contribute to the acclimation of T. bicornis to oxidative stress, which is modulated by MeJA.
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This study was supported by the grants from the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, China (No. 2015-1098), the Key University Science Research Project of Anhui Province, China (No. KJ2016A225), the Provincial Quality Engineer Fund of Anhui Education Department (No. 2015GXK015), and the Subject-Talents Program of Anhui Agricultural University (No. 2014XKPY-43).
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Conflict of interest
The authors declare that they have no conflict of interest.
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