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 K_m and V_max 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.

Caffeoyl shikimate esterase (CSE) hydrolyzes caffeoyl shikimate into caffeate and shikimate in the phenylpropanoid pathway. In this study, we performed a systematic analysis of the CSE gene family and investigated the possible roles of CSE and CSE-like genes in Populus. We conducted a genome-wide analysis of the CSE gene family, including functional and phylogenetic analyses of CSE and CSE-like genes, using the poplar (Populus trichocarpa) genome. Eighteen CSE and CSE-like genes were identified in the Populus genome, and five phylogenetic groups were identified from phylogenetic analysis. CSEs in Group Ia, which were proposed as bona fide CSEs, have probably been lost in most monocots except Oryza sativa. Primary functional classification showed that PoptrCSE1 and PoptrCSE2 had putative function in lignin biosynthesis. In addition, PoptrCSE2, along with PoptrCSE12, might also respond to stress with a function in cell wall biosynthesis. Enzymatic assay of PoptoCSE1 (Populus tomentosa), -2 and -12 showed that PoptoCSE1 and -2 maintained CSE activity. PoptoCSE1 and 2 had similar biochemical properties, tissue expression patterns and subcellular localization. Most of the PoptrCSE-like genes are homologs of AtMAGL (monoacylglycerol lipase) genes in Arabidopsis and may function as MAG lipase in poplar. Our study provides a systematic understanding of this novel gene family and suggests the function of CSE in monolignol biosynthesis in Populus.

Dehydrins (DHN) belong to the late embryogenesis abundant II family and have been found to enhance plant tolerance to abiotic stress. In the present study, we reported four DHNs in Larix kaempferi (LkDHN) which were identified from the published transcriptome. Alignment analysis showed that these four LkDHNs shared close relationships and belonged to SK₃-type DHNs. The electrophoretic mobility shift assay indicated that these four LkDHNs all possess sequence-independent binding capacity for double-strands DNAs. The subcellular localizations of the four LkDHNs were in both the nucleus and cytoplasm, indicating that these LkDHNs enter the nucleus to exert the ability to bind DNA. The preparation of tobacco protoplasts with different concentrations of mannitol showed that LkDHNs enhanced the tolerance of plant cells under osmotic stress. The overexpression of LkDHNs in yeasts enhanced their tolerance to osmotic stress and helped the yeasts to survive severe stress. In addition, LkDHNs in the nucleus of salt treated tobacco increased. All of these results indicated that the four LkDHNs help plants survive from heavy stress by participating in DNA protection. These four LKDHNs played similar roles in the response to osmotic stress and assisted in the adaptation of L. kaempferi to the arid and cold winter of northern China.

Larix olgensis is an economically important tree species native to northeastern China. The use of somatic embryogenesis (SE) is efficient and enables the rapid production of varieties with desirable qualities. Here, isobaric labeling via tandem mass tags was used to conduct a large-scale quantitative proteomic analysis of proteins in three critically important stages of SE in L. olgensis: the primary embryogenic callus, the single embryo, and the cotyledon embryo. We identified 6269 proteins, including 176 shared differentially expressed proteins across the three groups. Many of these proteins are involved in glycolipid metabolism, hormone response/signal transduction, cell synthesis and differentiation, and water transport; proteins involved in stress resistance and secondary metabolism, as well as transcription factors, play key regulatory roles in SE. The results of this study provide new insights into the key pathways and proteins involved in SE in Larix. Our findings have implications for the expression of totipotency, the preparation of synthetic seeds, and genetic transformation.