A chromosome-level genome assembly for Dracaena cochinchinensis reveals the molecular basis of its longevity and formation of dragon's blood.

Dracaena, a remarkably long-lived and slowly maturing species of plant, is world famous for its ability to produce dragon's blood, a precious traditional medicine used by different cultures since ancient times. However, there is no detailed and high-quality genome available for this species at present; thus, the molecular mechanisms that underlie its important traits are largely unknown. These factors seriously limit the protection and regeneration of this rare and endangered plant resource. Here, we sequenced and assembled the genome of Dracaena cochinchinensis at the chromosome level. The D. cochinchinensis genome covers 1.21 Gb with a scaffold N50 of 50.06 Mb and encodes 31 619 predicted protein-coding genes. Analysis showed that D. cochinchinensis has undergone two whole-genome duplications and two bursts of long terminal repeat insertions. The expansion of two gene classes, cis-zeatin O-glucosyltransferase and small auxin upregulated RNA, were found to account for its longevity and slow growth. Two transcription factors (bHLH and MYB) were found to be core regulators of the flavonoid biosynthesis pathway, and reactive oxygen species were identified as the specific signaling molecules responsible for the injury-induced formation of dragon's blood. Our study provides high-quality genomic information relating to D. cochinchinensis and significant insight into the molecular mechanisms responsible for its longevity and formation of dragon's blood. These findings will facilitate resource protection and sustainable utilization of Dracaena.

Genetic diversity of native populations of Croton tetradenius Baill. using ISSR markers.

Brazil has about 300 Croton species in different types of vegetation. Croton tetradenius Baill., which is endemic to the Northeast region and predominant in the Caatinga vegetation, stands out among the several species of this genus. Considering the importance of knowing the genetic variability of a species, the objective of this study was to analyze the genetic diversity of the genotypes of natural populations of C. tetradenius in the State of Sergipe, using ISSR molecular markers. Forty individuals were sampled in four natural populations of the State of Sergipe, Brazil. Thirteen primers were used for DNA amplification using ISSR-PCR, totaling 77 amplified fragments, of which 94.8% were polymorphic. Results of the cluster analysis obtained by the Jaccard's similarity index, using the UPGMA method, resulted in the formation of six distinct clusters. Analysis of molecular variance (AMOVA), used to estimate the genetic variability among populations, revealed significant genetic variance (P < 0.01) between and within the studied populations, and most of the genetic diversity was found (87%) within populations. According to the Jaccard's similarity index, none of the studied plants was genetically identical. CTE210 and CTE305 presented high similarity index (0.76), while CTE105 presented low similarity index (<0.16) with all related individuals. ISSR markers were efficient and allowed the formation of a molecular profile, and had sufficient polymorphism to estimate the genetic variability between the accessions of the studied populations.

Genetic diversity analysis of Croton antisyphiliticus Mart. using AFLP molecular markers.

Croton antisyphiliticus Mart. is a medicinal plant native to Cerrado vegetation in Brazil, and it is popularly used to treat urogenital tract infections. The objective of the present study was to assess the genetic variability of natural C. antisyphiliticus populations using AFLP molecular markers. Accessions were collected in the states of Minas Gerais, São Paulo, and Goiás. The genotyping of individuals was performed using a LI-COR® DNA Analyzer 4300. The variability within populations was found to be greater than the variability between them. The F(ST) value was 0.3830, which indicated that the populations were highly structured. A higher percentage of polymorphic loci (92.16%) and greater genetic diversity were found in the population accessions from Pratinha-MG. Gene flow was considered restricted (N(m) = 1.18), and there was no correlation between genetic and geographic distances. The populations of C. antisyphiliticus exhibited an island-model structure, which demonstrates the vulnerability of the species.

Refining DNA Barcoding Coupled High Resolution Melting for Discrimination of 12 Closely Related Croton Species.

DNA barcoding coupled high resolution melting (Bar-HRM) is an emerging method for species discrimination based on DNA dissociation kinetics. The aim of this work was to evaluate the suitability of different primer sets, derived from selected DNA regions, for Bar-HRM analysis of species in Croton (Euphorbiaceae), one of the largest genera of plants with over 1,200 species. Seven primer pairs were evaluated (matK, rbcL1, rbcL2, rbcL3, rpoC, trnL and ITS1) from four plastid regions, matK, rbcL, rpoC, and trnL, and the nuclear ribosomal marker ITS1. The primer pair derived from the ITS1 region was the single most effective region for the identification of the tested species, whereas the rbcL1 primer pair gave the lowest resolution. It was observed that the ITS1 barcode was the most useful DNA barcoding region overall for species discrimination out of all of the regions and primers assessed. Our Bar-HRM results here also provide further support for the hypothesis that both sequence and base composition affect DNA duplex stability.

Functional expression of a putative geraniol 8-hydroxylase by reconstitution of bacterially expressed plant CYP76F45 and NADPH-cytochrome P450 reductase CPR I from Croton stellatopilosus Ohba.

While attempting to isolate the enzyme geranylgeraniol 18-hydroxylase, which is involved in plaunotol biosynthesis in Croton stellatopilosus (Cs), the cDNAs for a cytochrome P450 monooxygenase(designated as CYP76F45) and an NADPH-cytochrome P450 reductase (designated as CPR I based on its classification) were isolated from the leaf. The CYP76F45 and CsCPR I genes have open reading frames (ORFs) encoding 507- and 711-amino acid proteins with predicted relative molecular weights of 56.7 and 79.0 kDa,respectively. Amino acid sequence comparison showed that both CYP76F45 (63­73%) and CsCPR I (79­83%) share relatively high sequence identities with homologous proteins in other plant species.Phylogenetic tree analysis confirmed that CYP76F45 belongs to the CYP76 family and that CsCPR I belongs to Class I of dicotyledonous CPRs, with both being closely related to Ricinus communis genes. Functional characterization of both enzymes, each expressed separately in Escherichia coli as recombinant proteins,showed that only simultaneous incubation of the membrane bound proteins with the substrate geraniol (GOH) and the coenzyme NADPH could form 8-hydroxygeraniol. The enzyme mixture could also utilize acyclic sesquiterpene farnesol (FOH) with a comparable substrate preference ratio (GOH:FOH) of 54:46. The levelsof the CYP76F45 and CsCPR I transcripts in the shoots, leaves and twigs of C. stellatopilosus were correlated with the levels of a major monoterpenoid indole alkaloid, identified tentatively as 19-Evallesamine,that accumulated in these plant parts. These results suggested that CYP76F45 and CPR I function as the enzyme geraniol-8-hydroxylase (G8H), which is likely to be involved in the biosynthesis of the indole alkaloid in C. stellatopilosus [corrected].

Molecular cloning, bacterial expression and functional characterisation of cytochrome P450 monooxygenase, CYP97C27, and NADPH-cytochrome P450 reductase, CPR I, from Croton stellatopilosus Ohba.

The cDNAs for cytochrome P450 monooxygenase (designated as CYP97C27 by D. Nelson's group) and NADPH-cytochrome P450 reductase (designated as CPR I based on its classification) were isolated from Croton stellatopilosus leaves, which actively biosynthesise plaunotol (18-OH geranylgeraniol). CYP97C27 and CPR I contain open reading frames encoding proteins of 471 and 711 amino acids with predicted molecular masses of 53 and 79kDa, respectively. By aligning the deduced sequences of CYP97C27 and CPR I with other plant species, all functional domains of CYP97C27 (heme and oxygen binding) and CPR I (CYP- and FMN, FAD, and NADPH cofactor binding) were identified. Amino acid sequence comparison indicated that both CYP97C27 (85-93%) and CPR I (79-83%) share high sequence identities with homologous proteins in other plant species, suggesting that CYP97C27 belongs to the CYP97C subfamily and that CPR I belongs to class I of the dicotyledonous CPR. Functional characterisation of both enzymes, produced in Escherichia coli (pET32a/BL21(DE3)) as recombinant proteins, showed that simultaneous incubation of CYP97C27 and CPR I with the substrate geranylgeraniol (GGOH) and coenzyme NADPH led to formation of the product plaunotol. In C. stellatopilosus, the levels of the CYP97C27 and CPR I transcripts were highly correlated with those of several mRNAs involved in the plaunotol biosynthetic pathway, suggesting that CYP97C27 and CPR I are the enzymes that catalyse the last hydroxylation step of the pathway.

RNAi mediated gene silencing against betasatellite associated with Croton yellow vein mosaic begomovirus.

Plant viruses encode suppressors of posttranscriptional gene silencing, an adaptive antiviral defense responses that confines virus infection. Previously, we identified single-stranded DNA satellite (also known as DNA-ß) of ~1,350 nucleotides in length associated with Croton yellow vein mosaic begomovirus (CYVMV) in croton plants. The expression of genes from DNA-ß requires the begomovirus for packaged, replication, insect transmission and movement in plants. The present study demonstrates the effect of the ßC1 gene on the silencing pathway as analysed by using both transgenic systems and transient Agrobacterium tumefaciens based delivery. Plants that carry an intron-hairpin construct covering the ßC1 gene accumulated cognate small-interfering RNAs and remained symptom-free after exposure to CYVMV and its satellite. These results suggest that ßC1 interferes with silencing mechanism.

Comparison of methods for estimates of molecular genetic diversity in genus Croton: influence of coefficients, clustering strategies and data projection.

We investigated 10 similarity (and disimilarity) coefficients in a set of 40 wild genotypes of Croton linearifolius subjected to analyses using hierarchical grouping methods, grouping methods by optimization and data projection in two-dimensional space. Genotypes were characterized by analyzing DNA polymorphism with the use of 15 ISSR and 12 RAPD markers. The distance measurements were compared by the Spearman correlation test, projection in two-dimensional space and grouping efficiency evaluation. The Spearman correlation coefficients between the 10 coefficients evaluated were significant (P < 0.001) and indicated significant changes in genotype ranking due to type of coefficient used (0.76 ≤ rs ≤ 1). Wide variation was also observed in the efficiency of clustering methods, where the unweighted pair group method with arithmetic mean was the most suitable (0.3 ≤ D ≤ 1.5 ; 0.41 ≤ rc ≤ 0.77; 5.99 ≤ S ≤ 12.61). Projection efficiencies in two-dimensional space showed high-stress values (65 < S < 89%). Similar to the results observed for hierarchical clustering methods and for projection in two-dimensional space, the formation of groups with grouping methods by optimization showed variations when using different coefficients. We believe that the results confirm the influence of coefficients in studies of genetic diversity, showing the need to use criteria and standards for selecting appropriate methods for genetic studies of the genus Croton.

Cytogenetics and characterization of microsatellite loci for a South American pioneer tree species, Croton floribundus.

Despite the recent advances in plant population genetic studies, the lack of information regarding pedigree, ploidy level, or mode of inheritance for many polyploids can compromise the analysis of the molecular data produced. The aim of this study was to examine both microsatellite and cytogenetic characteristics of the pioneer tree Croton floribundus Spreng. (Euphorbiaceae) to test for the occurrence of polyploidy in the species and to evaluate its implications for the appropriate use of SSR markers. Seven microsatellite markers were developed and screened for 62 individuals from a semi-deciduous tropical forest in Brazil. Chromosome number, meiotic behavior, and pollen viability were evaluated from male flower buds. All SSR loci were highly polymorphic. The number of bivalents observed in meiosis n = 56 (2n = 8× = 112) and the maximum number of alleles per individual (Ni = 8) demonstrated the occurrence of polyploidy in C. floribundus. The normal meiotic pairing and the high pollen viability suggested that C. floribundus is a regular and stable polyploid, most likely an allopolyploid. The combined SSR and cytogenetic data provided new evidence on the origin and evolution of the species as well as assured the accurate use of SSR loci for population genetic studies of the polyploid pioneer species.

Molecular cloning and catalytic activity of a membrane-bound prenyl diphosphate phosphatase from Croton stellatopilosus Ohba.

Geranylgeraniol (GGOH), a bioactive acyclic diterpene with apoptotic induction activity, is the immediate precursor of the commercial anti-peptic, plaunotol (18-hydroxy geranylgeraniol), which is found in Croton stellatopilosus (Ohba). From this plant, a cDNA encoding a prenyl diphosphate phosphatase (CsPDP), which catalyses the dephosphorylation of geranylgeranyl diphosphate (GGPP) to GGOH, was isolated using a PCR approach. The full-length cDNA contained 888bp and encoded a 33.6 kDa protein (295 amino acids) that was phylogenetically grouped into the phosphatidic acid phosphatase (PAP) enzyme family. The deduced amino acid sequence showed 6 hydrophobic transmembrane regions with 57-85% homology to the sequences of other plant PAPs. The recombinant CsPDP and its 4 truncated constructs exhibited decreasing dephosphorylation activities relative to the lengths of the N-terminal deletions. While the full-length CsPDP successfully performed the two sequential monodephosphorylation steps on GGPP to form GGOH, the larger N-terminal deletion in the truncated enzymes appeared to specifically decrease the catalytic efficiency of the second monodephosphorylation step. The information presented here on the CsPDP cDNA and factors affecting the dephosphorylation activity of its recombinant protein may eventually lead to the discovery of the specific GGPP phosphatase gene and enzyme that are involved in the formation of GGOH in the biosynthetic pathway of plaunotol in C. stellatopilosus.

Molecular phylogenetics and character evolution of the "sacaca" clade: novel relationships of Croton section Cleodora (Euphorbiaceae).

Phylogenetic relationships of Croton section Cleodora (Klotzsch) Baill. were evaluated using the nuclear ribosomal ITS and the chloroplast trnL-F and trnH-psbA regions. Our results show a strongly supported clade containing most previously recognized section Cleodora species, plus some other species morphologically similar to them. Two morphological synapomorphies that support section Cleodora as a clade include pistillate flowers in which the sepals overlap to some degree, and styles that are connate at the base to varying degrees. The evolution of vegetative and floral characters that have previously been relied on for taxonomic decisions within this group are evaluated in light of the phylogenetic hypotheses. Within section Cleodora there are two well-supported clades, which are proposed here as subsections (subsection Sphaerogyni and subsection Spruceani). The resulting phylogenetic hypothesis identifies the closest relatives of the medicinally important and essential oil-rich Croton cajucara Benth. as candidates for future screening in phytochemical and pharmacological studies.

Genetic variation of Croton stellatopilosus Ohba based on non-coding DNA sequences of ITS, trnK and trnL-F regions.

Croton stellatopilosus Ohba (Plau-noi), a well-known Thai medicinal plant, was investigated for its genetic variation by analyzing three DNA regions, one nuclear internal transcribed spacer (ITS) region and two chloroplast trnL-F intergenic spacer and trnK intron regions. The results of ITS sequencing from 30 leaf samples showed that there were two major genotypes of C. stellatopilosus which were designated as STEL Type A and B. In addition, various nucleotide additive sequences which had presumably arisen from these two groups were also found. These so-called "putative hybrids", interestingly, displayed trnK intron sequences identical to the STEL Type B but different from the Type A. For the trnL-F region, all the 30 samples showed identical sequences. Thus, it was suggested that in the hybridization of C. stellatopilosus, the Type A genotype acts as paternal parent whereas the Type B genotype acts as maternal parent. In addition, all C. stellatopilosus samples were analyzed for their plaunotol content using TLC densitometry. We found that the Type A genotype, hybrid group and Type B genotype had plaunotol content in the ranges 0.209-0.492, 0.319-0.896 and 0.442-1.000% (w/w) dry weight, respectively. The results indicated that there is a correlation between the plaunotol contents and non-coding DNA sequences of ITS, trnK and trnL-F regions of C. stellatopilosus.

Cytotoxic 3,4-seco-Atisane Diterpenoids from Croton barorum and Croton goudotii.

In a screening program directed to the discovery of new anticancer agents from Madagascan plants, ethyl acetate extracts of Croton barorum and C. goudotii showed strong cytotoxic activity, with 100% inhibition at 10 µg/mL in a primary screen using the murine lymphocytic leukemia P388 cell line. Bioassay-guided fractionation led to the isolation of two new 3,4-seco-atisane diterpenoids, crotobarin (1) and crotogoudin (2). Their structures were elucidated by spectroscopic data interpretation. Compounds 1 and 2 produced a net progression in the number of cells arrested at the G2/M growth stage in the cell cycle of the K562 human leukemia cell line at 4 µM.

Cloning and expression of 1-deoxy-d-xylulose 5-phosphate synthase cDNA from Croton stellatopilosus and expression of 2C-methyl-d-erythritol 4-phosphate synthase and geranylgeranyl diphosphate synthase, key enzymes of plaunotol biosynthesis.

1-Deoxy-d-xylulose 5-phosphate synthase (DXS, EC: 4.1.3.37), the first enzyme in the 2C-methyl-d-erythritol 4-phosphate (MEP) pathway, is known to be responsible for the rate-limiting step of isoprenoid biosynthesis in Escherichia coli and Arabidopsis thaliana. In this study, the dxs gene from Croton stellatopilosus, designated csdxs, was cloned from leaf tissue using the rapid amplification of cDNA ends (RACE) technique. Leaves of C. stellatopilosus contain plaunotol, an acyclic diterpene alcohol. The csdxs cDNA containing the open reading frame of 2163 base pairs appeared to encode a polypeptide of 720 amino acids. Analysis of the deduced amino acid sequence revealed that the NH(2)-terminus of CSDXS carried a chloroplast transit peptide, a thiamine diphosphate binding site, and a transketolase motif, which are the important characteristics of DXS enzymes in higher plants. Multiple alignments of CSDXS with other plant DXSs have indicated that CSDXS has identity ranging between 68% and 89%. Expression levels of csdxs and genes encoding key enzymes in the plaunotol biosynthetic pathway, namely 2C-methyl-d-erythritol 4-phosphate synthase (meps) and geranylgeranyl diphosphate synthase (ggpps), were analysed by measuring transcript levels in leaves of different developmental stages. The results showed that dxs, meps, and ggpps are all active in young leaves prior to full expansion when plaunotol is synthesised from the DXP precursor in chloroplasts. The dense presence of chloroplasts and oil globules in the palisade cells of these leaves support the view that these genes are involved in plaunotol biosynthesis in chloroplast-containing tissues.

Revisão taxonômica de Croton sect. Lamprocroton (Müll. Arg.) Pax (Euphorbiaceae s.s.) / Taxonomic revision of Croton sect. Lamprocroton (Müll. Arg.) Pax (Euphorbiaceae s.s.)

Croton is the second bigger and more diverse genus in the family Euphorbiaceae, with about 1,200 species distributed in 40 sections, occurring in all tropical areas, most of them in Americas. In South America, Brazil is the country in which a larger number of taxa are found, ca. 356. According to recent classification, the genus belongs to the tribe Crotoneae, and despite the wide and morphological diversity, it would be a monophyletic taxon. However, a phylogenetic analysis using markers of ITS region from nuclear ribosomal DNA, and of trnL-F from plastidial DNA, showed that Croton, like traditionally circumscribed, is not a monophyletic taxon. A taxonomic revision of Croton section Lamprocroton (Müll. Arg.) Pax is presented here. It is a Neotropical group with most of its species occurring from Southeast and South Brazil to southern South America (Uruguay and Argentina). Morphologically, the members of Lamprocroton are characterized as monoecious or dioecious shrubs or subshrubs, with a lepidote indumentum at least in part of foliage, entire leaves with no glands. The staminate flowers have 9 to 16 stamens and the pistillate flowers may have equal or unequal sepals, reduced to absent petals, and styles once or twice bifid. Overall, are recognized 26 species in the group, three of them new to the science. Identification key, morphological descriptions, illustrations, phenological period, as well as data on geographic distribution and general comments of each species are presented. Four taxa were excluded from C. sect. Lamprocroton because they do not show the morphological features that are diagnostics of the section. Four species that are poorly known were not included in the taxonomic treatment.

O gênero Croton L. é o segundo maior e mais diverso da família Euphorbiaceae, possuindo cerca de 1.200 espécies, agrupadas em 40 seções, com distribuição pantropical, das quais a maioria ocorre nas Américas. Na América o Sul, o Brasil é o país que congrega o maior número de espécies, aproximadamente 356. De acordo com a mais recente classificação, o gênero pertence à tribo Crotoneae e, apesar do grande número de espécies e da grande diversidade morfológica, seria um táxon monofilético. Entretanto, uma análise filogenética recente, que utilizou dados moleculares das regiões ITS, do DNA nuclear ribossômico, e do fragmento trnL-F, do DNA plastidial, demonstrou que Croton, como tradicionalmente circunscrito, não é um táxon monofilético. Neste trabalho, é apresentada uma revisão taxonômica de Croton sect. Lamprocroton (Müll. Arg.) Pax. Trata-se de um grupo neotropical com a maioria das espécies ocorrendo nas regiões Sudeste e Sul do Brasil e sul da América do Sul. Seus representantes caracterizam-se por ser plantas arbustivas ou subarbustivas, monóicas ou dióicas, com indumento lepidoto presente em pelo menos parte da folhagem e folhas inteiras e sem glândulas. As flores estaminadas possuem androceu composto por 9 a 16 estames e as flores pistiladas apresentam sépalas iguais ou desiguais no tamanho, pétalas reduzidas ou ausentes e estiletes uma ou duas vezes bífidos. Neste trabalho são reconhecidas 26 espécies na seção sendo três novas para a ciência. Chave de identificação, descrições morfológicas, ilustrações, período fenológico, distribuição geográfica e comentários gerais de cada uma das espécies são apresentados. Quatro táxons foram excluídos de C. sect. Lamprocroton por não possuírem os caracteres morfológicos diagnósticos da seção. Quatro espécies não foram incluídas no tratamento taxonômico por falta de informação sobre as mesmas.