Functional Characteristics of Caffeoyl Shikimate Esterase in Larix Kaempferi and Monolignol Biosynthesis in Gymnosperms

Abstract

Caffeoyl shikimate esterase (CSE) has been reported to be involved in lignin biosynthesis; however, studies of CSE in gymnosperms are lacking. In this study, CSE was successfully cloned from Larix kaempferi (LkCSE) based on Larix laricina transcriptome screening. LkCSE was likely to have catalytic activity based on homologous sequence alignment and phylogenetic analyses of CSEs from different species. In vitro assays with the recombinant enzyme validated the catalytic activity of LkCSE, indicating its function in converting caffeoyl shikimate into caffeate and shikimate. Additionally, the optimum reaction pH and temperature of LkCSE were determined to be 6.0 and 30 °C, respectively. The values of Km and Vmax of CSE for caffeoyl shikimate were 98.11 μM and 14.44 nM min−1, respectively. Moreover, LkCSE was observed to have tissue expression specificity and was abundantly expressed in stems and leaves, especially stems, which was 50 times higher than the expression levels of roots. Lastly, translational fusion assays using LkCSE fused with green fluorescent proteins (GFP) in tobacco leaves indicated that LkCSE was localized in the plasma membrane and endoplasmic reticulum (ER). These results revealed that CSE clearly functions in gymnosperms and it is possible for LkCSE to interact with other ER-resident proteins and regulate mass flux in the monolignol biosynthesis pathway.

Systematic Analysis and Biochemical Characterization of the Caffeoyl Shikimate Esterase Gene Family in Poplar

“…(Kim et al 2016;Gao et al 2010)In addition, subcellular localization and the enzymatic assay of PoptoCSE1 and PoptoCSE2 provided further evidence to con rm the existence of genuine CSE involved in monolignol biosynthesis in poplar. CSE in genuine clade was also identi ed in gymnosperm which is corresponding to our previous study in Larix Kaempferi (Wang et al 2019). The de ciency of CSE in most monocots are corresponding with the studies in Brachypodium distachyon and Zea may (Ha et al 2016).…”

“…Two bona de CSEs were characterized in hybrid poplar and down-regulation of CSE in hybrid poplar resulted in the accumulation of feruloyl shikimate and sinapoyl shikimate indicating that these compounds also are in vivo CSE substrates. In addition, genuine CSE has also been characterized in Pinus in our previous study (Wang et al 2019).…”

“…The method used for subcellular location of PoptoCSE is according to our previous study (Wang et al 2019). The pBI121 vector with PoptoCSE1 and PoptoCSE2 gene and green uorescent protein (GFP) fusion expression was constructed by restriction enzymes and then ligation with T4 ligase (Takara, Dalian), and then the recombinant plasmids pBI121-35S-PoptoCSE1 and pBI121-35S-PoptoCSE2 were transferred into Agrobacterium tumefaciens strain GV3101 which were transferred into tobacco leaves via Agrobacterium-mediated transient transformation (Morita et al 2015).…”

Functional Characteristics Analysis of Dehydrins in Larix kaempferi under Osmotic Stress

“…The pET28a-LkDHNs vectors were constructed using the Onestep Seamless Kloning kit (Aidlab, Beijing, China) and transformed into E.coli strain BL21 (DE3). The recombinant strain was cultured overnight at 37 °C in lysogeny broth (LB) medium containing 500 mg/L kanamycin, and cultured in 1 L LB medium at a ratio of 1:20 until the OD 600 reached 0.6, following which 0.4 mM isopropyl β- d-thiogalactoside (IPTG) was used to induce protein expression at 28 °C for 4 h. For protein purification, the recombinant protein was washed with a wash buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, and 20 mM imidazole) and then eluted with an elution buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, and 150 mM imidazole) [ 28 ]. The partial sequence of the caffeoyl shikimate esterase (CSE) (MK211161) and the partial end of pET28a vector (about 200 bp) were selected as probes and labeled by a Beyotime EMSA probe biotin labeling kit (Beyotime, Shang Hai, China).…”

Two UGT84A Family Glycosyltransferases Regulate Phenol, Flavonoid, and Tannin Metabolism in Juglans regia (English Walnut)

“…Alternatively, 4-HCA can be converted to caffeic acid directly by C4H ( Chen et al, 2011 ). Ferulic and sinapinic are biosynthesized successively by CMT with the methyl group being incorporated by S-adenosylmethionine ( Urban et al, 1994 ; Wang et al, 2018 , 2019 ). CHS constitutes the first committed step of flavonoid biosynthesis by condensation of 4-coumaroyl-CoA (from 4-HCA) and malonyl-CoA and thus is linked tightly to phenylpropanoid metabolism ( Nakayama et al, 2019 ) ( Figure 1 ).…”