Chalcone Synthase Transcripts Research Articles

Integrated transcriptome and metabolome provide insight into flavonoid variation in goji berries (Lycium barbarum L.) from different areas in China

Goji berries (Lycium barbarum L.) were rich in flavonoids, showing high nutritional and medicinal value. However, a thorough evaluation and comparison of the flavonoids in goji berries from various regions and the possible biological regulation pathways with differences are scanty. Here, we investigated the flavonoid metabolites and gene expression levels of goji berries from three major production areas in China using transcriptomics sequencing and metabolomics. The total flavonoid content and total polyphenol content of goji berry in Ningxia (57.87 μg/g and 183.41 μg/g, respectively) were higher than in Qinghai (50.77 μg/g and 156.81 μg/g) and Gansu (47.86 μg/g and 111.17 μg/g). We identified the 105 differentially accumulated flavonoids (DAFs) and 1858 differentially expressed genes (DEGs) from the goji berries in three habitats. Interestingly, gossypetin-3-O-rutinoside and isorhamnetin were significantly expressed between Ningxia and Qinghai berries. The chalcone isomerase (CHI), chalcone synthase (CHS), and flavonol synthase (FLS) genes also played key roles in the regulation of flavonoid synthesis. In addition, MYB1 positively regulated the expression of quercetin-3-O-glucoside, quercetin-7-O-glucoside and isohyperoside. As a result, we speculated that CHI, CHS, FLS genes, and related transcription factors jointly controlled the variation of flavone accumulation in goji berries. These findings may provide a new perspective for understanding the accumulation and molecular mechanisms of goji flavonoids.

Responses of flavonoids to solar UV radiation and gradual soil drying in two Medicago truncatula accessions

Ground level UV-B (290–315 nm) and UV-A (315–400 nm) radiation regulates multiple aspects of plant growth and development. In a natural environment, UV radiation interacts in a complex manner with other environmental factors (e.g., drought) to regulate plants’ morphology, physiology, and growth. To assess the interactive effects of UV radiation and soil drying on plants’ secondary metabolites and transcript abundance, we performed a field experiment using two different accessions of Medicago truncatula (F83005-5 French origin and Jemalong A17 Australian origin). Plants were grown for 37 days under long-pass filters to assess the effects of UV short wavelength (290–350 nm, UVsw) and UV-A long wavelength (350–400 nm, UV-Alw). Soil–water deficit was induced by not watering half of the plants during the last seven days of the experiment. The two accessions differed in the concentration of flavonoids in the leaf epidermis and in the whole leaf: F83005-5 had higher concentration than Jemalong A17. They also differed in the composition of the flavonoids: a greater number of apigenin derivatives than tricin derivatives in Jemalong A17 and the opposite in F83005-5. Furthermore, UVsw and soil drying interacted positively to regulate the biosynthesis of flavonoids in Jemalong A17 through an increase in transcript abundance of CHALCONE SYNTHASE (CHS). However, in F83005-5, this enhanced CHS transcript abundance was not detected. Taken together the observed metabolite and gene transcript responses suggest differences in mechanisms for acclimation and stress tolerance between the accessions.Graphical abstract

Ultraviolet-B exposure and exogenous hydrogen peroxide application lead to cross-tolerance toward drought in Nicotiana tabacum L.

Acclimation of plants to water deficit involves biochemical and physiological adjustments. Here, we studied how ultraviolet (UV)-B exposure and exogenously applied hydrogen peroxide (H2 O2 ) potentiates drought tolerance in tobacco (Nicotiana tabacum L. cv. xanthi nc). Separate and combined applications for 14 days of 1.75 kJ m-2 day-1 UV-B radiation and 0.2 mM H2 O2 were assessed. Both factors, individually and combined, resulted in inhibition of growth. Furthermore, the combined treatment led to the most compacted plants. UV-B- and UV-B + H2 O2 -treated plants increased total antioxidant capacity and foliar epidermal flavonol index. H2 O2 - and UV-B + H2 O2 -pre-treated plants showed cross-tolerance to a subsequent 7-day moderate drought treatment, which was assessed as the absence of negative impact on growth, leaf wilting, and leaf relative water content. Plant responses to the pre-treatment were notably different: (1) H2 O2 increased the activity of catalase (EC 1.11.1.6), phenylalanine ammonia lyase (EC 4.3.1.5), and peroxidase activities (EC 1.11.1.7), and (2) the combined treatment induced epidermal flavonols which were key to drought tolerance. We report synergistic effects of UV-B and H2 O2 on transcription accumulation of UV RESISTANCE LOCUS 8, NAC DOMAIN PROTEIN 13 (NAC13), and BRI1-EMS-SUPPRESSOR 1 (BES1). Our data demonstrate a pre-treatment-dependent response to drought for NAC13, BES1, and CHALCONE SYNTHASE transcript accumulation. This study highlights the potential of combining UV-B and H2 O2 to improve drought tolerance which could become a useful tool to reduce water use.

Deciphering the role of stress elicitors on the differential modulation of chalcone synthase gene and subsequent production of secondary metabolites in micropropagated Coelogyne ovalis Lindl., a therapeutically important medicinal orchid

Coelogyne ovalis Lindl. is one of the important medicinal orchid, has been used in traditional medicines for the treatment of various human aliments. The therapetical uses of this orchid may be attributed to the presence of various secondary metabolites ultimately produced via the phenylpropanoid pathway. Amongst the various genes involved in phenylpropanoid pathway, chalcone synthase (CHS) deserves a special mention as it plays a pivotal role in the synthesis of secondary metabolites. In the present research, an attempt has been made to modulate the expressions of CHS transcript and subsequent enhancement in the production of secondary metabolites (phenolics, flavonoids and anthocyanins) and antioxidant activity by the application of biotic (chitosan and yeast extracts) and abiotic (salicylic acid, methyl jasmonate and abscisic acid) stresses to the in vitro-raised plantlets. Amongst the various stress factors taken into consideration, salicylic acid-treated leaves showed the highest tissue-specific expression pattern (13.53-fold at 50 μM), followed by methyl jasmonate (10.80-fold at 100 μM), chitosan (9.04-fold at 100 μM) as compared to control plantlets. The correlation between CHS expression and the presence of secondary metabolites was also studied to find out a suitable condition which would result in higher accumulation of secondary metabolites. Further, elicitations with best three stresses (namely salicylic acid, methyl jasmonate and chitosan) significantly modulated the accumulation of secondary metabolites. Deviations were also detected in terms of phytochemical contents; the salicylic acid-treated leaf tissues showed the highest contents of flavonoids (34.18 mg QE/gm DW) and anthocyanins (24.08 mg / gm FW) whereas phenolic contents were recorded to be highest in pseudobulbs (44.61 mg GAE/ gm DW). The plantlets treated with salicylic acid resulted in enhanced production of secondary metabolites and significantly higher antioxidant activity (DPPH and metal chelating). The present result suggests that stress stimulates CoCHS expression and secondary metabolites production in the treated tissues of C. ovalis.

Identification of MOS9 as an interaction partner for chalcone synthase in the nucleus.

Plant flavonoid metabolism has served as a platform for understanding a range of fundamental biological phenomena, including providing some of the early insights into the subcellular organization of metabolism. Evidence assembled over the past three decades points to the organization of the component enzymes as a membrane-associated complex centered on the entry-point enzyme, chalcone synthase (CHS), with flux into branch pathways controlled by competitive protein interactions. Flavonoid enzymes have also been found in the nucleus in a variety of plant species, raising the possibility of alternative, or moonlighting functions for these proteins in this compartment. Here, we present evidence that CHS interacts with MOS9, a nuclear-localized protein that has been linked to epigenetic control of R genes that mediate effector-triggered immunity. Overexpression of MOS9 results in a reduction of CHS transcript levels and a metabolite profile that substantially intersects with the effects of a null mutation in CHS. These results suggest that the MOS9–CHS interaction may point to a previously-unknown mechanism for controlling the expression of the highly dynamic flavonoid pathway.

Increased chalcone synthase (CHS) expression is associated with dicamba resistance in Kochia scoparia.

Resistance to the synthetic auxin herbicide dicamba is increasingly problematic in Kochia scoparia. The resistance mechanism in an inbred dicamba-resistant K. scoparia line (9425R) was investigated using physiological and transcriptomics (RNA-Seq) approaches. No differences were found in dicamba absorption or metabolism between 9425R and a dicamba-susceptible line, but 9425R was found to have significantly reduced dicamba translocation. Known auxin-responsive genes ACC synthase (ACS) and indole-3-acetic acid amino synthetase (GH3) were transcriptionally induced following dicamba treatment in dicamba-susceptible K. scoparia but not in 9425R. Chalcone synthase (CHS), the gene regulating synthesis of the flavonols quertecin and kaemperfol, was found to have twofold higher transcription in 9425R both without and 12 h after dicamba treatment. Increased CHS transcription co-segregated with dicamba resistance in a forward genetics screen using an F2 population. Prior work has shown that the flavonols quertecin and kaemperfol compete with auxin for intercellular movement and vascular loading via ATP-binding cassette subfamily B (ABCB) membrane transporters. The results of this study support a model in which constitutively increased CHS expression in the meristem produces more flavonols that would compete with dicamba for intercellular transport by ABCB transporters, resulting in reduced dicamba translocation. © 2017 Society of Chemical Industry.

Nitric oxide as a bioactive molecule in the regulation of chalcone synthase during jasmonic acid mediated defense signaling in ginger

Nitric oxide (NO) and elicitors have been found to enhance the defense responses in plants. In this study, the role of NO in the regulation of chalcone synthase (CHS) gene during jasmonic acid (JA)-mediated defense response under wound stress in ginger were investigated. It was observed that NO is responsible for the up-regulation of CHS during wound stress, which further stimulates the JA-mediated stress responses. Confocal microscopy and qRT-PCR were used to study the accumulation of NO and CHS (ZoCHS) transcript levels, respectively, at the wound site. Relative expression of ZoCHS was found to increase by double fold in wounded plants upon treatment with NO and JA. Confocal studies confirmed the accumulation of NO at the wound sites which further triggered the JA-mediated stress responses. Overall results in this work implicate the role of NO in positive modulation of JA defense signaling responses and expression of CHS in ginger during wounding. These findings will help in the development of plants with improved metabolic and defense traits.

Genetic diversity of stilbene metabolism in Vitis sylvestris.

Stilbenes, as important secondary metabolites of grapevine, represent central phytoalexins and therefore constitute an important element of basal immunity. In this study, potential genetic variation in Vitis vinifera ssp. sylvestris, the ancestor of cultivated grapevine, was sought with respect to their output of stilbenes and potential use for resistance breeding. Considerable variation in stilbene inducibility was identified in V. vinifera ssp. sylvestris. Genotypic differences in abundance and profiles of stilbenes that are induced in response to a UV-C pulse are shown. Two clusters of stilbene 'chemovars' emerged: one cluster showed quick and strong accumulation of stilbenes, almost exclusively in the form of non-glycosylated resveratrol and viniferin, while the second cluster accumulated fewer stilbenes and relatively high proportions of piceatannol and the glycosylated piceid. For all 86 genotypes, a time dependence of the stilbene pattern was observed: piceid, resveratrol, and piceatannol accumulated earlier, whereas the viniferins were found later. It was further observed that the genotypic differences in stilbene accumulation were preceded by differential accumulation of the transcripts for chalcone synthase (CHS) and stilbene-related genes: phenylalanine ammonium lyase (PAL), stilbene synthase (StSy), and resveratrol synthase (RS). A screen of the population with respect to susceptibility to downy mildew of grapevine (Plasmopara viticola) revealed considerable variability. The subpopulation of genotypes with high stilbene inducibility was significantly less susceptible as compared with low-stilbene genotypes, and for representative genotypes it could be shown that the inducibility of stilbene synthase by UV correlated with the inducibility by the pathogen.

Epimutant Induction as a New Plant Breeding Technology

A small interfering RNA (siRNA) can be custom-produced in plant cells when a transgene comprising an inverted-repeat sequence is used to form the corresponding double-stranded (ds)RNA. This siRNA induces methylation of the homologous DNA through the RNA-directed DNA methylation (RdDM) pathway. As methylation of the promoter region causes transcriptional gene silencing (TGS), dsRNA for the promoter sequence of a gene can induce TGS of the gene, accompanied by modification of chromatin. Such epigenetic variation provides a novel technique to induce silencing of a target gene. Furthermore, once epigenetic variation has occurred, the siRNA need not be present to maintain the stable repression of transcription even in the subsequent generation. Thus, the induction of epigenetic changes is a potentially new plant-breeding technology to improve crops. Discipline: Plant breeding Additional key words: DNA methylation, RNA silencing, transcriptional gene silencing, siRNA longdistance transport *Corresponding author: e-mail tharada@cc.hirosakiu.ac.jp Received 10 March 2014; accepted 9 January 2015. Introduction Napoli et al. (1990) and van der Krol et al. (1990) reported an RNA interference (RNAi) phenomenon for the first time. They overexpressed a transgene of chalcone synthase (CHS), a key enzyme in flavonoid biosynthesis and the rate-limiting enzyme in anthocyanin biosynthesis, to generate a darker violet petunia flower, but unexpectedly obtained a white flower. Because the CHS transcription level was 50-fold lower than that in the wild-type flower, they concluded that as well as the transgene, the endogenous CHS gene had also been silenced. A similar phenomenon in a fungus (Neurospora crassa), termed “quelling”, was reported by Romano & Macino (1992). Furthermore, in an animal species (Caenorhabditis elegans), it was reported that introducing sense or antisense RNA of a gene to cells resulted in the mRNA degrading (Guo et al. 1995). Fire et al. (1998), Nobel Prize winner in 2006, clarified the RNAi mechanism by which dsRNA could cause endogenous mRNA to degrade. A number of subsequent studies revealed that dsRNA initiates gene silencing through inhibition at the post-transcriptional level (post-transcriptional gene silencing; PTGS) or the transcriptional level (transcriptional gene silencing; TGS). In the latter case, a small interfering RNA (siRNA) 24 nt long induces methylation of the fifth carbon cytosine residue within the region of sequence identity between the triggering RNA and the homologous DNA (Matzke et al. 2004). Although cytosine methylation is mostly limited to CG dinucleotide sequence contexts in animal genomes, in plants the methylation occurs not only in the symmetrical CG context but also in the CHG and CHH sequence contexts (where H=A, C or T) throughout their genomes. The cytosine methylation of each context is performed by respective methylation enzymes. The process of de novo DNA methylation is triggered by 24 nt siRNAs produced by RdDM. Furthermore, two plant-specific RNA polymerases, PolIV and PolV and the RDR2, DCL3 and AGO4 proteins operate in this pathway (Matzke et al. 2009, Haag & Pikaad 2011). Chemical modifications of DNA influence chromatin structure through histone methylation, while the methylation of histone H3 lysine (H3K9) plays an important role in establishing and maintaining DNA methylation (Grewal & Jia 2007). Epigenetic changes play pivotal roles in the developmental responses of plants. Recent studies have suggested

A 22‐nt artificial microRNA mediates widespread RNA silencing in Arabidopsis

It is known that 22-nucleotide (nt) microRNAs (miRNAs) derived from asymmetric duplexes trigger phased small-interfering RNA (phasiRNA) production from complementary targets. Here we investigate the efficacy of 22-nt artificial miRNA (amiRNA)-mediated RNA silencing relative to conventional hairpin RNA (hpRNA) and 21-nt amiRNA-mediated RNA silencing. CHALCONE SYNTHASE (CHS) was selected as a target in Arabidopsis thaliana due to the obvious and non-lethal loss of anthocyanin accumulation upon widespread RNA silencing. Over-expression of CHS in the pap1-D background facilitated visual detection of both local and systemic RNA silencing. RNA silencing was initiated in leaf tissues from hpRNA and amiRNA plant expression vectors under the control of an Arabidopsis RuBisCo small subunit 1A promoter (SSU). In this system, hpRNA expression triggered CHS silencing in most leaf tissues but not in roots or seed coats. Similarly, 21-nt amiRNA expression from symmetric miRNA/miRNA* duplexes triggered CHS silencing in all leaf tissues but not in roots or seed coats. However, 22-nt amiRNA expression from an asymmetric duplex triggered CHS silencing in all tissues, including roots and seed coats, in the majority of plant lines. This widespread CHS silencing required RNA-DEPENDENT RNA POLYMERASE6-mediated accumulation of phasiRNAs from the endogenous CHS transcript. These results demonstrate the efficacy of asymmetric 22-nt amiRNA-directed RNA silencing and associated phasiRNA production and activity, in mediating widespread RNA silencing of an endogenous target gene. Asymmetric 22-nt amiRNA-directed RNA silencing requires little modification of existing amiRNA technology and is expected to be effective in suppressing other genes and/or members of gene families.

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.

Anthocyanin Accumulation and Expression of Anthocyanin Biosynthetic Genes in Radish (Raphanus sativus)

Radish [Raphanus sativus (Rs)] is an important dietary vegetable in Asian countries, especially China, Japan, and Korea. To elucidate the molecular mechanisms of anthocyanin accumulation in radish, the gene expression of enzymes directly involved in anthocyanin biosynthesis was analyzed. These genes include phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol reductase (DFR), and anthocyanidin synthase (ANS). RsDFR and RsANS were found to accumulate in the flesh or skin of two radish cultivars (Man Tang Hong and Hong Feng No.1). Radish skin contained higher CHS, CHI, and F3H transcript levels than radish flesh in all three cultivars. In the red radish, 16 anthocyanins were separated and identified by high-performance liquid chromatography (HPLC) and elctrospray ionization-tandem mass spectrometry (ESI-MS/MS). Some of them were acylated with coumaroyl, malonoyl, feruoyl, and caffeoyl moieties. Furthermore (-)-epicatechin and ferulic acid were also identified in the three cultivars.

Stilbene synthase gene transfer caused alterations in the phenylpropanoid metabolism of transgenic strawberry (Fragaria x ananassa).

The gene encoding stilbene synthase is frequently used to modify plant secondary metabolism with the aim of producing the self-defence phytoalexin resveratrol. In this study, strawberry (Fragaria×ananassa) was transformed with the NS-Vitis3 gene encoding stilbene synthase from frost grape (Vitis riparia) under the control of the cauliflower mosaic virus 35S and the floral filament-specific fil1 promoters. Changes in leaf metabolites were investigated with UPLC-qTOF-MS (ultra performance liquid chromatography-quadrupole time of flight mass spectrometry) profiling, and increased accumulation of cinnamate, coumarate, and ferulate derivatives concomitantly with a decrease in the levels of flavonols was observed, while the anticipated resveratrol or its derivatives were not detected. The changed metabolite profile suggested that chalcone synthase was down-regulated by the genetic modification; this was verified by decreased chalcone synthase transcript levels. Changes in the levels of phenolic compounds led to increased susceptibility of the transgenic strawberry to grey mould fungus.

Quantitative Real-time Reverse Transcriptase-PCR Profiling of Anthocyanin Biosynthetic Genes during Orange Fruit Ripening

Genes encoding chalcone synthase (CHS), anthocyanidin synthase (ANS), and UDP-glucose-flavonoid 3-O-glucosyltransferase (UFGT), some of the enzymes of anthocyanin biosynthetic pathway, were assayed in two different experiments using quantitative real-time reverse transcriptase (RT)-PCR, in order to test their transcription levels in the flesh of blood and common orange [Citrus sinensis (L.) Osbeck] fruit, and to investigate their role in anthocyanin accumulation in the same tissue. The first experiment compared a blood orange and a common orange cultivar during seven different fruit maturation stages. This was followed by the test of 11 different genotypes at the end of the winter season. Data collected from the first experiment, over the blood orange cultivar, were statistically analyzed using the Pearson correlation coefficient. Results show that CHS, ANS, and UFGT mRNA transcripts are up- and co-regulated in the blood orange cultivar, whereas they are down-regulated in the common orange cultivar. There is evidence of correspondence between the target genes expression level and the content of the pigment assessed. The second test confirms this correlation and shows that enzyme synthesis levels and pigment accumulation, in plants grown under the same environmental conditions, are dependent on the differences occurring among the genotypes tested. These results suggest that the absence of pigment in the common orange cultivars may be caused by the lack of induction on the structural genes expression. This is the first report on the characterization of the relationships between biosynthetic genes expression and fruit flesh anthocyanin content in blood oranges.

Silencing the flavonoid pathway in Medicago truncatula inhibits root nodule formation and prevents auxin transport regulation by rhizobia.

Legumes form symbioses with rhizobia, which initiate the development of a new plant organ, the nodule. Flavonoids have long been hypothesized to regulate nodule development through their action as auxin transport inhibitors, but genetic proof has been missing. To test this hypothesis, we used RNA interference to silence chalcone synthase (CHS), the enzyme that catalyzes the first committed step of the flavonoid pathway, in Medicago truncatula. Agrobacterium rhizogenes transformation was used to create hairy roots that showed strongly reduced CHS transcript levels and reduced levels of flavonoids in silenced roots. Flavonoid-deficient roots were unable to initiate nodules, even though normal root hair curling was observed. Nodule formation and flavonoid accumulation could be rescued by supplementation of plants with the precursor flavonoids naringenin and liquiritigenin. The flavonoid-deficient roots showed increased auxin transport compared with control roots. Inoculation with rhizobia reduced auxin transport in control roots after 24 h, similar to the action of the auxin transport inhibitor N-(1-naphthyl)phthalamic acid (NPA). Rhizobia were unable to reduce auxin transport in flavonoid-deficient roots, even though NPA inhibited auxin transport. Our results present genetic evidence that root flavonoids are necessary for nodule initiation in M. truncatula and suggest that they act as auxin transport regulators.

Chalcone synthase as a reporter in virus-induced gene silencing studies of flower senescence

Agrobacterium-mediated infection of petunia (Petunia hybrida) plants with tobacco rattle virus (TRV) bearing fragments of Petunia genes resulted in systemic infection and virus-induced gene silencing (VIGS) of the homologous host genes. Infection with TRV containing a phytoene desaturase (PDS) fragment resulted in reduced abundance of PDS transcripts and typical photobleaching of photosynthetic tissues. Infection with TRV containing a chalcone synthase (CHS) fragment resulted in silencing of anthocyanin production in infected flowers. The silencing phenotype ranged from scattered white spots on the normal purple background to entirely white flowers. Symptoms in the V26 cultivar were a diffuse mosaic, but infection of some purple-flowered commercial cultivars resulted in large white sectors and even entirely white flowers. Abundance of CHS transcripts in the white flowers was less than 4% of that in purple flowers on the same plant. Infection with TRV containing a tandem construct of PDS and CHS resulted in leaf photobleaching and white patterns on the flowers. Transcripts of CHS and PDS were reduced both in leaves and in flowers confirming simultaneous silencing of both genes by the tandem construct. We tested the effects of infection with TRV containing CHS and a fragment of a petunia gene encoding for 1-aminocyclopropane-1-carboxylate oxidase (ACO4) Abundance of transcripts encoding ACO4 and ACO1 were reduced (by 5% and 20%, respectively) in infected flowers. Whether the flowers were treated with ACC or pollinated, the white (silenced) flowers or flower sectors produced less ethylene and senesced later than purple (non-silenced) tissues. These results indicate the value of VIGS with tandem constructs containing CHS as reporter and a target gene as a tool for examining the function of floral-associated genes.

Induced responses to pathogen infection in Norway spruce phloem: changes in polyphenolic parenchyma cells, chalcone synthase transcript levels and peroxidase activity.

Polyphenolic parenchyma cells (PP cells) in Norway spruce (Picea abies (L.) Karst.) stem phloem play important roles in constitutive and inducible defenses. To determine whether anatomical and molecular changes in PP cells are correlated with tree resistance, we infected two Norway spruce clones with the pathogenic fungus Ceratocystis polonica (Siem.) C. Moreau. The fungus induced significantly different lesion lengths in the two clones, indicating that one clone was more resistant to the fungus (short lesions) than the other (long lesions). After infection, the cross-sectional area of PP cells and their vacuolar polyphenol bodies increased in the three most recent annual rings of PP cells in both clones. The more resistant clone had larger PP cells with denser polyphenol bodies than the less resistant clone, whereas the less resistant clone accumulated relatively more polyphenols after infection. Compared with the less resistant clone, the more resistant clone contained higher starch concentrations before infection that were reduced more quickly after infection before returning to original values. Low transcript levels of chalcone synthase were detected in uninfected tissues of both clones, but the levels increased dramatically after infection. Transcript levels were higher and peaked 6 days earlier in the more resistant clone than in the less resistant clone. The activity of at least one highly basic peroxidase isoform was greatly enhanced after infection, and this increase occurred earlier in the more resistant clone.

Tissue-specific gene silencing mediated by a naturally occurring chalcone synthase gene cluster in Glycine max.

Chalcone synthase, a key regulatory enzyme in the flavonoid pathway, constitutes an eight-member gene family in Glycine max (soybean). Three of the chalcone synthase (CHS) gene family members are arranged as inverted repeats in a 10-kb region, corresponding to the I locus (inhibitor). Spontaneous mutations of a dominant allele (I or i(i)) to a recessive allele (i) have been shown to delete promoter sequences, paradoxically increasing total CHS transcript levels and resulting in black seed coats. However, it is not known which of the gene family members contribute toward pigmentation and how this locus affects CHS expression in other tissues. We investigated the unusual nature of the I locus using four pairs of isogenic lines differing with respect to alleles of the I locus. RNA gel blots using a generic open reading frame CHS probe detected similar CHS transcript levels in stems, roots, leaves, young pods, and cotyledons of the yellow and black isolines but not in the seed coats, which is consistent with the dominant I and i(i) alleles mediating CHS gene silencing in a tissue-specific manner. Using real-time RT-PCR, a variable pattern of expression of CHS genes in different tissues was demonstrated. However, increase in pigmentation in the black seed coats was associated with release of the silencing effect specifically on CHS7/CHS8, which occurred at all stages of seed coat development. These expression changes were linked to structural changes taking place at the I locus, shown to encompass a much wider region of at least 27 kb, comprising two identical 10.91-kb stretches of CHS gene duplications. The suppressive effect of this 27-kb I locus in a specific tissue of the G. max plant represents a unique endogenous gene silencing mechanism.

Influence of PTGS on Chalcone Synthase Gene Family in Yellow Soybean Seed Coat

Most commercial soybean varieties have yellow seeds due to loss of pigmentation in the seed coat. We previously showed that inhibition of seed coat pigmentation in yellow soybeans is controlled by post-transcriptional gene silencing (PTGS) of chalcone synthase (CHS) genes. Soybean CHS genes are composed of at least eight members, CHS1-CHS8. In this study, we compared the steady state mRNA level of each CHS member in the seed coat between a yellow soybean cultivar displaying CHS PTGS and its pigmented seed coat mutant in which the CHS PTGS is abrogated. Northern hybridization analysis suggested that in the pigmented seed coat, the transcripts of CHS7 and CHS8 were abundant among the total CHS transcripts, whereas those of other CHS members (CHS1-CHS6) were less abundant. Reverse transcription and subsequent real time PCRs using member-specific primers revealed reduction of the amount of mRNA of most CHS members in the non-pigmented seed coat of the yellow soybean as compared to the pigmented seed coat of the mutant, indicating the influence of PTGS on the CHS gene family.

Biosynthesis of secondary metabolites in sugarcane

A set of genes related to secondary metabolism was extracted from the sugarcane expressed sequence tag (SUCEST) database and was used to investigate both the gene expression pattern of key enzymes regulating the main biosynthetic secondary metabolism pathways and the major classes of metabolites involved in the response of sugarcane to environmental and developmental cues. The SUCEST database was constructed with tissues in different physiological conditions which had been collected under varied situation of environmental stress. This database allows researchers to identify and characterize the expressed genes of a wide range of putative enzymes able to catalyze steps in the phenylpropanoid, isoprenoid and other pathways of the special metabolic mechanisms involved in the response of sugarcane to environmental changes. Our results show that sugarcane cDNAs encoded putative ultra-violet induced sesquiterpene cyclases (SC); chalcone synthase (CHS), the first enzyme in the pathway branch for flavonoid biosynthesis; isoflavone synthase (IFS), involved in plant defense and root nodulation; isoflavone reductase (IFR), a key enzyme in phenylpropanoid phytoalexin biosynthesis; and caffeic acid-O-methyltransferase, a key enzyme in the biosynthesis of lignin cell wall precursors. High levels of CHS transcripts from plantlets infected with Herbaspirillum rubri or Gluconacetobacter diazotroficans suggests that agents of biotic stress can elicit flavonoid biosynthesis in sugarcane. From this data we have predicted the profile of isoprenoid and phenylpropanoid metabolism in sugarcane and pointed the branches of secondary metabolism activated during tissue-specific stages of development and the adaptive response of sugarcane to agents of biotic and abiotic stress, although our assignment of enzyme function should be confirmed by careful biochemical and genetic supporting evidence.