Complete plastid genome structure of 13 Asian Justicia (Acanthaceae) species: comparative genomics and phylogenetic analyses.

BACKGROUND: Justicia L. is the largest genus in Acanthaceae Juss. and widely distributed in tropical and subtropical regions of the world. Previous phylogenetic studies have proposed a general phylogenetic framework for Justicia based on several molecular markers. However, their studies were mainly focused on resolution of phylogenetic issues of Justicia in Africa, Australia and South America due to limited sampling from Asia. Additionally, although Justicia plants are of high medical and ornamental values, little research on its genetics was reported. Therefore, to improve the understanding of its genomic structure and relationships among Asian Justicia plants, we sequenced complete chloroplast (cp.) genomes of 12 Asian plants and combined with the previously published cp. genome of Justicia leptostachya Hemsl. for further comparative genomics and phylogenetic analyses. RESULTS: All the cp. genomes exhibit a typical quadripartite structure without genomic rearrangement and gene loss. Their sizes range from 148,374 to 151,739 bp, including a large single copy (LSC, 81,434-83,676 bp), a small single copy (SSC, 16,833-17,507 bp) and two inverted repeats (IR, 24,947-25,549 bp). GC contents range from 38.1 to 38.4%. All the plastomes contain 114 genes, including 80 protein-coding genes, 30 tRNAs and 4 rRNAs. IR variation and repetitive sequences analyses both indicated that Justicia grossa C. B. Clarke is different from other Justicia species because its lengths of ndhF and ycf1 in IRs are shorter than others and it is richest in SSRs and dispersed repeats. The ycf1 gene was identified as the candidate DNA barcode for the genus Justicia. Our phylogenetic results showed that Justicia is a polyphyletic group, which is consistent with previous studies. Among them, J. grossa belongs to subtribe Tetramerinae of tribe Justicieae while the other Justicia members belong to subtribe Justiciinae. Therefore, based on morphological and molecular evidence, J. grossa should be undoubtedly recognized as a new genus. Interestingly, the evolutionary history of Justicia was discovered to be congruent with the morphology evolution. CONCLUSION: Our study not only elucidates basic features of Justicia whole plastomes, but also sheds light on interspecific relationships of Asian Justicia plants for the first time.

Transcriptome analysis used to identify and characterize odorant binding proteins in Agasicles hygrophila (Coleoptera: Chryspmelidae).

The transcriptomes of Agasicles hygrophila eggs and first instar larvae were analyzed to explore the olfactory mechanism of larval behavior. The analysis resulted in 135,359 unigenes and the identification of 38 odorant-binding proteins (OBPs), including 23 Minus-C OBPs, 8 Plus-C OBPs, and 7 Classic OBPs. Further analysis of differentially expressed genes (DEGs) revealed 10 DEG OBPs, with 5 (AhygOBP5, AhygOBP9, AhygOBP12, AhygOBP15 and AhygOBP36) up-regulated in first instar larvae. Verification of expression patterns of these 5 AhygOBPs using qPCR showed that AhygOBP9 and AhygOBP36 were mainly expressed in the adult stage with gradually increasing expression in the larval stage. AhygOBP5, AhygOBP12, and AhygOBP15 were not expressed in eggs and pupae, and their expression in larvae and adults showed no clear pattern. These 5 AhygOBPs may play an olfactory role in larval behavior, providing a basis for further investigation of their specific functions and clarifying the olfactory mechanism of A. hygrophila.

Chloroplast genome analysis of three Acanthus species reveal the adaptation of mangrove to intertidal habitats.

Acanthus is a distinctive genus that covers three species with different ecological niches including Acanthus mollis (arid terrestrial), Acanthus leucostachyus (damp forest) and Acanthus ilicifolius (coastal intertidal). It is an intriguing question how these species evolved from terrestrial to coastal intertidal. In the present study, we assembled chloroplast genomes of A. ilicifolius, A. leucostachyus and A. mollis, which exhibited typical quadripartite structures. The sizes were 150,758, 154,686 and 150,339 bp that comprised a large single copy (LSC, 82,963, 86,461 and 82,612 bp), a small single copy (SSC, 17,191, 17,511 and 17,019 bp), and a pair of inverted repeats (IRs, 25,302, 25,357 and 25,354 bp), respectively. Gene annotation revealed that A. ilicifolius, A. leucostachyus and A. mollis contained 113, 112 and 108 unique genes, each of which contained 79, 79 and 74 protein-coding genes, 30, 29 and 30 tRNAs, and 4 rRNA genes, respectively. Differential gene analysis revealed plenty of ndhs gene deletions in the terrestrial plant A. mollis. Nucleotide diversity analysis showed that the psbK, ycf1, ndhG, and rpl22 have the highest nucleotide variability. Compared to A. leucostachyus and A. mollis, seven genes in A. ilicifolius underwent positive selection. Among them, the atpF gene showed a strong positive selection throughout terrestrial to marine evolution and was important for adaptation to coastal intertidal habitats. Phylogenetic analysis indicated that A. ilicifolius has a closer genetic relationship with A. leucostachyus than A. mollis which further confirmed the evolutionary direction of Acanthus going from terrestrial to coastal intertidal zones.

The Complete Chloroplast Genome of Hypoestes forskaolii (Vahl) R.Br: Insights into Comparative and Phylogenetic Analyses within the Tribe Justiceae.

Hypoestes forskaolii is one of the most important species of the family Acanthaceae, known for its high economic and medicinal importance. It is well distributed in the Arab region as well as on the African continent. Previous studies on ethnomedicine have reported that H. forskaolii has an anti-parasitic effect as well as antimalarial and anthelmintic activities. Previous studies mainly focused on the ethnomedicinal properties, hence, there is no information on the genomic architecture and phylogenetic positions of the species within the tribe Justiceae. The tribe Justicieae is the most taxonomically difficult taxon in Acanthoideae due to its unresolved infratribal classification. Therefore, by sequencing the complete chloroplast genome (cp genome) of H. forskaolii, we explored the evolutionary patterns of the cp genome and reconstructed the phylogeny of Justiceae. The cp genome is quadripartite and circular in structure and has a length of 151,142 bp. There are 130 genes (86 coding for protein, 36 coding for tRNA and 8 coding for rRNA) present in the plastome. Analyses of long repeats showed only three types of repeats: forward, palindromic and reverse were present in the genome. Microsatellites analysis revealed 134 microsatellites in the cp genome with mononucleotides having the highest frequency. Comparative analyses within Justiceae showed that genomes structure and gene contents were highly conserved but there is a slight distinction in the location of the genes in the inverted repeat and single copy junctions. Additionally, it was discovered that the cp genome includes variable hotspots that can be utilized as DNA barcodes and tools for determining evolutionary relationships in the Justiceae. These regions include: atpH-atpI, trnK-rps16, atpB-rbcL, trnT-trnL, psbI-trnS, matK, trnH-psbA, and ndhD. The Bayesian inference phylogenetic tree showed that H. forskaolii is a sister to the Dicliptra clade and belongs to Diclipterinae. The result also confirms the polyphyly of Justicia and inclusion of Diclipterinae within justicioid. This research has revealed the phylogenetic position of H. forskaolii and also reported the resources that can be used for evolutionary and phylogenetic studies of the species and the Justicieae.

Chloroplast Genome Analysis for Genetic Information and Authentication in Five Barleria Species.

In order to authenticate the genomic information of Barleriacristata L., B. lupulina Lindl., B. repens Nees, B. siamensis Craib, and B. strigosa Willd, cp genomes were investigated. They revealed a general structure with a total size of 151,997-152,324 bp. The genomes encoded a total of 131 genes, including 86 CDS, 37 tRNA, and 8 rRNA genes. Other details found were as follows: different numbers and types of SSRs; identical gene content, which is adjacent to the border regions, except for B. strigosa, that revealed a shorter ndhF gene sequence and lacked the ycf1 gene; slightly different genetic distance values, which can be used for species identification; three distinct gaps of nucleotide variations between the species located at the intergenic spacer regions of the LSC and CDS of the SSC; three effective molecular markers derived from divergent hotspot regions, including the ccsA-ndhD, ndhA-ndhH-rps15, and ycf1. The genetic relationships derived from the cp genome and the CDS phylogenetic trees of Barleria and the 13 genera in Acanthaceae and different families, Scrophulariaceae and Phrymaceae, showed similar results. The six Barleria species as monophyletic groups with inner and outer outgroups were found to have perfect discrimination. These results have helped to authenticate the five Barleria species and the six genera in Acanthaceae.

Comparative transcriptome analysis on the mangrove Acanthus ilicifolius and its two terrestrial relatives provides insights into adaptation to intertidal habitats.

Acanthus is a unique genus covering both mangroves and terrestrial species, and thus is an ideal system to comparatively analyze the mechanisms of mangrove adaptation to intertidal habitats. We performed RNA sequencing of the mangrove plant Acanthus ilicifolius and its two terrestrial relatives, Acanthus leucostachyus and Acanthus mollis. A total of 91,125, 118,290, and 141,640 unigenes were obtained. Simple sequence repeats (SSR) analysis showed that A. ilicifolius had more SSRs, the highest frequency of distribution, and higher in polymorphism potential compared to the two terrestrial relatives. Phylogenetic analyses suggested a relatively recent split between A. ilicifolius and A. leucostachyus, i.e., about 16.76 million years ago (Mya), after their ancestor divergence with A. mollis (32.11 Mya), indicating that speciation of three Acanthus species occurred in the Early to Middle Miocene. Gene Ontology (GO) enrichment revealed that the unique unigenes in A. ilicifolius are predominantly related to rhythmic process, reproductive process and response to stimuli. The accelerated evolution and positive selection analyses indicated that the genus Acanthus migrated from terrestrial to intertidal habitats, where 311 pairs may be under positive selection. Functional enrichment analysis revealed that these genes associated with essential metabolism and biosynthetic pathways such as oxidative phosphorylation, plant hormone signal transduction, photosynthetic carbon fixation and arginine and proline metabolism, are related to the adaptation of A. ilicifolius to intertidal habitats, which are characterized by high salinity and hypoxia. Our results indicate the evolutionary processes and the mechanisms underlying the adaptability of Acanthus to various harsh environments from the arid terrestrial to intertidal habitats.

Co-expression network analyses of anthocyanin biosynthesis genes in Ruellia (Wild Petunias; Acanthaceae).

BACKGROUND: Anthocyanins are major pigments contributing to flower coloration and as such knowledge of molecular architecture underlying the anthocyanin biosynthetic pathway (ABP) is key to understanding flower color diversification. To identify ABP structural genes and associated regulatory networks, we sequenced 16 transcriptomes generated from 10 species of Ruellia and then conducted co-expression analyses among resulting data. RESULTS: Complete coding sequences for 12 candidate structural loci representing eight genes plus nine candidate regulatory loci were assembled. Analysis of non-synonymous/synonymous (dn/ds) mutation rates indicated all identified loci are under purifying selection, suggesting overall selection to prevent the accumulation of deleterious mutations. Additionally, upstream enzymes have lower rates of molecular evolution compared to downstream enzymes. However, site-specific tests of selection yielded evidence for positive selection at several sites, including four in F3'H2 and five in DFR3, and these sites are located in protein binding regions. A species-level phylogenetic tree constructed using a newly implemented hybrid transcriptome-RADseq approach implicates several flower color transitions among the 10 species. We found evidence of both regulatory and structural mutations to F3'5'H in helping to explain the evolution of red flowers from purple-flowered ancestors. CONCLUSIONS: Sequence comparisons and co-expression analyses of ABP loci revealed that mutations in regulatory loci are likely to play a greater role in flower color transitions in Ruellia compared to mutations in underlying structural genes.

A RADseq phylogeny of Barleria (Acanthaceae) resolves fine-scale relationships.

Barleria is a genus of approximately 300 species of herbs, shrubs or, rarely, trees, that is broadly distributed across the Paleotropics. The genus is especially diverse in Tanzania, Angola, and Madagascar. A recent molecular study sampled 53 Barleria species and gathered data for five molecular markers (i.e., four chloroplast loci and the nuclear nrITS) to find support for the recognition of two subgenera previously circumscribed based on morphology, subg. Barleria and subg. Prionitis. That study further reconstructed four previously recognized sections (i.e., Fissimura, Prionitis, Somalia, Stellatohirta) as monophyletic, while three others (i.e., Barleria, Cavirostrata, Chrysothrix) were recovered as para- or polyphyletic. The present study aimed to reconstruct phylogenetic relationships within Barleria based on a broader sample of taxa and many more characters. We sampled 190 accessions representing 184 taxa, including varieties and subspecies. The dataset includes 167 of the ca. 300 species currently recognized or about 56% of total species diversity. We relied heavily on herbarium specimens to sample across the taxonomic breadth and geographic range of Barleria. Single nucleotide polymorphism data were generated using double-digest restriction-site associated DNA sequencing (ddRADseq). The maximum likelihood phylogeny corroborated the topology estimated from the chloroplast and nrITS data, but with greatly increased resolution and support for fine-scale relationships. A coalescent analysis failed to resolve distant evolutionary relationships across Barleria and between Barleria and outgroups, but recovered the same or similar topologies within each Barleria section. Importantly, the ddRADseq phylogeny recovered seven major lineages within subg. Barleria and resolved a polytomy that included B. cristata, the type species of the genus. The topology suggests at least four independent dispersal events to Madagascar followed by three subsequent radiations. Our results broadly inform our understanding of diversity and evolution in one of the largest genera of Acanthaceae, representing an important step towards a stable subgeneric classification for the genus.

Efficient micropropagation of Thunbergia coccinea Wall. and genetic homogeneity assessment through RAPD and ISSR markers.

Thunbergia coccinea Wall. ex D. Don being a rare, ornamental and medicinal plant of India, is needed to propagate for conserving the germplasm and analyzing its phytochemical compounds in the future. A reliable protocol for direct in vitro propagation using nodal shoot meristem of T. coccinea as explant was standardized. The highest number of shoots per explant (22.17 ± 0.54) with maximum shoot length (2.36 ± 0.28) in cm was obtained in Murashige and Skoog (MS) medium supplemented with 9.70 µM of 6-furfurylaminopurine (Kinetin) and 0.053 µM of α-naphthaleneacetic acid (NAA) combination, among all the different plant growth regulators (PGR's) and concentrations tested. The aforesaid PGR's combination was optimum for axillary shoot bud induction and multiplication in T. coccinea. The best rooting was observed on the half-strength MS medium fortified with 2.68 µM NAA with the highest number of roots per shoot (3.75 ± 0.12) and maximum length (5.22 ± 0.32) in cm. All the in vitro raised plantlets were acclimatized in sterile sand and soil mixture (1:1) with a survival rate of 70% on earthen pots under greenhouse conditions. PCR-based RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeat) molecular markers were employed to determine the genetic homogeneity amongst the plantlets. Twelve (12) RAPD and nine (9) ISSR primers developed a total of 104 and 91 scorable bands, respectively. The band profiles of micropropagated plantlets were monomorphic to the mother, donor in vivo plant, and similarity values varied from 0.9542-1.000. The dendrogram generated through UPGMA (unweighted pair group method with arithmetic mean) showed 99% similarities amongst all tested plants confirming the genetic uniformity of in vitro raised plants.

Regioselectivity mechanism of the Thunbergia alata Δ6-16:0-acyl carrier protein desaturase.

Plant plastidial acyl-acyl carrier protein (ACP) desaturases are a soluble class of diiron-containing enzymes that are distinct from the diiron-containing integral membrane desaturases found in plants and other organisms. The archetype of this class is the stearoyl-ACP desaturase which converts stearoyl-ACP into oleoyl (18:1Δ9cis)-ACP. Several variants expressing distinct regioselectivity have been described including a Δ6-16:0-ACP desaturase from black-eyed Susan vine (Thunbergia alata). We solved a crystal structure of the T. alata desaturase at 2.05 Å resolution. Using molecular dynamics (MD) simulations, we identified a low-energy complex between 16:0-ACP and the desaturase that would position C6 and C7 of the acyl chain adjacent to the diiron active site. The model complex was used to identify mutant variants that could convert the T. alata Δ6 desaturase to Δ9 regioselectivity. Additional modeling between ACP and the mutant variants confirmed the predicted regioselectivity. To validate the in-silico predictions, we synthesized two variants of the T. alata desaturase and analyzed their reaction products using gas chromatography-coupled mass spectrometry. Assay results confirmed that mutants designed to convert T. alata Δ6 to Δ9 selectivity exhibited the predicted changes. In complementary experiments, variants of the castor desaturase designed to convert Δ9 to Δ6 selectivity lost some of their Δ9 desaturation ability and gained the ability to desaturate at the Δ6 position. The computational workflow for revealing the mechanistic understanding of regioselectivity presented herein lays a foundation for designing acyl-ACP desaturases with novel selectivities to increase the diversity of monoenes available for bioproduct applications.

Mangrove tree (Avicennia marina): insight into chloroplast genome evolutionary divergence and its comparison with related species from family Acanthaceae.

Avicennia marina (family Acanthaceae) is a halotolerant woody shrub that grows wildly and cultivated in the coastal regions. Despite its importance, the species suffers from lack of genomic datasets to improve its taxonomy and phylogenetic placement across the related species. Here, we have aimed to sequence the plastid genome of A. marina and its comparison with related species in family Acanthaceae. Detailed next-generation sequencing and analysis showed a complete chloroplast genome of 150,279 bp, comprising 38.6% GC. Genome architecture is quadripartite revealing large single copy (82,522 bp), small single copy (17,523 bp), and pair of inverted repeats (25,117 bp). Furthermore, the genome contains 132 different genes, including 87 protein-coding genes, 8 rRNA, 37 tRNA genes, and 126 simple sequence repeats (122 mononucleotide, 2 dinucleotides, and 2 trinucleotides). Interestingly, about 25 forward, 15 reversed and 14 palindromic repeats were also found in the A. marina. High degree synteny was observed in the pairwise alignment with related genomes. The chloroplast genome comparative assessment showed a high degree of sequence similarity in coding regions and varying divergence in the intergenic spacers among ten Acanthaceae species. The pairwise distance showed that A. marina exhibited the highest divergence (0.084) with Justicia flava and showed lowest divergence with Aphelandra knappiae (0.059). Current genomic datasets are a valuable resource for investigating the population and evolutionary genetics of family Acanthaceae members' specifically A. marina and related species.

Karyomorphology in two species of the genus Phlogacanthus Nees of Assam: some new karyological insights.

The genus Phlogacanthus Nees belongs to the family Acanthaceae and is represented by herbs or shrubs species. The present work shows detailed karyomorphological studies in two species, Phlogacanthus quadrangularis (Hook.) Heine and Phlogacanthus guttatus Nees. Both the species grow as undergrowth vegetation. The conservation status of both the species are yet to be determined. The somatic chromosome counts of both the mentioned species are available for the first time, 2n = 40 for P. quadrangularis and 2n = 34 for P. guttatus. The karyomorphological observations showed that both plant species show dominance of sub-telocentric chromosomes with a few metacentric chromosomes. P. quadrangularis have telocentric chromosomes which is absent in the other species. The range of length of chromosomes in P. quadrangularis is from 0.340 lm to 1.32 lm and that of P. guttatus is from 0.560 lm to 1.878 lm. The karyotype type of the two species are classified as 3B type. But the dispersion index (DI) value of both the species are different. P. guttatus shows higher value of DI than P. quadrangularis. Higher the DI value more specialized is the karyotype. By comparing the chromosome length, size, DI value and idiogram of both the species, it is concluded that the karyotype of P. guttatus is more asymmetric and advanced than karyotype of P. quadrangularis. The karyomorphological findings of the present study will aid in determining the importance and utility, ex-situ conservation, protection, preservation and regeneration of germplasm.

Blue genome: chromosome-scale genome reveals the evolutionary and molecular basis of indigo biosynthesis in Strobilanthes cusia.

Natural plant dyes have been developed and used across many traditional societies worldwide. The blue pigment indigo has seen widespread usage across South America, Egypt, Europe, India and China for thousands of years, mainly extracted from indigo-rich plants. The utilization and genetic engineering of indigo in industries and ethnobotanical studies on the effects of cultural selection on plant domestication are limited due to lack of relevant genetic and genomic information of dye plants. Strobilanthes cusia (Acanthaceae) is a typical indigo-rich plant important to diverse ethnic cultures in many regions of Asia. Here we present a chromosome-scale genome for S. cusia with a genome size of approximately 865 Mb. About 79% of the sequences were identified as repetitive sequences and 32 148 protein-coding genes were annotated. Metabolic analysis showed that the main indigoid pigments (indican, indigo and indirubin) were mainly synthesized in the leaves and stems of S. cusia. Transcriptomic analysis revealed that the expression level of genes encoding metabolic enzymes such as monooxygenase, uridine diphosphate-glycosyltransferase and ß-glucosidase were significantly changed in leaves and stems compared with root tissues, implying their participation in indigo biosynthesis. We found that several gene families involved in indigo biosynthesis had undergone an expansion in number, with functional differentiation likely facilitating indigo biosynthesis in S. cusia. This study provides insight into the physiological and molecular bases of indigo biosynthesis, as well as providing genomic data that provide the basis for further study of S. cusia cultivation by Asia's traditional textile producers.

Complete chloroplast genome sequence of Barleria prionitis, comparative chloroplast genomics and phylogenetic relationships among Acanthoideae.

BACKGROUND: The plastome of medicinal and endangered species in Kingdom of Saudi Arabia, Barleria prionitis was sequenced. The plastome was compared with that of seven Acanthoideae species in order to describe the plastome, spot the microsatellite, assess the dissimilarities within the sampled plastomes and to infer their phylogenetic relationships. RESULTS: The plastome of B. prionitis was 152,217 bp in length with Guanine-Cytosine and Adenine-Thymine content of 38.3 and 61.7% respectively. It is circular and quadripartite in structure and constitute of a large single copy (LSC, 83, 772 bp), small single copy (SSC, 17, 803 bp) and a pair of inverted repeat (IRa and IRb 25, 321 bp each). 131 genes were identified in the plastome out of which 113 are unique and 18 were repeated in IR region. The genome consists of 4 rRNA, 30 tRNA and 80 protein-coding genes. The analysis of long repeat showed all types of repeats were present in the plastome and palindromic has the highest frequency. A total number of 98 SSR were also identified of which mostly were mononucleotide Adenine-Thymine and are located at the non coding regions. Comparative genomic analysis among the plastomes revealed that the pair of the inverted repeat is more conserved than the single copy region. In addition high variation is observed in the intergenic spacer region than the coding region. The genes, ycf1and ndhF and are located at the border junction of the small single copy region and IRb region of all the plastome. The analysis of sequence divergence in the protein coding genes indicates that the following genes undergo positive selection (atpF, petD, psbZ, rpl20, petB, rpl16, rps16, rpoC, rps7, rpl32 and ycf3). Phylogenetic analysis indicated sister relationship between Ruellieae and Justcieae. In addition, Barleria, Justicia and Ruellia are paraphyletic, suggesting that Justiceae, Ruellieae, Andrographideae and Barlerieae should be treated as tribes. CONCLUSIONS: This study sequenced and assembled the first plastome of the taxon Barleria and reported the basics resources for evolutionary studies of B. prionitis and tools for phylogenetic relationship studies within the core Acanthaceae.

Establishment of an Agrobacterium mediated transformation protocol for the detection of cytokinin in the heterophyllous plant Hygrophila difformis (Acanthaceae).

KEY MESSAGE: This is the first report of a highly efficient Agrobacterium tumefaciens-mediated transformation protocol for Acanthaceae and its utilization in revealing important roles of cytokinin in regulating heterophylly in Hygrophila difformis. Plants show amazing morphological differences in leaf form in response to changes in the surrounding environment, which is a phenomenon called heterophylly. Previous studies have shown that the aquatic plant Hygrophila difformis (Acanthaceae) is an ideal model for heterophylly study. However, low efficiency and poor reproducibility of genetic transformation restricted H. difformis as a model plant. In this study, we reported successful induction of callus, shoots and the establishment of an efficient stable transformation protocol as mediated by Agrobacterium tumefaciens LBA4404. We found that the highest callus induction efficiency was achieved with 1 mg/L 1-Naphthaleneacetic acid (NAA) and 2 mg/L 6-benzyladenine (6-BA), that efficient shoot induction required 0.1 mg/L NAA and 0.1 mg/L 6-BA and that high transformation efficiency required 100 µM acetosyringone. Due to the importance of phytohormones in the regulation of heterophylly and the inadequate knowledge about the function of cytokinin (CK) in this process, we analyzed the function of CK in the regulation of heterophylly by exogenous CK application and endogenous CK detection. By using our newly developed transformation system to detect CK signals, contents and distribution in H. difformis, we revealed an important role of CK in environmental mediated heterophylly.

Molecular responses to freshwater limitation in the mangrove tree Avicennia germinans (Acanthaceae).

Environmental variation along the geographical space can shape populations by natural selection. In the context of global warming and changing precipitation regimes, it is crucial to understand the role of environmental heterogeneity in tropical trees adaptation, given their disproportional contribution to water and carbon biogeochemical cycles. Here, we investigated how heterogeneity in freshwater availability along tropical wetlands has influenced molecular variations of the black mangrove (Avicennia germinans). A total of 57 trees were sampled at seven sites differing markedly in precipitation regime and riverine freshwater inputs. Using 2,297 genome-wide single nucleotide polymorphic markers, we found signatures of natural selection by the association between variations in allele frequencies and environmental variables, including the precipitation of the warmest quarter and the annual precipitation. Additionally, we found candidate loci for selection based on statistical deviations from neutral expectations of interpopulation differentiation. Most candidate loci within transcribed sequences were functionally associated with central aspects of drought tolerance or plant response to drought. Moreover, our results suggest the occurrence of the rapid evolution of a population, probably in response to sudden and persistent limitations in plant access to soil water, following a road construction in 1974. Observations supporting rapid evolution included the reduction in tree size and changes in allele frequencies and in transcript expression associated with increased drought tolerance through the accumulation of osmoprotectants and antioxidants, biosynthesis of cuticles, protection against protein degradation, stomatal closure, photorespiration and photosynthesis. We describe a major role of spatial heterogeneity in freshwater availability in the specialization of this typically tropical tree.

Molecular cloning and metabolomic characterization of the 5-enolpyruvylshikimate-3-phosphate synthase gene from Baphicacanthus cusia.

BACKGROUND: Indigo alkaloids, such as indigo, indirubin and its derivatives, have been identified as effective antiviral compounds in Baphicacanthus cusia. Evidence suggests that the biosynthesis of indigo alkaloids in plants occurs via the shikimate pathway. The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is involved in plant metabolism; however, its underlying putative mechanism of regulating the production of indigo alkaloids is currently unknown. RESULTS: One gene encoding EPSPS was isolated from B. cusia. Quantitative real-time PCR analysis revealed that BcEPSPS was expressed at the highest level in the stem and upregulated by methyl jasmonate (MeJA), salicylic acid (SA) and abscisic acid (ABA) treatment. The results of subcellular localization indicated that BcEPSPS is mainly expressed in both the plastids and cytosol, which has not been previously reported. An enzyme assay revealed that the heterogeneously expressed BcEPSPS protein catalysed the generation of 5-enolpyruvyl shikimate-3-phosphate. The overexpression of BcEPSPS in Isatis indigotica hairy roots resulted in the high accumulation of indigo alkaloids, such as indigo, secologanin, indole and isorhamnetin. CONCLUSIONS: The function of BcEPSPS in catalysing the production of EPSP and regulating indigo alkaloid biosynthesis was revealed, which provided a distinct view of plant metabolic engineering. Our findings have practical implications for understanding the effect of BcEPSPS on active compound biosynthesis in B. cusia.

Complete Chloroplast Genome Sequence of Justicia flava: Genome Comparative Analysis and Phylogenetic Relationships among Acanthaceae.

The complete chloroplast genome of J. flava, an endangered medicinal plant in Saudi Arabia, was sequenced and compared with cp genome of three Acanthaceae species to characterize the cp genome, identify SSRs, and also detect variation among the cp genomes of the sampled Acanthaceae. NOVOPlasty was used to assemble the complete chloroplast genome from the whole genome data. The cp genome of J. flava was 150, 888bp in length with GC content of 38.2%, and has a quadripartite structure; the genome harbors one pair of inverted repeat (IRa and IRb 25, 500bp each) separated by large single copy (LSC, 82, 995 bp) and small single copy (SSC, 16, 893 bp). There are 132 genes in the genome, which includes 80 protein coding genes, 30 tRNA, and 4 rRNA; 113 are unique while the remaining 19 are duplicated in IR regions. The repeat analysis indicates that the genome contained all types of repeats with palindromic occurring more frequently; the analysis also identified total number of 98 simple sequence repeats (SSR) of which majority are mononucleotides A/T and are found in the intergenic spacer. The comparative analysis with other cp genomes sampled indicated that the inverted repeat regions are conserved than the single copy regions and the noncoding regions show high rate of variation than the coding region. All the genomes have ndhF and ycf1 genes in the border junction of IRb and SSC. Sequence divergence analysis of the protein coding genes showed that seven genes (petB, atpF, psaI, rpl32, rpl16, ycf1, and clpP) are under positive selection. The phylogenetic analysis revealed that Justiceae is sister to Ruellieae. This study reported the first cp genome of the largest genus in Acanthaceae and provided resources for studying genetic diversity of J. flava as well as resolving phylogenetic relationships within the core Acanthaceae.

De novo characterization of the Baphicacanthus cusia(Nees) Bremek transcriptome and analysis of candidate genes involved in indican biosynthesis and metabolism.

Baphicacanthus cusia (Nees) Bremek is an herb widely used for the clinical treatment of colds, fever, and influenza in Traditional Chinese Medicine. The roots, stems and leaves can be used as natural medicine, in which indigo and indirubin are two main active ingredients. In this study, quantification of indigo, indirubin, indican and adenosine among various tissues of B. cusia was conducted using HPLC-DAD. Leaves have significantly higher contents than stems and roots (380.66, 315.15, 20,978.26, 4323.15 µg/g in leaves, 306.36, 71.71, 3,056.78, 139.45 µg/g in stems, and 9.31, 7.82, 170.45, 197.48 µg/g in roots, respectively). De novo transcriptome sequencing of B. cusia was performed for the first time. The sequencing yielded 137,216,248, 122,837,394 and 140,240,688 clean reads from leaves, stems and roots respectively, which were assembled into 51,381 unique sequences. A total of 33,317 unigenes could be annotated using the databases of Nr, Swiss-Prot, KEGG and KOG. These analyses provided a detailed view of the enzymes involved in indican backbone biosynthesis, such as cytochrome P450, UDP-glycosyltransferase, glucosidase and tryptophan synthase. Analysis results showed that tryptophan synthase was the candidate gene involved in the tissue-specific biosynthesis of indican. We also detected sixteen types of simple sequence repeats in RNA-Seq data for use in future molecular mark assisted breeding studies. The results will be helpful in further analysis of B. cusia functional genomics, especially in increasing biosynthesis of indican through biotechnological approaches and metabolic regulation.

Should DNA sequence be incorporated with other taxonomical data for routine identifying of plant species?

BACKGROUND: A variety of plants in Acanthaceae have long been used in traditional Thai ailment and commercialised with significant economic value. Nowadays medicinal plants are sold in processed forms and thus morphological authentication is almost impossible. Full identification requires comparison of the specimen with some authoritative sources, such as a full and accurate description and verification of the species deposited in herbarium. Intake of wrong herbals can cause adverse effects. Identification of both raw materials and end products is therefore needed. METHODS: Here, the potential of a DNA-based identification method, called Bar-HRM (DNA barcoding coupled with High Resolution Melting analysis), in raw material species identification is investigated. DNA barcode sequences from five regions (matK, rbcL, trnH-psbA spacer region, trnL and ITS2) of Acanthaceae species were retrieved for in silico analysis. Then the specific primer pairs were used in HRM assay to generate unique melting profiles for each plants species. RESULTS: The method allows identification of samples lacking necessary morphological parts. In silico analyses of all five selected regions suggested that ITS2 is the most suitable marker for Bar-HRM in this study. The HRM analysis on dried samples of 16 Acanthaceae medicinal species was then performed using primer pair derived from ITS2 region. 100% discrimination of the tested samples at both genus and species level was observed. However, two samples documented as Clinacanthus nutans and Clinacanthus siamensis were recognised as the same species from the HRM analysis. Further investigation reveals that C. siamensis is now accepted as C. nutans. CONCLUSIONS: The results here proved that Bar-HRM is a promising technique in species identification of the studied medicinal plants in Acanthaceae. In addition, molecular biological data is currently used in plant taxonomy and increasingly popular in recent years. Here, DNA barcode sequence data should be incorporated with morphological characters in the species identification.