Current biodiversity status, distribution, and prospects of seaweed in Indonesia: A systematic review.

Seaweeds are a valuable component of marine biodiversity that play multiple essential roles in Indonesia's coastal ecology and economy. This systematic review (1993-2023) aimed to provide an updated overview of seaweed distribution, biodiversity, cultivation, and industry in Indonesia. The literature search derived from major databases, Scopus, Web of Science (WoS) and ResearchGate (RG), and Google Scholar (GS) retrieved 794 studies, after removing 80 duplicates, identified 646 studies passed title and abstract screening that satisfied all criteria: Indonesia, seaweed, seaweed biodiversity and composition, which consisted of 80 exclusion studies. Full text screening decided 194 studies were selected based on the specific inclusion criteria (at least two criteria passed: seaweed distribution site, species, cultivation, and habitat). After additional filtering, 137 studies were included for extraction and analysis. We found that Indonesia is rich in seaweed biodiversity, with at least 325 identified species consisting of 103 Chlorophyceae (green algae), 167 Rhodophyceae (red algae), and 55 Phaeophyceae (brown algae), respectively. Seaweed distribution and abundance in Indonesia are influenced by environmental factors, including nutrients, grazing, competition, physical tolerance, light intensity, and degree of water circulation. Seaweed species are predominantly found in mangrove forests and coral reefs on the islands of Sumatra, Java, Kalimantan, and Sulawesi. This review provides an up-to-date and comprehensive overview of the distribution and biodiversity of seaweeds in Indonesia, highlighting the ecological, economic, and cultivation of marine resources. In addition, we identify knowledge gaps and areas for further research, which can inform sustainable seaweed management and utilization in Indonesia. This review also emphasizes the significance of this marine resource to Indonesia's environment and economy.

Drought stress induced by polyethylene glycol (PEG) in local maize at the early seedling stage.

Drought stress adversely impacts growth, crop production, reproductive organ development, and yield characteristics in maize. As a drought-sensitive crop, maize (Zea mays L.) shows considerable varietal differences. A study was conducted at the Tissue Culture Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, University of North Sumatra, Medan, Indonesia in order to identify drought-tolerant maize varieties. During germination and early seedling growth, 16 local accessions were evaluated for drought tolerance. Based on local climate and soil conditions, these specific accessions were chosen. The varieties were tested against five levels of drought stress imposed by Polyethylene glycol 6000 (PEG-6000) at 10, 20, 30, 40, and 50%. An experiment with three replications was conducted using a completely randomized design. In the study, local maize accessions (BI3, SB5, DS2, and MN3) and the hybrid variety (H) showed the capability of tolerating drought stress. Generally, germination time, germination percent and vigor index, root and shoot length, shoot ratio, and fresh and dry weight were decreased by increasing PEG concentrations (up to 50%). According to ANOVA results, shoot water content was not significantly affected by the PEG, nor was the interaction between the PEG and the accessions. The root water content, however, was significantly affected by PEG, and the interaction between PEG and accessions. Although interactions between accessions with low PEG concentrations improved germination characteristics, the root histology of the accessions varied. According to drought tolerance indexes, five maize accessions are drought-tolerant, including H (0.683), SB5 (0.617), DS2 (0.565), MN3 (0.512), and BI3 (0.504). The drought-tolerant varieties are recommended in regions with low rainfall or low water sources since they are less water-intensive and produce higher yields.

Briquettes Made of Branches Wood of Three Mangrove Species Bonded by Starch Adhesive.

The development and utilization of wood briquettes is one of the efforts to reduce dependence on fossil fuels, including to fulfill overseas market need. This study aimed to evaluate the properties of wood briquettes made of the branches of three mangrove species and to analyze the effect of different wood species of mangrove branches, and the types of starch adhesive, on the quality of wood briquettes. The wood briquettes made in this study were 3 cm × 4 cm in a cylindrical shape using three wood species of mangrove branches, namely mata buaya (Bruguiera sexangula), buta-buta (Excoecaria agallocha), and bakau minyak (Rhizophora apiculata), while the adhesives used were tapioca starch, maize starch, and potato starch. The results showed that the moisture, ash content, and calorific value of the wood briquettes mostly met the ISO 17225-3:2-2020 class A2 standard and the specification and quality standards of wood briquettes for Grade A2 issued by the Korea Forest Research Institute, except the density. Wood briquettes made of mata buaya by using the three types of starch adhesives generally had better properties than all other types of wood briquettes. The interaction of mangrove wood species and the types of starch adhesive had a significant effect on the properties of wood briquettes, except for volatile matter and calorific value for which they had no significant effect. The use of wood briquettes from mangrove wood branches contributes to sustainable forest management and maintains the ecological function of mangrove forests while providing environmentally friendly alternative energy for households as a source of fuel/energy. Furthermore, future research is needed, such as investigating the optimal pressing pressure needed to achieve higher density of the wood briquettes.

Challenges and opportunities for achieving Sustainable Development Goals through restoration of Indonesia's mangroves.

Indonesia, the most mangrove-rich nation in the world, has proposed the most globally ambitious mangrove rehabilitation target (600,000 ha) of any nation, to be achieved by 2024 to support multiple Sustainable Development Goals (SDG 1-3, 6, 13 and 14). Yet, mangrove restoration and rehabilitation across the world have often suffered low success rates and been applied at small scales. Here, we identify 193,367 ha (estimated costs at US$0.29-1.74 billion) that have the potential to align with the national mangrove rehabilitation programme. Despite being only 30% of the national target, our robust assessment considered biogeomorphology, 20 years of land-use and land-cover change and state forest land status, all key factors moderating mangrove restoration success which have often been neglected in Indonesia. Increasing subnational government representation in mangrove governance as well as improving monitoring and evaluation will increase the likelihood of achieving the mangrove rehabilitation targets and reduce risks of failure. Rehabilitating and conserving mangroves in Indonesia could benefit 74 million coastal people and can potentially contribute to the national land-sector emissions reduction of up to 16%.

Effectiveness and Efficacy of Vaccine on Mutated SARS-CoV-2 Virus and Post Vaccination Surveillance: A Narrative Review.

The ongoing COVID-19 pandemic, as a result of the SARS-CoV-2 virus, since December 2019, is a major health problem and concern worldwide. The pandemic has impacted various fields, from the social to the development of health science and technology. The virus has been mutating and thus producing several new variants, rushing research in the field of molecular biology to develop rapidly to overcome the problems that occur. Vaccine clinical studies are developing promptly with the aim of obtaining vaccines that are effective in suppressing the spread of the virus; however, the development of viral mutations raises concerns about the decreasing effectiveness of the resulting vaccine, which also results in the need for more in-depth studies. There have been 330 vaccines developed, including 136 clinical developments and 194 pre-clinical developments. The SARS-CoV-2 variant continues to evolve today, and it poses a challenge in testing the effectiveness of existing vaccines. This is a narrative review describing the emergence of the COVID-19 pandemic, development of vaccine platforms, identification of concerning mutations and virus variants in various countries of the world, and real-world monitoring of post-vaccination effectiveness and surveillance.

Current Status, Distribution, and Future Directions of Natural Products against Colorectal Cancer in Indonesia: A Systematic Review.

In 2020, an estimated 19.3 million new cancer cases and nearly 10 million cancer deaths have occurred worldwide, with colorectal cancer ranking as the third most frequently diagnosed (10.0%). Several attempts have been conducted against cancer, including surgery, radiation, monoclonal antibodies, and chemotherapy. Many people choose natural products as alternatives against cancer. These products will not only help in human life preservation but also work as a source of up-to-date information, leading people away from incorrect information. We discuss the current status, distribution, and future implications of protecting populations with natural products as an alternative against colorectal cancer in Indonesia. Thirty-eight studies were included in this review for data extraction. The distribution of natural products in Indonesia that have potential activity against colorectal cancer cells was predominated by terpenoids, followed by phytosterols, phenolics, alkaloids, and polyisoprenoids. The type of cell line utilized in the cytotoxic activity analysis of natural products was the WiDr cell line, followed by HT-29 cells and HCT-116 cells. This review showed that MTT in vitro assay is a general method used to analyze the cytotoxic activity of a natural product against colorectal cancer cells, followed by other in vitro and in vivo methods. The systematic review provided predictions for several secondary metabolites to be utilized as an alternative treatment against colorectal cancer in Indonesia. It also might be a candidate for a future co-chemotherapy agent in safety, quality, and standardization. In addition, computational methods are being developed to predict the drug-likeness of compounds, thus, drug discovery is already on the road towards electronic research and development.

Chemical analysis and anticancer activity of sesterterpenoid from an endophytic fungus Hypomontagnella monticulosa Zg15SU and its host Zingiber griffithii Baker.

Zingiber griffithii Baker is one of the native Zingiberaceous species in a tropical forest of North Sumatra, Indonesia. Zingiberaceous species have been intensively studied and reported as herbal ingredients in ethnomedicine and currently their endophytic fungal associates were studied for pharmacological importance. Fifteen endophytic fungi were isolated from Zingiber griffithii following morphological and molecular characterization. All isolates exhibited antibacterial properties to at least one of the tested pathogenic bacteria Staphylococcus aureus, Escherichia coli, Methicilin-resistant S. aureus (MRSA), and Enteropathogenic E. coli (EPEC). The isolate, identified as Hypomontagnella monticulosa strain Zg15SU (syn. Hypoxylon monticulosum Mont.) based on its rDNA/ITS sequence, displayed antibacterial activities to all tested pathogens. The EtOAc extract of the H. monticulosum Zg15SU showed the highest activity for gram-negative bacteria, the E. coli and EPEC, while the extract of Z. griffithii rhizome displayed activity only for E. coli. The gas chromatography-mass spectrometry analysis (GC-MS) indicated a major portion of similar compounds found in both the endophytic fungus and plant extract, revealing the compounds of oleic acid, cyclononasiloxane, octadecamethyl, and eicosanoic acid Furthermore, purification and structural elucidation on the EtOAc extract of both Z. griffithii rhizome and H. monticulos a Zg15SU yielded two bioactive compounds: a novel compound, griffithiiene, a terpenoid-alkaloid bearing the skeleton of a scalarane (1) and scalaradial (2) which were confirmed by 1H- (500 MHz) and 13C-NMR (125 MHz) spectroscopy. Importantly, the elucidated compounds showed a cytotoxicity activity against cancer cell lines, the Panc-1, NBT-T2, and HCT116 based on in vitro MTT proliferation assay. This is the first report of Z. griffithii harboring an endophytic fungus, H. monticulosa, which produced potential antibacterial and anticancer metabolites along with its host to be utilized for future prospects.

Potential compounds from several Indonesian plants to prevent SARS-CoV-2 infection: A mini-review of SARS-CoV-2 therapeutic targets.

The outbreak of coronaviruses (CoVs) presents an enormous threat to humans. To date, no new therapeutic drugs or vaccines licensed to treat human coronaviruses remain undiscovered. This mini-review briefly reports the number of potential plants widely distributed in Indonesia for further research and development as anti-SARS-CoV-2 agents and the critical targets for SARS-CoV-2 therapy, such as angiotensin-converting enzyme 2 (ACE-2) receptor, spike protein, 3-chymotrypsin-like protease (3CLpro), papain-like protease (PLpro), RNA-dependent RNA polymerase (RdRp), helicase, and serine protease. Indonesia is rich in medicinal plants (herbal); it also has a long history of using plants to treat various hereditary diseases. However, since SARS-CoV-2 is a new disease, it has no history of plant-based treatment anywhere in the world. This mini-review describes natural products from several Indonesian plants that contain compounds that could potentially prevent or reduce SARS-CoV-2 infection, act as potential targeted therapy, and provide new therapeutic strategies to develop SARS-CoV-2 countermeasures.

Properties of wood composite plastics made from predominant Low Density Polyethylene (LDPE) plastics and their degradability in nature.

To address concerns over plastics in the global environment, this project produced three wood plastics composites (WPCs) which could divert plastics from the waste stream into new materials. The three materials made had a ratio of 85%:15%, 90%:10%, and 95%:5% low density polyethylene (LDPE) to wood powder and were produced using the dissolution method. Physical and mechanical properties of each WPC were evaluated according to Japanese Industrial Standard (JIS) A 5908:2003. Their degradation in nature was evaluated through a graveyard test and assay test conducted in Coptotermes curvignathus termites. Results showed that density, moisture content, thickness swelling and water absorption of the WPCs fulfilled the JIS standard. The mechanical properties of these composites also met the JIS standard, particularly their modulus of elasticity (MOE). Modulus of rupture (MOR) and internal bonding (IB) showed in lower values, depending on the proportion of wood filler they contained. Discoloration of the WPCs was observed after burial in the soil with spectra alteration of attenuated transmission reflectance (ATR) in the band of 500-1000 cm-1 which could be assigned to detach the interphase between wood and plastics. As termite bait, the WPCs decreased in weight, even though the mass loss was comparatively small. Micro Confocal Raman Imaging Spectrometer revealed that termite guts from insects feeding on WPCs contained small amounts of LDPE. This indicated termite can consume plastics in the form of WPCs. Thus WPCs made predominantly of plastics can be degraded in nature. While producing WPCs can assist in decreasing plastics litter in the environment, the eventual fate of the LDPE in termites is still unknown.

Anticancer Activity of Polyisoprenoids from Avicennia alba Blume. in WiDr Cells.

Colorectal cancer is the third most common cancer world wide and has been occurred more in developing regions. The use of conventional chemotherapy agents may lead to various adverse effects. Therefore, it is required to find the potential drug for anticancer from alternative source of natural product including mangrove plants. The present study was conducted to determine the anticancer activity of polyisoprenoids from Avicennia alba Blume. leaves (PAL) in WiDr cells. Cell cycle inhibition, apoptosis activity, and suppression of cyclooxygenase-2 (COX-2) were also evaluated. The anticancer activity of PAL was determined by observing the activity of these compounds against WiDr cells using the [3-(4,5-dimetiltiazol-2-il)-2,5-difenil tetrazolium bromida] MTT assay. Inhibition of the cell cycle and increased apoptosis were analysed by flowcytometry. Suppression of COX-2 was analysed using immunocytochemistry. PAL exhibited anticancer activity against WiDr cells with an IC50 of 173.775 µg/mL. Cell cycle analysis revealed that the inhibition occurred in the G0-G1 phase, and apoptosis occurred in the early apoptosis phase. Furthermore, the result of an analysis of COX-2 expression showed that PAL enabled the suppression of COX-2 expression. PAL can be used as anticancer agents against WiDr colon cancer cells. However, in-vivo studies is required to confirm the in-vitro finding of the anticancer activity of polyisoprenoid extract.

Prominent Secondary Metabolites from Selected Genus of Avicennia Leaves.

BACKGROUND: Mangrove plants distributed in the intertidal of the tropical and subtropical region including in North Sumatra, Indonesia. The production of secondary metabolite compounds is well known to mangroves. Characterisation of prominent compounds from mangrove plants such as genus of Avicennia is required to explore for their biological and pharmacological properties of these compounds. AIM: The purpose of this research was to analyse the prominent secondary metabolites through the characterisation of phytochemical, physicochemical, and microscopic of the mangrove genus Avicennia leaves, particularly Avicennia alba, A. lanata, A. marina, and A. officinalis. METHODS: Phytochemical screening was carried out on Avicennia spp leaves to the established process. Physicochemical characters of mangrove leaves were investigated by simplicial powder consisting of moisture content, water-soluble, ethanol-soluble, ash content and ash soluble acid according to the WHO formula. Microscopic analysis on the simplicial powder was carried out based on the WHO procedure. RESULTS: The result showed that physicochemical feature displays diversity among the species and important findings on the water concentration was less than 10% as a prerequisite for the drug. The phytochemical search of simplified grain also depicted divergence among the species, only alkaloid, saponin, and triterpenoid or phytosterol were found entirely in Avicennia spp leaves. Microscopic search found a similar type of stoma in Avicennia spp leaves, namely diacytic. CONCLUSION: The prominent secondary metabolites in Avicennia spp leaves consisting of alkaloid and saponin in simplicial and triterpenoid/sterol was either in simplicial or hexane extract. The present study may provide significant pharmacological properties from mangrove Avicennia genus green foliages, which could accelerate another prospect for non-wood mangrove utilisation.

Effect of Salt and Fresh Water Concentration on Polyisoprenoid Content in Bruguiera cylindrica Seedlings.

BACKGROUND: Mangrove forest is a typical forest found along the coast or river mouth which is affected by tides and salinity. Although polyisoprenoid was widespread in the plant kingdom, the physiological roles of these compounds are not well understood, especially from mangrove plants. It is therefore essential to characterize the polyisoprenoid content under abiotic stress. AIM: This study aimed to determine the effect of salinity and subsequent fresh water change on polyisoprenoids concentration in Bruguiera cylindrica seedlings. METHODS: Bruguiera cylindrica planted in a greenhouse for three months under various salinity concentrations. After three months grew under variable salinity, these seedlings were then divided into two treatment groups, and grown for another three months: one continuously in a salt solution and another in fresh water to relieve salt stress. The leaves and roots of B. cylindrica seedlings were harvested after six months of cultivation. The leaves and roots of B. cylindrica seedlings were extracted for polyisoprenoids content and composition analyzed using two-dimensional thin layer chromatography. RESULTS: Polyisoprenoids composition under salinity and subsequent fresh water with dominating dolichols (more than 90%) were found in leaves and roots of B. cylindrica seedlings referring type I of polyisoprenoid composition. The carbon chain length of dolichols located in the leaves and roots were ranging from C75-C100 and C75-C105, respectively. CONCLUSON: Dolichol dominated over polyprenol both in B. cylindrical leaves and roots under salinity and subsequent relief supported the previous finding on the predominance dolichols over polyprenols in mangrove plants. The present study suggested the significance of dolichols in the adaptation to cope with salt stress and or water stress.

Cytotoxic and Antiproliferative Activity of Polyisoprenoids in Seventeen Mangroves Species Against WiDr Colon Cancer Cells

Background: Secondary metabolites from the group of isoprenoid compounds are widely distributed in mangrove plants. Polyisoprenoids (dolichol and polyprenol) are known to have benefits as anticancer agents. The present study was conducted to determine the cytotoxic potential of polyisoprenoids in leaves from seventeen selected mangrove species against colon cancer (WiDr) cells. Methods: Cytotoxic activity was evaluated by MTT assay in vitro using WiDr human colon cancer cells and 3T3 fibroblasts from Swiss albino mouse embryo tissue as controls. Mechanisms of action were approached by assessing apoptosis and the cell cycle using flow cytometry and fluorescence microscopy with annexin V-FITC, as well as expression of Bcl-2 and cyclin D1 by immunocytochemistry. Results: Polyisoprenoids from N. fruticans leaves demonstrated the highest anticancer activity, with an IC50 of 180.2 µg/mL, as compared to 397.7 µg/mL against 3T3 normal cells. Significant decrease in the expression of Bcl-2 and cyclin D1 was also noted, facilitating apoptosis and arrest of the cell cycle in the G0-G1 phase in WiDr cells. The present study showed for the first time that polyisoprenoids from N. fruticans exhibit concrete anticancer activity in vitro, decreasing cell proliferation and inducing apoptosis in colon cancer cells. Conclusions: Polyisoprenoids isolated from N. fruticans leaves may have promise as a source of anticancer agents.

Triterpenoid modulates the salt tolerance of lanosterol synthase deficient Saccharomyces cerevisiae, GIL77.

This study examined the effect of triterpenoid on the salt tolerance of lanosterol synthase deficient yeast mutant GIL77. The expression of the triterpenoid synthase gene under GAL1 promoter in GIL77 increased the triterpenoid concentration of both whole cell and plasma membrane fractions. Without the induction of the genes, the growth curve of BgbAS or RsM1 transformant depicted patterns similar to control cells in both the presence and absence of salt with growth inhibition at 500 mM NaCl. The induction of BgbAS and RsM1 gene expression slightly repressed growth compared with control cells in the absence of NaCl. The growth of GIL77 was significantly suppressed by the expression of BgbAS or RsM1 under salinity conditions. Of the triterpenoid synthase genes, BgbAS rather than RsM1 was found to strongly inhibit the growth of GIL77 cells under salt stressed conditions. The expression of the triterpenoid synthase gene in GIL77 also influenced their tolerance to other abiotic stresses. In contrast to the endogenous synthesis, the exogenous supply of triterpenoid in the culture medium appeared to occur in the plasma membrane fraction and enhanced the salt tolerance of GIL77. This study thus discussed the physiological significance of triterpenoid in relation to its possible role in modulating salt tolerance.

Salt-dependent increase in triterpenoids is reversible upon transfer to fresh water in mangrove plants Kandelia candel and Bruguiera gymnorrhiza.

This study examined the salinity dependence of triterpenoid content and triterpenoid synthase gene expression in mangrove plants, Kandelia candel and Bruguiera gymnorrhiza (Rhizophoraceae) after long-term exposure to salinity and subsequent re-adaptation. Seedlings of the two mangrove species grown in varying salt concentrations for 4 months were divided into two treatment groups and grown for another 4 months, one group continued under the respective saline condition and the other in fresh water for re-adaptation. The total content of triterpenoids increased with increasing salinity in roots and leaves of K. candel, but only in roots in B. gymnorrhiza. This increase was reversed to a variable extent, depending on the species and organ, after transfer to fresh water. In contrast, the total content of phytosterols showed no correlation with salinity throughout the experiment. The increase in total triterpenoids was accompanied by an up-regulation of several triterpenoid synthase genes: KcMS, a multifunctional triterpenoid synthase, in roots and leaves of K. candel and BgLUS, a lupeol synthase, and BgbAS, a ß-amyrin synthase, in roots of B. gymnorrhiza. The expression of root KcCAS, a cycloartenol synthase, which is involved in phytosterol biosynthesis, was not modulated by the salinity conditions but decreased with increasing salinity in leaves, followed by the restoration to the initial level after transfer to fresh water. The concentrations of individual triterpenoids, but not of phytosterols, in the roots positively correlated with the salinity. These results reinforced the importance of triterpenoids in the adaptation of mangroves to withstand salt and/or water stress.

Expression of terpenoid synthase mRNA and terpenoid content in salt stressed mangrove.

The halophytic Kandelia candel and Bruguiera gymnorrhiza are ideal model for studying the molecular mechanisms of salinity tolerance in mangrove plants. The correlation between mRNA expression of four oxidosqualene cyclase (OSC) genes namely, KcMS multifunctional terpenoid synthase and KcCAS cyloartenol synthase (K. candel), BgbAS beta-amyrin synthase and BgLUS lupeol synthase (B. gymnorrhiza) and salt concentration was examined. mRNA level of KcMS was increased with salt concentration in both roots and leaves of K. candel. Similarly, salt stress increased the mRNA levels of BgLUS and BgbAS in the root of B. gymnorrhiza. This result suggests that the function of terpenoids in root is associated with the salt stress. In contrast to these observations, the mRNA level of KcCAS was not modulated by salt stress in the roots, and decreased in the leaves. These results therefore suggest that the terpenoids but not phytosterols play an important role to cope with the salt stress in mangrove root. The content and proportion of beta-amyrin and lupeol increased with salinity in the root of K. candel and B. gymnorrhiza, providing additional evidence for the protective role of terpenoids. However, beta-amyrin and lupeol in B. gymnorrhiza leaves decreased with salt concentration, suggesting that the physiological significance for the terpenoids in the leaf may differ from that for the root.

Triterpene synthases from the Okinawan mangrove tribe, Rhizophoraceae.

Oleanane-type triterpene is one of the most widespread triterpenes found in plants, together with the lupane type, and these two types often occur together in the same plant. Bruguiera gymnorrhiza (L.) Lamk. and Rhizophora stylosa Griff. (Rhizophoraceae) are known to produce both types of triterpenes. Four oxidosqualene cyclase cDNAs were cloned from the leaves of B. gymnorrhiza and R. stylosa by a homology-based PCR method. The ORFs of full-length clones termed BgbAS (2280 bp, coding for 759 amino acids), BgLUS (2286 bp, coding for 761 amino acids), RsM1 (2280 bp, coding for 759 amino acids) and RsM2 (2316 bp coding for 771 amino acids) were ligated into yeast expression plasmid pYES2 under the control of the GAL1 promoter. Expression of BgbAS and BgLUS in GIL77 resulted in the production of beta-amyrin and lupeol, suggesting that these genes encode beta-amyrin and lupeol synthase (LUS), respectively. Furthermore, RsM1 produced germanicol, beta-amyrin, and lupeol in the ratio of 63 : 33 : 4, whereas RsM2 produced taraxerol, beta-amyrin, and lupeol in the proportions 70 : 17 : 13. This result indicates that these are multifunctional triterpene synthases. Phylogenetic analysis and sequence comparisons revealed that BgbAS and RsM1 demonstrated high similarities (78-93%) to beta-amyrin synthases, and were located in the same branch as beta-amyrin synthase. BgLUS formed a new branch for lupeol synthase that was closely related to the beta-amyrin synthase cluster, whereas RsM2 was found in the first branch of the multifunctional triterpene synthase evolved from lupeol to beta-amyrin synthase. Based on these sequence comparisons and product profiles, we discuss the molecular evolution of triterpene synthases and the involvement of these genes in the formation of terpenoids in mangrove leaves.

Cloning and functional expression of cycloartenol synthases from mangrove species Rhizophora stylosa Griff. and Kandelia candel (L.) Druce.

To obtain cDNAs encoding oxidosqualene cyclase (OSC), we cloned two cDNAs, KcCAS and RsCAS, from roots of Kandelia candel (L.) Druce and leaves of Rhizophora stylosa Griff. by homology based PCR method respectively. The deduced amino acid sequences of both OSCs showed 82% homology to cycloartenol synthases from Lotus japonicus (OSC5) and Ricinus cummunis (RcCAS), suggesting that these are cycloartenol synthases of K. candel and R. stylosa. The genes obtained were expressed in a lanosterol synthase deficient Saccharomyces cerevisiae (ERG7) strain, GIL77. GC-MS analysis identified the accumulated reaction product in the yeast transformant to be cycloartenol, indicating that both KcCAS and RsCAS encode cycloartenol synthase.

Molecular cloning and functional expression of a multifunctional triterpene synthase cDNA from a mangrove species Kandelia candel (L.) Druce.

Homology based PCRs with degenerate primers designed from the conserved sequences among the known oxidosqualene cylases (OSCs) have resulted in cloning of a triterpene synthase (KcMS) from the young roots of Kandelia candel (L.) Druce (Rhizophoraceae). KcMS consists of a 2286 bp open reading frame, which codes for 761 amino acids. The deduced amino acid sequence showed 79% homology to a lupeol synthase from Ricinus communis suggesting it to be a lupeol synthase of K. candel. KcMS was expressed in a lanosterol synthase deficient yeast with the expression vector pYES2 under the control of GAL1 promoter. GC-MS analysis showed that the transformant accumulated a mixture of lupeol, beta-amyrin and alpha-amyrin in a 2:1:1 ratio, indicating that KcMS encodes a multifunctional triterpene synthase, although it showed high sequence homology to a R. communis lupeol synthase. This is the first OSC cloning from mangrove tree species.