Abstract Mangrove plants are known to produce secondary metabolites including isoprenoid and long chain polyisoprenoid. SonneratiaalbaandS. caseolaris, are true mangroves that contain alkane (C25-C33), triterpenoid, phytosterol, dolichol, polyprenol, and polyprenyl acetone. Here we reported new compounds of probably epoxy polyprenol and epoxy dolichol from S. albaandS. caseolarisold leaves using two-dimensional thin layer chromatography (2D-TLC). In the S. albaold leaves, having the occurrence of polyprenol, dolichol, epoxy polyprenol, and epoxy dolichol with a dominating pattern of polyprenol. A similar result obtained in the S. caseolarisold leaves, displaying the presence of polyprenol, dolichol, epoxy polyprenol, and epoxy dolichol with a dominating carbon chain length of dolichol. By contrast, no epoxy polyprenol or epoxy dolichol were detected in S. alba, and S. caseolarisleaves. The present study indicated a new type of polyisoprenoid distribution and further investigation is needed to clarify the chemical structure of this newcompound.

Abstract Hevea brasiliensis Mull. Arg. (rubber plant) is now used utterly for the commercial production of natural rubber, mainly of cis-1, 4-polyisoprenoid. The current study assesses the bioinformatics approaches to assay four probably polyprenol reductase genes from Hevea brasileinsis on NCBI database as well as expected the physicochemical, subcellular localisation, and phylogenetic of polyprenol reductase. Several parameters of physicochemical polyprenol reductase in H. brasiliensis were varied among the genes observed. The predictable half-life of polyprenol reductase in this study was similar to polyprenols reductase from other majorities of plant species. Based on stability coefficients, there were no stable proteins; all polyprenol reductase genes were non-stable proteins. It is notable that mitochondria target peptide value diverse from 0.053 to 0.101, signifying that is expected to be a presence. To clarify the homology in the midst of the polyprenol reductase gene in Euphorbiaceae family, a dendrogram tree was constructed. The close relationship among polyprenol reductase genes: environs may interpret Kandelia obovata, Ricinus communis, Manihot esculenta with rubber plant in the polyprenol reductase environs in the tropical rain forests. The present results indicated the prominence of understanding the variation and role of physical and chemical characteristics of the distinct amino acids in plant polyprenol reductase genes in H. brasiliensis.

This present study describes the polyisoprenoids (polyprenol/dehydrodolichol and dolichol) profile, and composition from selected mangrove associates leaves namely Amorphophallus paeoniifolius, Borassus flabellifer, Finlaysonia maritima, and Pandanus tectorius. The occurrence and distribution of polyisoprenoids were investigated using a convenient two-plate thin layer chromatography method. The polyisoprenoid profile in the leaves was determined and classified into two types. Type-I, having domination of dolichols over polyprenols (100%) was found in P. tectorius. These dolichols occurred one dolichol family (C60–C95). Type-II, displaying the existence of both dehydrodolichols and dolichols, was confirmed in A. paeoniifolius, B. flabellifer, and F. maritima. Dolichol contents were slightly more abundance found than polyprenols (in the ratio of approximately 60%:40%) in these three species. Polyprenols and dolichols with the chain length of C80–C90, respectively, detected in A. paeoniifolius. Ficaprenol (C50–C70) was only found in B. flabellifer. Dolichols also occurred longer-chains in B. flabellifer (C50–C105) and F. maritime (C65–C130). The present study suggested that the patterns of shorter-chain and longer-chain polyprenol, shorter and longer dolichols are regulated in mangrove associates.

Cloning of Kandelia obovata KcCAS gene (previously known as Kandelia candel) and Rhizophora stylosa RsCAS have already have been reported and encoded cycloartenol synthases In this study, the predicted KcCAS and RsCAS protein were analyzed using online software of Phyre2 and Swiss-model. The protein modelling for KcCAS and RsCAS cycloartenol synthases was determined using Pyre2 had similar results with slightly different in sequence identity. By contrast, the Swiss-model for KcCAS slightly had higher sequence identity (47.31%) and Qmean (0.70) compared to RsCAS. No difference of ligands binding site which is considered as modulators for both cycloartenol synthases The range of predicted protein derived from 91-757 amino acid residues with coverage sequence similarities 0.86, respectively from template model of lanosterol synthase from the human. Homology modelling revealed that 706 residues (93% of the amino acid sequence) had been modelled with 100.0% confidence by the single highest scoring template for both KcCAS and RsCAS using Phyre2. This coverage was more elevated than swiss-model predicted (86%). The present study suggested that both genes are responsible for the genesis of cycloartenol in these mangrove plants.

A. floridum and L. littorea are the members of significant mangroves which are abundant in Indonesia that has been reported to have biological properties. The pattern and existence of polyisoprenoid alcohols (polyprenols/dehydrodolichol and dolichols) inthe leaves and roots of A. floridum and L. littorea were investigated using a convenient two-plate thin layer chromatography method. The polyprenols and dolichols distribution was determined and classified into two groups. Group-I, having a dominating of dolichols over dehydrodolichols, was found in the roots of L. littorea with one dolichol family (C90-C95). Type-II, showing the existence of both dehydrodolichols and dolichols, was observed in the leaves A. floridum (with polyprenol and dolichol chain length C60-C80 and C90-C95, respectively). Furthermore detected in leaves of L. Littorea, polyprenol and dolichol occurred longer chain length (C60-C140 and C70-C140, respectively) and roots of A. floridum with a chain length of dolichol (C75-C100) few longer that polyprenol (C85-C95). The composition of polyisoprenoids in leaves and roots tissues of A. floridum and L. littorea is the first description to extend our previous finding on the occurrence polyisoprenoids in mangrove plants.

It has been previously reported that dolichols but not polyprenols were predominated in mangrove leaves and roots. Therefore, the occurrence of larger amounts of dolichol in leaves of mangrove plants implies that polyprenol reductase is responsible for the conversion of polyprenol to dolichol may be active in mangrove leaves. Here we report the early assessment of probably polyprenol reductase gene from genome sequence of mangrove plant Kandelia obovata. The functional assignment of the gene was based on a homology search of the sequences against the non-redundant (nr) peptide database of NCBI using Blastx. The degree of sequence identity between DNA sequence and known polyprenol reductase was confirmed using the Blastx probability E-value, total score, and identity. The genome sequence data resulted in three partial sequences, termed c23157 (700 bp), c23901 (960 bp), and c24171 (531 bp). The c23157 gene showed the highest similarity (61%) to predicted polyprenol reductase 2-like from Gossypium raimondii with E-value 2e(-100). The second gene was c23901 to exhibit high similarity (78%) to the steroid 5-alpha-reductase Det2 from J. curcas with E-value 2e(-140). Furthermore, the c24171 gene depicted highest similarity (79%) to the polyprenol reductase 2 isoform X1 from Jatropha curcas with E- value 7e(-21). The present study suggested that the c23157, c23901, and c24171, genes may encode predicted polyprenol reductase. The c23157, c23901, c24171 are therefore the new type of predicted polyprenol reductase from K. obovata.

Molecular cloning of Kandelia candel KcMS gene has previously been cloned and encoded a multifunctional triterpene synthase. In this study, the KcMS gene promoter was cloned through Genome walking, sequenced, and analyzed. A 1,358 bp genomic DNA fragment of KcMS promoter was obtained. PLACE and PlantCARE analysis of the KcMS promoter revealed that there was some regulatory elements in response to environmental signals and involved in the regulation of gene expression. Results showed that four kinds of elements are regulated by hormone binding, namely 2 MeJA-responsiveness elements (CGTCA-motif and TGACG-motif), the ABRE (TACGTG) involved in abscisic acid responsiveness, gibberellin-related GARE-motif (AAACAGA), and the TGA-element (AACGAC) as an auxin-responsive element. Several elements in the KcMS have been shown in other plants to be responsive to abiotic stress. These motifs were MBS (CAACTG), TC-rich repeats, and eight light responsive elements. The KcMS promoter was also involved in the activation of defense genes in plants such as HSE (AAAAAATTC) and four circadian control elements (CAANNNNATC). The presence of multipotential regulatory motifs suggested that KcMS may be involved in regulation of plant tolerance to several types of stresses.

Molecular cloning of five oxidosqualene cyclases (OSC) genes from Bruguiera gymnorrhiza, Kandelia candel, and Rhizophora stylosa had previously been cloned, characterized, and encoded mono and -multi triterpene synthases. The present study analyzed protein modelling of triterpene synthase genes from mangrove using Phyre2 and Swiss-model. The diversity was noted within protein modelling of triterpene synthases using Phyre2 from sequence identity (38-43%) and residue (696-703). RsM2 was distinguishable from others for template structure; it used lanosterol synthase as a template (PDB ID: w6j.1.A). By contrast, other genes used human lanosterol synthase (1w6k.1.A). The predicted bind sites were correlated with the product of triterpene synthase, the product of BgbAS was β-amyrin, while RsM1 contained a significant amount of β-amyrin. Similarly BgLUS and KcMS, both main products was lupeol, on the other hand, RsM2 with the outcome of taraxerol. Homology modelling revealed that 696 residues of BgbAS, BgLUS, RsM1, and RsM2 (91-92% of the amino acid sequence) had been modelled with 100% confidence by the single highest scoring template using Phyre2. This coverage was higher than Swiss-model (85-90%). The present study suggested that molecular cloning of triterpene genes provides useful tools for studying the protein modelling related regulation of isoprenoids biosynthesis in mangrove forests.

Coastal plants are known to produce secondary metabolites including polyisoprenoid alcohols. Coastal plants have been shown to have phytomedicinal, biological, and pharmacological properties. The present study reports the detection of polyisoprenoids composition from roots and stems of selected coastal medicinal grasses, Cyperus rotundus, Distichlis spicata, and Spinifex littoreus. A two-dimensional thin layer chromatography (2D-TLC) method was used to analyse the content and distribution of polyisoprenoid alcohols (polyprenols/dehydrodolihols and dolichols) in coastal grasses. The presence of polyprenols and dolichols in the roots and stems were identified and grouped into two types. Type-I, showing a preponderance of dolichols over polyprenols, was detected in the roots of C. rotundus and D. spicata. Type-II, displaying the existence of both polyprenols and dolichols, was traced in S. littoreusroots and the stems of C. rotundus, D. spicata, and S. littoreus. The diversity of polyisoprenoids in the root and stem tissues even in the same species, suggesting the chemotaxonomic criterion of polyisoprenoids in coastal grasses.

A. floridum and L. littorea are the members of significant mangroves which are abundant in Indonesia that has been reported to have biological properties. The pattern and existence of polyisoprenoid alcohols (polyprenols/dehydrodolichol and dolichols) inthe leaves and roots of A. floridum and L. littorea were investigated using a convenient two-plate thin layer chromatography method. The polyprenols and dolichols distribution was determined and classified into two groups. Group-I, having a dominating of dolichols over dehydrodolichols, was found in the roots of L. littorea with one dolichol family (C90-C95). Type-II, showing the existence of both dehydrodolichols and dolichols, was observed in the leaves A. floridum (with polyprenol and dolichol chain length C60-C80 and C90-C95, respectively). Furthermore detected in leaves of L. Littorea, polyprenol and dolichol occurred longer chain length (C60-C140 and C70-C140, respectively) and roots of A. floridum with a chain length of dolichol (C75-C100) few longer that polyprenol (C85-C95). The composition of polyisoprenoids in leaves and roots tissues of A. floridum and L. littorea is the first description to extend our previous finding on the occurrence polyisoprenoids in mangrove plants.

This present study describesthe bioinformatics approach to analyze three partial polyprenol reductase genes from mangrove plant, Kandeliaobovataas well aspredictedphysical and chemical properties, potential peptide, subcellular localization, and phylogenetic. The diversity was noted in the physical and chemical properties of three partial polyprenol reductase genes. The values of chloroplast were relatively high, showed that chloroplast transit peptide occurred in mangrove polyprenol reductase. The target peptide value of mitochondria varied from 0.088 to 0.198 indicated it was possible to be present. These results suggested the importance of understanding the diversity of physicochemical properties of the different amino acids in polyprenol reductase. The subcellular localization of two partial genes located in the plasma membrane. To confirm the homology among the polyprenol reductase in the database, a dendrogram was drawn. The phylogenetic tree depicts that there are three clusters, the partial genes of K obovata joined the largest one: C23157 was close to Ricinus communis polyprenol reductase. Whereas, C23901 and C24171 were grouped with Ipomoea nil polyprenol reductase, suggested that these polyprenol reductase genes form distinct separation into tropical habitat plants.

Dolichols isolated from leaves of the fern Matteucia struthiopteris were present as a mixture of prenologues composed of 14 up to 20 isoprene units with Dol-16 dominating. They comprised approximately 0.004% of the fresh weight of fresh plant tissue and were accompanied by traces of polyprenols (Pren-14 up to Pren-17, Pren-16 dominating). Their structure was confirmed by electropray ionization mass spectrometry (ESI-MS). This is the first time that dolichols have been reported as dominating polyisoprenoid alcohols in plant photosynthetic tissue.

Lipids extracted from the shiitake mushroom Lentinus edodes contain dolichols composed of 15 up to 19 isoprene units with Dol-17 as the dominating prenologue. Identification of dolichols was achieved by the application of 2D-TLC, HPLC and electrospray ionization mass spectrometry. Additionally a family of polyprenols (α-unsaturated counterparts) with the same chain-length was also detected. Dolichols comprised approximately 0.002% of the fresh weight of the mushroom. Dolichols accompanied by traces of polyprenols are for the first time found in the mushroom tissue.

Geranylgeranyl diphosphate synthase (GGPS) is a branch point enzyme in the mevalonate pathway that catalyzes the synthesis of geranylgeranyl diphosphate used for the geranylgeranylation of Rho, Rac and Rab proteins. The current study showed the production of multiple forms of GGPS mRNA from a single GGPS gene in rat. The mRNAs resulted from combinations of multiple alternative introns and two poly(A) sites in the 3'-translated and 3'-untranslated regions. These are classified into 1a-type and 1b-type mRNAs, based on the splicing of intron 4b resulting in the difference in deduced amino acid sequence between the C-terminal regions. The 1a-type and 1b-type proteins expressed in both Escherichia coli and HeLa cells were active and inactive, respectively. In the case of HeLa cells, the latter protein expression level was about 10% relative to the former one. This was also observed for Cos-7 and 293 cells. When fusions of beta-galactosidase with C-terminal regions differing between the 1a-type and 1b-type proteins were expressed in HeLa cells, the expressed fusion proteins were both found to be active but the latter fusion protein expression level was considerably low compared with the former one. The expression level of 1a-type mRNA was higher than that of 1b-type mRNA in brain, liver, heart, and thymus, but the two expression levels were the same in testis and ovary. During testis development the total GGPS mRNA expression level increased, accompanied by an increase in 1b-type mRNA, the expression level of 1a-type mRNA encoding active GGPS remaining kept unchanged. These results indicate that the expression level of rat active GGPS is at least regulated through the splicing of intron 4b of its gene.

We synthesized three water-soluble biotin-tagged compounds with different prenyl chain lengths, biotinylated farnesal (BF), biotinylated C(55)-polyprenal (BP55), and biotinylated C(80)-polyprenal (BP80), and examined their effects on in vitro dolichol synthesis from farnesyl diphosphate. BF and BP55 did not affect the dolichol synthesis, whereas BP80 inhibited the reduction pathway from polyprenol to dolichol, accompanied by a decrease in the entire polyprenol and dolichol synthesis. Comparison of BP80 with eighteen detergents, including Triton X-100, CHAPS, octylglucoside, deoxycholate, and Tween 80, revealed the specific effect of BP80 on the reduction pathway. On SDS-polyacrylamide gel electrophoresis, BP80 was detected in an associated form with a 50 kDa protein. These results suggest that the reduction of polyprenol to dolichol in the dolichol biosynthetic pathway proceeds with the recognition of the polyprenol chain length by a 50 kDa protein.

To understand the dolichol biosynthetic pathway in detail, we performed complementation analysis of the yeast temperature-sensitive mutant #64 that exhibits defects in protein glycosylation with a reduced pool of endogenous dolichyl phosphate. Three transformants (#64-1, #64-2, and #64-4) were isolated and analyzed for possession of a fragment of chromosome IV, XIII, and VII, respectively. Endogenous dolichyl-P content of the wild type, #64, #64-1, #64-2, and #64-4 was 10.0, 2.2, 3.4, 3.0, and 1.8 μg/1010 cells, respectively. In the case of endogenous dolichol, the content was 2.4, 3.8, 4.0, 0.8, and 3.2 μg/1010 cells, respectively. Shorter-chain (C55–C60) polyprenol (dehydrodolichol) was also detected in mutant #64 (1.0 μg/1010 cells), #64-1 (1.5 μg/1010 cells), and #64-2 (1.9 μg/1010 cells), but not in #64-4 and the wild type. These facts imply that not only dolichyl-P, but also unknown shorter-chain polyprenyl-derived compounds, play the important role in dolichol biosynthesis.

Polyisoprenoid alcohols occurring in spinach leaves were analyzed by a two-plate TLC method. Z,E-mixed polyprenols (C(55-60)), glycinoprenols (C(50-55)), and solanesol (C(45)) were mainly found in chloroplasts, whereas dolichols (C(70-80)) were mainly found in microsomes. Analysis of enzymatic products derived from [1-(14)C]isopentenyl diphosphate and farnesyl diphosphate (FPP) with subcellular fractions revealed that chloroplasts and microsomes had the ability to synthesize Z,E-mixed polyprenyl (C(50-65)) and all E-polyprenyl (C(45-50)) diphosphates, and Z,E-mixed polyprenyl (C(70-85)) diphosphates, respectively. FPP and geranylgeranyl diphosphate (GGPP) were both accepted for these enzymatic reactions, the former being a better substrate than the latter. NMR analysis of naturally occurring spinach Z,E-mixed polyprenol (C(55)) and dolichol (C(75)) revealed that the number of internal trans isoprene residues in the former was three in comparison with two internal trans residues found for the latter. These results indicate that two kinds of polyprenyl diphosphate synthases occur in spinach: One is the chloroplast enzyme involved in the synthesis of the shorter-chain (C(50-65)) Z,E-mixed polyprenols and the other is the microsomal enzyme involved in the synthesis of longer-chain (C(70-85)) Z,E-mixed polyprenols, which is converted to dolichols.

Using a two-plate thin-layer chromatography method, we analyzed polyisoprenoid alcohols (dolichols and polyprenols) of the rubber plant Hevea brasiliensis (angiosperm), and of ginkgo Ginkgo biloba and pine Pinus sylvestris (gymnosperms). Special attention was paid to the occurrence of dolichol in various tissues of different plants. Dolichols were found to occur in all of the tissues examined except for flowers of the rubber plant. The chain length distributions of dolichols in seeds, young roots, young shoots, young leaves and old leaves of the rubber plant were C70-C95, C85-C105, C80-C105, C75-C105 and C65-C90, respectively. In the case of ginkgo, the chain length distributions of dolichols in seeds, embryos, young and old leaves were C70-C90, C70-C85, C70-C90 and C80-C95, respectively. Pine seeds were found to contain dolichols with the chain length distribution of C70-C90. Two kinds of polyprenol families were detected in leaves of the rubber plant and ginkgo. The longer chain polyprenol family was also detected in seeds of the rubber plant, in seeds and embryos of ginkgo and in seeds of pine. The chain length distributions of the polyprenols were not necessarily the same as those of dolichols occurring in the same tissues.