Cloning and characterization of a novel chalcone synthase gene from Phalaenopsis hybrida orchid flowers

Chalcone synthase (CHS, EC 2.3.1.74) is the key enzyme involved in flavonoid and anthocyanin biosynthesis. A complete DNA sequence of chalcone synthase gene designated Pchs1 was isolated by means of usual and then inverse polymerase chain reactions from genomic DNA of an orchid, Phalaenopsis hybrida, cv. Formosa rose. Nucleotide sequence analysis based on alignment with published Phalaenopsis chs cDNA revealed that Pchs1 contained an intact open reading frame of 1173-bp with one 109-bp intron at the conserved site. The deduced polypeptide (PCHS1) from Pchs1 comprised 390 amino acids with a predicted mol wt of 42.5 kD. PCHS1 showed 61–65% identities with CHS from other plants and retained most of the conserved residues. Some putative cis-regulatory elements were present at the 5′ and 3′ flanking regions of Pchs1. Southern blot analysis predicted at least four chs-like genes, thus indicating the presence of a small multigene chs family in P. hybrida. Relative quantitative RT-PCR showed that Pchs1 is expressed in petals at early flower development as well as in lip tissue when the flower has just opened. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 2, pp. 250–258. The text was submitted by the authors in English.     

The Flavonoid Pathway Regulates the Petal Colors of Cotton Flower

Although biochemists and geneticists have studied the cotton flower for more than one century, little is known about the molecular mechanisms underlying the dramatic color change that occurs during its short developmental life following blooming. Through the analysis of world cotton germplasms, we found that all of the flowers underwent color changes post-anthesis, but there is a diverse array of petal colors among cotton species, with cream, yellow and red colors dominating the color scheme. Genetic and biochemical analyses indicated that both the original cream and red colors and the color changes post-anthesis were related to flavonoid content. The anthocyanin content and the expression of biosynthesis genes were both increased from blooming to one day post-anthesis (DPA) when the flower was withering and undergoing abscission. Our results indicated that the color changes and flavonoid biosynthesis of cotton flowers were precisely controlled and genetically regulated. In addition, flavonol synthase (FLS) genes involved in flavonol biosynthesis showed specific expression at 11 am when the flowers were fully opened. The anthocyanidin reductase (ANR) genes, which are responsible for proanthocyanidins biosynthesis, showed the highest expression at 6 pm on 0 DPA, when the flowers were withered. Light showed primary, moderate and little effects on flavonol, anthocyanin and proanthocyanidin biosynthesis, respectively. Flavonol biosynthesis was in response to light exposure, while anthocyanin biosynthesis was involved in flower color changes. Further expression analysis of flavonoid genes in flowers of wild type and a flavanone 3-hydroxylase (F3H) silenced line showed that the development of cotton flower color was controlled by a complex interaction between genes and light. These results present novel information regarding flavonoids metabolism and flower development.     

Flavonoid synthesis in Petunia hybrida: partial characterization of dihydroflavonol-4-reductase genes

In this paper we describe the organization and expression of the genes encoding the flavonoid-biosynthetic enzyme dihydroflavonol-4-reductase (DFR) in Petunia hybrida. A nearly full-size DFR cDNA clone (1.5kb), isolated from a corolla-specific cDNA library was compared at the nucleotide level with the pallida gene from Antirrhinum majus and at the amino acid level with enzymes encoded by the pallida gene and the A1 gene from Zea mays.The P. hybrida and A. majus DFR genes transcribed in flowers contain 5 introns, at identical positions; the three introns of the A1 gene from Z. mays coincide with first three introns of the other two species. P. hybrida line V30 harbours three DFR genes (A, B, C) which were mapped by RFLP analysis on three different chromosomes (IV, II and VI respectively).Steady-state levels of DFR mRNA in the line V30 follow the same pattern during development as chalcone synthase (CHS) and chalcone flavanone isomerase (CHI) mRNA. Six mutants that accumulate dihydroflavonols in mature flowers were subjected to Northern blot analysis for the presence of DFR mRNA. Five of these mutants lack detectable levels of DFR mRNA. Four of these five also show drastically reduced levels of activity for the enzyme UDPG: flavonoid-3-O-glucosyltransferase (UFGT), which carries out the next step in flavonoid biosynthesis; these mutants might be considered as containing lesions in regulatory genes, controlling the expression of the structural genes in this part of the flavonoid biosynthetic pathway. Only the an6 mutant shows no detectable DFR mRNA but a wild-type level for UFGT activity. Since both an6 and DFR-A are located on chromosome IV and DFR-A is transcribed in floral tissues, it is postulated that the An6 locus contains the DFR structural gene. The an9 mutant shows a wild-type level of DFR mRNA and a wild-type UFGT activity.     

The transcriptome of flower development provides insight into floral scent formation in <Emphasis Type="Italic">Freesia hybrida</Emphasis>

Freesia hybrida is a popular cut flower that is globally cultivated. Despite fragrance being an important floral trait, little is known of its associated molecular mechanisms in F. hybrida. In this study, cDNA libraries were constructed for three floral developmental stages in F. hybrida. A total of 74,660 unigenes were obtained from RNA sequencing and de novo assembly, of which 72.20% were annotated by seven public protein databases. Approximately 12,420 differentially expressed genes were identified during flower development. Gas chromatography–mass spectrometry analysis detected a total of 34 floral volatile compounds, primarily volatile monoterpenes, which accounted for approximately 90% of the volatiles. Using a system analysis-based approach, 36 candidate genes related to volatile terpenes were identified, in which the majority of up-regulated genes associated with the biosynthesis of monoterpenes and the majority of down-regulated genes associated with the biosynthesis of sesquiterpenes. Of these, FhDXS2A, FhGPPs and FhTPSs were considered to be important in floral scent formation. This study provides an in-depth assessment of floral scent and a reference for the future molecular breeding of floral scent in ornamental plants.     

A chalcone isomerase‐like protein enhances flavonoid production and flower pigmentation

Flavonoids are major pigments in plants, and their biosynthetic pathway is one of the best‐studied metabolic pathways. Here we have identified three mutations within a gene that result in pale‐colored flowers in the Japanese morning glory (Ipomoea nil). As the mutations lead to a reduction of the colorless flavonoid compound flavonol as well as of anthocyanins in the flower petal, the identified gene was designated enhancer of flavonoid production (EFP). EFP encodes a chalcone isomerase (CHI)‐related protein classified as a type IV CHI protein. CHI is the second committed enzyme of the flavonoid biosynthetic pathway, but type IV CHI proteins are thought to lack CHI enzymatic activity, and their functions remain unknown. The spatio‐temporal expression of EFP and structural genes encoding enzymes that produce flavonoids is very similar. Expression of both EFP and the structural genes is coordinately promoted by genes encoding R2R3‐MYB and WD40 family proteins. The EFP gene is widely distributed in land plants, and RNAi knockdown mutants of the EFP homologs in petunia (Petunia hybrida) and torenia (Torenia hybrida) had pale‐colored flowers and low amounts of anthocyanins. The flavonol and flavone contents in the knockdown petunia and torenia flowers, respectively, were also significantly decreased, suggesting that the EFP protein contributes in early step(s) of the flavonoid biosynthetic pathway to ensure production of flavonoid compounds. From these results, we conclude that EFP is an enhancer of flavonoid production and flower pigmentation, and its function is conserved among diverse land plant species.     

Activation of galactolipid biosynthesis in development of pistils and pollen tubes

Galactolipids such as monogalactosyldiacylglycerol and digalactosyldiacylglycerol are essential lipids for the proper functioning of photosynthetic membranes. However, the function of galactolipids in flowers is unknown. Previously, we reported that pistils have higher galactolipid-producing activity than leaves. The present study investigated galactolipid biosynthesis in pistils in more detail using Petunia hybrida and Lilium longiflorum. The results showed that digalactosyldiacylglycerol levels increased during flower development. In addition, the galactose incorporation activity into galactolipids was induced, suggesting that the pathway for the production of digalactosyldiacylglycerol was stimulated. Interestingly, a significant increase in galactolipids was also observed in elongated pollen tubes. Therefore, pistils are the main site of galactolipid biosynthesis and whose galactolipid biosynthesis activity is induced during flower development, and this induction includes considerable galactolipid biosynthesis in pollen tubes.     

Characterization of tt15, a novel transparent testa mutant of Arabidopsis thaliana (L.) Heynh.

The Arabidopsis thaliana seed coat typically has a brown color due to the accumulation of flavonoid pigments in the testa. Mutants of A. thaliana with defects in pigment biosynthesis often produce seeds that are olive brown or even yellow in appearence, and the responsible genetic loci are referred to as TRANSPARENT TESTA (TT). Large-scale screening for mutants affected in seed development and complementation analysis of a candidate mutant line with all published A. thalianatt mutants identified a new tt locus designated tt15. The tt15 mutation maps to the lower part of chromosome 1. Mutant plants produced pale greenish-brown seeds whose dormancy was slightly reduced. The phenotype was consistent with the maternal origin of the testa. Analysis of pigment accumulation and the study of expression patterns of genes involved in flavonoid biosynthesis in tt15 plants and seeds indicated a seed-specific phenotype. Most notable was a reduction of the cyanidin and quercetin content of tt15 seeds. Received: 2 October 1998 / Accepted: 10 October 1998     

The chalcone synthase multigene family of Petunia hybrida (V30): differential,light-regulated expression during flower development and UV light induction

We have analysed the expression of the 8–10 members of the gene family encoding the flavonoid biosynthetic enzyme chalcone synthase (CHS) from Petunia hybrida. During normal plant development only two members of the gene family (CHS-A and CHS-J) are expressed. Their expression is restricted to floral tissues mainly. About 90% of the total CHS mRNA pool is transcribed from CHS-A, wheares CHS-J delivers about 10% in flower corolla, tube and anthers. Expression of CHS-A and CHS-J during flower development is coordinated and (red) light-dependent. In young seedlings and cell suspension cultures expression of CHS-A and CHS-J can be induced with UV light. In addition to CHS-A and CHS-J, expression of another two CHS genes (CHS-B and CHS-G) is induced in young seedlings by UV light, albeit at a low level. In contrast to CHS genes from Leguminoseae, Petunia CHS genes are not inducible by phytopathogen-derived elicitors. Expression of CHS-A and CHS-J is reduced to a similar extent in a regulatory CHS mutant, Petunia hybrida Red Star, suggesting that both genes are regulated by the same trans-acting factors. Comparison of the promoter sequences of CHS-A and CHS-J reveals some striking homologies, which might represent cis-acting regulatory sequences.     

Flower color diversity revealed by differential expression of flavonoid biosynthetic genes and flavonoid accumulation in herbaceous peony (Paeonia lactiflora Pall.)

Herbaceous peony (Paeonia lactiflora Pall.) is an important ornamental plant which contains different flower colors. In this paper, eight genes encoding phenylalanine ammonialyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), UDP-glucose: flavonoid 3-o-glucosyltransferase (UF3GT) were isolated. Moreover, the expression patterns of these eight genes and UF5GT in the flowers were investigated in three cultivars, that is, ‘Hongyanzhenghui’, ‘Yulouhongxing’ and ‘Huangjinlun’ with purplish-red, white and yellow flower respectively. Furthermore, flavonoid accumulation in the flowers was also analyzed. The results showed that in different organs, most of genes expressed higher in flowers than in other organs. During the development of flowers, all genes could be divided into four groups. The first group (PlPAL) was highly expressed in S1 and S4. The second group (PlCHS and PlCHI) was at a high expression level throughout the whole developmental stages. The third group (PlF3H, PlF3′H, PlDFR, PlANS and PlUF5GT) gradually decreased with the development of flowers. The fourth group (PlUF3GT) gradually increased during the flower development. In addition, anthoxanthins and anthocyanins were detected in ‘Hongyanzhenghui’ and ‘Yulouhongxing’, chalcones and anthoxanthins were found in ‘Huangjinlun’. When different color flowers were concerned, low expression level of PlCHI induced most of the substrate accumulation in the form of chalcones and displaying yellow, changing a small part of substrates to anthoxanthins, and there was no anthocyanin synthesis in ‘Huangjinlun’ because of low expression level of DFR. In ‘Yulouhongxing’, massive expressions of upstream genes and low expression of DFR caused synthesis of a great deal of anthoxanthins and a small amount of colorless anthocyanins. In ‘Hongyanzhenghui’, a large number of colored anthocyanins were changed from anthoxanthins because of PlDFR, PlANS and PlUF3GT high expressions. These results would provide us a theoretical basis to understand the formation of P. lactiflora flower colors.     

Cloning and Analysis of Promoter Regions of Flavonoid Biosynthesis Genes in Safflower

The flower of the safflower plant Carthamus tinctorius L. has been widely used in traditional Chinese medicine because of its ability to improve cerebral blood flow. Flavonoids are the main active ingredient of safflower plants. Analyses of the regulation of flavonoids are of great significance for the artificial regulation of their biosynthesis, and previous studies have mainly focused on their metabolic synthesis in safflower. For a better understanding of the regulation of flavonoid biosynthesis in safflower, promoters of flavonoid biosynthesis genes in safflower were cloned and analysed. First, the expression of the flavonoid genes in safflower was analysed. Twenty-four genes involved in flavonoid biosynthesis exhibited large differences. Four promoters of flavonoid genes that were mainly expressed in the flowers of safflower (termed pctCHI-1, pctCHS-2, pctF3H-2 and pctGT-1) were cloned and analysed. Elements that respond to light, i.e. phytohormones and the MYB regulator, were identified. The promoter activities were analysed using a protoplast system. All four promoters had activities in the flower protoplast, and the pctCHS-2 activity was the highest. Our results also demonstrate that pctCHS-2 is a flower-specific promoter. All of these results provide a basis for improved analyses of flavonoid regulation in safflower.     

Serologically-Related Anionic Peroxidases from Petunia and Cucumber can Substitute Flavonoid Antioxidants

In this study we have investigated whether naturally occurring flavonoid-deficient mutant Red Star of Petunia hybrida is capable of metabolizing H₂O₂ by invoking other antioxidant enzyme system. We demonstrated that reduced flower pigmentation due to a reduction in the chalcone synthase mRNA expression results in strong H₂O₂ accumulation accompanied by the induction of a specific set of anionic peroxidase (PRX), serologically-related to main cucumber srPRX. We found correlation between rate of H₂O₂ accumulation and qualitative, as well as quantitative changes in the srPRX expression which seems to be determined by flower phenotype. In detached flower buds cultured in vitro both abscisic acid and anther extirpation prevented anthocyanin pigmentation, and thus flavonoid biosynthesis, resulting in a marked accumulation of immunoprecipitable srPRX. In contrast, pigmented flowers cultivated under the same conditions did not accumulate corresponding srPRX. The results suggest that a specific set of anionic PRX can substitute for the absence of flavonoid antioxidants.