Identification and in vivo functional analysis of furanocoumarin-responsive cytochrome P450s in a Rutaceae-feeding Papilio butterfly.

The Order Lepidoptera contains nearly 160,000 described species and most of them are specialist herbivores that use restricted plant species as hosts. Speciation that originated from host shift is one of the important factors for the diversification of Lepidoptera. Because plants prepare secondary metabolites for defense against herbivores, with varying profiles of the components among different plant taxa, the specialist herbivores need to be adapted to the toxic substances unique to their host plants. Swallowtail butterflies of the genus Papilio consist of over 200 species. Approximately 80% of them utilize Rutaceae plants, and among the remaining species, a specific subgroup uses phylogenetically distant Apiaceae plants as larval hosts. Rutaceae and Apiaceae commonly contain toxic secondary metabolites, furanocoumarins, and molecular phylogenetic studies support the concept that Apiaceae feeders were derived from Rutaceae feeders. Molecular mechanisms underlying furanocoumarin tolerance in Papilio butterflies have been investigated almost exclusively in an Apiaceae feeder by an in vitro assay. In contrast, there is little information regarding the Rutaceae feeders. Here, we focused on a Rutaceae feeder, Papilio xuthus, and identified two furanocoumarin-responsive cytochrome P450-6B (CYP6B) genes, of which one was an ortholog of a furanocoumarin-metabolizing enzyme identified in the Apiaceae-feeding Papilio while the other was previously unreported. We further conducted in vivo functional analysis using the CRISPR/Cas9 system, revealing a contribution of these CYP6Bs to furanocoumarin tolerance of P. xuthus larvae. Our findings suggest that co-option of furanocoumarin-metabolizing CYP6B enzymes at least partially contributed to the host shift from Rutaceae to Apiaceae in Papilio butterflies.

Comparative analysis of codon usage bias in chloroplast genomes of ten medicinal species of Rutaceae.

Rutaceae family comprises economically important plants due to their extensive applications in spices, food, oil, medicine, etc. The Rutaceae plants is able to better utilization through biotechnology. Modern biotechnological approaches primarily rely on the heterologous expression of functional proteins in different vectors. However, several proteins are difficult to express outside their native environment. The expression potential of functional genes in heterologous systems can be maximized by replacing the rare synonymous codons in the vector with preferred optimal codons of functional genes. Codon usage bias plays a critical role in biogenetic engineering-based research and development. In the current study, 727 coding sequences (CDSs) obtained from the chloroplast genomes of ten Rutaceae plant family members were analyzed for codon usage bias. The nucleotide composition analysis of codons showed that these codons were rich in A/T(U) bases and preferred A/T(U) endings. Analyses of neutrality plots, effective number of codons (ENC) plots, and correlations between ENC and codon adaptation index (CAI) were conducted, which revealed that natural selection is a major driving force for the Rutaceae plant family's codon usage bias, followed by base mutation. In the ENC vs. CAI plot, codon usage bias in the Rutaceae family had a negligible relationship with gene expression level. For each sample, we screened 12 codons as preferred and high-frequency codons simultaneously, of which GCU encoding Ala, UUA encoding Leu, and AGA encoding Arg were the most preferred codons. Taken together, our study unraveled the synonymous codon usage pattern in the Rutaceae family, providing valuable information for the genetic engineering of Rutaceae plant species in the future.

Novel studies on Isolation, purification and characterization of dibenzonitro compound from Glycosmis pentaphylla (Retz.) DC. and effect in downregulating neuronal cancers.

To isolate Letrozole from Glycosmis pentaphylla (Retz.) DC. and to determine its effect on regulating the proliferation, cell cycle distribution, apoptosis and key mechanisms in human neuroblastoma cell lines. Letrozole was isolated through column chromatographic technique and its effect was checked on human neuroblastoma cell lines, IMR 32. The effects of Letrozole on cell viability were measured by MTT assay, and the cell cycle distribution was determined by flow cytometry. The expression changes in mRNA of proliferating cell nuclear antigen (PCNA), cyclin D1 and Bcl-xL were taken from real-time PCR analysis and the protein levels were detected by Western blotting. The results of the present study showed that Letrozole, isolated from leaves of G. pentaphylla could cause significant inhibitory effect on proliferation of IMR 32 cells in a dose dependent manner. Cell arrest was obtained at S phase with the treatment of Letrozole. Apart from this, the expression of PCNA, cyclin D1 and Bcl-xL were decreased both at mRNA and protein levels for the same treatment. Letrozole can inhibit proliferation, induce cell arrest and cause apoptosis in IMR 32 cell lines. The decreased expression of PCNA, cyclin D1 and Bcl-xL induced by Letrozole contributes to the above effects in vitro. This is the first report on the isolation of Letrozole from G. pentaphylla.

Identification of genes associated with soluble sugar and organic acid accumulation in 'Huapi' kumquat (Fortunella crassifolia Swingle) via transcriptome analysis.

BACKGROUND: The levels and ratios of sugar and acid are important contributors to fruit taste. Kumquat is one of the most economically important citrus crops, but information on the soluble sugar and organic acid metabolism in kumquat is limited. Here, two kumquat varieties - 'Rongan' (RA) and its mutant 'Huapi' (HP) - were used to assess soluble sugar and organic acid accumulation and the related genes. RESULTS: Soluble sugars include sucrose, glucose and fructose, while malate, quinic acid and citrate are the dominant organic acids in the fruits of both kumquat varieties. HP accumulated more sugars but fewer organic acids than did RA. Transcriptome analysis revealed 63 and 40 differentially expressed genes involved in soluble sugar and organic acid accumulation, respectively. The genes associated with sugar synthesis and transport, including SUS, SPS, TST, STP and ERD6L, were up-regulated, whereas INVs, FRK and HXK genes related to sugar degradation were down-regulated in HP kumquat. For organic acids, the up-regulation of PEPC and NAD-MDH could accelerate malate accumulation. In contrast, high expression of NAD-IDH and GS resulted in citric acid degradation during HP fruit development. Additionally, the PK, PDH, PEPCK and FBPase genes responsible for the interconversion of soluble sugars and organic acids were also significantly altered in the early development stages in HP. CONCLUSION: The high sugar accumulation in HP fruit was associated with up-regulation of SUS, SPS, TST, STP and ERD6L genes. The PEPCK, PEPC, NAD-MDH, NADP-IDH, GS and FBPase genes played important roles in acid synthesis and degradation in HP kumquat. These findings provide further insight into understanding the mechanisms underlying metabolism of sugars and organic acids in citrus. © 2021 Society of Chemical Industry.

Natural variations of TFIIAγ gene and LOB1 promoter contribute to citrus canker disease resistance in Atalantia buxifolia.

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating diseases in citrus industry worldwide. Most citrus cultivars such as sweet orange are susceptible to canker disease. Here, we utilized wild citrus to identify canker-resistant germplasms, and found that Atalantia buxifolia, a primitive (distant-wild) citrus, exhibited remarkable resistance to canker disease. Although the susceptibility gene LATERAL ORGAN BOUNDARIES 1 (LOB1) could also be induced in Atalantia after canker infection, the induction extent was far lower than that in sweet orange. In addition, three of amino acids encoded by transcription factor TFIIAγ in Atalantia (AbTFIIAγ) exhibited difference from those in sweet orange (CsTFIIAγ) which could stabilize the interaction between effector PthA4 and effector binding element (EBE) of LOB1 promoter. The mutation of AbTFIIAγ did not change its interaction with transcription factor binding motifs (TFBs). However, the AbTFIIAγ could hardly support the LOB1 expression induced by the PthA4. In addition, the activity of AbLOB1 promoter was significantly lower than that of CsLOB1 under the induction by PthA4. Our results demonstrate that natural variations of AbTFIIAγ and effector binding element (EBE) in the AbLOB1 promoter are crucial for the canker disease resistance of Atalantia. The natural mutations of AbTFIIAγ gene and AbLOB1 promoter in Atalantia provide candidate targets for improving the resistance to citrus canker disease.

Phylogenetic relationship of Paramignya trimera and its relatives: an evidence for the wide sexual compatibility.

The genus Paramignya (Rutaceae) comprises about 30 species typically distributing in tropical Asia. Like other genera of the family Rutaceae, the significant variation in the morphology of Paramignya species makes the taxonomic study and accurate identification become difficult. In Vietnam, Paramignya species have been mostly found in Khanh Hoa and Lam Dong provinces and used as traditional medicines. Recently, Paramignya trimera, a species of the genus Paramignya with local name "Xao tam phan" has been drawn attention and intensively exploited to treat liver diseases and cancers. However, the significant variations in the morphology and different local names of P. trimera have caused confusion and difficulty in the accurate identification and application of this plant for medicine. In this study, the combination of both morphological and DNA sequence data has effectively supported the taxonomic identification of P. trimera and some relatives collected in Khanh Hoa and Lam Dong provinces. The comparison of the morphology and analysis of the phylogenetic trees suggested that there was a significant variation of P. trimera. In addition, some accessions of P. trimera with morphological characteristics similar and Atalantia buxifolia were likely the intergeneric hybrids between the two species. Analysis of genetic variation, interspecific and intraspecific distances using ITS, matK and rbcL sequences shown that P. trimera was closely related to A. buxifolia, Severinia monophylla and Luvunga scandens. In addition, matK sequences represented as the effective candidate DNA barcode to identify and distinguish Paramignya species from others of the family Rutaceae.

The genetic origin of evolidine, the first cyclopeptide discovered in plants, and related orbitides.

Cyclic peptides are reported to have antibacterial, antifungal, and other bioactivities. Orbitides are a class of cyclic peptides that are small, head-to-tail cyclized, composed of proteinogenic amino acids and lack disulfide bonds; they are also known in several genera of the plant family Rutaceae. Melicope xanthoxyloides is the Australian rain forest tree of the Rutaceae family in which evolidine, the first plant cyclic peptide, was discovered. Evolidine (cyclo-SFLPVNL) has subsequently been all but forgotten in the academic literature, so to redress this we used tandem MS and de novo transcriptomics to rediscover evolidine and decipher its biosynthetic origin from a short precursor just 48 residues in length. We also identified another six M. xanthoxyloides orbitides using the same techniques. These peptides have atypically diverse C termini consisting of residues not recognized by either of the known proteases plants use to macrocyclize peptides, suggesting new cyclizing enzymes await discovery. We examined the structure of two of the novel orbitides by NMR, finding one had a definable structure, whereas the other did not. Mining RNA-seq and whole genome sequencing data from other species of the Rutaceae family revealed that a large and diverse family of peptides is encoded by similar sequences across the family and demonstrates how powerful de novo transcriptomics can be at accelerating the discovery of new peptide families.

Antinociceptive compounds and LC-DAD-ESIMSn profile from Dictyoloma vandellianum leaves.

Limonoids, quinolone alkaloids and chromones have been reported as constituents of Dictyoloma vandellianum Adr. Juss. (Rutaceae). Although those compounds are known for their biological activities, only the anti-inflammatory activity of chromones isolated from the underground parts has been evaluated. There are no studies of the pharmacological properties of the aerial parts of D. vandellianum. The present study was carried out to determine the phytochemical profile and antinociceptive activity of the methanol extract, fractions and isolated compounds of leaves of D. vandellianum. The phytochemical profile was performed by HLPC-DAD-ESIMSn and pure substances obtained were characterized by MS and NMR spectroscopy. The antinociceptive activity was assessed using the formalin assay in mice, and the motor function in the rotarod test. ME and all the fractions obtained from ME produced antinociceptive effects. Among them, the ethyl ether fraction was the most active. Data from HPLC-DAD-ESIMSn showed that the ethyl ether fraction presented 42 compounds. The major compounds isolated from this fraction-gallic acid, methyl gallate and 1,2,6-tri-O-galloyl-ß-d-glucopyranose-were tested and produced antinociceptive effects. Gallic acid, methyl gallate and 1,2,6-tri-O-galloyl-ß-d-glucopyranose at antinociceptive doses did not affect the motor performance in mice in the rotarod test. This work is the first report of the occurrence of gallotanins in D. vandellianum. In addition, the pharmacological study showed that D. vandellianum leaves present antinociceptive activity, probably induced by gallic acid, methyl gallate and 1,2,6-tri-O-galloyl-ß-d-glucopyranose.

Conflicting selection on Cneorum tricoccon (Rutaceae) seed size caused by native and alien seed dispersers.

The disappearance of native seed dispersers due to anthropogenic activities is often accompanied by the introduction of alien species, which may to some extent replace the ecological service provided by the extinct ones. Yet, little empirical evidence exists demonstrating the evolutionary consequences of such alien "replacement." Here, we document the conflicting selection exerted on seed size by two native lizards (Podarcis lilfordi and P. pityusensis) and an alien mammal species (Martes martes), all acting as legitimate seed dispersers of the Mediterranean relict Cneorum tricoccon. While lizards mostly exerted a negative directional selection on seed diameter, especially P. pityusensis, the much larger pine marten exerted positive selection on seed size. Our findings suggest that this among-disperser variation in the selection regimes, together with the occurrence of spatial variation in the presence of each seed disperser, help to create the geographical variation observed for seed size of C. tricoccon. To our knowledge, this is the first empirical evidence showing opposing selective pressures between native and alien species in the seed dispersal process in an invaded ecosystem.

Evolution of phytochemical diversity in Pilocarpus (Rutaceae).

The evolution of phytochemical diversity and biosynthetic pathways in plants can be evaluated from a phylogenetic and environmental perspective. Pilocarpus Vahl (Rutaceae), an economically important medicinal plant in the family Rutaceae, has a great diversity of imidazole alkaloids and coumarins. In this study, we used phylogenetic comparative methods to determine whether there is a phylogenetic signal for chemical traits across the genus Pilocarpus; this included ancestral reconstructions of continuous and discrete chemical traits. Bioclimatic variables found to be associated with the distribution of this genus were used to perform OLS regressions between chemical traits and bioclimatic variables. Next, these regression models were evaluated to test whether bioclimatic traits could significantly predict compound concentrations. Our study found that in terms of compound concentration, variation is most significantly associated with adaptive environmental convergence rather than phylogenetic relationships. The best predictive model of chemical traits was the OLS regression that modeled the relationship between coumarin and precipitation in the coldest quarter. However, we also found one chemical trait was dependent on phylogenetic history and bioclimatic factors. These findings emphasize that consideration of both environmental and phylogenetic factors is essential to tease out the intricate processes in the evolution of chemical diversity in plants. These methods can benefit fields such as conservation management, ecology, and evolutionary biology.

Transcriptome analysis of the curry tree (Bergera koenigii L., Rutaceae) during leaf development.

The curry tree (Bergera koenigii L.) is a widely cultivated plant used in South Asian cooking. Next-generation sequencing was used to generate the transcriptome of the curry leaf to detect changes in gene expression during leaf development, such as those genes involved in the production of oils which lend the leaf its characteristic taste, aroma, and medicinal properties. Using abundance estimation (RSEM) and differential expression analysis, genes that were significantly differentially expressed were identified. The transcriptome was annotated with BLASTx using the non-redundant (nr) protein database, and Gene Ontology (GO) terms were assigned based on the top BLAST hit using Blast2GO. Lastly, functional enrichment of the assigned GO terms was analyzed for genes that were significantly differentially expressed. Of the most enriched GO categories, pathways involved in cell wall, membrane, and lignin synthesis were found to be most upregulated in immature leaf tissue, possibly due to the growth and expansion of the leaf tissue. Terpene synthases, which synthesize monoterpenes and sesquiterpenes, which comprise much of the curry essential oil, were found to be significantly upregulated in mature leaf tissue, suggesting that oil production increases later in leaf development. Enzymes involved in pigment production were also significantly upregulated in mature leaves. The findings were based on computational estimates of gene expression from RNA-seq data, and further study is warranted to validate these results using targeted techniques, such as quantitative PCR.

Study of Some Zanthoxylum Species by Chemical and DNA Analysis Approaches.

The authentication and traceability of spices is a major concern for industrials and consumers. We focused on species from Zanthoxylum genera which are used for many different applications by local populations and also for trading as spices (dried pericarps or whole fruits). In this case, literature gives contradictory data about botanical names, and commercial labelling is often confusing. We studied commercial fruits pericarps extracts obtained by supercritical CO2 and analyzed them by GC/MS. The very complex volatile and semi volatile fractions composition of each extract is described. The barcoding method including molecular biology and phylogenetic analyses was also developed in order to check the commercial botanical identification of the raw material. This is a robust method to identify species in berries samples. We used one genetic marker to identify two Rutaceae clusters, including several species of Zanthoxylum genus. These results indicate that Fagara and Zanthoxylum groups could be considered as two different genera. Combination of chemical analysis and DNA analysis provides an original approach to increase chemical and botanical Zanthoxylum genus knowledge.

Phylogenetic relationships of Vepris (Rutaceae) inferred from chloroplast, nuclear, and morphological data.

The tribe Toddalieae Hook. F. (Rutaceae) has been controversial since its inception by Bentham and Hooker. The nine taxa examined, Acronychia J.R. & G.Foster, Diphasia Pierre, Diphasiopsis Mendonca, Fagaropsis Mildbr.ex. Siebenl., Oricia Pierre, Teclea Delile, Toddaliopsis Engl., Toddalia Juss. and Vepris Comm. ex. A. Juss, have been recognized under the tribe Toddalieae or Tribes Acronychia, Phellodendron and Toddalia. More recently Araliopsis Engl., Diphasia, Diphasiopsis, Oricia, Teclea, and Toddaliopsis have been incorporated into the genus Vepris, while Toddalia and Fagaropsis have continued to be recognized as closely related. For this study, sequence data of one non-coding chloroplast region (trnL-F) and one nuclear region (ITS) and various morphological characters, based on Mziray's taxonomic studies were examined to try to elucidate these relationships. This study found that the taxa Diphasia, Diphasiopsis, Oricia, Teclea, Toddaliopsis, Vepris, Toddalia eugeniifolia Engl. and Toddalia glomerata F. Hoffm. form a monophyletic group. Due to the amount of intrageneric and intraspecific variation, species delimitations were difficult to determine; however, these genera should be united into Vepris. The analyses also confirmed that Toddalia asiatica (L.) Lam., Zanthoxylon sp. and Fagaropsis angolensis (Engl.) H.M. Gardner are the closest relatives to this group.

Analysis of whole chloroplast genomes from the genera of the Clauseneae, the curry tribe (Rutaceae, Citrus family).

The Clauseneae (Aurantioideae, Rutaceae) is a tribe in the Citrus family that, although economically important as it contains the culinary and medicinally-useful curry tree (Bergera koenigii), has been relatively understudied. Due to the recent significant taxonomic changes made to this tribe, a closer inspection of the genetic relationships among its genera has been warranted. Whole genome skimming was used to generate chloroplast genomes from six species, representing each of the four genera (Bergera, Clausena, Glycosmis, Micromelum) in the Clauseneae tribe plus one closely related outgroup (Merrillia), using the published plastome sequence of Citrus sinensis as a reference. Phylogenetically informative character (PIC) data were analyzed using a genome alignment of the seven species, and variability frequency among the species was recorded for each coding and non-coding region, with the regions of highest variability identified for future phylogenetic studies. Non-coding regions exhibited a higher percentage of variable characters as expected, and the phylogenetic markers ycf1, matK, rpoC2, ndhF, trnS-trnG spacer, and trnH-psbA spacer proved to be among the most variable regions. Other markers that are frequently used in phylogenetic studies, e.g. rps16, atpB-rbcL, rps4-trnT, and trnL-trnF, proved to be far less variable. Phylogenetic analyses of the aligned sequences were conducted using Bayesian inference (MrBayes) and Maximum Likelihood (RAxML), yielding highly supported divisions among the four genera.

Towards a molecular taxonomic key of the Aurantioideae subfamily using chloroplastic SNP diagnostic markers of the main clades genotyped by competitive allele-specific PCR.

BACKGROUND: Chloroplast DNA is a primary source of molecular variations for phylogenetic analysis of photosynthetic eukaryotes. However, the sequencing and analysis of multiple chloroplastic regions is difficult to apply to large collections or large samples of natural populations. The objective of our work was to demonstrate that a molecular taxonomic key based on easy, scalable and low-cost genotyping method should be developed from a set of Single Nucleotide Polymorphisms (SNPs) diagnostic of well-established clades. It was applied to the Aurantioideae subfamily, the largest group of the Rutaceae family that includes the cultivated citrus species. RESULTS: The publicly available nucleotide sequences of eight plastid genomic regions were compared for 79 accessions of the Aurantioideae subfamily to search for SNPs revealing taxonomic differentiation at the inter-tribe, inter-subtribe, inter-genus and interspecific levels. Diagnostic SNPs (DSNPs) were found for 46 of the 54 clade levels analysed. Forty DSNPs were selected to develop KASPar markers and their taxonomic value was tested by genotyping 108 accessions of the Aurantioideae subfamily. Twenty-seven markers diagnostic of 24 clades were validated and they displayed a very high rate of transferability in the Aurantioideae subfamily (only 1.2 % of missing data on average). The UPGMA from the validated markers produced a cladistic organisation that was highly coherent with the previous phylogenetic analysis based on the sequence data of the eight plasmid regions. In particular, the monophyletic origin of the "true citrus" genera plus Oxanthera was validated. However, some clarification remains necessary regarding the organisation of the other wild species of the Citreae tribe. CONCLUSIONS: We validated the concept that with well-established clades, DSNPs can be selected and efficiently transformed into competitive allele-specific PCR markers (KASPar method) allowing cost-effective highly efficient cladistic analysis in large collections at subfamily level. The robustness of this genotyping method is an additional decisive advantage for network collaborative research. The availability of WGS data for the main "true citrus" species should soon make it possible to develop a set of DSNP markers allowing very fine resolution of this very important horticultural group.

Phylogeny of Acronychia (Rutaceae) and First Insights into Its Historical Biogeography and the Evolution of Fruit Characters.

BACKGROUND: The genus Acronychia (Citrus family, Rutaceae) contains 49 species of trees and shrubs that are found mainly in rain forest. The genus has a large distributional range from mainland southern Asia to Australia and New Caledonia, but most species are endemic to either New Guinea or Australia. This study aimed to provide the first detailed molecular phylogeny of Acronychia and use it to test the taxonomic value of fruit morphological characters, and infer the historical biogeography of the genus. METHODOLOGY: Phylogenetic analyses (Bayesian Inference, Maximum Likelihood) were undertaken on nucleotide sequence data from two plastid (psbA-trnH, trnL-trnF) and three nuclear markers (ETS, ITS, NIAi3) from 29 Acronychia species (59% of the genus) and representatives of related genera. RESULTS AND CONCLUSIONS: The results indicate that the South-East Asian genus Maclurodendron is nested phylogenetically within Acronychia and must be synonymized to render Acronychia monophyletic. Fruit morphological characters have been used previously to infer relationships within Acronychia and our analyses show that these characters are informative for some subclades but are homoplasious for the group as a whole. Apocarpous fruits are the ancestral state in Acronychia and subapocarpous and fully syncarpous fruits are derived. The unisexual flowers of Maclurodendron are derived from bisexual flowers, which are found in all species of Acronychia as well as its relatives. Acronychia probably first evolved on Australia with range expansion to New Guinea via stepping-stone dispersal or direct land connections within the Sahul Shelf, followed by two independent dispersals to areas west of New Guinea. Most species of Acronychia occur in either Australia or New Guinea, but no species occurs in both regions. This is surprising given the close proximity of the landmasses, but might be explained by ecological factors.

Almeidea A. St.-Hil. belongs to Conchocarpus J.C. Mikan (Galipeinae, Rutaceae): evidence from morphological and molecular data, with a first analysis of subtribe Galipeinae.

Subtribe Galipeinae (tribe Galipeeae, subfamily Rutoideae) is the most diverse group of Neotropical Rutaceae, with 28 genera and approximately 130 species. One of its genera is Almeidea, whose species are morphologically similar to those of the genus Conchocarpus. Species of Almeidea occur in the Atlantic Rain Forest of Eastern Brazil, with one species (Almeidea rubra) also present in Bolivia. The objective of this study was to perform a phylogenetic analysis of Almeidea, using a broader sampling of Galipeinae and other Neotropical Rutaceae, the first such study focused on this subtribe. To achieve this objective, morphological data and molecular data from the nuclear markers ITS-1 and ITS-2 and the plastid markers trnL-trnF and rps16 were obtained. Representatives of eight genera of Galipeinae and three genera of Pilocarpinae (included also in Galipeeae) and Hortia (closely related to Galipeeae) were used. Five species of Almeidea and seven of Conchocarpus were included, given the morphological proximity between these two genera. Individual (for each molecular marker) and combined phylogenetic analyses were made, using parsimony and Bayesian inference as optimization criteria. Results showed Galipeinae as monophyletic, with the species of Almeidea also monophyletic (supported by the presence of pantocolporate pollen) and nested in a clade with a group of species of Conchocarpus, a non-monophyletic group. Additionally, C. concinnus appeared in a group with Andreadoxa, Erythrochiton, and Neoraputia, other members of Galipeinae. As a result, Conchocarpus would be monophyletic only with the exclusion of a group of species related to C. concinnus and with the inclusion of all species of Almeidea with the group of species of Conchocarpus that includes its type species, C. macrophyllus. Thus, species of Almeidea are transferred to Conchocarpus, and the new combinations are made here.

Fragmentation effects and genetic diversity of the key semidecidual forest species Metrodorea nigra in Southwestern Brazil.

Studies of genetic diversity in plant species present in the remaining fragments of the Atlantic Forest are very important for understanding their resilience to such a degraded ecosystem. We analyzed the genetic diversity of 3 populations of the high-density understory species Metrodorea nigra St. Hill. (Rutaceae) located in forest remnants in the region of Ribeirão Preto, SP, Brazil (M13-Rib, BSQ-Rib, and FAC-Crav), by using simple sequence repeat (SSR) and inter-simple sequence repeat (ISSR) molecular markers for conservation purposes. A total of 133 polymorphic loci were observed in 136 inter-simple sequence repeat loci (average of 17 per primer). The Nei genetic diversity (HE) was relatively high considering all populations (0.31). The BSQ-Rib population exhibited the highest value (0.27), followed by the M13-Rib (0.26) and FAC-Crav (0.24) populations. The simple sequence repeat markers analyzed showed a high number of alleles (K = 104), with an average of 14.85 alleles per locus. The average observed heterozygosity was 0.516 and the average expected heterozygosity was 0.771, ranging from 0.688 (FAC-Crav) to 0.765 (BSQ-Rib). The fixation indexes showed positive and significant differences from zero for all sample sets, indicating inbreeding, which may have resulted from the species' mating patterns and the barochoric seed dispersal system of M. nigra. Both markers indicated differentiation among populations, with higher values observed for inter-simple sequence repeat markers. No significant differences between juvenile and adult generations in any of the fragments were observed, indicating the resilience of M. nigra to the effects of fragmentation and reduced habitat.

FcWRKY70, a WRKY protein of Fortunella crassifolia, functions in drought tolerance and modulates putrescine synthesis by regulating arginine decarboxylase gene.

WRKY comprises a large family of transcription factors in plants, but most WRKY members are still poorly understood. In this study, we report functional characterization of a Group III WRKY gene (FcWRKY70) from Fortunella crassifolia. FcWRKY70 was greatly induced by drought and abscisic acid, but slightly or negligibly by salt and cold. Overexpression of FcWRKY70 in tobacco (Nicotiana nudicaulis) and lemon (Citrus lemon) conferred enhanced tolerance to dehydration and drought stresses. Transgenic tobacco and lemon exhibited higher expression levels of ADC (arginine decarboxylase), and accumulated larger amount of putrescine in comparison with wild type (WT). Treatment with D-arginine, an inhibitor of ADC, caused transgenic tobacco plants more sensitive to dehydration. Knock-down of FcWRKY70 in kumquat down-regulated ADC abundance and decreased putrescine level, accompanied by compromised dehydration tolerance. The promoter region of FcADC contained two W-box elements, which were shown to be interacted with FcWRKY70. Taken together, our data demonstrated that FcWRKY70 functions in drought tolerance by, at least partly, promoting production of putrescine via regulating ADC expression.

Carotenoid accumulation affects redox status, starch metabolism, and flavonoid/anthocyanin accumulation in citrus.

BACKGROUND: Carotenoids are indispensable plant secondary metabolites that are involved in photosynthesis, antioxidation, and phytohormone biosynthesis. Carotenoids are likely involved in other biological functions that have yet to be discovered. In this study, we integrated genomic, biochemical, and cellular studies to gain deep insight into carotenoid-related biological processes in citrus calli overexpressing CrtB (phytoene synthase from Pantoea agglomerans). Fortunella hindsii Swingle (a citrus relative) and Malus hupehensis (a wild apple) calli were also utilized as supporting systems to investigate the effect of altered carotenoid accumulation on carotenoid-related biological processes. RESULTS: Transcriptomic analysis provided deep insight into the carotenoid-related biological processes of redox status, starch metabolism, and flavonoid/anthocyanin accumulation. By applying biochemical and cytological analyses, we determined that the altered redox status was associated with variations in O2 (-) and H2O2 levels. We also ascertained a decline in starch accumulation in carotenoid-rich calli. Furthermore, via an extensive cellular investigation of the newly constructed CrtB overexpressing Fortunella hindsii Swingle, we demonstrated that starch level reducation occurred in parallel with significant carotenoid accumulation. Moreover, studying anthocyanin-rich Malus hupehensis calli showed a negative effect of carotenoids on anthocyanin accumulation. CONCLUSIONS: In citrus, altered carotenoid accumulation resulted in dramatic effects on metabolic processes involved in redox modification, starch degradation, and flavonoid/anthocyanin biosynthesis. These findings provided new perspectives to understand the biological importance of carotenogenesis and of the developmental processes associated with the nutritional and sensory qualities of agricultural products that accumulate carotenoids.