Differential transcriptome and metabolome analysis of Plumbago zeylanica L. reveal putative genes involved in plumbagin biosynthesis.

Plumbagin is a pharmacologically active naphthoquinone present in the Plumbago zeylanica L. having important medicinal properties. The root of P. zeylanica is rich and primary tissue of the plumbagin biosynthesis and accumulation. The complete biosynthetic pathway of plumbagin in plant is still obscure. The present study attempts to understand the plumbagin biosynthetic pathway with the help of differential transcriptome and metabolome analysis of P. zeylanica leaf and root. The transcriptome data showed co-expression of Aldo-keto reductase (PzAKR), Polyketide cyclase (Pzcyclase) and Cytochrome P450 (PzCYPs) transcripts along with the Polyketide synthase (PzPKS) transcripts. Their higher expression in root as compared to leaf supports their possible involvement in plumbagin biosynthesis. The metabolome data of leaf and root revealed naphthalene derivative isoshinanolone that could be potential precursor of plumbagin. Pathway elucidation and transcriptome data of P. zeylanica, will enable and accelerate research on naphthoquinone biosynthesis in plants.

Cis-Regulatory Elements Determine Germline Specificity and Expression Level of an Isopentenyltransferase Gene in Sperm Cells of Arabidopsis.

Flowering plant sperm cells transcribe a divergent and complex complement of genes. To examine promoter function, we chose an isopentenyltransferase gene known as PzIPT1. This gene is highly selectively transcribed in one sperm cell morphotype of Plumbago zeylanica, which preferentially fuses with the central cell during fertilization and is thus a founding cell of the primary endosperm. In transgenic Arabidopsis (Arabidopsis thaliana), PzIPT1 promoter displays activity in both sperm cells and upon progressive promoter truncation from the 5'-end results in a progressive decrease in reporter production, consistent with occurrence of multiple enhancer sites. Cytokinin-dependent protein binding motifs are identified in the promoter sequence, which respond with stimulation by cytokinin. Expression of PzIPT1 promoter in sperm cells confers specificity independently of previously reported Germline Restrictive Silencer Factor binding sequence. Instead, a cis-acting regulatory region consisting of two duplicated 6-bp Male Gamete Selective Activation (MGSA) motifs occurs near the site of transcription initiation. Disruption of this sequence-specific site inactivates expression of a GFP reporter gene in sperm cells. Multiple copies of the MGSA motif fused with the minimal CaMV35S promoter elements confer reporter gene expression in sperm cells. Similar duplicated MGSA motifs are also identified from promoter sequences of sperm cell-expressed genes in Arabidopsis, suggesting selective activation is possibly a common mechanism for regulation of gene expression in sperm cells of flowering plants.

Phylogeography and modes of reproduction in diploid and tetraploid halophytes of Limonium species (Plumbaginaceae): evidence for a pattern of geographical parthenogenesis.

BACKGROUND AND AIMS: The genus Limonium (Plumbaginaceae) has long been recognized to have sexual and apomictic (asexual seed formation) modes of reproduction. This study aimed to elucidate phylogeographical patterns and modes of reproduction in diploid and tetraploid Limonium species, namely three putative sexual diploid species with morphological affinities (L. nydeggeri, L. ovalifolium, L. lanceolatum) and three related, probably apomict tetraploid species (L. binervosum, L. dodartii, L. multiflorum). METHODS: cpDNA diversity and differentiation between natural populations of the species were investigated using two chloroplast sequence regions (trnL intron and trnL-trnF intergenic spacer). Floral heteromorphies, ovule cytoembryological analyses and pollination and crossing tests were performed in representative species of each ploidy group, namely diploid L. ovalifolium and tetraploid L. multiflorum, using plants from greenhouse collections. KEY RESULTS AND CONCLUSIONS: Genetic analyses showed that diploid species have a higher haplotype diversity and a higher number of unique (endemic) haplotypes than tetraploid species. Network analysis revealed correlations between cpDNA haplotype distribution and ploidy groups, species groups and geographical origin, and haplotype sharing within and among species with distinct ploidy levels. Reproductive biology analyses showed that diploid L. ovalifolium mainly forms meiotically reduced tetrasporic embryo sacs of Gagea ova, Adoxa and Drusa types. Limonium multiflorum, however, has only unreduced, diplosporic (apomictic) embryo sacs of Rudbeckia type, and autonomous apomictic development seems to occur. Taken together, the findings provide evidence of a pattern of 'geographical parthenogenesis' in which quaternary climatic oscillations appear to be involved in the geographical patterns of coastal diploid and tetraploid Limonium species.

Analysis of genetic variability and population structure of the endemic medicinal Limonium sinense using molecular markers.

Limonium sinense is an endemic medicinal herb used to treat fever, hemorrhage and other disorders. In the present study, population genetic diversity was elucidated using random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) primers. Percentage of polymorphic bands, Nei's gene diversity and Shannon's information index revealed a high level of genetic diversity at species level. The analysis of molecular variance revealed that 69.88% (RAPD), 71.19% (ISSR) and 70.97% (AFLP) of variability were partitioned among individuals within populations, which indicated the coherent trend by Gst (0.3849/0.3577/0.3670). Gene flow number (Nm) was 0.581/0.618/0.612, which indicated that there was a limited gene exchange between populations. The UPGMA clustering results showed that the genetic distance had no significant correlation with geographic distance. These results indicate that these markers were reliable tools for the differentiation and determination of the genetic diversity among the populations of L. sinense and the conservation of existing natural population is necessary.

Male fertility versus sterility, cytotype, and DNA quantitative variation in seed production in diploid and tetraploid sea lavenders (Limonium sp., Plumbaginaceae) reveal diversity in reproduction modes.

The genus Limonium Miller, a complex taxonomic group, comprises annuals and perennials that can produce sexual and/or asexual seeds (apomixis). In this study, we used diverse cytogenetic and cytometric approaches to analyze male sporogenesis and gametogenesis for characterizing male reproductive output on seed production in Limonium ovalifolium and Limonium multiflorum. We showed here that the first species is mostly composed of diploid cytotypes with 2n = 16 chromosomes and the latter species by tetraploid cytotypes with 2n = 32, 34, 35, 36 chromosomes and had a genome roughly twice as big as the former one. In both species, euploid and aneuploid cytotypes with large metacentric chromosomes having decondensed interstitial sites were found within and among populations, possibly involved in chromosomal reconstructions. L. ovalifolium diploids showed regular meiosis resulting in normal tetrads, while diverse chromosome pairing and segregation irregularities leading to the formation of abnormal meiotic products are found in balanced and non-balanced L. multiflorum tetraploids. Before anther dehiscence, the characteristic unicellular, bicellular, or tricellular pollen grains showing the typical Limonium micro- or macro-reticulate exine ornamentation patterns were observed in L. ovalifolium using scanning electron microscopy. Most of these grains were viable and able to produce pollen tubes in vitro. In both balanced and unbalanced L. multiflorum tetraploids, microspores only developed until the "ring-vacuolate stage" with a collapsed morphology without the typical exine patterns, pointing to a sporophytic defect. These microspores were unviable and therefore never germinated in vitro. L. ovalifolium individuals presented larger pollen grains than those of L. multiflorum, indicating that pollen size and ploidy levels are not correlated in the Limonium system. Cytohistological studies in mature seeds from both species revealed that an embryo and a residual endosperm were present in each seed. Flow cytometric seed screens using such mature seeds showed quantitative variations in seeds ploidy level. It is concluded that male function seems to play an important role in the reproduction modes of Limonium diploids and tetraploids.

Isolation of powerful antioxidants from the medicinal halophyte Limoniastrum guyonianum.

The antioxidant capacities of the crude extract, aqueous and ethyl acetate partitions of Limoniastrum guyonianum Boiss. (Zita) were investigated in this study. The ethyl acetate phase exhibited a significant antioxidant activity as judged by total antioxidant activity, DPPH test and reducing power. Fractionation of this extract by centrifugal partition chromatography (CPC) using quaternary Arizona solvent systems composed of n-heptane/ethyl acetate/methanol/water led to ten fractions. The antioxidant capacities of these fractions were assessed using the same previous tests. Fraction 8 showed the highest antioxidant capacity (1291.1mg GAE/g DR), the power ability to quench DPPH radical (IC(50)=2µg/ml) and to reduce Fe(3+) (EC(50)=65µg/ml). From this fraction, three powerful flavonoids were isolated (1-3): gallocatechin (1), epigallocatechin (2) and epigallocatechin-3-O-gallate (3). These findings suggest that the antioxidative property of L. guynianum is may be related to the presence of these flavonoids, which can be used in various industrial fields.

Mutagenic screening of some commonly used medicinal plants in Nigeria.

The uses of medicinal plants have always been part of human culture. The World Health Organization estimates that up to 80% of the world's population relies on traditional medicinal system for some aspect of primary health care. However, there are few reports on the toxicological properties of most medicinal plants especially, their mutagenicity and carcinogenicity. Therefore, this research is to determine the mutagenic potentials of Morinda lucida [Oruwo (Root)], Azadirachta indica [Dongoyaro (Leaf)], Terapluera tetraptera [Aridan (Fruit)], Plumbago zeylanica [Inabiri (Root)], Xylopia aethiopica [Erunje (Fruit)], Newbouldia laevis [Akoko (Leaf)], Alstonia boonei [Ahun (Bark)], Enantia chlorantha [Awopa (Bark)], and Rauvolfia vomitoria [Asofeyeje (Root)] using the Allium cepa Linn. model and the modified Ames assay. Allium cepa model was used to determine the mean root length, mitotic index and chromosomal aberrations effects of these plants on onion bulbs using 0.1, 1, 5 and 10mg/ml concentration of the plant extracts. The modified Ames test which is a modification of the standard Ames test as described by Ames et al. [Ames, B.N., McCann, J., Yamasaki, E., 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutation Research 31, 347-364] was done using Escherichia coli (0157:H7) that has the phenotypic characteristics of glucose and lactose fermentation, motile, urease negative, indole positive and citrate negative. The results obtained from Allium cepa assay showed increasing root growth inhibition with increased concentration, decreasing mitotic index with increased concentration and chromosomal aberrations. The modified Ames test showed an alteration in the biochemical characteristics of Escherichia coli (0157:H7) for all plants except Rauvolfia vomitoria and Plumbago zeylanica. Three of the medicinal plants altered at least three of the normal biochemical characteristics thus demonstrating mutagenic potentials. The results of internationally accepted Allium cepa were comparable with the modified Ames test. However, a long term in vivo and dose dependent study should be carried out to validate these results and the findings should be communicated to drug and food regulatory body and also to the general public.

Gene expression in the dimorphic sperm cells of Plumbago zeylanica: transcript profiling, diversity, and relationship to cell type.

Plumbago zeylanica produces cytoplasmically dimorphic sperm cells that target the egg and central cell during fertilization. In mature pollen, the larger sperm cell contains numerous mitochondria, is associated with the vegetative nucleus (S(vn)), and fuses preferentially with the central cell, forming endosperm. The other, plastid-enriched sperm cell (S(ua)) fuses with the egg cell, forming the zygote and embryo. Sperm expressed genes were investigated using ESTs produced from each sperm type; differential expression was validated through suppression subtractive hybridization, custom microarrays, real-time RT-PCR and in situ hybridization. The expression profiles of dimorphic sperm cells reflect a diverse and broad complement of genes, including high proportions of conserved and unknown genes, as well as distinct patterns of expression. A number of genes were highly up-regulated in the male germ line, including some genes that were differentially expressed in either the S(ua) or the S(vn). Differentially up-regulated genes in the egg-targeted S(ua) showed increased expression in transcription and translation categories, whereas the central cell-targeted S(vn) displayed expanded expression in the hormone biosynthesis category. Interestingly, the up-regulated genes expressed in the sperm cells appeared to reflect the expected post-fusion profiles of the future embryo and endosperm. As sperm cytoplasm is known to be transmitted during fertilization in this plant, sperm-contributed mRNAs are probably transported during fertilization, which could influence early embryo and endosperm development.

beta-alanine N-methyltransferase of Limonium latifolium. cDNA cloning and functional expression of a novel N-methyltransferase implicated in the synthesis of the osmoprotectant beta-alanine betaine.

Beta-alanine (Ala) betaine, an osmoprotectant suitable under saline and hypoxic environments, is found in most members of the halophytic plant family Plumbaginaceae. In Limonium latifolium (Plumbaginaceae), it is synthesized via methylation of beta-Ala by the action of a trifunctional S-adenosyl L-methionine (Ado-Met): beta-Ala N-methyltransferase (NMTase). Peptide sequences from purified beta-Ala NMTase were used to design primers for reverse transcriptase-PCR, and several cDNA clones were isolated. The 5' end of the cDNA was cloned using a 5'-rapid amplification of cDNA ends protocol. A 500-bp cDNA was used as a probe to screen a lambda-gt10 L. latifolium leaf cDNA library. Partial cDNA clones represented two groups, NMTase A and NMTase B, differing only in their 3'-untranslated regions. The full-length NMTase A cDNA was 1,414 bp and included a 1128-bp open reading frame and a 119-bp 5'-untranslated region. The deduced amino acid sequence of 375 residues had motifs known to be involved in the binding of Ado-Met. The NMTase mRNA was expressed in L. latifolium leaves but was absent in Limonium sinuatum, a member of the genus that lacks the synthetic pathway for beta-Ala betaine. NMTase mRNA expression was high in young and mature leaves and was enhanced by light. NMTase cDNA was expressed in yeast (Saccharomyces cerevisiae) under the control of a galactose-inducible promoter. Protein extracts of galactose-induced recombinant yeast had Ado-Met-specific NMTase activities that were highly specific to beta-Ala, N-methyl beta-Ala, and N,N-dimethyl beta-Ala as methyl acceptors. NMTase activities were not detectable in comparable protein extracts of yeast, transformed with vector control. The NMTase protein sequence shared homology with plant caffeic acid O-methyltransferases and related enzymes. Phylogenetic analyses suggested that beta-Ala NMTase represents a novel family of N-methyltransferases that are evolutionarily related to O-methyltransferases.