Diversity analysis of endophytes with antimicrobial and antioxidant potential from Viola odorata: an endemic plant species of the Himalayas.

Viola odorata, also known as "Banafshah" in high altitudes of Himalayas, is well known for its pharmaceutical importance in Ayurvedic and Unani medicinal system. The plant is a source of various drugs for its anti-inflammatory, diaphoretic, diuretic, emollient, expectorant, antipyretic, and laxative properties. The endophytes of plants have been reported for their role in modulating various physiological and biological processes of the host plants. In the present study, a total of 244 endophytes were isolated in pure cultures from the roots of Viola odorata, and genetic diversity was evaluated using amplified ribosomal DNA restriction analysis (ARDRA) and enterobacterial repetitive intergenic consensus (ERIC). The molecular fingerprinting revealed variation among various rRNA types among morphologically different endophytes based on ARDRA and ERIC-PCR. The screening of endophytes showed antimicrobial activity of 11 bacterial isolates and one actinomycete SGA9 against various pathogens Bacillus cereus, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis. The antioxidant activity revealed the majority of the bacterial isolates able to scavenge the free radical in the range of 10-50% and 8 bacterial isolates in the range of 50-85%. Principal component analysis separated eight isolates away from the central eclipse and form a separate group based on antimicrobial and antioxidant potential. The identification of these eight isolates showed affiliation with different species of the genus Enterobacter, Microbacterium, Pseudomonas, Rhizobium, and Streptomyces. This is the first report on the characterization of endophytic bacteria and actinomycetes from endemic Viola odorata. Results suggested that these endophytes could be explored for the production of antimicrobial and antioxidant products.

Application of chloroplast genome in the identification of Traditional Chinese Medicine Viola philippica.

BACKGROUND: Viola philippica Cav. is the only source plant of "Zi Hua Di Ding", which is a Traditional Chinese Medicine (TCM) that is utilized as an antifebrile and detoxicant agent for the treatment of acute pyogenic infections. Historically, many Viola species with violet flowers have been misused in "Zi Hua Di Ding". Viola have been recognized as a taxonomically difficult genera due to their highly similar morphological characteristics. Here, all common V. philippica adulterants were sampled. A total of 24 complete chloroplast (cp) genomes were analyzed, among these 5 cp genome sequences were downloaded from GenBank and 19 cp genomes, including 2 "Zi Hua Di Ding" purchased from a local TCM pharmacy, were newly sequenced. RESULTS: The Viola cp genomes ranged from 156,483 bp to 158,940 bp in length. A total of 110 unique genes were annotated, including 76 protein-coding genes, 30 tRNAs, and four rRNAs. Sequence divergence analysis screening identified 16 highly diverged sequences; these could be used as markers for the identification of Viola species. The morphological, maximum likelihood and Bayesian inference trees of whole cp genome sequences and highly diverged sequences were divided into five monophyletic clades. The species in each of the five clades were identical in their positions within the morphological and cp genome tree. The shared morphological characters belonging to each clade was summarized. Interestingly, unique variable sites were found in ndhF, rpl22, and ycf1 of V. philippica, and these sites can be selected to distinguish V. philippica from samples all other Viola species, including its most closely related species. In addition, important morphological characteristics were proposed to assist the identification of V. philippica. We applied these methods to examine 2 "Zi Hua Di Ding" randomly purchased from the local TCM pharmacy, and this analysis revealed that the morphological and molecular characteristics were valid for the identification of V. philippica. CONCLUSIONS: This study provides invaluable data for the improvement of species identification and germplasm of V. philippica that may facilitate the application of a super-barcode in TCM identification and enable future studies on phylogenetic evolution and safe medical applications.

Selective forces on the maintenance of outcrossing in an almost exclusively cleistogamous violet species.

PREMISE: Cleistogamous species constitute interesting study systems to resolve the longstanding question of how outcrossing is maintained given that seed production is ensured through selfing. In this work, we investigate the selective forces that allow the persistence of producing self-pollinated cleistogamous (CL) and chasmogamous (CH) flowers in Viola jaubertiana Marès & Vigin. METHODS: We monitored three populations at different elevation for two years, and studied the flowering phenology and the relative contribution of each flower morph to parental fitness. We tested whether allocation to CH and CL flowers differed across populations and if it covaried with herbivory and water stress conditions. We also performed hand-pollination and bagging experiments in CH flowers to estimate inbreeding depression and heterosis. RESULTS: The CH flowers open in winter under unfavorable conditions for pollination, show high pollen limitation and no-delayed selfing, and thus produce a low amount of seeds. Conversely, CL flowers appear in early spring, are physiologically cheaper to produce (i.e., dry weight is 3.4 times lower than that of CH flowers), and yield approximately 100 times more seeds than CH flowers. The CH flowers were favored under water stress and low herbivory. Crosses between populations showed up to 25% greater fitness than those within populations. CONCLUSIONS: Despite the great pollen limitation in CH flowers, we suggest that the interaction among different environmental determinants and heterosis are probably sufficient forces to maintain chasmogamy in this long-lived species, reducing deleterious fixed mutations in the selfed lines.

Tropical vibes from Sri Lanka - cyclotides from Viola betonicifolia by transcriptome and mass spectrometry analysis.

Cyclotides are an extremely stable class of peptides, ubiquitously distributed in Violaceae. The aim of the present study was to investigate the presence of cyclotides in Sri Lankan Violaceae plants, using combined tools of transcriptomics and mass spectrometry. New cyclotides were discovered for the first time in the wild flora of Sri Lanka, within Viola betonicifolia, a plant used in traditional medicine as an antimicrobial. Plant extracts prepared in small scale from Viola betonicifolia were first subjected to LC-MS analysis. Subsequent transcriptome de novo sequencing of Viola betonicifolia uncovered 25 new (vibe 1-25) and three known (varv A/kalata S, viba 17, viba 11) peptide sequences from Möbius and bracelet cyclotide subfamilies as well as hybrid cyclotides. Among the transcripts, putative linear acyclotide sequences (vibe 4, vibe 10, vibe 11 and vibe 22) that lack a conserved asparagine or aspartic acid vital for cyclisation were also present. Four asparagine endopeptidases (AEPs), VbAEP1-4 were found within the Viola betonicifolia transcriptome, including a peptide asparaginyl ligase (PAL), potentially involved in cyclotide backbone cyclisation, showing >93% sequence homology to Viola yedoensis peptide asparaginyl ligases, VyPALs. In addition, we identified two protein disulfide isomerases (PDIs), VbPDI1-2, likely involved in cyclotide oxidative folding, having high sequence homology (>74%) with previously reported Rubiaceae and Violaceae PDIs. The current study highlights the ubiquity of cyclotides in Violaceae as well as the utility of transcriptomic analysis for cyclotides and their putative processing enzyme discovery. The high variability of cyclotide sequences in terms of loop sizes and residues in V. betonicifolia showcase the cyclotide structure as an adaptable scaffold as well as their importance as a combinatorial library, implicated in plant defense.

Lack of correlation between pollen aperture number and environmental factors in pansies (Viola L., sect. Melanium Ging.) - pollen heteromorphism re-examined.

The development of different pollen morphs by one specimen - pollen heteromorphism - occurs in ca. one-third species of the genus Viola. Melanium section species (pansies) stand out in producing the widest range of apertures among Viola species. Aperture number decreases with elevation increase, and faster germination of five-aperturate pollen as compared with three-aperturate has previously been postulated. We re-examined pollen heteromorphism in the context of its viability, and made correlations with elevation (>1500 m a.s.l. versus <1500 m a.s.l.), soil type (metalliferous versus non-metalliferous; MET versus NMET) and chromosome number based on selected study criteria of ca. 20% karyologically and morphologically strongly differentiated but genetically closely related pansies. A total of 79% of analysed species were heteromorphic, forming three- to six-colp or ate pollen per individual flower. Mean aperture number and pollen viability were not affected by soil type (MET versus NMET). Mean aperture number was also not influenced by elevation or species chromosome number. Positive correlations were established between aperture number and pollen viability, negative between pollen viability and elevation (increasing altitude of 100 m decreased pollen viability by 0.4%) and lack of correlation between chromosome number and pollen viability. The varied frequencies of different pollen morphs among species are not under the general pressure of ecological conditions, as previously postulated for the species of Melanium section. Rather, this trait in pansies, similar to other floral characters (e.g. long, curved nectar spur, 'landing platform', posterior petals with nectar guides), is adaptive but dependent on the breeding system (inbreeding versus outbreeding) of the individual species.

Transcriptome sequencing and differential gene expression analysis in Viola yedoensis Makino (Fam. Violaceae) responsive to cadmium (Cd) pollution.

Viola yedoensis Makino is an important Chinese traditional medicine plant adapted to cadmium (Cd) pollution regions. Illumina sequencing technology was used to sequence the transcriptome of V. yedoensis Makino. We sequenced Cd-treated (VIYCd) and untreated (VIYCK) samples of V. yedoensis, and obtained 100,410,834 and 83,587,676 high quality reads, respectively. After de novo assembly and quantitative assessment, 109,800 unigenes were finally generated with an average length of 661 bp. We then obtained functional annotations by aligning unigenes with public protein databases including NR, NT, SwissProt, KEGG and COG. In addition, 892 differentially expressed genes (DEGs) were investigated between the two libraries of untreated (VIYCK) and Cd-treated (VIYCd) plants. Moreover, 15 randomly selected DEGs were further validated with qRT-PCR and the results were highly accordant with the Solexa analysis. This study firstly generated a successful global analysis of the V. yedoensis transcriptome and it will provide for further studies on gene expression, genomics, and functional genomics in Violaceae.

Establishing the phylogenetic origin, history, and age of the narrow endemic Viola guadalupensis (Violaceae).

PREMISE OF THE STUDY: Climate change and shifts in land use are two major threats to biodiversity and are likely to disproportionately impact narrow endemics. Understanding their origins and the extent of their genetic diversity will enable land managers to better conserve these unique, highly localized gene pools. Viola guadalupensis is a narrow endemic of the Guadalupe Mountains (west Texas, USA). Its affinities within Viola section Chamaemelanium have been the subject of some debate. Furthermore, the polyploid and presumably reticulate relationships within this section remain largely unknown. METHODS: We counted chromosomes for V. guadalupensis. Phylogenies for the chloroplast trnL-F region and the low-copy nuclear gene GPI for 24 Viola taxa were generated and used to produce a polyploid phylogenetic network. Divergence dates were obtained by fossil calibration. KEY RESULTS: Meiotic chromosome counts revealed that V. guadalupensis is tetraploid (n = 12), and the presence of two GPI homoeologs further suggested allotetraploidy. Phylogenetic reconstructions showed that it originated through hybridization between unidentified members of subsection Canadenses (paternal parent) and subsection Nuttallianae (maternal parent). A fossil-calibrated relaxed clock dating analysis of GPI estimated the maximum age of V. guadalupensis to be 8.6 (5.7-11.6) Myr, suggesting the species evolved after the Guadalupe Mountains formed 12-13 Ma. CONCLUSIONS: Viola guadalupensis originated by intersubsectional hybridization followed by polyploidization. Within section Chamaemelanium, this phenomenon has occurred repeatedly in the last 9 Myr (at least for V. bakeri, V. douglasii, V. glabella, and V. sempervirens). Consequences for the systematics of the section are discussed.

A transcriptional profile of metallophyte Viola baoshanensis involved in general and species-specific cadmium-defense mechanisms.

Viola baoshanensis Shu, Liu et Lan is a newly identified metallophyte, and its defensive strategies against heavy metals are still unclear. In the present study, we firstly constructed a root cDNA library of the plant subjected to 300muM Cd for 48h by using suppression subtractive hybridization (SSH), and 43 unique cDNA fragments were further isolated from the library. Sequence homology analysis showed that half of the identified genes were involved in general stress defense, such as antioxidative enzymes, protein degradation and stress signal transduction. After RT-PCR and RACE analysis, a Cd-responsive gene Vb40 was identified, which could deduce a novel cysteine-rich mini-protein. Meanwhile, five cyclotide precursor genes (VbCP1-VbCP5) were also identified. The Vb40 and the VbCP1-VbCP5 were further investigated by yeast expression analysis, and they could improve copper (Cu) tolerance in hosted yeast, indicating that these species-specific genes possibly functioned in V. baoshanensis heavy metals tolerance. Our results suggested that heavy metal tolerance in V. baoshanensis relied on both general and species-specific defense.

Intraspecific variation in Viola suavis in Europe: parallel evolution of white-flowered morphotypes.

BACKGROUND AND AIMS: Viola species are commonly grown for their ornamental flowers, but their evolutionary history and taxonomy are often complicated and have been poorly explored so far. This is a study of the polymorphic, typically blue-flowered species Viola suavis, concentrating on the white-flowered populations of uncertain taxonomic assignment that occur in Spain and central and south-eastern Europe. The aim was to resolve their origin and taxonomic status and to study the intraspecific structure and (post)glacial history of this species. METHODS: Viola suavis and five close relatives were sampled from multiple locations and subjected to molecular (AFLP, sequencing of nrDNA ITS) and morphometric analyses. Data on ploidy level and pollen fertility were also obtained, to address an assumed hybrid origin of the white-flowered populations. KEY RESULTS: In V. suavis a strong intraspecific genetic split into two groups was observed, indicating that there has been a long-term isolation and survival in distinct glacial refugia. The white-flowered populations could be placed within the variation range of this species, and it is clear that they evolved independently in two distant areas. Their parallel evolution is supported by both morphological and genetic differentiation. The strongly reduced genetic variation and absence of unique AFLP fragments suggest their derived status and origin from the typical, blue-flowered populations. CONCLUSIONS: These results suggest that intraspecific variation in V. suavis has been largely shaped by population isolations during the last glaciation and subsequent recolonizations, although cultivation and vegetative spread by humans have affected the present picture as well.