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.     

Chalcone isomerase gene from rice (Oryza sativa) and barley (Hordeum vulgare): physical,genetic and mutation mapping

The barley and rice chalcone flavonone isomerase (Cfi) genes were isolated and identified by homology to the maize Cfi gene. Structure analysis indicated high similarity except that the barley gene lacked intron 3. The maize Cfi gene has been mapped to three loci, but only a single locus was detected in barley and rice. This explains the lack of observed mutants in maize while a single locus anthocyanin-less 30 (ant30), with four alleles ant30-245, ant30-310, ant30-272 and ant30-287 has been described in barley. Based on biochemical analysis it has been suggested that these mutants are in the Cfi gene resulting in absence of anthocyanin. In order to provide molecular evidence for or against this hypothesis we sequenced the four ant30 alleles and compared them to their respective wild-type alleles. The three sodium azide induced mutants ant30-245, ant30-272 and ant30-287 showed single base changes resulting in two non-sense and one mis-sense mutations affecting the protein function. The 1-nitroso-5,6-dihydrouracil induced mutant ant30-310 had one base substitution and a 25 bp deletion. These observations are in accordance with the conclusion that the ant30 phenotype is caused by mutations in the Cfi gene. The nature of the mutants induced is in line with the proposed mechanism of action for the mutagens used.     

Nucleotide sequence of the triosephosphate isomerase gene from Aspergillus nidulans: implications for a differential loss of introns

A functional cDNA from Aspergillus nidulans encoding triosephosphate isomerase (TPI) was isolated by its ability to complement a tpi1 mutation in Saccharomyces cerevisiae. This cDNA was used to obtain the corresponding gene, tpiA. Alignment of the cDNA and genomic DNA nucleotide sequences indicated that tpiA contains five introns. The intron positions in the tpiA gene were compared with those in the TPI genes of human, chicken, and maize. One intron is present at an identical position in all four organisms, two other introns are located in similar positions in A. nidulans and maize, and the remaining two introns are unique to A. nidulans. These Aspergillus-specific introns are located in regions of the protein that were predicted to be interrupted by introns based on analysis of a Go plot of chicken TPI. These comparisons are discussed in relation to the evolution of introns within TPI genes.     

对水稻4号染色体上一个编码二氢黄酮醇还原酶类似蛋白基因簇的结构和进化分析

通过对水稻 4号染色体一段 32 3kb的序列测定和分析 ,在其中 56kb的区域内发现了一个由 7个编码二氢黄酮醇还原酶 (DFR)类似蛋白基因组成的基因簇。这 7个基因在基因簇中串联排列 ,每个基因都由 6个外显子和 5个内含子组成。这 7个基因的预测蛋白质序列都和DFR以及BANYULS蛋白序列类似。DFR和BANYULS都是植物次生代谢类黄酮醇生物合成途径中的结构基因 ,它们的缺失或突变都会造成植物花色素合成代谢的不正常。RT PCR实验证明这 7个基因在水稻的 5个组织中表达不同。文中讨论了这 7个基因的结构和功能特性以及它们的进化关系。     

Nucleotide sequence of the B1-hordein gene of barley Hordeum vulgare L

The gene encoding B1 hordein of Hordeum vulgare (cv. Donetsky 4) was cloned and entirely sequenced. It contains no introns and codes for of 293 amino acids long polypeptide with molecular weight 33418. Our clone differs from the previously sequenced B1 hordein genes in some positions within the coding region (there are 4 nucleotide changes and a 12 bp deletion, as compared with the pB11 cDNA clone, and 5 nucleotide changes, as compared with the pBHP184 genomic clone). These changes result in polymorphism of amino acid sequences at 5 positions. 5'-flanking region contains putative regulatory and promoter sequences and differs from that of the pBHP184 clone in 3 positions.     

Zea mays ZmMybst1 cDNA,encodes a single Myb-repeat protein with the VASHAQKYF motif

A cDNA clone from a 4 DAP dissected maize embryo sac encoding a novel Zea mays single-repeat Myb protein is reported here. This full-length cDNA contains an ORF of 948 bp. The gene ZmMybst1 contains two introns (1166 and 706 bp) and is a single copy gene. The ZmMybst1 protein shares high sequence identity with the potato Mybst1 protein (58%). Northern blot, RT-PCR and electronic northern analysis shows that ZmMybst1 is expressed in endosperm between 4 and 30 DAP, coinciding with the period of aleurone cell differentiation and development.     

Accumulation of the chalcone isosalipurposide in primary leaves of barley flavonoid mutants indicates a defective chalcone isomerase

Mutants defective in flavonoid biosynthesis have become increasingly useful in elucidating the potential functions of these compounds in plants. To define the role of flavonoids as UV-B protectants in barley, we have screened part of the collection of proanthocyanidin-free barley mutants at the Carlsberg Research Laboratory, Copenhagen, Denmark. The four mutants ant 30–245, ant 30–272, ant 30–287 and ant 30–310 showed drastically reduced flavonoid levels in the primary leaf as compared to their corresponding parent varieties, and in addition accumulated a new mutant-specific phenolic compound which was identified as the chalcone glucoside isosalipurposide. Results from diallelic crosses indicate that all four mutants belong to the same new complementation group, which is designated as the Ant 30 locus. This gene has not earlier been described in barley. The data presented suggest a defective chalcone isomerase gene for the observed flavonoid pattern in leaves of ant 30 mutants.     

Intron-exon structure and gene copy number of a gene encoding for a membrane-intrinsic light-harvesting polypeptide of the red alga Galdieria sulphuraria

Genes for light-harvesting proteins (lhc genes) of higher plants are well examined. However, little is known about the corresponding genes of algae, although this knowledge might give valuable information about the evolution of photosynthetic antennae. In the case of rhodophytes only two cDNA sequences from a single organism, Porphyridium cruentum, have been published. Here we describe an additional sequence from another species, the thermo-acidophilic red alga Galdieria sulphuraria. For the first time also a genomic sequence for a red algal lhc gene is presented. From a cDNA library of G. sulphuraria we isolated a clone containing an open reading frame for a protein of 302 amino acids with a deduced molecular mass of 33.86kDa. It shares major structural features with eukaryotic light-harvesting polypeptides. A proposed cleavage site between transit peptide and mature protein gives rise to a transit peptide of 119 amino acids and a mature protein of 183 residues. Hydropathy analysis suggests that the mature protein consists of three transmembrane helices. Several amino acid residues supposed to bind chlorophyll a and chlorophyll b in higher plants are conserved. The protein shows up to 69% identity and 81% similarity to the Porphyridium polypeptides in the transmembrane helices 1 and 3. Using oligonucleotides annealing in the regions of the start and stop codons of the gene as primers, a DNA sequence was amplified from nuclear G. sulphuraria DNA by PCR. Compared with the cDNA clone, this sequence contains five additional intervening DNA strings of 50-74bp length. Four of them show typical features of spliceosomal introns with GT-AG borders, and the fifth differs by starting with GC. Three of the supposed introns are located in similar positions as introns of higher plant light-harvesting proteins. Southern blotting and hybridization experiments indicate that G. sulphuraria contains at least three copies of this gene.     

Construction of the seed-coat cDNA microarray and screening of differentially expressed genes in barley

Proanthocyanidins are dimeric or polymeric conden-sation products of the flavonoids, including catechin,epicatechin or gallocatechin with leucocyanidin, leuco-pelargonidin or leucodelphinidin [1]. They are prominentcolorless compounds, and are found widely existed inthe bark of trees, leaves, fruits, flowers and seed coats.They have many natural functions, such as antioxidantproperties [2] and insect resistance [3]. In forage, theycan bind and precipitate dietary proteins, thus protectthe anim…     

The petunia homologue of the Antirrhinum majus candi and Zea mays A2 flavonoid genes; homology to flavanone 3-hydroxylase and ethylene-forming enzyme

The synthesis of anthocyanins in higher plants involves many enzymatic steps. Here we describe the isolation and characterization of a cDNA, ant17, which encodes a protein that has 73% amino acid sequence identity with the candi gene product of Antirrhinum majus and 48% with that of the maize a2 gene. This protein may therefore be involved in the synthesis of anthocyanins in the steps after the action of dihydroflavonol 4-reductase. This is consistent with the absence of ant17 expression in the regulatory anthocyanin mutants of petunia an1, an2 and an11. Furthermore, ant17 is predominantly expressed in corollas and anthers and is induced by gibberellic acid.     

Sequence and tissue-specific expression of a putative peroxidase gene from wheat (Triticum aestivum L.)

We have used a cDNA clone encoding a pathogen-induced putative wheat peroxidase to screen a genomic libary of wheat (Triticum aestivum L. cv. Cheyenne) and isolated one positive clone, lambda POX1. Sequence analysis revealed that this clone contains a gene encoding a putative peroxidase with a calculated pI of 8.1 which exhibits 58% and 83% sequence identity to the amino acid sequence of the turnip (Brassica rapa) peroxidase and a pathogen-induced putative wheat peroxidase, respectively. The two introns in the wheat gene are at the same positions as introns in the peroxidase genes of tomato and horseradish. Results of S1-mapping experiments suggest that this gene is neither pathogen-nor wound-induced in leaves but is constitutively expressed in roots.