Encoding Chalcone Synthase Research Articles

The characterization of anther-specific promoter encoding chalcone synthase isolated from Lilium Oriental hybrid cv. Acapulco

The characterization of anther-specific promoter encoding chalcone synthase isolated from Lilium Oriental hybrid cv. Acapulco

Ectopic expression and functional characterization of type III polyketide synthase mutants from Emblica officinalis Gaertn.

Functional characterization and ectopic expression studies of chalcone synthase mutants implicate the role of phenylalanine in tailoring the substrate specificity of type III polyketide synthase. Chalcone synthase (CHS) is a plant-specific type III polyketide synthase that catalyzes the synthesis of flavonoids. Native CHS enzyme does not possess any functional activity on N-methylanthraniloyl-CoA, which is the substrate for acridione/quinolone alkaloid biosynthesis. Here, we report the functional transformation of chalcone synthase protein from Emblica officinalis (EoCHS) to quinolone and acridone synthase (ACS) with single amino acid substitutions. A cDNA of 1173bp encoding chalcone synthase was isolated from E. officinalis and mutants (F215S and F265V) were generated by site-directed mutagenesis. Molecular modeling studies of EoCHS did not show any active binding with N-methylanthraniloyl-CoA, but the mutants of EoCHS showed strong affinity to the same. As revealed by the modeling studies, functional analysis of CHS mutants showed that they could utilize p-coumaroyl-CoA as well as N-methylanthraniloyl-CoA as substrates and yield active products such as naringenin, 4-hydroxy 1-methyl 2(H) quinolone and 1,3-dihydroxy-n-methyl acridone. Exchange of a single amino acid in EoCHS (F215S and F265V) resulted in functionally active mutants that preferred N-methylanthraniloyl-CoA over p-coumaroyl-CoA. This can be attributed to the increase in the relative volume of active sites in mutants by mutation. Moreover, metabolomic and MS analyses of tobacco leaves transiently expressing mutant genes showed high levels of naringenin, acridones and quinolone derivatives compared to wild-type CHS. This is the first report demonstrating the functional activity of EoCHS mutants with N-methylanthraniloyl-CoA and these results indicate the role of phenylalanine in altering the substrate specificity and in the evolution of type III PKSs.

Mushrooms do not contain flavonoids

A literature and database review and several experiments were carried out because of the perturbing increase and exponential spreading of a major mistake thought scientific publications. Up to October 2015, more than 136 reports ensured that mushrooms contain flavonoids. However, 91% of the publications used an unspecific colorimetric method that was developed to quantify them in plants. However, no sequences encoding chalcone synthase or chalcone isomerase (key enzymes involved in the flavonoid biosynthetic pathway) were found in genomic databases even in completely sequenced mushrooms indicating that they do not have the enzymes needed for their production. Fruiting bodies obtained from substrates containing onion wastes and mycelia grown on media supplemented with specific flavonoids showed no flavonoid content after HPLC DAD/MS determinations indicating that they were also unable of absorbing them.

Molecular cloning and transgenic characterization of the genes encoding chalcone synthase and chalcone isomerase from the Tibetan herbal plant Mirabilis himalaica.

Mirabilis himalaica is an endangered medicinal plant species in the Tibetan Plateau. The two genes respectively encoding chalcone synthase (MhCHS) and chalcone isomerase (MhCHI) were isolated and characterized from M. himalaica. The sequence analysis revealed that the two genes were similar with their corresponding homologous genes in other plants. The tissue profiles showed that both MhCHS and MhCHI had higher expression levels in roots than in stems and leaves. Transgenic hairy root cultures respectively with overexpressing MhCHS and MhCHI were established. The genomic PCR detection confirmed the authority of transgenic hairy root lines, in which either MhCHS or MhCHI expression levels were much higher than that in non-transgenic hairy root line. Finally, the HPLC detection results demonstrated that the rotenoid contents in MhCHS/MhCHI-transformed hairy root lines were enhanced. This study provided two candidate genes that could be used to genetic engineering rotenoid biosynthesis in M. himalaica and an alternative method to produce rotenoid using transgenic hairy root cultures.

AtROS1 overexpression provides evidence for epigenetic regulation of genes encoding enzymes of flavonoid biosynthesis and antioxidant pathways during salt stress in transgenic tobacco.

In plants, epigenetic changes have been identified as regulators of developmental events during normal growth as well as environmental stress exposures. Flavonoid biosynthetic and antioxidant pathways play a significant role in plant defence during their exposure to environmental cues. The aim of this study was to unravel whether genes encoding enzymes of flavonoid biosynthetic and antioxidant pathways are under epigenetic regulation, particularly DNA methylation, during salt stress. For this, a repressor of silencing from Arabidopsis, AtROS1, was overexpressed in transgenic tobacco. Generated transgenics were evaluated to examine the influence of AtROS1 on methylation status of promoters as well as on coding regions of genes encoding enzymes of flavonoids biosynthesis and antioxidant pathways. Overexpression of AtROS1 increases the demethylation levels of both promoters as well as coding regions of genes encoding chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonol synthase, dihydroflavonol 4-reductase, and anthocyanidin synthase of the flavonoid biosynthetic pathway, and glutathione S-transferase, ascorbate peroxidase, glutathione peroxidase, and glutathione reductase of the antioxidant pathway during control conditions. The level of demethylation was further increased at promoters as well as coding regions of these genes during salt-stress conditions. Transgenic tobacco overexpressing AtROS1 showed tolerance to salt stress that could have been due to the higher expression levels of the genes encoding enzymes of the flavonoid biosynthetic and antioxidant pathways. This is the first comprehensive study documenting the epigenetic regulation of flavonoid biosynthetic and antioxidant pathways during salt-stress exposure of plants.

The possible involvement of salicylic acid and hydrogen peroxide in the systemic promotion of phenolic biosynthesis in clover roots colonized by arbuscular mycorrhizal fungus

Arbuscular mycorrhizal fungal (AMF) colonization can induce both the local and the systemic increase in phenolic accumulation in hosts. However, the signaling molecules responsible for the systemic induction is still unclear. In this study, a split-root rhizobox system was designed to explore these molecules, with one half of clover (Trifolium repense) roots colonized by AMF, Funneliformis mosseae (formerly known as Glomus mosseae), and the other not (NM/M). Plants with two halves both (M/M) or neither (NM/NM) inoculated were also established for comparison. The contents of phenols and the accumulation of salicylic acid (SA), hydrogen peroxide (H2O2) and nitric oxide (NO) in roots were monitored, the activities of l-phenylalanine ammonia-lyase (PAL) and nitric oxide synthase (NOS) in roots were assayed, and the expressions of pal and chs (gene encoding chalcone synthase) genes in roots were also quantified using qRT-PCR. Results indicated that when phenolic content in NM/NM plants was lower than that in M/M plants, AMF colonization systemically induced the increase in phenolic content in NM/M plants. Similarly, the accumulations of SA and H2O2 were increased by AMF both locally and systemically, while that of NO was only increased locally. Moreover, enzyme assay and qRT-PCR were in accordance with these results. These data suggest that AMF colonization can systemically increase the phenolic biosynthesis, and SA and H2O2 are possibly the signaling molecules involved. The role of MeSA, a signaling molecule capable of long distance transport in this process, is also discussed.

Isolation and expression analysis of anthocyanin biosynthetic genes in Morus alba L.

Anthocyanins from mulberry fruits are used in medicine. However, little anthocyanin can be detected in other tissues and sometimes also mulberry fruits are colorless. The aim of this study was to investigate which gene or genes have the strongest correlation with the anthocyanin biosynthesis. The expression of several anthocyanin synthesis genes were determined in different tissues of two white and two purple fruit cultivars. Genes encoding dihydroflavonol reductase (MaDFR) and anthocyanidin synthase (MaANS) showed a high expression only in fruit tissue of purple-fruit cultivars. During the development of mulberry fruits, the anthocyanin content was well correlated with the transcripts abundance of MaDFR, MaANS, and MaCHS (encoding chalcone synthase). The skin of female mulberry flowers turns red under irradiance because of up-regulated expressions of MaCHS, MaDFR, and MaANS. These three genes may control the anthocyanin biosynthesis in mulberry and up-regulation of them may greatly increase the anthocyanin content.

Regulation of POD activity by pelargonidin during vegetative growth in radish (Raphanus sativus L.)

Peroxidase (POD) can degrade anthocyanin in planta. Either positive or negative correlation between POD activity and anthocyanin content has been reported in plants. Given the occurrence of conflicting results, we investigated the relationship between POD activity and anthocyanin content during vegetative growth of three radish (Raphanus sativus L.) cultivars (Man Tang Hong, Da Hong Pao and Si Ji Da Gen). Significant positive correlations were observed between POD activity and pelargonidin content in the flesh throughout the vegetative growth stage and in the skin from 50 to 90 days after sowing in all three cultivars. A high-level constitutive expression of genes encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) directly involved in anthocyanin biosynthesis was observed in the flesh, while, the expression of these genes was decreased in the skin with the growth of cultivar Man Tang Hong. The expression of anthocyanin biosynthesis genes requires coordinated transcripts of a bHLH transcription factor, Transparent Testa 8 (RsTT8), and a MYB transcription factor. RsMYB was mainly expressed in the flesh of Man Tang Hong, while RsTT8 was expressed in both the skin and flesh of Man Tang Hong. The expression kinetics of POD gene Rsprx1 was similar to those of anthocyanin biosynthesis genes in Man Tang Hong during growth. These results indicated that the activity and expression of POD can be regulated by anthocyanin content in the plant during development.

Deep sequencing uncovers commonality in small RNA profiles between transgene-induced and naturally occurring RNA silencing of chalcone synthase-A gene in petunia

BackgroundIntroduction of a transgene that transcribes RNA homologous to an endogenous gene in the plant genome can induce silencing of both genes, a phenomenon termed cosuppression. Cosuppression was first discovered in transgenic petunia plants transformed with the CHS-A gene encoding chalcone synthase, in which nonpigmented sectors in flowers or completely white flowers are produced. Some of the flower-color patterns observed in transgenic petunias having CHS-A cosuppression resemble those in existing nontransgenic varieties. Although the mechanism by which white sectors are generated in nontransgenic petunia is known to be due to RNA silencing of the CHS-A gene as in cosuppression, whether the same trigger(s) and/or pattern of RNA degradation are involved in these phenomena has not been known. Here, we addressed this question using deep-sequencing and bioinformatic analyses of small RNAs.ResultsWe analyzed short interfering RNAs (siRNAs) produced in nonpigmented sectors of petal tissues in transgenic petunia plants that have CHS-A cosuppression and a nontransgenic petunia variety Red Star, that has naturally occurring CHS-A RNA silencing. In both silencing systems, 21-nt and 22-nt siRNAs were the most and the second-most abundant size classes, respectively. CHS-A siRNA production was confined to exon 2, indicating that RNA degradation through the RNA silencing pathway occurred in this exon. Common siRNAs were detected in cosuppression and naturally occurring RNA silencing, and their ranks based on the number of siRNAs in these plants were correlated with each other. Noticeably, highly abundant siRNAs were common in these systems. Phased siRNAs were detected in multiple phases at multiple sites, and some of the ends of the regions that produced phased siRNAs were conserved.ConclusionsThe features of siRNA production found to be common to cosuppression and naturally occurring silencing of the CHS-A gene indicate mechanistic similarities between these silencing systems especially in the biosynthetic processes of siRNAs including cleavage of CHS-A transcripts and subsequent production of secondary siRNAs in exon 2. The data also suggest that these events occurred at multiple sites, which can be a feature of these silencing phenomena.

A pair of transposons coordinately suppresses gene expression, independent of pathways mediated by siRNA in Antirrhinum

Our knowledge is limited regarding mechanisms by which transposable elements control host gene expression. Two Antirrhinum lines, HAM2 and HAM5, show different petal colors, pale-red and white, respectively, although these lines contain the same insertion of transposon Tam3 in the promoter region of the nivea (niv) locus encoding chalcone synthase. Among 1000 progeny from HAM5 grown under the preferred conditions for the Tam3 transposition, a few showed an intermediate petal color between HAM2 and HAM5. Transposon tagging using these progeny identified a causative insertion of Tam3 for the HAM5 type (white) petal color, which was found 1.6 kb downstream of the niv gene. Insertion of Tam3 at the position 1.6 kb downstream of niv alone showed nearly wildtype petal pigmentation, and the niv expression reduced by only 50%. Severe suppression of niv observed in HAM5 required interaction of two Tam3 copies on either side of the niv coding sequence. DNA methylation and small interfering RNAs (siRNAs) were not associated with the suppression of niv expression in HAM5. Insertion of a pair of transposons in close proximity can interfere with the expression of gene located between the two copies, and also provide evidence that this interference is not directly associated with pathways mediated by siRNAs.

Anthocyanin accumulation and expression analysis of biosynthesis-related genes during chili pepper fruit development

Chili pepper (Capsicum annuum L.) cv. Arbol and Uvilla fruits differing in anthocyanin contents were analyzed to characterize the accumulation patterns. The maximum accumulation of the aglycon delphinidin occurred 20 days postanthesis (DPA) with higher content in Uvilla than in Arbol fruits. Regarding the cDNA library, 9 186 cDNA clones were selected. The clones with high homology to genes concerning anthocyanin biosynthesis, such as encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3′,5′-hydroxylase (F3′5′H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), UDP Glc-flavonoid 3-O-gluco-syl transferase (UFGT), and also those possibly involved in anthocyanin transport into the vacuoles, an anthocyanin permease (ANP) and a glutathione S-transferase (GST) were used for gene expression analysis. In general, the expression of all investigated genes was developmentally regulated in both Arbol and Uvilla. CHS and CHI transcripts were expressed at the maximal level at 10 DPA, and then consistently declined throughout fruit development. F3′5′H, DFR, UFGT and GST expression exhibited a positive correlation with anthocyanin accumulation, and the highest transcript levels were detected prior to or by the time of maximum anthocyanin accumulation, depending on the chili pepper type. Pericarp fruit tissues from cv. Tampiqueno 74, an anthocyanin non-accumulator, also showed CHS, CHI, F3H, ANS and ANP expression at some developmental stages.

Molecular mechanism for cadmium-induced anthocyanin accumulation in Azolla imbricata

Anthocyanins inducibly synthesized by Cd treatment showed high antioxidant activity and might be involved in internal detoxification mechanisms of Azolla imbricata against Cd toxicity. In order to understand anthocyanin biosynthesis mechanism during Cd stress, the cDNAs encoding chalcone synthase (CHS) and dihydroflavonol reductase (DFR), two key enzymes in the anthocyanin synthesis pathway, were isolated from A. imbricata. Deduced amino acid sequences of the cDNAs showed high homology to the sequences from other plants. Expression of AiDFR, and to a lesser extent AiCHS, was significantly induced in Cd treatment plant in comparison with the control. CHS and DFR enzymatic activities showed similar pattern changes with these genes expression during Cd stress. These results strongly indicate that Cd induced anthocyanin accumulation is probably mediated by up-regulation of structural genes including CHS and DFR, which might further increase the activities of enzymes encoded by these structural genes that control the anthocyanin biosynthetic steps.

Resistance to Elsinoë ampelina and expression of related resistant genes in Vitis rotundifolia Michx. grapes.

Muscadine grapes (Vitis rotundifolia Michx) are considered as excellent genetic resources for grape breeding programs as they are known for their hardiness and resistance to pests and diseases. However, contrary to popular belief, our study indicated that not all muscadine cultivars are resistant to anthracnose disease. In order to identify a source of genetic tolerance towards anthracnose among muscadine cultivars, a series of in-situ and ex-situ experiments were conducted through strict and sensitive screening processes. Two consecutive years of field evaluation of 54 grape cultivars showed various levels of anthracnose incidence among the cultivars between a scale of 0 (tolerant) to 5 (highly-susceptible). Resistance bioassay by inoculation of different spore densities of Elsinoë ampelina on 40 cultivars presented similar results and was consistent with those obtained from the field test. A real-time PCR analysis was conducted to investigate differences of gene expression between susceptible and tolerant cultivars and to confirm results by phenotypic identification. Expression of genes encoding chalcone synthase, stilbene synthase, polygalacturonase-inhibiting protein, chitinase and lipid transfer-protein was only detected in tolerant cultivars. Resistant muscadine cultivars identified in this study could be excellent candidates for grape disease resistance breeding programs.

Differential expression of anthocyanin biosynthetic genes in relation to anthocyanin accumulation in the pericarp of Litchi chinensis Sonn.

Litchi has diverse fruit color phenotypes, yet no research reflects the biochemical background of this diversity. In this study, we evaluated 12 litchi cultivars for chromatic parameters and pigments, and investigated the effects of abscisic acid, forchlorofenron (CPPU), bagging and debagging treatments on fruit coloration in cv. Feizixiao, an unevenly red cultivar. Six genes encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) were isolated from the pericarp of the fully red litchi cv. Nuomici, and their expression was analyzed in different cultivars and under the above mentioned treatments. Pericarp anthocyanin concentration varied from none to 734 mg m−2 among the 12 litchi cultivars, which were divided into three coloration types, i.e. non-red (‘Kuixingqingpitian’, ‘Xingqiumili’, ‘Yamulong’and ‘Yongxing No. 2′), unevenly red (‘Feizixiao’ and ‘Sanyuehong’) and fully red (‘Meiguili’, ‘Baila’, Baitangying’ ’Guiwei’, ‘Nuomici’ and ‘Guinuo’). The fully red type cultivars had different levels of anthocyanin but with the same composition. The expression of the six genes, especially LcF3H, LcDFR, LcANS and LcUFGT, in the pericarp of non-red cultivars was much weaker as compared to those red cultivars. Their expression, LcDFR and LcUFGT in particular, was positively correlated with anthocyanin concentrations in the pericarp. These results suggest the late genes in the anthocyanin biosynthetic pathway were coordinately expressed during red coloration of litchi fruits. Low expression of these genes resulted in absence or extremely low anthocyanin accumulation in non-red cultivars. Zero-red pericarp from either immature or CPPU treated fruits appeared to be lacking in anthocyanins due to the absence of UFGT expression. Among these six genes, only the expression of UFGT was found significantly correlated with the pericarp anthocyanin concentration (r = 0.84). These results suggest that UFGT played a predominant role in the anthocyanin accumulation in litchi as well as pericarp coloration of a given cultivar.

Transcription profiling of the isoflavone phenylpropanoid pathway in soybean in response to Bradyrhizobium japonicum inoculation

Isoflavones are legume-specific secondary metabolites that function as defence compounds, signal molecules and regulators of gene expression during both pathogen attack and beneficial plant-microbe interactions. They are synthesised by a branch of the core phenylpropanoid pathway, using several isoenzymes within each enzymatic step. Gene-specific quantitative real-time reverse transcriptase PCR (qRT-PCR) was used to quantify expression of isoflavone synthesis genes in soybean (Glycine max L). Genes encoding chalcone synthase 7 (CHS7), chalcone synthase 8 (CHS8) and isoflavone synthase 1 (IFS1) displayed high basal expression levels in roots compared with hypocotyls, suggesting they could be the gene family members encoding the isoenzyme that contributes the most to the principal substrate flux towards specific isoflavone synthesis in roots. The genes encoding phenylalanine ammonia lyase 1 (PAL1) and IFS1 showed induction in root tissue after inoculation with Bradyrhizobium japonicum (Kirchner) Jordan, suggesting a control point. The absence of a functional nodulation regulator, GmNARK (G. max nodulation autoregulation receptor kinase), in the soybean mutant nts1007 resulted in significantly increased basal expression of PAL1 compared with levels induced by B. japonicum, suggesting that GmNARK is a negative regulator for isoflavone phenylpropanoid pathway genes during nodulation and that distinct genes, as opposed to the complete pathway, are coordinately regulated by the nodulation status of the mutant.

Engineering traditional monolignols out of lignin by concomitant up-regulation of F5H1 and down-regulation of COMT in Arabidopsis

Lignin engineering is a promising strategy to optimize lignocellulosic plant biomass for use as a renewable feedstock for agro-industrial applications. Current efforts focus on engineering lignin with monomers that are not normally incorporated into wild-type lignins. Here we describe an Arabidopsis line in which the lignin is derived to a major extent from a non-traditional monomer. The combination of mutation in the gene encoding caffeic acid O-methyltransferase (comt) with over-expression of ferulate 5-hydroxylase under the control of the cinnamate 4-hydroxylase promoter (C4H:F5H1) resulted in plants with a unique lignin comprising almost 92% benzodioxane units. In addition to biosynthesis of this particular lignin, the comt C4H:F5H1 plants revealed massive shifts in phenolic metabolism compared to the wild type. The structures of 38 metabolites that accumulated in comt C4H:F51 plants were resolved by mass spectral analyses, and were shown to derive from 5-hydroxy-substituted phenylpropanoids. These metabolites probably originate from passive metabolism via existing biochemical routes normally used for 5-methoxylated and 5-unsubstituted phenylpropanoids and from active detoxification by hexosylation. Transcripts of the phenylpropanoid biosynthesis pathway were highly up-regulated in comt C4H:F5H1 plants, indicating feedback regulation within the pathway. To investigate the role of flavonoids in the abnormal growth of comt C4H:F5H1 plants, a mutation in a gene encoding chalcone synthase (chs) was crossed in. The resulting comt C4H:F5H1 chs plants showed partial restoration of growth. However, a causal connection between flavonoid deficiency and this restoration of growth was not demonstrated; instead, genetic interactions between phenylpropanoid and flavonoid biosynthesis could explain the partial restoration. These genetic interactions must be taken into account in future cell-wall engineering strategies.

The molecular basis of incomplete dominance at the A locus of CHS-D in the common morning glory, Ipomoea purpurea

The mutable a(flaked (a(f)) allele at the A locus of the common morning glory (Ipomoea purpurea) confers incomplete dominance in flower pigmentation and is caused by insertion of the DNA transposon Tip100 into CHS-D, which encodes chalcone synthase and is required for anthocyanin biosynthesis. Levels of CHS-D transcripts, CHS-D protein, and anthocyanin pigment in heterozygous flowers were about half that in homozygous flowers, indicating that dosage-dependent expression of CHS-D is the primary cause of the observed incomplete dominance. This contrasts with the Nivea locus in snapdragon (Antirrhinum majus) in which incomplete dominance is caused by semi-dominant CHS alleles.

Improvement of the design and generation of highly specific plant knockdown lines using primary synthetic microRNAs (pri-smiRNAs)

BackgroundmicroRNAs (miRNAs) are endogenous small non-coding RNAs that post-transcriptionally regulate gene expression. In plants, they typically show high complementarity to a single sequence motif within their target mRNAs and act by catalyzing specific mRNA cleavage and degradation. miRNAs are processed from much longer primary transcripts via precursor miRNAs containing fold-back structures. Leaving these secondary structures intact, miRNAs can be re-designed experimentally to target mRNAs of choice.ResultsWe designed primary synthetic miRNAs (pri-smiRNAs) on the basis of the primary transcript of the Arabidopsis MIR159A gene by replacing the original miR159a and the corresponding miR159a* with novel sequences, keeping the overall secondary structure as predicted by the program RNAfold. We used the program RNAhybrid to optimize smiRNA design and to screen the complete Arabidopsis transcriptome for potential off-targets. To improve the molecular cloning of the pri-smiRNA we inserted restriction sites in the original MIR159A primary transcript to easily accommodate the smiRNA/smiRNA* DNA fragment. As a proof-of-concept, we targeted the single gene encoding chalcone synthase (CHS) in Arabidopsis. We demonstrate smiRNA(CHS) expression and CHS mRNA cleavage in different transgenic lines. Phenotypic changes in these lines were observed for seed color and flavonol derivatives, and quantified with respect to anthocyanin content. We also tested the effect of mismatches and excess G:U base pairs on knockdown efficiency.ConclusionsRNAhybrid-assisted design of smiRNAs and generation of pri-smiRNAs using a novel vector containing restriction sites greatly improves specificity and speed of the generation of stable knockdown lines for functional analyses in plants.

Molecular cloning and expression profiling of a chalcone synthase gene from hairy root cultures of Scutellaria viscidula Bunge

A cDNA encoding chalcone synthase (CHS), the key enzyme in flavonoid biosynthesis, was isolated from hairy root cultures of Scutellaria viscidula Bunge by rapid amplification of cDNA ends (RACE). The full-length cDNA of S. viscidula CHS, designated as Svchs (GenBank accession no. EU386767), was 1649 bp with a 1170 bp open reading frame (ORF) that corresponded to a deduced protein of 390 amino acid residues, a calculated molecular mass of 42.56 kDa and a theoretical isoelectric point (pI) of 5.79. Multiple sequence alignments showed that SvCHS shared high homology with CHS from other plants. Functional analysis in silico indicated that SvCHS was a hydrophilic protein most likely associated with intermediate metabolism. The active sites of the malonyl-CoA binding motif, coumaroyl pocket and cyclization pocket in CHS of Medicago sativa were also found in SvCHS. Molecular modeling indicated that the secondary structure of SvCHS contained mainly α-helixes and random coils. Phylogenetic analysis showed that SvCHS was most closely related to CHS from Scutellaria baicalensis. In agreement with its function as an elicitor-responsive gene, the expression of Svchs was induced and coordinated by methyl jasmonate. To our knowledge, this is the first report to describe the isolation and expression of a gene from S. viscidula.

NICOTIANA MUTABILIS – A NOVEL SYSTEM FOR STUDYING ETHYLENE-MEDIATED FLORAL SENESCENCE

Nicotiana mutabilis, a tobacco species recently discovered in southern Brazil, has flowers that change color during their display life. Opening buds are white, and as the flower ages, the color changes, at first gradually, and then rapidly through light pink to dark pink and even red. The onset of the pigment change is associated with a marked increase in ethylene production, and the change is delayed by treating the flowers with 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action. Expression of the gene encoding chalcone synthase increased slightly in association with a substantial rise in concentrations of anthocyanins in the petals. Likewise, expression of a homolog of SAG12, a leaf senescence-associated gene first isolated from Arabidopsis, increased as the color change advanced and ethylene production increased. The numerous genetic and experimental tools available for N. tabacum can readily be applied to this close relative to provide an interesting new model for studying flower senescence.