ISOPENTENYLTRANSFERASE (IPT) genes play important roles in the initial steps of cytokinin synthesis, exist in plant and pathogenic bacteria, and form a multigene family in plants. Protein domain searches revealed that bacteria and plant IPT proteins were to assigned to different protein domains families in the Pfam database, namely Pfam IPT (IPTPfam) and Pfam IPPT (IPPTPfam) families, both are closely related in the P-loop NTPase clan. To understand the origin and evolution of the genes, a species matrix was assembled across the tree of life and intensively in plant lineages. The IPTPfam domain was only found in few bacteria lineages, whereas IPPTPfam is common except in Archaea and Mycoplasma bacteria. The bacterial IPPTPfam domain miaA genes were shown as ancestral of eukaryotic IPPTPfam domain genes. Plant IPTs diversified into class I, class II tRNA-IPTs, and Adenosine-phosphate IPTs; the class I tRNA-IPTs appeared to represent direct successors of miaA genes were found in all plant genomes, whereas class II tRNA-IPTs originated from eukaryotic genes, and were found in prasinophyte algae and in euphyllophytes. Adenosine-phosphate IPTs were only found in angiosperms. Gene duplications resulted in gene redundancies with ubiquitous expression or diversification in expression. In conclusion, it is shown that IPT genes have a complex history prior to the protein family split, and might have experienced losses or HGTs, and gene duplications that are to be likely correlated with the rise in morphological complexity involved in fine tuning cytokinin production. Agrobacterium tumefaciens , the cyscoideum .IPT genes were first identified in A. tumefaciens [Arabidopsis thaliana [IPT genes were identified in the genome [IPTs have been studied in several angiosperms and mosses  and tRNA-IPTs [Agrobacterium tumefaciens also retaining AP-IPT, preferentially uses AMP whereas those in plants prefer ATP and ADP as substrates [efaciens , 10 and thaliana , 12, aftthaliana , and nine genome . To datethaliana ; Oryza sa sativa ; Physcoma patens ; Solanumpersicum ), and wepersicum , 20, 21.RNA-IPTs . Agrobacbstrates .IPT genes into five groups: `bacterial adenylate IPTs`, `plant adenylate IPTs`, `eukaryotic origin plant tRNA IPTs`, `bacterial tRNA IPTs`, and `prokaryotic origin plant tRNA IPTs`, based on an unrooted gene tree reconstructed from full sequence lengths, where members of two plant families  were included. Lindner et al. [IPTs into `class I tRNA-IPTs`, `class II tRNA-IPTs`and `adenylate-IPTs`, and bacteria IPTs into `bacterial tRNA-IPTs`and `bacterial AMP-IPTs`, using a midpoint rooted Bayesian inference tree. The cytokinin synthesizing genes of the bacteria A. tumefaciens and the slime-mold D. discoideum were found to belong to the AMP-IPT clade and were separated from plant IPT clades in Lindner et al. [IPTs are closely related to bacteria tRNA-IPTs, and class II tRNA-IPTs to adenylate-IPTs [In previous studies, Frébort et al.  classifir et al.  carried r et al. . The autate-IPTs .IPTs was not fully explained in the two studies since the phylogenetic trees were unrooted, and the direction of evolution as well as the origin of the gene family unexplored. A further complication might have been that the full sequence and protein sequences between the different groups of IPTs are highly divergent and their alignment might have included ambiguous alignment information, obscuring the phylogenetic signal [The two different classifications by Frébort et al.  and Lindc signal .IPTs, to infer the deep origin and evolution of this gene family. The conserved protein domain of IPT genes across kingdoms were assembled with a focus on plants and the matrix included 37 plants (of 21 families), three animals, two fungi, one amoeba, and one zooplankton species, selected across the evolutionary breadth of the tree of life [IPT genes in plants in a phylogenetic context.Therefore, this study focused on the conserved protein domain of the  of life , 25, 26.IPT genes were retrieved from publicly accessible genome or transcriptome databases. The list of species analysed and the databases used in this study are listed in IPTs are mostly single-domain proteins and retain either IPTPfam (Pfam family IPT) or IPPTPfam (Pfam family IPPT) domains, these were searched across kingdoms, including Archaea, bacteria, plants, yeast, animals, and slime-molds -isopentenylpyrophosphate transferases and modify tRNA to stabilize codon recognition in a wide range of lineages . They use AMP/ADP/ATP as substrates and contribute to cytokinin synthesis in plants [E < 0.1) using IPPTPfam and IPTPfam domains from A. thaliana and A. tumefaciens. Sequence matches were re-evaluated in Pfam searches, and only gene sequences clearly showing IPPTPfam and IPTPfam domain sequences were used for this study (Domain searches were carried out using deduced amino acid sequences in Pfam v.31.0 . Since Ime-molds . Proteinis study .Pfam (PF01745) and IPPTPfam (PF01715) protein domain families, were analysed. This clan included 217 protein domain families in Pfam v.31.0, and their Hidden Markov Model (HMM) profiles were downloaded from the Pfam website. HMM profiles estimate the true frequency of protein residues from the observed frequency by a Markov process with hidden status [The protein families in the clan P-loop NTPase (CL0023), including IPTn status . The HMMn status .Pfam (PF01715) and IPPTPfam (PF01745) domain sequences were analysed using VirEPfam (PF05272) domain sequences as outgroup to focus on the phylogenetic relationship between IPTPfam and IPPTPfam. Sequences in the seed alignments of the three families were combined into a matrix. The seed alignment of IPTPfam contains seven, and that of VirEPfam six sequences and all were used in the analyses. The IPPTPfam seed alignment is large and contains 1247 sequences, and only representative sequences were selected for the analyses: to select sequences, preliminary phylogenetic analyses were carried out on the IPPTPfam seed alignment using all sequences. Hypervariable regions of the original IPPTPfam seed alignment were trimmed with BMGE v.1.12 [Pfam seed alignment (162 sequences), IPTPfam seed alignment (7 sequences), and VirEPfam seed alignment (6 sequences), were combined with the MAFFT-merge subprogram in MAFFT v.7 [Pfam sequences. For branch support, values of an approximate likelihood ratio test with non-parametric branch support based on a Shimodaira-Hasegawa-like procedure (αLRT SH-like support) were estimated using PhyML. Additionally, an ultrafast bootstrap (UFBT) analysis of 1,000 replicates was carried out in W-IQ-TREE [Following the topology of the pHMM-tree, IPTE v.1.12 , and a pE v.1.12 , and 162AFFT v.7 , and theAFFT v.7 . An ML tAFFT v.7  with SmaAFFT v.7  with the-IQ-TREE .Pfam original seed alignment with 1247 sequences was reduced to 103 representative sequences as described above. To confirm the similarity between the original (1247 sequences) and the representative sequences, HMM profiles were built for the 1247 and 103 sequences respectively, with hmmerbuild in HMMER v.3.0 [Pfam HMM , for IPTPfam from Agrobacterium tumefaciens (PDBsum accession number: 2ze5) and for IPPTPfam from Escherichia coli (3foz) as references.The IPPTER v.3.0 , and HMMER v.3.0  and the Pfam HMM . A new HPfam domains were found in species in a few plant families in the Pfam database . Those plant IPTPfam domain genes registered in Pfam were retrieved and assessed with hmmersearch in HMMER v.3.0, which compares the protein sequences with IPPTPfam.hmm and IPTPfam.hmm from Pfam, and IPPTPfam_N40.hmm built in this study, to examine the similarities between the domain sequences and the HMM profiles.Fragmental IPTPfam domains in the database. The matrix was assembled with plant IPTPfam domain genes together with the bacterial IPTPfam domain genes, the bacterial IPPTPfam genes (miaA), and IPPTPfam genes from P. patens, A. thaliana, O. sativa, S. lycopersicum, S. tuberosum, M. acuminata. The IPPTPfam and IPTPfam domain sequences were first aligned separately using the hmmeralign in the HMMER v.3.0 with IPTPfam.hmm or IPPTPfam_N40.hmm. The two alignments were merged using MAFFT merge v.7 [In addition, a phylogenetic analysis was carried out with plant genes registered under IPTerge v.7  and trimerge v.7 . The WAGerge v.7  using Prerge v.7 , and an erge v.7 .Pfam and IPTPfam domain genes in bacteria and slime-mold, a species tree based on Battistuzzi et al. [http://tree.bio.ed.ac.uk/software/figtree/).To show the presence or absence of IPPTi et al.  and Tomii et al.  was genei et al.  and FigTPfam and IPTPfam domain sequences of bacteria, slime-mold and yeast was build alongside the species tree generated above. IPPTPfam and IPTPfam domain sequences were aligned separately using hmmeralign with IPPTPfam_N40.hmm or IPTPfam.hmm. The two alignments were merged using MAFFT merge v.7 and trimmed using Gblocks [The phylogenetic tree of IPPT Gblocks . The LG  Gblocks .Pfam domain gene tree, IPPTPfam domain genes were retrieved from genome databases from algae to angiosperms. bacteria, animals, yeast, slime-molds, and zooplankton genes were also included in the analyses  analyses were carried. Gene subtrees containing plant IPTs were reconciled and rearranged with plant species trees separately for class I tRNA IPTs and class II tRNA-IPT/AP-IPTs , and a Intron-exon structures were also examined, by interrogating databases and comparing genome and transcribed sequences. The number of nucleotides in exons and introns were determined and schematic illustrations based on their number, size and position drawn with GSDS v.2.0 .IPT genes. For interspecific comparisons, the expression data were categorised into root, leaf, flower, and fruit. For mosses, the protonema, mature gametophytic stage, and sporophytic stage were reported and these categories were used here. The literature used in this study regarding gene expression patterns are summarized in Literature searches were carried out to obtain an overview of gene expression patterns in relation to the duplication history of The accession numbers of the sequences used in this study are listed in http://purl.org/phylo/treebase/phylows/study/TB2:S22409). The files include a FastTree inferred approximately-ML tree of the IPPTPfam domain seed alignment with 1247 sequences (M46567), a IPPTPfam_N40.hmm new seed alignment with 103 sequences (M46568), the IPPTPfam/IPTPfam/VirEPfam merged matrix (with 175 sequences) and tree shown in Pfam domain matrix (with 101 sequences) and tree file shown in Pfam/IPPTPfam domain matrix (with 64 sequences) and tree shown in Pfam/IPPTPfam domain matrix (with 215 sequences) and tree across kingdoms shown in The matrixes and tree files used in this study are deposited in TreeBASE  are single domain proteins of about 250–460 amino acid (AA) length  length . We founm domain . Thus, cPfam has a 228 AA long Hidden Markov Model (HMM) profile, and AP-IPT in A. thaliana has a truncated IPPTPfam domain lacking ca. 75–140 AA of the IPPTPfam HMM profile, while those of tRNA-IPT have almost the full length of the IPPTPfam HMM profile  in the Pfam database v.31.0 [Pfam and IPTPfam HMM profiles are closely related and appear as sisters in the Neighbor Joining tree  and were highly supported sister to each other , suggesting that the origin of IPPTPfam and IPTPfam proteins could be traced back to before the emergence of the protein families , and not in the IPTPfam domain gene clade  . IPTPfam domain genes were only found in a few species: in Proteobacteria  they formed a clade , and with Cyanobacteria (Cy) and Actinobacteria (Ac) in sister grades (D. discoideum (amoeba: Am) was also assigned to the IPTPfam domain clade.Across bacteria, T = 100) , S7 Fig.r grades , S4 Fig.IPTs in the plant species examined here all retained IPPTPfam domains , the IPPTPfam domain genes formed a maximally supported clade  and could be divided into two grades and four clades with mostly high branch support  of 48, and suggested 18 duplications and 21 losses. Rearranging the tree topology around poorly supported branches resulted in a greatly reduced DL score of 23.5, with 13 duplications and 4 losses  contains 16,712 protein families and 604 clans. Each family is based on the manually curated seed-alignment of protein domains and thus each has a unique Hidden Markov Model (HMM) profile. A Pfam clan is a structural unit of families that share a related structure, function, and significantly matching HMM profile, suggesting that they have a single evolutionary origin , 58. Them family .Pfam domain genes are only found in a few bacteria, whereas IPPTPfam domain genes are found in most organisms except the Archaea and Mycoplasma lineages. It is unclear whether IPPTPfam is lost in Archaea or gained in bacteria since the relationships between the two groups are still unclear . It appar (e.g. ). The Fines here .Pfam domain genes are phylogenetically scattered and found only in some members of Actinobacteria, Cyanobacteria, ɑ-Proteobacteria, β-Proteobacteria, and γ-Proteobacteria and in the eukaryote D. discoideum. The IPTPfam domain clade showed long branches and its topology was mostly congruent with the species tree. One could hypothesize that they were present in the ancestor of bacteria, and as a result of a strong evolutionary selection only the plant pathogenic lineages retained the IPTPfam domain genes, perhaps because of the importance of cytokinins in plant pathogenicity . Howeveorganism .IPTs appear to be only distantly related to plant IPTs. Plant IPPTPfam domain IPTs were found indeed closer related to bacteria IPPTPfam domain miaA genes that however, do not synthesise cytokinins  are still unresolved which somewhat hampers the clarification of the origin of IPT genes as well as the limited sampling in non-plant lineages in this study. However, based on the distribution of the genes among lineages  and SAR , bryophytes, and lycophytes retained class II tRNA-IPTs and AP-IPTs . The time of acquisition of a second set of tRNA-IPTs in euphyllophytes was estimated to around 411 MYA, sometime after the emergence of land plants [IPT and AP-IPT. The latter was apparently lost in monilophytes and gymnosperms . Consides (e.g. . It can es (e.g. ). A uniqytokinin . Howevern Poales , 74 and n Poales . It mighISOPENTENYLTRANSFERASEs, key genes for the production of cytokinins, have been studied intensively over the last two decades. The accumulating genome knowledge of model and non-model plants and an accompanying advancement in statistical analytical methodology applied here allowed us to reveal the phylogenetic origin and evolution of these genes across the tree of life. This study revealed that plant IPTs are closely related to bacteria miaA genes (IPPTPfam) and not to bacteria IPT genes (IPTPfam). Further, plants possess two independent IPTs, class I tRNA-IPTs and class II tRNA-IPT/AP-IPTs. Their exact deep origin could not be fully resolved due to uncertain relationships in basal eukaryotes. However, class II tRNA-IPTs and AP-IPTs are the consequence of a gene duplication event at the onset of euphyllophyte diversification. Further gene duplication events in the plant lineage were inferred with increasing frequency towards angiosperms, coinciding with emerging increased specialisation of functions. This study is an example for the elucidation of the deep history of cytokinin synthesis genes that involved an interplay of possible horizontal gene transfers, gene duplications, losses and diversification in function in the evolution of a multigene family.The roles and functions of S1 FigDomains are shown as green boxes. Coordinates to the Pfam HMM profiles are shown below the boxes.(PDF)Click here for additional data file.S2 FigPfam and IPPTPfam families appeared as sister clades (arrow).The tree was calculated by pHMM-tree. IPT(PDF)Click here for additional data file.S3 FigPfam sequences. The αLRT SH-like values (left) and UFBT values (right) are shown on major branches leading to each protein family. Branches with above 70% support values are emphasized by a thick line.The tree is rooted on VirE(PDF)Click here for additional data file.S4 FigPfam domain gene are shown in black, for species with both IPPTPfam and IPTPfam domain genes in orange, and for species lacking IPPTPfam and IPTPfam domain genes in grey.Species retaining the IPPT(PDF)Click here for additional data file.S5 FigPfam.hmm and the original IPPTPfam.hmm (1247 seed seq) were retrieved from Pfam v.31.0. IPPTPfam.hmm (103 seed seq) built with 103 representative out of 1247 seed sequences. Logos build from 1247 sequences in the original seed alignment and the 103 representative sequences were very similar. New HMM profile with extended N-terminus (IPPTPfam_N40.hmm) built in this study. N40: additional N-terminus region in IPPTPfam_N40.hmm.IPT(PDF)Click here for additional data file.S6 FigPfam family of Pfam indicated by red arrows, and those shown in the IPPTPfam domain clade but not in the IPTPfam clade (αLRT SH-like = 1).Plant sequences in IPT(PDF)Click here for additional data file.S7 FigThe αLRT (left) and UFBT values (right) are shown along major branches. An asterisk indicates support values < 0.5 and < 50%. Thickened branches indicate support values > 0.9 and > 90%, medium-thick branches indicate > 0.7 and > 70%. The classification of the species is indicated by two characters at the end of the gene names; Ac: Actinobacteria, Al: α-Proteobacteria, Am: Amoebozoa, Aq: Aquficae, As: Ascomycota, Be: β-Proteobacteria, Ch: Chlamydiae, Cy: Cyanobacteria, Ep: ε-Proteobacteria, Fi: Firmicutes, Fu: Fusobacteria, Ga: γ-Proteobacteria, Sp: Spirochaetes, Th: Thermotogae.(PDF)Click here for additional data file.S8 FigPfam domain genes were used as outgroup. The αLRT (left) and UFBT support values (right), are shown along the major branches. An asterisk indicates support values < 0.5 and < 50%. Thickened branches indicate support values > 0.9 and > 90%, medium-thick branches indicate > 0.7 and > 70%.IPT(PDF)Click here for additional data file.S9 Fig(PDF)Click here for additional data file.S10 Fig(PDF)Click here for additional data file.S11 FigWeak edges highlighted yellow. Gene duplications marked by red ‘D’.(PDF)Click here for additional data file.S12 Fig(PDF)Click here for additional data file.S13 Fig(PDF)Click here for additional data file.S14 FigWeak edges highlighted yellow. Gene duplication marked by red ‘D’.(PDF)Click here for additional data file.S15 FigThe tree is a cladogram of the tree shown in (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 TableGene ID is the ID used in the large phylogeny in (PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.


Tengella perfuga, a spider that spins sheet webs lined with cribellar silk. Cribellar silk is a type of adhesive capture thread composed of numerous fibrils that originate from a specialized plate-like spinning organ called the cribellum. The predominant components of spider silks are spidroins, members of a protein family synthesized in silk glands. Here, we use silk gland RNA-Seq and cDNA libraries to infer T. perfuga silks at the protein level. We show that T. perfuga spiders express 13 silk transcripts representing at least five categories of spider silk proteins (spidroins). One category is a candidate for cribellar silk and is thus named cribellar spidroin (CrSp). Studies of ontogenetic changes in web construction and spigot morphology in T. perfuga have documented that after sexual maturation, T. perfuga females continue to make capture webs but males halt web maintenance and cease spinning cribellar silk. Consistent with these observations, our candidate CrSp was expressed only in females. The other four spidroin categories correspond to paralogs of aciniform, ampullate, pyriform, and tubuliform spidroins. These spidroins are associated with egg sac and web construction. Except for the tubuliform spidroin, the spidroins from T. perfuga contain novel combinations of amino acid sequence motifs that have not been observed before in these spidroin types. Characterization of T. perfuga silk genes, particularly CrSp, expand the diversity of the spidroin family and inspire new structure/function hypotheses.Most spiders spin multiple types of silk, including silks for reproduction, prey capture, and draglines. Spiders are a megadiverse group and the majority of spider silks remain uncharacterized. For example, nothing is known about the silk molecules of  Tengella perfuga, Dahl 1901 (Zoropsidae) uses copious amounts of silk to build sheet webs with deep retreats in high elevation remnant cloud forest habitats in Nicaragua [T. perfuga spiders belong to the RTA  clade, which diverged approximately 191–247 million years ago from orb-web weaving spiders, and are cribellate spiders [Spiders are widely distributed and abundant in most terrestrial communities, and their evolutionary success is partly associated with diversification of silk usage –3. Silk icaragua ,6. T. pe spiders . Cribell spiders . Cribell spiders –15. To a spiders ,14,16. C spiders ,18. Crib spiders .Silk production in spiders involves a combination of highly specialized genes, structures, and behaviors. Spider silk genes are expressed in silk glands, which are located in the abdomen. Spider silk genes encode proteins known as spidroins (spidroin is a contraction of spider fibroin ), and thT. perfuga spiders, with their large body size and ease of rearing in captivity, provide an opportunity to investigate the genetics of cribellar silk [T. perfuga has recently been examined [T. perfuga spiders use silk for foraging, building retreats, and constructing egg sacs. Spiderlings make small sheet webs without cribellar silk. As the spiderlings mature, their webs become more complex with the addition of cribellar silk.lar silk ,6. Additexamined . Adult fT. perfuga [T. perfuga, the “modified spigot” produces the axial lines the cribellar silk is combed out on [T. perfuga spigot ontogeny also involve the cribellum. With successive molts, the number of cribellar spigots and size of the cribellum increases as the spiders molt to adulthood, except that males lose their cribellar spigots in the final molt [Cribellar silk fibers fill the sheet of adult female webs, lining the retreat and knockdown lines that extend from the substrate to the sheet. By contrast, after becoming sexually mature, males abandon their webs and adopt a wandering life style. Based on scanning electron microscopy, spigots corresponding to aciniform, cribellate, major ampullate, minor ampullate, pyriform, and tubuliform silk glands have been imaged for  perfuga . Additio perfuga ,28. The  perfuga . In T. pnal molt .T. perfuga. Based on studies of their silk usage and silk spigot ontogeny [T. perfuga will express spidroin genes with orthology to known aciniform, major ampullate, minor ampullate, pyriform, and tubuliform silk genes. If true, then T. perfuga spidroins will group with corresponding orthologous genes in phylogenetic analyses. Additionally, because T. perfuga uses extensive amounts of cribellar silk for capture web construction, we hypothesize that there will be an additional spidroin that is a candidate constituent of cribellar silk. To our knowledge, a cribellar silk spidroin has yet to be described at the molecular level. Because mature males lose the ability to produce cribellar silk, we would expect mature females but not mature males to express this spidroin. Finally, we predict that the main silk genes associated with capture web construction will be highly expressed compared to other spidroin genes because T. perfuga spiders use copious amounts of silk in their capture webs.Here, we use expression libraries to characterize the silk genes of ontogeny ,26,27, woN, 85.9105oW) in May 2012 and 2014 [T. perfuga adult individuals were used for all silk gland dissections. T. perfuga adult individuals were used for all silk gland dissections. Spiders were anesthetized with CO2 and euthanized by separating the cephalothorax from the abdomen. Immediately after euthanization, silk glands were dissected from each individual, flash frozen in liquid nitrogen, and stored at -80°C. From the total silk gland complement of a T. perfuga spider, the following silk glands were identified and dissected based on shape, size, and position: ampullate-shaped, tubuliform-shaped , and an assortment of small silk glands which were close to and left attached to the spinnerets. These small silk glands were presumed to include the cribellate silk glands.All spiders used in this study were part of a lab-reared spider colony. The colony was started with mature spiders collected in Nicaragua , was fully sequenced  using the transposon-based EZ-Tn5 <TET-1> insertion kit (Epicentre). T. perfuga cDNA clones were Sanger sequenced at the University of California Riverside (UCR) Genomics Core Facility.Using the ampullate-shaped silk glands and all the smaller silk glands from mature male and female spiders; and the tubuliform silk glands from mature females, silk gland type-specific plasmid-based cDNA libraries were constructed and screened following the methods described in Garb et al. . The libpidroins ,32. To dT. perfuga females raised by R. Alfaro. The glands were flash frozen in liquid nitrogen and stored at -80°C. Separate RNA extractions were done for the total set of silk glands from each individual spider following the methods of Starrett et al. [The total set of silk glands was dissected from each of two t et al. . In short et al.  with theT. perfuga reads were combined to assemble a de novo female silk gland transcriptome with Trinity v2.1.1 using default parameters [T. perfuga assembly was approximated using N50 and completeness determined by comparison to the arthropod v9 set of Universal Single-Copy Orthologs .Raw sequencing reads from each FASTQ file were processed by clipping the adaptors and removing low quality reads with Trimmomatic . Qualityrameters . See S1 O v 3.0; ). 96.8% BLASTX searches  to both NCBI nr and UniProtKB were used to automatically annotate transcripts . PutativSpidroin gene family members identified from the automatic annotation were further examined with additional BLASTX searches  against a protein database with spidroin genes downloaded from NCBI nr proteins and UniProtKB/Swiss-Prot databases (September 2016) in Geneious v8.1.8 . Visual T. perfuga spidroin contigs with published spidroin sequences from araneomorph (true spider) spiders , was used to root each analysis (GenBank accessions EU117162 and HM752562). Amino acid alignments were done with MUSCLE [http://tree.bio.ed.ac.uk/software/Fig.tree/).Phylogenetic analyses of spidroin family members were done by aligning the N- and C-terminal region translations of  spiders . A spidrh MUSCLE  as impleh MUSCLE . JTT andT. perfuga silk glands were quantified by mapping filtered sequencing reads from T. perfuga RNA-Seq libraries  to our female T. perfuga transcriptome using TopHat2 v2.1.1 with default parameters [The relative levels of spidroin gene expression in rameters . Reads PT. perfuga spiders that contain N- or C- terminal coding regions and partial adjacent repetitive regions (T. perfuga ampullate (AmSp), aciniform (AcSp), pyriform (PySp), and tubuliform (TuSp) sequences group together with spidroins of the same respective type from the comparison species  and major ampullate (major ampullate spidroin1 and 2-MaSp1 and MaSp2) spidroins of orb-web and cob-web weaving spiders , with a version letter to distinguish them from each other following the nomenclature used by Collin et al [The repetitive region of  spiders . Althougin et al  , the presumed main component of aciniform silk, has a similar repetitive region to the AcSp from other species [T. perfuga AcSp has a repeat length of 190 amino acids (aa), which is similar to that of orb-web weavers in the genus Argiope  and the presence of poly-serine motifs  and species, ). The T.e motifs . Unlike d motifs . Indeed,T. perfuga PySp contains a novel combination of known PySp amino acid sequence motifs. In other species, PySp is the main component of pyriform silk, which is used to anchor silk fibers to a substrate [T. perfuga PySp sequence contains one short (10 aa) stretch of alternating prolines (PX). This amino acid motif has also been identified in PySp from the cribellate spider S. mimosarum, orb-web weaving species, and one cob-web weaving spider (Parasteatoda tepidariorum). In addition to PX, T. perfuga PySp also contains motifs with short runs of alanines .T. perfuga female TuSp has extraordinary sequence conservation among the four repeat units represented in our contig, which despite being nearly 3 kb is still a partial transcript (T. per_TuSp_C). The tandem arrayed, 194 aa repeat units in T. perfuga TuSp had >94% average pairwise identity at the amino acid and nucleotide levels. This high sequence similarity among tandem repeats within a molecule has been observed in TuSp from other species [T. perfuga TuSp repeats are similar in length and amino acid composition, largely composed of serine and alanine, to TuSp repeats from orb-web and cob-web weaving spiders. species ,52. AddiT. perfuga had two novel spidroin transcripts, one containing a C-terminal region and the other an N-terminal region . Phylogenetic analysis of C-terminal regions provides support for the annotation of this T. perfuga transcript as a CrSp. T. perfuga_CrSp_C formed a clade with S. mimosarum_Sp1 and the two C-termini shared 55% aa identity  by genes associated with egg sac construction (T. per_TuSp_C) and web construction . Ampullate spidroin genes  were found to have the highest combined relative transcript levels compared to other spidroin genes in T. perfuga female silk glands (T. per_AmSp_C_vA) had the highest relative abundance when compared to other spidroins , to account for ~ 7% of total T. perfuga silk gene expression.To investigate the relationship of silk gene expression and silk use in female k glands . Differepidroins . We alsoT. perfuga ampullate spidroins have sequence similarity to MiSp and MaSp spidroins of orb-web and cob-web weaving spiders. Similar amino acid motifs include poly-alanine (A)n, glycine-alanine (GA)n, and glycine-glycine-X (GGX)n, where X is a subset of all amino acids. These motifs have been related to differences in tensile properties between silk types that are primarily composed of MaSp1, MaSp2, or MiSp [T. perfuga silks have similar functional demands as the silks of orb-web and cob-web weaving spiders. In orb-web weavers, MiSp is the primary component of minor ampullate silk, which is used in the temporary spiral during orb-web construction  contain novel combinations of amino acid sequence motifs that have not been observed before in these spidroin types . The gro or MiSp –61. One on e.g. ,63). In . In T. pon  and alanine-rich amino acid motifs. The PX motif found in T. perfuga PySp is shared with other spider species , and has been hypothesized to provide extensibility to pyriform silk fibers in orb-web weaving spiders [L. hesperus [L. hesperus lacks PX, while S. mimosarum, P. tepidariorum, and orb-web weaving species lack these alanine-rich motifs [T. perfuga PySp sequences suggests similar functional constraints on T. perfuga PySp and PySp from orb-web and cob-web weaving spiders. Moreover, T. perfuga has the first PySp that we know of that combines the PX extensibility motif and the alanine-rich motifs in the same repeat, which has structure/function implications. spiders –73. The hesperus . L. hesph motifs –74. The T. perfuga Sp_N, has phylogenetic affinities with flagelliform spidroins, the main component of the core fiber of the orb-web capture spiral called flagelliform silk [Tengella is homologous to the modified/pseudoflagelliform silk spigot in other cribellate species. Thus, T. per_Sp_N may be associated with pseudoflagelliform glands. More specific annotation of this T. perfuga spidroin beyond “Sp”  requires future work to obtain more complete sequence and more closely related spidroins.One of the novel spidroin transcripts, orm silk . While oorm silk ,75,76. Rorm silk  proposedT. perfuga spidroin with a silk gland type. Recent studies describing the web-building ontogeny of T. perfuga found that females deploy vast amounts of cribellar silk during web and retreat construction [T. perfuga males were found to use cribellar silk as juveniles and then lose the spigots associated with cribellar silks at their final molt [T. per_CrSp_C was present in the female tissue cDNA library constructed from the small glands attached to the spinnerets, which is where cribellar glands are expected to be located. This transcript was not present in our male (mature) tissue cDNA libraries .We can be more definitive about associating another novel truction . By contnal molt ,27. Consibraries . The ideanalysis , CrSp orT. perfuga use multiple ampullate spidroin variants that collectively account for most of the spidroin expression in females  have new combinations of amino acid motifs never described before for the same spidroin types from different species.We identified 13 new spidroin contigs from the cribellate spider ragments . This meT. perfuga is a cribellate spider, although males lose the ability to spin cribellar silk when they mature. We show evidence that T. perfuga CrSp is expressed by T. perfuga mature females but not mature males. T. perfuga CrSp has distinctive repetitive and C-terminal region sequences and gene tree analysis and pairwise alignments show an affinity with a spidroin from S. mimosarum, another cribellate species . Bootstrap percentages ≥ 50% are shown. Scale bar represents substitutions per site.Shaded boxes indicate spidroin types as in (PDF)Click here for additional data file.S2 FigA) Repetitive sequence adjacent to N-terminal region. (B) Repetitive sequence adjacent to C-terminal region. Spidroin names abbreviated as in ((PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


The evolution of antimicrobial resistance (AMR) in pathogens has prompted extensive research to find alternative therapeutics. Plants rich with natural secondary metabolites are one of the go-to reservoirs for discovery of potential resources to alleviate this problem. Terpenes and their derivatives comprising of hydrocarbons, are usually found in essential oils (EOs). They have been reported to have potent antimicrobial activity, exhibiting bacteriostatic and bactericidal effects against tested pathogens. This brief review discusses the activity of terpenes and derivatives against pathogenic bacteria, describing the potential of the activity against AMR followed by the possible mechanism exerted by each terpene class. Finally, ongoing research and possible improvisation to the usage of terpenes and terpenoids in therapeutic practice against AMR are discussed. The increase of antimicrobial resistance (AMR) in microbiological pathogens has spurred a global mandate to identify potentially effective alternatives . AMR is Staphylococcus aureus (MRSA), Escherichia coli ST131 and Klebsiella ST258; this further contributes to the dissemination of resistant genes such as Klebsiella pneumoniae carbapenemase (KPC), NDM-1, and Enterobacteriaceae-producing extended-spectrum β-lactamases (ESBL) [Laxminarayan et al.  reporteds (ESBL) .Klebsiella spp. and E. coli was common in the hospitals of developing countries [Enterobacteriaceae and Acinetobacter spp. in neonatal nurseries. It was also noted by Saleem [E. coli, bacteria commonly associated with urinary infection. Barbieri [The continuous dissemination of AMR not only contributes to new resistance mechanisms; it will also have a detrimental impact whereby the efficacy of current antibiotics are drastically reduced, leading to therapeutic failure . It is wountries . This way Saleem  that in Barbieri  stated iEnterococcus spp. can be disseminated to susceptible strains via horizontal transfer mediated by conjugative plasmids [Bacterial resistance is commonly mediated by transfer of resistance genes . Overallplasmids . Finallyplasmids . The up-plasmids .Strategies to curb the ongoing emergence of AMR require the involvement from various parties. They include policy makers to develop antibiotic regulations, pharmacists and physicians to practice proper antibiotic stewardship, the pharmaceutical industry to invest in new antibiotic discovery, as well as academics to provide adequate public education . Some stAntimicrobial agents may comprise of naturally-occurring compounds such as phytochemicals and essential oils (EOs) . They ca2, subcritical water), distillation , solvent-free (microwave) and combination method (solvent + steam) [Mentha x piperita L. Carl) essential oil was added to meropenem against resistant E. coli [EOs are naturally produced from aromatic plants such as herbs as their secondary metabolites. Usually EOs exist in liquid form, are volatile and exhibit good solubility in lipids and inorganic compounds that are less dense compared to water. They can be extracted from various parts of the plant organ such as flowers, buds, leave, bark, twigs, stem, wood, seed, or root  by vario+ steam) . General+ steam) . In part+ steam) . Previou+ steam) . A list  E. coli . Recentl E. coli . There aTerpenes are large hydrocarbon groups that consist of 5-carbon isoprene (C5H8) units as their basic building block. They are synthesized via two pathways which are the non-mevalonate pathway; the Methylerythritol Phosphate (MEP) and the mevalonate pathway from Acetyl CoA precursor. Their backbones can be reorganized into cyclic structure by cyclases. The commonly found terpenes which differ in numbers of isoprene units are the monoterpenes and sesquiterpenes; however longer chains such as diterpenes and triterpenes also exist ,54. P-CyTerpenoids represent a large group of phytochemicals with promising antimicrobial activity . The cheAlthough the antibacterial mode of action of terpenes remains largely unknown, Griffin et al.  reportedEnterobacter aerogenes by becoming a potent efflux pump inhibitor [S. aureus and E. coli. Other compounds such as carvacrol, trans-cinnamaldehyde and (+)-carvone were reported to possess potent inhibitory activity against E. coli and S. typhimurium [Monoterpenes comprise of two isoprene units and exist in many plants. It has been reported by Griffin et al.  that monnhibitor . Trombetnhibitor  claimed himurium . In addihimurium .cis/trans citral, eugenol, geraniol, Geranyl acetate, cis-hex-3-en-1-ol, R(+)limonene, (2)-linalool, menthone, nerol, α-pinene, β-pinene, (+)sabinene, α-terpinene, terpinen-4-ol, α-terpineole, (−)-thujone, thymol) which were previously reported regarding their antimicrobial activity against 25 bacterial strains [S.aureus and S. typhimurium [K. pneumoniae [In fact, in as early as 1979, Kurita et al.  had list strains . Phenol himurium . Recentleumoniae . The proeumoniae .Herpes simplex virus type 1 (HSV-1) were made by Thompson in 1989. Later in 2010, Dunkic et al. [Tobacco mosaic virus (TMV) and Cucumber mosaic virus (CMV) [Monoterpene ketones were also found to exhibit antimicrobial properties . In compc et al.  listed aus (CMV) .Bacillus spp. [Streptococcus mutans and Streptococcus sobrinus biofilm formation [S. aureus and S. epidermidis whereby it inhibited the biofilm development [S. aureus without altering the microbial flora and successfully inhibited the biofilm production of S. aureus. Sesquiterpenes were also incorporated in the combination therapy using existing drugs.It has been long recognized that sesquiterpenes possess antimicrobial activites . Back inlus spp. . Farnesoormation . Farnesoelopment . Two stuelopment  evidenceB. pseudomallei. A phenol sesquiterpene, xanthorrhizol was found to reduce 60% of Staphylococcus mutans cell adherence ability [Mycobacterium smegmatis [S. aureus with a combination of sets of available antibiotics such as tetracycline, erythromycin, penicillin, and vancomycin.A recent study conducted by Castelo-Branco et al.  showed p ability , and inhmegmatis . Recentlmegmatis . In 2011megmatis  reportedP. aeruginosa, E. coli, S. aureus, and C. albicans [S. aureus and S. epidermidis [Diterpene is a class of terpene with broad biological activities . Previoualbicans . They wealbicans  suggestealbicans . Additiodermidis .Polyathia longifolia enhanced the efficacy of oxacillin, tetracycline, daptomycin, and linezolid against clinical isolates of MRSA. All MICs of the antibiotic dropped significantly between 10–80, 4–16, 2–8 and 2–4-folds respectively when combined with CD. Gupta et al. [Besides sesquiterpenes, diterpenes also function as a good antibiotic enhancer against MRSA. Moreover, diterpenes have been widely used in combination therapy with antibiotics . For insa et al.  then proSalvia sclarea [S. aureus, Enterococcus faecalis and S. epidermidis. Both salvipisone and aethiopinone were also tested for their synergistic activity when combined with antimicrobial drugs alongside oxacillin, vancomycin, and linezolid against MRSA and Methicillin resistant Staphylococcus epidermidis (MRSE). It was discovered that they were either bactericidal or bacteriostatic against planktonic cultures of tested MRSA and MRSE [Salvipisone and aethiopinone are diterpenoids isolated from roots of  sclarea . They weand MRSE . RemarkaPandanaceae containing triterpenes in the form of 24, 24-dimethyl-5β-tirucall-9 [Mycobacterium tuberculosis. OA also had promising synergy against MDR when combined with rifampicin, isoniazide, and ethambutol with significant MIC reduction of 128–16 fold, 32–4 fold, and from 128 to 16 fold, respectively [Triterpenes comprises of six isoprene units. It was reported that rucall-9 , 25-dienRadermachera boniana. Both compounds were found to be active against M. tuberculosis. There are at least six known molecules which include OA, ergosterol peroxide, and ursolic acid (UA). In fact, the combination of both ergosterol peroxide and UA showed synergistic activity against M. tuberculosis [Miconia ligustroides, resulted in significant antibacterial activity when tested on selected bacteria . When UA was used against B. cereus, the MIC value was 20 µg/mL and OA showed MIC value of 80 µg/mL against B. cereus and S. pneumoniae. In 2013, a study conducted by Zhou et al. [S. mutans and S. sobrinus. Moreover, OA and UA were reported to enhance antimicrobial activity against Listeria monocytogenes without affecting toxin secretion; this influenced the virulence factors of L. monocytogenes and inhibited the capacity of biofilm production from these bacteria [Besides OA, bonianic acid A and B are two triterpenoids that were extracted from rculosis . The reprculosis  depictedu et al.  showed tu et al.  expandedbacteria .A. baumanii, but not with other classes of which ampicillin, norfloxacin, chloramphenicol, tetracycline, and rifampicin are examples [Callicarpa farinose Roxb (Verbenaceae) and were shown to exhibit potent antimicrobial activity against clinical methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) with MICs ranging from 2 to 512 µg/mL [OA also exhibited strong interactions alongside aminoglycoside (gentamicin and kanamycin) against examples . Based oexamples . Three t12 µg/mL .While there is no firm report specifically on modes of action by terpenoids, the mechanisms of action of phytochemicals found in nature have been proposed. Typically, phytochemicals aim either for disruption of the bacterial cell membranes, modulation of bacterial efflux pump, suppression of bacterial biofilm development or inhibition of some virulence factors which include enzymes and toxins . For insC. albicans, C. tropicalis, and C. glabrata and 11 fluconazole-resistant C. albicans, C. krusei, C. glabrata, C. tropicalis, and C. parapsilopsis. The combination analysis showed that of the strains tested, 32 out of 38 strains and eight out of 10 strains have obtained a Fractional Inhibitory Concentration (FIC) index of less than 0.5 [Baccharis extract synergistically reduced the dose of the anti-fungal drug Terbinafine against Trichophyton rubrum [C. albicans strains [Combination therapy with terpenes have been widely seen in current therapeutic practice especially in antifungal drugs . It was than 0.5 . FIC is than 0.5 . The seqthan 0.5 . Previoun rubrum . Another strains . It was Malaleuca alternifolia Cheel (tea tree) oil against azole-susceptible and resistant human pathogenic candida species. In this demonstration, terpinen-4-ol was able to clear a well-established model of rat vaginal candidiasis. Terpenes which were found in Cassia occidentalis and Phyllanthus niruri showed antimalarial activity in vivo using mice against Plasmodium berghei [Prior to the development of novel drugs, in vitro and in vivo testing are usually performed to ascertain the safety and efficacy of the compound to better understand the physiological effects. Despite a number of published in vitro reports pertaining to terpenes antimicrobial testing, incorporation of various terpenes in clinical trials focusing on antimicrobial activity is still lacking due to insufficient data on the in vivo system. Most in vivo testing for terpenes and its derivatives has been conducted for human health associated with anti-inflammatory, anti-tumorigenic, anti-cancer, transdermal delivery medium and neuroprotective  aspects. berghei . In addi berghei .Ginkgo biloba extract (GBE) which constitutes of the terpene, lactone, was improved significantly with 2–4 times greater plasma concentration compared to a non-phytosome delivery method [In order to ensure greater therapeutic effect from drugs, terpene bioavailability should be determined. It was reported that while natural volatile terpenes from 1,8-cineole of uncrushed capsule from the plasma yielded relatively 100% of bioavailability, limomene and α-pinene were only detectable for a few subjects . In 2017y method .50 respectively; these are mixed with two-fold dilutions of both agents with a fixed ratio. The CI will resolve synergistic (CI < 1), additive (CI = 1) and antagonistic effects (CI > 1). In addition, another method that contributed significantly in synergistic activity was to calculate the Dose Reduction Index (DRI), also known as the reversal enhancement ratio, that measures how many folds the dose of antimicrobial drugs may be cut down when used in combination rather than alone. One of the measures that can be used to evaluate synergism is through checkerboard assay.Combination therapies using natural products such as the terpenoids may synergistically, additively, or antagonistically affect the treatments. Zacchino et al.  reportedIn our previous study, through this method, peppermint essential oil was proven to synergistically react with meropenem. MIC of individual peppermint oil and meropenem were 8% and 4 µg/mL respectively. Meanwhile when used in combination, the MIC of peppermint oil and meropenem were reduced to 1% and 0.5 µg/mL respectively. The CI value of 0.26 obtained in checkerboard assay had portrayed a high synergism. Last year, our group carried out extensive analysis to investigate the additive interaction of cinnamon bark oil and meropenem. The shift of attention towards synergism between compounds and antibiotics have caused researchers to overlook the additive effects, thus we conducted the study to understand additive interaction which focused on the effect on the bacterial membrane .p-Iodonitrotetrazolium chloride (INT) colorimetric assay, micro- or macro-dilution and disc diffusion methods. However, Griffin et al. [Both in vitro and in vivo experimental systems can be used to evaluate the antimicrobial activity of either synthetic compounds or naturally-acquired compounds. Nevertheless, in vitro approaches have been more commonly used due to their feasibility. In vivo studies, however, are seldom applied due to limitations in detecting the actual mechanism of action. Susceptibility testing which determines the MIC of a compound against bacteria is routinely done using variations in the methods of MIC assay such as rapid n et al.  stated in et al. . By perfn et al. . The Dosn et al.  can alson et al. . More exSenegalia nigrescens are among the novel terpene derivatives discovered recently. Both in vitro and in silico anti-quorum sensing evaluation have demonstrated potential anti-quorum sensing against Chromobacterium violaceum [Effective management and treatment of microbial resistance are among the main priorities in healthcare. Terpene derivatives are an important and promising source of novel antibiotics. Indeed, ent-kaurenoids  extracted from iolaceum . In addiiolaceum . Additioiolaceum .Caesalpinia pulcherrima, Lawsonia inermis, Pithecellobium dulce, Euphorbia tithymaloides, Punica granatum, Plumeria obtusa, Carica papaya, Cassia fistula, Cordia dichotoma, Euphorbia prostrate, Nerium oleander, and Cyanthillium cinereum [Cupressus torulosa essential oil. The compounds are effective against pathogens including B.subtilis, Pseudomonas alcaligenes, M. luteus, and B. cereus [Ganoderma lucidum (Reishi) which is a medicinal mushroom, contains several triterpenoid substances such as ganoderic acid and lucidenic acid. The compounds were then evaluated for their therapeutic effects whereby they exhibited anti-human immunodeficiency virus (HIV) activity by inhibiting the effects of HIV progression [With the success of terpenoids in the treatment of microbial resistance, the hunt for new terpenoids has been an important quest amongst the scientific community as a potential application. For instance, screening for terpenoids was conducted on semi-arid plants such as cinereum . A separ. cereus . This cagression .Dermanyssus gallinae at 20% of the pathogen population. Eugenol, geraniol, and citral found in plant essential oils were able to administer 100% mortality when used undiluted. This shows that some undiluted terpenes are highly toxic upon direct usage [As mentioned previously, terpenes and terpenoids had been known to exert antimicrobial activity against a wide variety of bacteria, both Gram-positive and Gram-negative. Clinical trials regarding highlighting the application of terpenes had been performed in several studies. In addition, β-sitosterol had also demonstrated immune enhancing ability in tuberculosis patients, demonstrating significant weight gain and higher white blood cell counts which resulted in faster recovery . Howeverct usage . Generalct usage . Due to The evidenced antimicrobial activity of terpenes and their derivatives need to be further expounded with the aid of automation and advancement in technology. Experimental analyses will need to be more streamlined to become more precise, resulting in less ambiguity so that the results obtained can be ensured and are consistent. This will reduce the time taken for experimental work and more time can be spent for extended analyses. Researchers may then study the modification effects of natural products with a special focus on terpenoids. Structural modifications of natural compounds produced either synthetically or via biotransformation may offer a new facet in finding novel AMR solutions as it explores new antimicrobial agents. In addition, delivery methods involving existing treatments against AMR should be improved and new inventions can be investigated. Combination therapeutics may be enhanced by exploring more antimicrobial adjuvants which will synergistically affect treatment outcomes with greater efficiency and less side effects. Natural terpenes and terpenoids which are available at very low prices such as carvacrol, thymol, and geraniol , should From this review, it has been evidenced that some terpenes and their derivatives were proven to be potent antimicrobial agents against drug resistant pathogens which mainly include bacteria and fungi. Specific mechanisms of each class of terpenes have also been highlighted and as a whole, terpenes provide a possible mitigation route for AMR and navigating the dead end of the diminishing antibiotic pipeline, hence, an appropriate match between terpenoids and existing antimicrobial agents may provide ultimate therapeutic options for AMR-associated infections.


Brassica rapa. We identified 60 SET (HKMTases), 53 JmjC, and 4 LSD (HDMases) genes in B. rapa. The domain composition analysis subcategorized them into seven and nine subgroups, respectively. Duplication analysis for paralogous pairs of SET and JmjC  exhibited variation. Interestingly, three pairs of SET exhibited Ka/Ks > 1.00 values, signifying positive selection, whereas the remaining underwent purifying selection with values less than 1.00. Furthermore, RT-PCR validation analysis and RNA-sequence data acquired on six different tissues  revealed dynamic expression patterns. This comprehensive study on the abundance, classification, co-regulatory network analysis, gene duplication, and responses to heat and cold stress of SET and JmjC provides insights into the structure and diversification of these family members in B. rapa. This study will be helpful to reveal functions of these putative SET and JmjC genes in B. rapa.Histone modifications, such as methylation and demethylation, are vital for regulating chromatin structure, thus affecting its expression patterns. The objective of this study is to understand the phylogenetic relationships, genomic organization, diversification of motif modules, gene duplications, co-regulatory network analysis, and expression dynamics of histone lysine methyltransferases and histone demethylase in  SUV39H1), was identified [Arabidopsis thaliana, which revealed both conserved and non-conserved modifications compared to animals [The role of histone methylation in transcription regulation was first reported in the 1960s [entified . This dientified , JmjC 3-9 members have H3K9 methyltransferase activity that plays a vital role in heterochromatin formation and gene silencing [A. thaliana.Histone lysine methyltransferase (HKMTases) contains the SET domain while histone demethylase (HDMases) is comprised of two major domains namely JmjC and LSD (lysine-specific demethylase domains) . There as of SET ,13. Thess of SET ,15. In Ailencing ,17,18. Wilencing . Similarilencing  and manyArabidopsis on the cold-responsive genes such as COR15A, and AtGolS3, decreased gradually in both a histone occupancy-dependent and independent manner during cold stress treatment [ZmDREB1 and ZmCOR413 increased [OsDREB1b genes was induced by cold stress [Arabidopsis to heat stress results in an increased global methylation and higher homologous recombination frequency [Arabidopsis, the upregulation of DRM2, nuclear RNA polymerase D 1(NRPD1) and NRPE1 in response to heat stress may contribute to increased genome methylation [Cork oak (Quercus suber L.) grown in higher temperatures [Brassica napus, the DNA methylation levels increase more in the heat-sensitive genotype compared to heat-tolerant genotype under heat treatment [Some of the recent studies highlighted the response of methylation in response to cold and heat stress. In reatment . In maizncreased . Similard stress . The glorequency . In Arabhylation . An increratures . In Brasreatment . Thus, eB. rapa will enhance understanding of their functions and regulatory mechanisms. Most angiosperms have undergone polyploidization during their long evolutionary history [B. rapa) is an excellent model plant for genome evolution study due to the availability of its sequenced and assembled genome [B. rapa also underwent two duplication (WGD α and β) and one whole-genome triplication events (WGT γ) in the Brassicaceae lineage [B. rapa genome, thus providing an opportunity to understand the molecular evolution of SET and JmjC genes in B. rapa. Its extensive data is also closely related to A. thaliana, estimating that about 93% of the predicted gene families of B. rapa appeared in A. thaliana and making it a vital model for evolutionary and genomic studies [The research detailed above emphasizes the need to further explore the importance of histone methylation and its complex nature. In addition, the evolutionary implications and functional dissection of these family members in  history . In para history . Chinesed genome . B. rapa lineage . The WGT studies .B. rapa were identified as SET candidates [HKMTases and HDMases from various clades are denoted as BraSET, BraJmjC, and BraLSD. A systematic and comprehensive description of the histone lysine modifiers in B. rapa was carried out through a comparative genome analysis to investigate copy variation, gene retention, syntenic region, collinear correlation, and expansion patterns following the WGT event. Furthermore, phylogenetic relationships, promoter sequence analysis, divergent tissue-specific expression patterns across six various tissues, co-regulatory network analysis, and the response of these genes to heat and cold stress will allow further understanding of functional analysis of SET and JmjC genes in B. rapa.To date, 198, 136, 124, and 71 genes coding for histone methylation proteins have been identified using bioinformatics surveys in apple, sweet orange, tomato, and strawberry, respectively ,34,35,36ndidates ,38, howeBraSET, 53 BraJmjC, and 4 BraLSD genes in B. rapa. To validate our results for domain confirmation, these proteins were submitted to the SMART and NCBI servers. These proteins were designated based on their respective family information and following the nomenclature of A. thaliana according to sequence similarities, i.e., BraSET1–BraSET60, BraJmjC1–BraJmjC53, and BraLSD1–BraLSD4 , according to previously reported study [B. rapa, while the other groups contained lower numbers of JmjC , medium fractionated (MF1), and most fractionated (MF2) and shares a diploid ancestor with A. thaliana [B. rapa and A. thaliana during a Brassica-specific WGT event. We similarly analyzed the syntenic relationship between paralogous and orthologous gene pairs by utilizing the BRAD database , followed by MF2 (26.67%) and MF1 (25%). We noted slight variation for JmjC as the LF share was only 43.86%, followed by MF1 (33.33%) and MF2 (22.81%). Overall, the results confirmed that large proportion of the genes (46.15%) were sited in the LF subgenome of B. rapa  (BraSET41 and BraSET42) and JmjC (BraJmjC53 and BraLSD4). In addition, we reconstructed the 24 conserved ancestral genomic blocks (GBs) mainly based on a previously reported study [To explore the evolutionary mechanism of both SET and JmjC, the chromosomal location and gene duplication events were further analyzed. For chromosomal localization, all SET and JmjC displayed an obvious uneven dispersal on the 10 different positions of 01–Br10) a. A larged study ,44, withB. rapa, the expansion of SET and JmjC was mainly driven by WGD events or segmental duplication. In SET genes, the Ks values ranged from 0.16 to 0.61 with an average divergence time of 11.13 MYA (million years ago). For JmjC, the Ks values ranged from 0.18 to 0.35 with an average divergence of 9.04 MYA  [For a gene family’s evolution, gene duplication is the driving force for gene activation . Using M9.04 MYA . Taken t6.1 MYA) .SET and JmjC genes in B. rapa growth and development [B. rapa. Among 60 SET genes, BraSET29, BraSET45, and BraSET53 are not expressed and BraSET17, BraSET52-55, and BraSET59 showed random involvement in a particular organ  was retrieved and used to gain insight into the divergence and putative functions of elopment . Resultsar organ . The remar organ . Among tJmjC genes were expressed highly compared to SET; only one gene (BraJmjC43) did not show any expression pattern, while the rest were expressed highly in one or more tissues , showing higher expression in all organs with positive correlation (>0.6). In addition, two pairs (BraSET45_BraSET46 and BraSET53_BraSET55) showed no correlation and the rest were expressed with mild positive correlation. These results indicated that due to pseudogenization, these genes might have lost function. On the other hand, for JmjC, four pairs  showed higher expression in all tissues with higher PCC values (>0.6), one pair (BraJmjC43_BraJmjC48) had no correlation, one (BraJmjC17_BraJmjC18) exhibited negative correlation, and the rest showed mild positive correlation. In addition, the clustering image showed that only one gene participated in siliques, while the rest were not co-expressed in other tissues , and their correlation values were calculated . Results tissues . The divThe promoter regions of SET and JmjC was used for the identification of cis-regulatory elements by using the PlantCARE database. A total of seven major groups, such as light, hormones, stress factors, enhancers, other regulatory stress factors, and circadian were the most prominent promoter region and found to be conserved among SET and JmjC domains. For both SET and JmjC, we identified the number of genes that were responsible for various cis-regulatory elements . These rJmjC and SET genes play a crucial role in developmental stages to tolerate abiotic stresses [In angiosperms and vertebrates, histone modifications, such as methylation and demethylation, have been identified as crucial factors for regulating chromatin structure. As a result, both the stresses ,47. The B. rapa genome, and a total of 60 and 57 full-length SET and JmjC genes were identified, respectively. These genes were further divided into seven SET and nine JmjC distinct subgroups based on domain organization and phylogenetic analysis. The phylogenetic characterization of SET and JmjC was highly conserved among subgroups, suggesting the importance of their roles in regulatory mechanisms for plant improvement. SET (Class V) and JmjC (PKDM3) groups were preferentially expanded in B. rapa compared to A. thaliana, implying that these group members evolved substantially to meet some unique regulatory needs [A. thaliana, O. sativa and Citrus [B. rapa and A. thaliana also demonstrated that BraSET, BraJmjC, and BraLSD were divergent from the model plant at a high frequency.To understand the gene structure, function, and evolution, a genome-wide gene family analysis is the first step . Moreovery needs . The resd Citrus ,50. ThisB. rapa is tremendous model plant to study evolutionary process as it has undergone WGD and WGT events, which allows the study of the relationships among gene family fractionation and discrepancies in morphotypes [SET and JmjC genes in B. rapa resulting from the WGT event to allow non-functionalization, sub-functionalization, and neo-functionalization [B. rapa from A. thaliana (9.6–16.1 MYA) [Ka/Ks is < 1, indicates gene pairs may have evolved from purifying selection ; Ka/Ks = 1 suggests neutral selection, while Ka/Ks > 1 means positive selection [Ka/Ks > 1, specifying positive selection, the remaining SET showed values less than 1.00. These results suggest that these pairs underwent purifying selection and thus mainly act the maintenance of B. rapa.Evolutionary history indicated that multiple polyploidization events have occurred in all extant angiosperms ,52,53. Dphotypes ,42. As dlization . For duplization . Further6.1 MYA) . Generalelection ,57,58. TSET and JmjC genes did not differ significantly among the three subgenomes  of B. rapa. Our study offers insight into the unique features and fairly high conservation in B. rapa. To comprehend plant functions, expression analysis of a gene can provide valuable clues. Some of the emerging evidence has supports that cellular processes and epigenetic regulations commonly occur during abiotic stress, emphasizing the significance of both DNA and histone modifications [Arabidopsis, the understanding of SUMOylation has been progressed under heat stress conditions and also various chromatin components including H2B, GCN5, HDA19, and the deubiquitinating enzyme UBP26, which removes ubiquinone bound to H2B, have been found to be SUMOylated [Arabidopsis, vernalization processes involved in epigenetic regulation induced by environmental stresses and can be achieved by long-term exposure to cold temperatures [Arabidopsis, it has been estimated that 3 to 20% of the transcription changes occurred in response to cold stress [Arabidopsis was induced by long-term low temperature treatments that resulted into mutation by showing sensitivity to freezing stress [B. rapa. The paralogous gene pairs respond differently. For most SET genes, the expression level increases after heat shock and 9 pairs showed a positive correlation. By contrast, the JmjC responded mostly negatively to cold treatment. In summary, some SET genes show active expression patterns upon heat and cold shock, which specifies that these genes may be participated in B. rapa responses to temperature stresses. The overall response of SET and JmjC across six various tissues was revealed by the combinatorial expression profiles against heat and cold stress. Certainly, the different organs/stages have dynamic expression patterns in specific tissues or treatments, and many of those SET and JmjC genes showed variation either up- or downregulation in definite tissues or treatments. Cis-elements and expression analysis results revealed that SET and JmjC genes expressed in various biotic, abiotic and hormone signaling, might have acquired new functions after duplication in the evolutionary processes. A common interacting pattern was observed in the expression profiling such as cis-acting element involved in heat stress-responsiveness (HSE) and in low-temperature responsiveness (LTR) as described in SET and JmjC genes showed variable responses to these factors and can be assumed to be responsive against heat and cold stress.Comparative structural analysis of both SET and JmjC shared common patterns among subgroups. It is worth mentioning that gene structure compositions might be useful for studying the origin of these genes. We observed in most of the subclasses contained more than one intron, and the rest exhibited consistency among the subgroups, indicating that the distribution pattern of introns and exons is pondered as the backbone of genes and the evolutionary fingerprint . Synonymications ,60. In pications ,62. The ications . In ArabMOylated . The heaMOylated . In Araberatures . In Arabd stress ,67. The g stress . ExpressCumulatively, our results provide fundamental information about SET-domain, JmjC-domain, and LSD-domain protein members that will assist in the identification and for functional studies in boosting abiotic stress-resistant crop plants.B. rapa genome (2.5 version), BRAD (http://brassicadb.org/brad/) [A. thaliana sequences were retrieved from TAIR (http://www.arabidopsis.org/) and the sequences of rice were extracted from the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/) [https://pfam.sanger.ac.uk/) [http://smart.embl-heidelberg.de/) [https://www.ncbi.nlm.nih.gov/). Proteins that contained both SWIRM and Amino_oxidase domains were identified as LSD genes. Sequences with obvious errors including gene length or domain compositions were eliminated.The sequences were downloaded from g/brad/) . The A. su.edu/) . The Hid.ac.uk/) . These perg.de/)  and NCBIFor MSA of SET and JmjC candidate genes, we executed MUSCLE  by MEGA Ka/Ks ratios for the paralogs of SET and JmjC were calculated using MEGA 7.0 [B. rapa genome. Gene pairs were selected among the subgenomes  of B. rapa. The rate of divergence was calculated by using the following formula: T = Ks/2r, where Ks represents the synonymous substitutions per site and r is the rate of divergence. For dicotyledonous plants, specifically B. rapa, the hypothesis is 1.5 synonymous substitutions per site of 108 years [The MEGA 7.0  software08 years .http://gsds.cbi.pku.edu.cn) for exon–intron structure [http://web.expasy.org/protparam/). Finally, WOLF PSORT (https://wolfpsort.hgc.jp/) server was used to predict subcellular localization.To identify the conserved motifs for both SET and JmjC proteins, Multiple Em for Motif Elicitation (MEME) software version 5.0.5 was used with the following parameters: maximum number of motifs 10, with a minimum width of 100 and a maximum of 120. The other parameters were set as default . We usedtructure . The phyB. rapa genome according to the generic file format (GFF). The various cis-elements were recognized by using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [The promoter sequences of SET and JmjC (selected as 2000 upstream bp) were retrieved from the e/html/) .A. thaliana and subgenomes of B. rapa  obtained from BRAD (http://brassicadb.org/brad/searchSynteny.php). Circos program was applied to demonstrate the syntenic relationships among the chromosomes of B. rapa and A. thaliana [B. rapa.The chromosomal location of SET and JmjC was illustrated from top to bottom with respect to their position in the genome annotation using Mapchart . For genthaliana . MoreovePearson correlation coefficient for both RNA-seq and qRT-PCR was performed by MS Excel (Ver. 2013) and figure was prepared using RStudio .The seeds of Chinese cabbage (Chiifu-401-42) were grown in plastic pots containing a mixture of soil and vermiculite (3:1) placed in a growth chamber for five weeks. The following growth conditions were maintained: the temperature was set to 24/16 ºC, the photoperiod was 16/8 h, and the relative humidity was 65–70%. Definite treatments (heat and cold) were provided to the seedlings as follows: for heat and cold treatments, seedlings were exposed to 38 ºC and 4 ºC, respectively and samples were collected with an interval of untreated (CK), 4 and 9 h, respectively. Finally, the samples were divided into three biological replicates and kept frozen in liquid nitrogen, and immediately stored at −70 ºC for further analysis.B. rapa genome. The RT-PCR assays were performed with three biological and three technical replicates by following the guidelines explained in our previous study with slight modifications [B. rapa actin gene Bra028615 (Forward: CTCAGTCCAAAAGAGGTATTCT and Reverse: GTAGAATGTGTGATGCCAGATC) was used as an internal control for normalization. The relative fold expression was calibrated by using the comparative Ct-method. The gene expression levels for SET and JmjC were analyzed by following the previously reported study [For RNA extraction, frozen leaves from both treatments and control plants were sampled. RNA was isolated by using Trizol  method, following the manufacturer’s protocols. cDNA was prepared from both treatment and control groups by reverse-transcribing the RNA through Primer Script RT reagent kit . The list and sequence of the primers used in our study are provided in ications . In brieed study ,82.B. rapa. We identified 60 SET and 57 JmjC genes by an in silico analysis of the B. rapa genome database. Phylogenetic analysis mapped closest putative orthologs of SET and JmjC from B. rapa and A. thaliana by sequence similarity, which were further divided into seven and nine subgroups, respectively. This classification was further supported by gene structure and motif analyses, with each group sharing a common junction of exon–intron and protein motifs. Transcriptomic analysis of B. rapa SET-domain, JmjC-domain, and LSD-domain proteins indicated that some of the family members exhibited tissue-specific expression. The expression analysis of SET and JmjC genes in response to heat and cold treatments indicated the co-occurrence of different signaling pathways. Our findings on genome-wide identification and expression analysis provide a foundation for the functional dissection of SET, JmjC, and LSD-domain in B. rapa.This study is an extensive genome-wide survey of the SET-domain, JmjC-domain, and LSD-domain proteins in


AbstractSchizoporaparadoxa, current name Xylodonparadoxus, is a white-rot fungus with certain useful biotechnological properties. The representative genome of Schizoporaparadoxa strain KUC8140 was published in 2015 as part of the 1000 Fungal Genomes Project. Multilocus phylogenetic analyses, based on three nuclear regions , confirmed a misidentification of S.paradoxa strain KUC8140 which should be identified as Xylodonovisporus. This wrong identification explains the unexpected geographical distribution of S.paradoxa, since this species has a European distribution, whereas the strain KUC8140 was recorded from Korea, Eastern Asia. Schizopora Velen., currently synonymous with Xylodon (Pers.) Fr. (Xylodon (=Schizopora) species, based on morphological characters, has led to inaccuracies due to a lack of clear diagnostic characters and it has been assumed that many Xylodon species have a worldwide distribution (The genus rs.) Fr. , includeribution . Howeverribution . Moreoveribution .Schizoporaparadoxa strain KUC8140, current name Xylodonparadoxus (Schrad.) Chevall., was sequenced in 2015 as part of the 1000 Fungal Genomes Project (http://jgi.doe.gov/fungi)  ; this stactivity . It has activity . Within Xylodon species were addressed. DNA from specimens of X.paradoxus, X.quercinus (Pers.) Gray, X.nothofagi (G. Cunn.) Hjorstam & Ryvarden, X.raduloides Riebesehl & E. Langer, X.flaviporus (Ber. & M.A. Curtis ex Cooke) Riebesehl & E. Langer and X.ovisporus (Corner) Riebesehl & E. Langer was extracted from herbaria specimens and culture collections  and the second largest subunit of RNA polymerase II (rpb2). Direct Polymerase chain reactions (PCRs) were performed to obtain sequences from ITS and LSU with the pair of primers ITS5/ITS4 . The same regions from X.paradoxus specimens FCUG-2425, MA-Fungi 70444 and MA-Fungi 81294 were used as reference sequences for BLAST searches, respectively . For rpb2, default BLAST settings were used . The best scoring sequence from the S.paradoxa KUC8140 strain genome for each region was extracted and downloaded.Using the BLAST tool from the JGI portal, ITS, LSU concatenated and rpb2, were created to compare the KUC8140 strain with other Xylodon species. The combination of novel, GenBank and KUC8140 sequences for each dataset were aligned in Geneious 9.0.2 with the MAFFT nucleotide sequence alignment function (Raw sequence data were processed and assembled with Geneious version 9.0.2. . Two indfunction . The autML) and Bayesian Inference (BI). ML and bootstrapping analyses were conducted in RAxML  values were above 200. The trees obtained were summarised in a maximum clade credibility tree by TreeAnnotator v.1.7. with a burn-in of 5000.Phylogenetic tree estimation for each alignment was performed using Maximum Likelihood  and the rpb2 dataset was 647 characters long. The results of phylogenetic analyses of ITS+LSU and rpb2 datasets are summarised in Fig. phytools R package (MLB) percentages ≥ 75% and Bayesian posterior probabilities (BPP) ≥ 0.99 are marked with empty circles in Fig. MLB/BPP); specimen vouchers and species names are provided on the tip labels.The ITS+ package . Each phparadoxa strain KUC8140, since sequences of this strain grouped in the X.ovisporus clade, showing a different evolutionary history from X.paradoxus. Therefore, S.paradoxa strain KUC8140, from Korea, must be identified as Xylodonovisporus, reported from Asia and West Pacific areas (S.paradoxa has a European distribution. This rectification helps to explain the biogeographical patterns of Xylodon and also sustains the idea that “not everything is everywhere” for wood-decay fungi (Our phylogenetic analyses confirmed the misidentification of S. ic areas . The neway fungi .Xylodonovisporus is the sister species of X.flaviporus and morphological characters confirm this relationship. The species can be discriminated by the spore size, shorter in the first one (Xylodonparadoxus, with useful biotechnological properties since, according to According to our phylogenetic analyses, irst one . This exirst one . It has irst one . Assessiirst one . It is e


Vangueria madagascariensis J. F. Gmel. is a plant species regarded as an important fruit tree and medicinal plant in sub-Saharan Africa. This study critically reviewed the nutritional value, phytochemistry, medicinal uses, and pharmacological properties of V. madagascariensis. Relevant information on food and medicinal uses of the species was collected from electronic databases such as ISI Web of Knowledge, ProQuest, ScienceDirect, OATD, Scopus, OpenThesis, PubMed, and Google Scholar, and preelectronic literatures were obtained from the university library covering the period 1966 to 2018. Literature studies revealed that V. madagascariensis has been integrated into farming systems as a fruit tree to support income and nutritional security of households in the region. Vangueria madagascariensis is used as a herbal medicine against diabetes, gastrointestinal problems, malaria, pain, parasitic worms, and skin diseases. Phytochemical compounds identified from the species include alcohols, aldehydes, esters, furanoids, ketones, and terpenoids. Pharmacological studies revealed that V. madagascariensis extracts have antibacterial, anticonvulsant, antidiabetic, antifungal, anti-inﬂammatory, antioxidant, cytotoxicity, antimalarial, and antiplasmodial properties. Vangueria madagascariensis should be subjected to detailed nutritional, pharmacological, and toxicological evaluations aimed at correlating the traditional uses of the species and the scientific evidence as well as establishing the efficacy, clinical relevance, safety, and mechanisms of action of the plant extracts and compounds. Vangueria madagascariensis J. F. Gmel. , a German naturalist and botanist. English common names of V. madagascariensis include common wild medlar, Spanish-tamarind, tamarind-of-the-Indies, voa vanga, and wild medlar  assay using human breast carcinoma (MCF-7), non-small cell lung cancer (A549), prostate adenocarcinoma (PC-3), human hepatocellular carcinoma (HepG2), human colon adenocarcinoma (HT-29), normal hepatic (WRL-68) and normal lung fibro blast (WI-38T) cells (50) values stretching from 22.8 μg/mL to 64.7 μg/mL [V. madagascariensis as herbal concoction for tumour in Sudan [Mustafa et al.  assessedT) cells . Test ag.7 μg/mL . These fin Sudan .in vivo antimalarial properties of water and methanol stem bark of V. madagascariensis against Plasmodium berghei strain ANKA using a four-day suppressive assay with chloroquine as positive control. The in vivo studies showed weak activities with chemo-suppression of parasitaemia in Plasmodium berghei infected mice of 26.0% to 39.0% which was much lower than 99.9% exhibited by chloroquine [V. madagascariensis against chloroquine sensitive (D6) and resistant (W2) Plasmodium falciparum clones using the [G-3H]hypoxanthine incorporation assay with artemisinin and chloroquine as positive controls  and chloroquine  [V. madagascariensis against chloroquine sensitive (D6) and resistant (W2) Plasmodium falciparum clones using the (G-3H) hypoxanthine incorporation assay with artemisinin and chloroquine as positive controls  and chloroquine  [V. madagascariensis extracts showed promising antimalarial and antiplasmodial activities and these findings corroborate the traditional usage of the bark, roots and stem bark of the species as remedies against malaria in Kenya and Tanzania [Muthaura et al.  assessedoroquine . Similaroroquine  assessedcontrols . The met3 ng/mL) . In anot3 ng/mL)  assessedcontrols . The met3 ng/mL) . TherefoTanzania , 53–57.V. madagascariensis by administering doses of 10 mg/kg, 100 mg/kg and 1000 mg/kg i.p. to male albino rats (50) values of 3.8 g/kg for both leaf and stem-bark extracts appear to suggest that the extracts of the species are safe to use as herbal medicines [V. madagascariensis stem bark extracts by oral administration in female Swiss mice. The behaviour of mice was observed for 1 hour, intermittently for 4 hours, 24 hours and 14 days noting for any signs of toxicity and the latency of death. The extracts did not cause any mortality or signs of toxicity at any dose level up to the highest dose tested of 5000 mg/kg [V. madagascariensis is widely utilized as both food and herbal medicine, there is need to ascertain toxicological properties of the species using different plant parts against several cell lines using both in vitro toxicological assays and in vivo studies.Bishay et al.  evaluateino rats . The treedicines . Muthauredicines  assessed00 mg/kg . Since VVangueria madagascariensis is an important functional food and source of nutraceutical ingredients in tropical Africa. Significant breakthrough has been made in the last 30 years elucidating the nutritional, phytochemical and pharmacological properties of the species. However, there are still some research gaps regarding correlating the nutritional and phytochemical properties of the species with its food value and medicinal applications. Detailed studies on the phytochemistry, pharmacokinetics, in vivo and clinical research are required. Further research on the antinutritive, enzymatic and molecular effects of V. madagascariensis fruits and kernel oil on human health will be needed to motivate further interest in the use of these products as food sources, additives and health promoting products. Given the situation that V. madagascariensis is used as herbal medicine in combination with other plant species such as Azadirachta indica, Ipomoea pes-caprae, Jatropha curcas, Sporobolus africanus and Toddalia asiatica, there is need to investigate the possibility of synergetic effects of the combined extracts. Since V. madagascariensis is a valuable functional food and nutraceutical plant species in tropical Africa, there is need to establish the toxicity and/or any side effects that can arise when the species and its products are used as functional food and sources of nutraceutical ingredients and/or as herbal medicines.


Croomine, epi-croomine, tuberostemonine, javastemonine A, and isoprotostemonine are isolated alkaloids from the roots of Stemona sp. and their inhibitory effect on FNRs from Plasmodium falciparum (PfFNR) was investigated. Croomine showed the highest level of inhibition (33.9%) of electron transfer from PfFNR to PfFd, while tuberstemonine displayed the highest level of inhibition (55.4%) of diaphorase activity of PfFNR. Docking analysis represented that croomine is located at the middle position of PfFNR and PfFd. Croomine from S. tuberosa appeared to have potential as an antimalarial agent.Ferredoxin-NADP Plasmodium species responsible for the majority of infections: P. falciparum and P. vivax [ P. falciparum in Cambodia, Thailand, Vietnam, and Myanmar, where artemisinin-resistant parasites have already been identified [ P. falciparum [ P. falciparum and P. vivax, respectively [ P. vivax dihydrofolate reductase (PvDHFR) found in Papua P. vivax isolates were 46 and 18%, respectively [Malaria is still considered as a threat in the world, especially in Asia and Africa. Almost 97 countries in the world are malaria endemic. More than 3.3 billion people  are at risk of malaria with twoP. vivax . Many anentified . In Papulciparum . Ratclifectively . Double Plasmodium sp. has a unique organelle, called apicoplast, and it possesses metabolic pathways, which is not found in human host [ P. falciparum and Toxoplasma [ Plasmodium [man host , 7. The xoplasma . The funasmodium , 9, 10.+ reductase (FNR) in apicoplast constitute a redox system [Ferredoxin (Fd) and Fd-NADPx system , 12 thatx system , 13.The x system  and isopx system , 15, fatx system . in silico analysis showed that croomine and epi-croomine could form hydrogen bonds with the amino acid residue of Ala9 of the DHFR enzyme [ Plasmodium, but FNR is found only in Plasmodium. Fd and FNR were cloned from P. falciparum and their interactions were characterized [ Stemona sp. on the PfFd/FNR redox system, and the results of their docking analysis.In a previous study,R enzyme . DHFR cacterized . There h S. tuberosa were cut in small size and ground into powder and extracted using ethanol. The filtrate was evaporated under vacuum and extracted by acid based method using 5% of HCl and conc of NH4OH. Water fraction was partitioned using dichloromethane (DCM) and evaporated. The crude alkaloid was separated using column chromatography under silica gel as stationary phase and DCM-methanol as mobile phase with polarity gradient elution. The alkaloids were purified using preparative chromatography to give tuberostemonine, croomine, and epi-croomine (epi-10-hydroxycroomine or 3-epi-tuberospironine A) [The dry roots ofonine A) . S. javanica utilized the same method as described for the isolation of alkaloids from S. tuberosa. Previous study exhibited that tuberostemonine, croomine, and epi-croomine were treated to antifolate target of DHFR enzymes. Isoprotostemonine and javastemonine A from S. javanica were carried out against P. falciparum multidrug resistant and wild type parasites at Medical Molecular Research Unit, Thailand Science Park, Pathumthani, Thailand [Isolation of isoprotostemonine and javastemonine A fromThailand . The str PfFd and PfFNR were performed as reported by Kimata-Ariga et al. [Production and purification of recombinanta et al. . PfFNR was measured using a grating microplate reader . Diaphorase activity of FNR with DCPIP as an electron acceptor  as an arbitrary electron acceptor as reported by Onda [μM cytc, and 10 nM PfFNR. Electron transfer reaction was initiated by adding 0.5-40 μM PfFd and 100 μM NADPH, and the absorbance was monitored for 5 min with 10-second intervals at 550 nm. The kinetic parameters for the Michaelis constant (Km) for these reactions were determined.Enzyme activity ofacceptor  was measNR to Fd  was meas by Onda  with mod PfFNR was measured in the reaction of  PfFNR to PfFd was measured in the reaction of  PfFd at 1 μM. A 5 mM solution of DMSO was used as a control.Inhibition of diaphorase activity ofhttp://dock.compbio.ucsf.edu/DOCK_6/index.htm) [Molecular docking was performed using Dock 6.8 software (dex.htm) . Dockinghttp://www.rcsb.org/) with ID 1GAQ [ Plasmodium falciparum (PfFNR), flavin adenine dinucleotide (FAD), and some crystals of water. Crystals of water were not involved in the reaction mechanism directly and were stripped out from the complex. In contrary, FAD was used as a part of the receptor. Hydrogen and charge atoms were added to PfFNR and FAD complex using molecular mechanics method with AMBERff14SB force field [ PfFNR and hydrogen-free PfFNR was saved in pdb format to generate molecular surface of the receptor.The receptor model was obtained from Protein Data Bank (PDB) server ( ID 1GAQ . This coce field . Hydrogehttp://www.hyper.com/). All the ligand models were preoptimized using MM+ molecular mechanics methods before being optimized utilizing semiempirical method AM1 to generate 3D conformation also in HyperChem 6 as final process. These optimized ligands were saved in MDL mol file format. Hydrogen and charge atoms were added and loaded using the AM1-BCC charge calculation [https://www.cgl.ucsf.edu/chimera/) [Ligand models were drawn using HyperChem6 software of HyperCube, Inc (culation  and UCSFhimera/)  methods,The molecular surface of the receptor was generated with the dms tools which was included in the DOCK 6.8 package program. The molecular surface was achieved by dms tool and spheres of the receptor were generated using program sphgen . Binding Stemona species are shown in The structures of alkaloids from the roots of Stemona spp. [ Stemona sp. were expected to bind to PfFNR.  PfFd (blue), PfFNR (brown), and ligand (red). FAD (green) is not involved in bonding receptors-ligand.The interaction between the alkaloids from theBID 1GAQ). Five al Plasmodium from PfFNR to PfFd through electrostatics interaction during respiration process [ PfFNR and PfFd, while epi-croomine, tuberostemonine, isoprotostemonine, and javastemonine A were on the edge of the receptors ( epi-croomine. Croomine could obstruct electron transfer from PfFNR to PfFd. This result was supported by research report foregoing that croomine inhibited competitively the human DHFR enzyme at 10 ppm [Electron transfer is going on in process . Positioeceptors . Croomint 10 ppm . epi-croomine are spironine, and isoprotostemonine and javastemonine A are stemoamide alkaloids types [Tuberostemonine is stenine alkaloid, while croomine andds types . The cha PfFNR to PfFd. Thus, croomine from S. tuberosa appeared to have potential as an antimalarial agent. Tuberostemonine inhibited diaphorase for redox reaction.Croomine could inhibit the activity of FNR enzyme and electron transfer from