Phylogeny of Alisma (Alismataceae) revisited: implications for polyploid evolution and species delimitation.

Alisma L. is a genus of aquatic and wetland plants belonging to family Alismataceae. At present, it is thought to contain ten species. Variation in ploidy level is known in the genus, with diploids, tetraploids and hexaploids recorded. Previous molecular phylogenetic studies of Alisma have generated a robust backbone that reveals important aspects of the evolutionary history of this cosmopolitan genus, yet questions remain unresolved about the formation of the polyploid taxa and the taxonomy of one particularly challenging, widely distributed species complex. Here we directly sequenced, or cloned and sequenced, nuclear DNA (nrITS and phyA) and chloroplast DNA (matK, ndhF, psbA-trnH and rbcL) of multiple samples of six putative species and two varieties, and conducted molecular phylogenetic analyses. Alisma canaliculatum and its two varieties known in East Asia and A. rariflorum endemic to Japan possess closely related but heterogeneous genomes, strongly indicating that the two species were generated from two diploid progenitors, and are possibly siblings of one another. This evolutionary event may have occurred in Japan. Alisma canaliculatum var. canaliculatum is segregated into two types, each of which are geographically slightly differentiated in Japan. We reconstructed a single phylogeny based on the multi-locus data using Homologizer and then applied species delimitation analysis (STACEY). This allowed us to discern A. orientale as apparently endemic to the Southeast Asian Massif and distinct from the widespread A. plantago-aquatica. The former species was most likely formed through parapatric speciation at the southern edge of the distribution of the latter.

The combination of Alisma and Atractylodes ameliorates cerebral ischaemia/reperfusion injury by negatively regulating astrocyte-derived exosomal miR-200a-3p/141-3p by targeting SIRT1.

ETHNOPHARMACOLOGICAL RELEVANCE: The combination of Alisma and Atractylodes (AA), a classical traditional Chinese herbal decoction, may protect against cerebral ischaemia/reperfusion injury (CIRI). However, the underlying mechanism has not been characterized. Intriguingly, exosomal microRNAs (miRNAs) have been recognized as vital factors in the pharmacology of Chinese herbal decoctions. AIM OF THE STUDY: The aim of the present study was to assess whether the neuroprotective effect of AA was dependent on the efficient transfer of miRNAs via exosomes in the brain. MATERIALS AND METHODS: Bilateral common carotid artery ligation (BCAL) was used to induce transient global cerebral ischaemia/reperfusion (GCI/R) in C57BL/6 mice treated with/without AA. Neurological deficits were assessed with the modified neurological severity score (mNSS) and Morris water maze (MWM) test. Western blot (WB) analysis was used to detect the expression of sirtuin 1 (SIRT1) in the cerebral cortex. The inflammatory state was quantitatively evaluated by measuring the expression of phospho-Nuclear factor kappa B (p-NF-κB), Interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) using WB analysis and glial fibrillary acidic protein (GFAP) immunohistochemical staining. The protein expression of zonula occluden-1 (ZO-1), occludin, caudin-5 and CD31 was examined by immunohistochemical staining to determine blood‒brain barrier (BBB) permeability. Exosomes were extracted from the brain interstitial space by ultracentrifugation and identified by transmission electron microscopy (TEM), WB analysis and nanoparticle tracking analysis (NTA). The origin of exosomes was clarified by measuring the specific mRNAs within exosomes via Real Time Quantitative PCR (RT‒qPCR). Differential miRNAs in exosomes were identified using microarray screening and were validated by RT‒qPCR. Exosomes were labelled with fluorescent dye (PKH26) and incubated with bEnd.3 cells, the supernatant was collected, IL-1ß/TNF-α expression was measured using enzyme-linked immunosorbent assay (ELISA), total RNA was extracted, and miR-200a-3p/141-3p expression was examined by RT‒qPCR. In addition, the levels of miR-200a-3p/141-3p in oxygen glucose deprivation/reoxygenation (OGD/R)-induced bEnd.3 cells were quantified. The direct interaction between miR-200a-3p/141-3p and the SIRT1 3' untranslated region (3'UTR) was measured by determining SIRT1 expression in bEnd.3 cells transfected with the miR-200a-3p/141-3p mimic/inhibitor. RESULTS: Severe neurological deficits and memory loss caused by GCI/R in mice was markedly ameliorated by AA treatment, particularly in the AA medium-dose group. Moreover, AA-treated GCI/R-induced mice showed significant increases in SIRT1, ZO-1, occludin, caudin-5, and CD31 expression levels and decreases in p-NF-κB, IL-1ß, TNF-α, and GFAP expression levels compared with those in untreated GCI/R-induced mice. Furthermore, we found that miR-200a-3p/141-3p was enriched in astrocyte-derived exosomes from GCI/R-induced mice and could be inhibited by treatment with a medium dose of AA. The exosomes mediated the transfer of miR-200a-3p/141-3p into bEnd.3 cells, promoted IL-1ß and TNF-α release and downregulated the expression of SIRT1. No significant changes in the levels of miR-200a-3p/141-3p were observed in OGD/R-induced bEnd.3 cells. The miR-200a-3p/141-3p mimic/inhibitor decreased/increased SIRT1 expression in bEnd.3 cells, respectively. CONCLUSION: Our findings demonstrated that AA attenuated inflammation-mediated CIRI by inhibiting astrocyte-derived exosomal miR-200a-3p/141-3p by targeting the SIRT1 gene, which provided further evidence and identified a novel regulatory mechanism for the neuroprotective effects of AA.

Comparative analysis of the structure and function of rhizosphere microbiome of the Chinese medicinal herb Alisma in different regions.

Rhizoma Alismatis, a commonly used traditional Chinese medicine, is the dried tuber of Alisma orientale and Alisma A. plantago-aquatica, mainly cultivated in Fujian and Sichuan provinces (China), respectively. Studies have shown that the rhizosphere microbiome is a key factor determining quality of Chinese medicinal plants. Here we applied metagenomics to investigate the rhizosphere microbiome of Alisma in Fujian and Sichuan, focusing on its structure and function and those genes involved in protostane triterpenes biosynthesis. The dominant phyla were Proteobacteria, Actinobacteria, Chloroflexi, Acidobacteria, and Gemmatimonadetes. Compared with Fujian, the rhizosphere of Sichuan has a greater α diversity and stronger microbial interactions but significantly lower relative abundance of archaea. Microbes with disease-suppressing functions were more abundant in Sichuan than Fujian, but vice versa for those with IAA-producing functions. Gemmatimonas, Anaeromyxobacter, and Pseudolabrys were the main contributors to the potential functional difference in two regions. Genes related to protostane triterpenes biosynthesis were enriched in Fujian. Steroidobacter, Pseudolabrys, Nevskia, and Nitrospira may contribute to the accumulation of protostane triterpenes in Alisma. This work fills a knowledge gap of Alisma's rhizosphere microbiome, providing a valuable reference for studying its beneficial microorganisms.

Proteomic insights into protostane triterpene biosynthesis regulatory mechanism after MeJA treatment in Alisma orientale (Sam.) Juz.

Protostane triterpenes in Alisma orientale (Sam.) Juz. have unique structural features with distinct pharmacological activities. Previously we have demonstrated that protostane triterpene biosynthesis could be regulated by methyl jasmonate (MeJA) induction in A. orientale. Here, proteomic investigation reveals the MeJA mediated regulation of protostane triterpene biosynthesis. In our study, 281 differentially abundant proteins were identified from MeJA-treated compared to control groups, while they were mainly associated with triterpene biosynthesis, α-linolenic acid metabolism, carbohydrate metabolism and response to stress/defense. Key enzymes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), squalene epoxidase (SE), oxidosqualene cyclase (OSC) and cytochrome P450s which potentially involved in protostane triterpene biosynthesis were significantly enriched in MeJA-treated group. Basic Helix-loop-helix (bHLH), MYB, and GRAS transcription factors were enhanced after MeJA treatment, and they also improved the expressions of key enzymes in Mevalonate pathway and protostane triterpene. Then, MeJA also could increase the expression of α-galactosidase (α-GAL), thereby promoting carbohydrate decomposition, and providing energy and carbon skeletons for protostane triterpene precursor biosynthesis. As well, exogenous MeJA treatment upregulated 13-lipoxygenase (13-LOX), allene oxide synthase (AOS) and allene oxide cyclase (AOC) involved in α-linolenic acid metabolism, leading to the accumulation of endogenous MeJA and activation of the protostane triterpene biosynthesis transduction. Finally, MeJA upregulated stress/defence-related proteins, as to enhance the defence responses activity of plants. These results were further verified by quantitative real-time PCR analysis of 19 selected genes and content analysis of protostane triterpene. The results provide some new insights into the role of MeJA in protostane triterpene biosynthesis.

Methyl jasmonate promote protostane triterpenes accumulation by up-regulating the expression of squalene epoxidases in Alisma orientale.

Protostane triterpenes, which are found in Alisma orientale, are tetracyclic triterpenes with distinctive pharmacological activities. The natural distribution of protostane triterpenes is limited mainly to members of the botanical family Alismataceae. Squalene epoxidase (SE) is the key rate-limiting enzyme in triterpene biosynthesis. In this study, we report the characterization of two SEs from A. orientale. AoSE1 and AoSE2 were expressed as fusion proteins in E. coli, and the purified proteins were used in functional research. In vitro enzyme assays showed that AoSE1 and AoSE2 catalyze the formation of oxidosqualene from squalene. Immunoassays revealed that the tubers contain the highest levels of AoSE1 and AoSE2. After MeJA induction, which is the main elicitor of triterpene biosynthesis, the contents of 2,3-oxidosqualene and alisol B 23-acetate increased by 1.96- and 2.53-fold, respectively. In addition, the expression of both AoSE proteins was significantly increased at four days after MeJA treatment. The contents of 2,3-oxidosqualene and alisol B 23-acetate were also positively correlated with AoSEs expression at different times after MeJA treatment. These results suggest that AoSE1 and AoSE2 are the key regulatory points in protostane triterpenes biosynthesis, and that MeJA regulates the biosynthesis of these compounds by increasing the expression of AoSE1 and AoSE2.

Comparative transcriptome and metabolite profiling of four tissues from Alisma orientale (Sam.) Juzep reveals its inflorescence developmental and medicinal characteristics.

Alisma orientale (Sam.) Juzep (A. orientale) is an important medicinal plant in traditional Chinese medicine. In this study, de novo RNA-seq of A. orientale was performed based on the cDNA libraries from four different tissues, roots, leaves, scapes and inflorescences. A total of 41,685 unigenes were assembled, 25,024 unigene functional annotations were obtained by searching against the five public sequence databases, and 3,411 simple sequence repeats in A. orientale were reported for the first time. 15,402 differentially expressed genes were analysed. The morphological characteristics showed that compared to the other tissues, the leaves had more chlorophyll, the scapes had more vascular bundles, and the inflorescences contained more starch granules and protein. In addition, the metabolic profiles of eight kinds of alisols metabolite profiling, which were measured by ultra-Performance liquid chromatography-triple quadrupole-mass spectrometry showed that alisol B 23-acetate and alisol B were the major components of the four tissues at amounts of 0.068~0.350 mg/g and 0.046~0.587 mg/g, respectively. In addition, qRT-PCR validated that farnesyl pyrophosphate synthase and 3-hydroxy-3-methylglutaryl-CoA reductase should be considered the critical candidate genes involved in alisol biosynthesis. These transcriptome and metabolic profiles of A. orientale may help clarify the molecular mechanisms underlying the medicinal characteristics of A. orientale.

[Correlation study between accumulation of triterpenoids and expression of relative genes in Alisma orientale].

To research the correlation between accumulation of triterpenoids and expression of key enzymes genes in triterpenoid biosynthesis of Alisma orientale,the study utilized UPLC-MS/MS method to detect eight triterpenoids content in the tuber of A. orientale from different growth stages,including alisol A,alisol A 24 acetate,alisol B,alisol B 23 acetate,alisol C 23 acetate,alisol F,alisol F 24 acetate and alisol G,and then the Real time quantitative PCR was used to analyze the expression of key enzymes genes HMGR and FPPS in triterpenoid biosynthesis. Correlation analysis showed that there was a significant positive relation between the total growth of these eight triterpenoids and the average relative expression of HMGR and FPPS(HMGR: r = 0. 998,P<0. 01; FPPS: r = 0. 957,P<0. 05),respectively. Therefore,the study preliminarily determined that HMGR and FPPS genes could regulate the biosynthesis of triterpenoids in A. orientale,which laid a foundation for further research on the biosynthesis and regulation mechanism of triterpenoids in A. orientale.

Characterization and function of the 3-hydroxy-3-methylglutaryl-CoA reductase gene in Alisma orientale (Sam.) Juz. and its relationship with protostane triterpene production.

Protostane triterpenes from Alisma orientale (Sam.) Juz. have exhibited distinct pharmacological properties that are currently in high demand. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is considered the first rate-limiting enzyme in isoprenoid biosynthesis via the mevalonic acid (MVA) pathway. In this study, we cloned a full-length cDNA of A. orientale (Sam.) Juz. HMGR (AoHMGR; 2252 bp; GenBank accession no. KP342318) with an open reading frame (ORF) of 1809 bp. The deduced protein sequence contained four conserved motifs and exhibited homology with HMGR proteins from other plants. We next expressed the cloned gene in Escherichia coli BL21 (Rosetta) cells, collected the expressed products, and incubated those with 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) to determine enzymatic activity. GC/MS analysis revealed that the products were able to catalyze HMG-CoA and NADPH to form MVA. The purified protein was used to immunize New Zealand rabbits and prepare an antibody against AoHMGR. Western blot results demonstrated that the antibodies specifically recognized AoHMGR protein in A. orientale (Sam.) Juz. We then established a rapid test to detect AoHMGR protein in the plant, and found the tuber to be the most AoHMGR protein-abundant organ in A. orientale (Sam.) Juz. Furthermore, we detected the expression level of AoHMGR and contents of the main active component, Alisol B 23-acetate, at different growth phases of A. orientale (Sam.) Juz. A significant positive correlation was identified, indicating that AoHMGR represents a key enzyme in the synthetic pathway of protostane triterpenes.

[Molecular cloning of farnesyl pyrophosphate synthase from Alisma orientale (Sam.) Juzep. and its distribution pattern and bioinformatics analysis].

Triterpenes, which have large application potential in the treatment of cancer, are the main active components of genuine medicinal material Alisma orientale (Sam.) Juzep. Farnesyl pyrophosphate synthase (FPPS) is one of the important rate-limiting enzymes in the synthetic pathway of triterpenes. In this study the FPPS full length cDNA of the A. orientale, was cloned via homology-based cloning approach and rapid amplification of cDNA ends (RACE). The full length of the FPPS cDNA was 1 531 bp (accession no. HQ724508), which contained a full 1 032 bp ORF that encoded 343 amino acids. The deduced protein sequence exhibited five conserved motifs, two of which is riched of Asp (DDXXD). The result of real-time quantitative PCR (QRT-PCR) showed that FPPS gene was expressed in different organs of A. orientale. The expression increased from October to the first ten-day period of December, and then decreased. The FPPS gene expression was higher in leaves but lower in leafstalk, tuber and root. HPLC analysis of active components 23-acetyl-alismol B of A. orientale. during different periods indicated that its change trend should be consistent with FPPS gene expression. It can be primarily deduced that FPPS gene should be an important control point in the synthetic pathway of Alisma terpenes. This study may facilitate the quality of medicinal plants through gene engineering in the future.

[Cloning and distribution pattern of HMGR gene conserved fragment in Alisma orientale].

OBJECTIVE: To clone and study the distribution pattern of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene conserved fragment in Alisma orientale. METHODS: The HMGR conserved fragment in A. orientale was amplified by RT-PCR strategy with the total RNA of young leaves as the template. HMGR mRNA expression in different organs was detected by real-time quantitative PCR (QRT-PCR). RESULTS: The conserved fragment was 458 bp (accession NO. HQ913638). NCBI Blast sequence analysis showed the resulting protein had high homology to HMGR with 86.8% similarity to Artemisia annua, 88.2% to Bupleurum chinense, 88.2% to Eucommia ulmoides, and 85.5% to Salvia miltiorrhiza. The result of QRT-PCR showed that the HMGR gene was expressed in different organs (i. e. leaves, petioles, tubers, and roots) which was higher in leaves relative to other tissues. CONCLUSION: The HMGR gene conserved fragment from A. orientale was cloned and its distribution pattern was detected for the first time. This work provides a foundation for exploring the synthetic pathway and bioengineering of Alisma terpenes.

[Comparative studies on genetic resource of characteristics of Alisma].

Based on the introduction and cultivation of Alisma germplasm which were from Fujian, Jiangxi and Sichuan provinces, the biological characteristics, morphological characteristics and quality were observed and studied. After three-year continuous experiment and monographic study, there were remarkable difference in the biological characteristics, morphological characteristics and product quality of Fujian Alisma, Sichuan Alisma and Jiangxi Alisma. Fujian Alisma and Jiangxi Alisma were the same plant species of A. orientalis, whereas Sichuan Alisma and Fujian Alisma were the different plant species of A. plantago-aquatica. The study results will provide the theoretical and practical basis for the genuine medicinal materials research and good agricultural practice (GAP) of Alisma.

Molecular authentication of Alisma orientale by PCR-RFLP and ARMS.

As a widely used medicinal plant, Alisma orientale is always a possible target for fraudulent labeling. The internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (nrDNA) of the six species of genus Alisma were sequenced, and two variant sites were found to be specific for A. orientale. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis and amplification refractory mutation system (ARMS) analysis were applied to the ITS region for the identification of A. orientale. A restriction site for PSTI useful for PCR-RFLP analysis was detected and a pair of diagnostic primers DFZX-JB02S and DFZX-JB02X were designed for ARMS.