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B W Shirley

Publications and source records attributed to B W Shirley.

13 recordsLinked to original sources
PubMed1998

A light-independent developmental mechanism potentiates flavonoid gene expression in Arabidopsis seedlings.

Throughout the plant kingdom expression of the flavonoid biosynthetic pathway is precisely regulated in response to developmental signals, nutrient status, and environmental stimuli such as light, heat and pathogen attack. Previously we showed that, in developing Arabidopsis seedlings, flavonoid genes are transiently expressed during germination in a light-dependent manner, with maximal mRNA levels occurring in 3-day-old seedlings. Here we describe the relationship between developmental and environmental regulation of flavonoid biosynthesis by examining phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR) mRNA levels in germinating Arabidopsis seedlings as a function of light, developmental stage and temperature. We show that seedlings exhibit a transient potential for induction of these four genes, which is distinct from that observed for chlorophyll a/b-binding protein(CAB). The potential for flavonoid gene induction was similar in seedlings grown in darkness and red light, indicating that induction potential is not linked to cotyledon expansion or the development of photosynthetic capacity. The evidence for metabolic regulation of flavonoid genes during seedling development is discussed.

Acyltransferases↗
PubMed1997

Expression of chalcone synthase and chalcone isomerase proteins in Arabidopsis seedlings.

Antibodies have been developed against the first two enzymes of flavonoid biosynthesis in Arabidopsis thaliana. Chalcone synthase (CHS) and chalcone isomerase (CHI) were overexpressed and purified from Escherichia coli as fusion proteins with glutathione S-transferase from Schistosoma japonicum. The recombinant proteins were then used to immunize chickens and the resulting IgY fraction was purified from egg yolks. Immunoblots of crude protein extracts from Arabidopsis seedlings carrying wild-type and null alleles for CHS and CHI showed that the resulting antibody preparations provide useful tools for characterizing expression of the flavonoid pathway at the protein level. An initial analysis of expression patterns in seedlings shows that CHS and CHI proteins are present at high levels during a brief period of early seedling germination that just precedes the transient accumulation of flavonoid end-products.

Acyltransferases↗
PubMed1997

Characterization of flavonol synthase and leucoanthocyanidin dioxygenase genes in Arabidopsis. Further evidence for differential regulation of "early" and "late" genes.

As part of an ongoing investigation into the organization and regulation of the flavonoid biosynthetic pathway, two Arabidopsis thaliana expressed sequence tag (EST) clones (153O10T7 and YAY780) with high homology to leucoanthocyanidin dioxygenase (LDOX) or flavonol synthase (FLS) were identified. EST YAY780 was sequenced and found to encode a protein 49 to 78% identical to all LDOX sequences in the database. EST 153O10T7 was used to isolate a genomic clone encoding a protein with 59 to 61% sequence identity to petunia (Petunia hybrida) and potato (Solanum tuberosum) FLS. DNA blot analysis was used to screen the Arabidopsis genome for sequences related to FLS and LDOX and to determine the positions of the two clones on the RI map. The expression patterns of FLS and LDOX in etiolated seedlings moved to white light and in two putative regulatory mutants (ttg and tt8) were determined by RNA blot analysis. These studies indicate that FLS is an "early" flavonoid gene in Arabidopsis seedlings, whereas LDOX is a "late" gene. Furthermore, FLS is the first flavonoid enzyme identified in Arabidopsis that may be encoded by a gene family.

Amino Acid Sequence↗
PubMed1996

Analysis of flavanone 3-hydroxylase in Arabidopsis seedlings. Coordinate regulation with chalcone synthase and chalcone isomerase.

A genomic clone encoding flavanone 3-hydroxylase (F3H) was isolated from Arabidopsis thaliana. The deduced amino acid sequence is 72 to 94% identical to all previously reported F3H proteins. Low-stringency DNA blot analysis indicated that F3H is encoded by a single gene in Arabidopsis. The F3H locus was mapped to the bottom of chromosome 3 and therefore does not correspond to any of the 13 flavonoid-deficient transparent testa mutants for which a map position is known. Analysis of gene expression in etiolated seedlings exposed to white light and in two putative regulatory mutants, ttg and tt8, demonstrated that the Arabidopsis F3H gene is coordinately expressed with chalcone synthase and chalcone isomerases is seedlings, whereas dihydroflavonol reductase expression is controlled by distinct regulatory mechanisms. The F3H gene may represent a pivotal point in the regulation of flavonoid biosynthesis because its expression is coordinated with different subsets of genes in different plant species.

Arabidopsis↗
PubMed1996

A null mutation in the first enzyme of flavonoid biosynthesis does not affect male fertility in Arabidopsis.

Flavonoids are a major class of secondary metabolites that serves a multitude of functions in higher plants, including a recently discovered role in male fertility. Surprisingly, Arabidopsis plants deficient in flavonoid biosynthesis appear to be fully fertile. Using RNA gel blot analysis and polymerase chain reaction-based assays, we have shown that a mutation at the 3' splice acceptor site in the Arabidopsis chalcone synthase gene completely disrupts synthesis of the active form of the enzyme. We also confirmed that this enzyme, which catalyzes the first step of flavonoid biosynthesis, is encoded by a single-copy gene. HPLC analysis of whole flowers and stamens was used to show that plants homozygous for the splice site mutation are completely devoid of flavonoids. This work provides compelling evidence that despite the high levels of these compounds in the pollen of most plant species, flavonoids are not universally required for fertility. The role of flavonoids in plant reproduction may therefore offer an example of convergent functional evolution in secondary metabolism.

Arabidopsis↗
PubMed1996

Are flavonoids synthesized by a multi-enzyme complex?

An enormous variety of metabolic processes are characterized by enzyme complexes, which are likely to play important roles in directing the efficient operation and specificity of cellular metabolism. In many cases membranes or cytoskeletal elements provide scaffolding for these highly ordered assemblies of enzymes. Biochemical and immunocytochemical studies indicate that the flavonoid biosynthetic pathway of higher plants involves a complex of sequentially-acting enzymes localized at the cytoplasmic face of the endoplasmic reticulum. This paper describes preliminary efforts to define the organization of this putative flavonoid biosynthetic complex and elucidate its role in controlling the synthesis of different flavonoid end-products in the model plant, Arabidopsis thaliana.

Acyltransferases↗
PubMed1995

Analysis of Arabidopsis mutants deficient in flavonoid biosynthesis.

Eleven loci that play a role in the synthesis of flavonoids in Arabidopsis are described. Mutations at these loci, collectively named transparent testa (tt), disrupt the synthesis of brown pigments in the seed coat (testa). Several of these loci (tt3, tt4, tt5 and ttg) are also required for the accumulation of purple anthocyanins in leaves and stems and one locus (ttg) plays additional roles in trichome and root hair development. Specific functions were previously assigned to tt1-7 and ttg. Here, the results of additional genetic, biochemical and molecular analyses of these mutants are described. Genetic map positions were determined for tt8, tt9 and tt10. Thin-layer chromatography identified tissue- and locus-specific differences in the flavonols and anthocyanidins synthesized by mutant and wild-type plants. It was found that UV light reveals distinct differences in the floral tissues of tt3, tt4, tt5, tt6 and ttg, even though these tissues are indistinguishable under visible light. Evidence was also uncovered that tt8 and ttg specifically affect dihydroflavonol reductase gene expression. A summary of these and previously published results are incorporated into an overview of the genetics of flavonoid biosynthesis in Arabidopsis.

Acyltransferases↗
PubMed1993

An Arabidopsis gene homologous to mammalian and insect genes encoding the largest proteasome subunit.

A gene encoding a protein with extensive homology to the largest subunit of the multicatalytic proteinase complex (proteasome) has been identified in Arabidopsis thaliana. This gene, referred to as AtPSM30, is entirely encompassed within a previously characterized radiation-induced deletion, which may thus provide the first example of a proteasome null mutation in a higher eukaryote. However, the growth rate and fertility of Arabidopsis plants do not appear to be significantly affected by this mutation, even though disruption experiments in yeast have shown that most proteasome subunits are essential. Analysis of mRNA levels in developing seedlings and mature plants indicates that expression of AtPSM30 is differentially regulated during development and is slightly induced in response to stress, as has been observed for proteasome genes in yeast, Drosophila, and mammals. Southern blot analysis indicates that the Arabidopsis genome contains numerous sequences closely related to AtPSM30, consistent with recent reports of at least two other proteasome genes in Arabidopsis. A comparison of the deduced amino acid sequences for all proteasome genes reported to date suggests that multiple proteasome subunits evolved in eukaryotes prior to the divergence of plants and animals.

Amino Acid Sequence↗
PubMed1992

Effects of ionizing radiation on a plant genome: analysis of two Arabidopsis transparent testa mutations.

Ionizing radiation is known to cause chromosomal alterations such as inversions and deletions and has been used extensively for inducing mutations. In Arabidopsis, two methods for the isolation of genes identified on the basis of mutant phenotypes--genomic subtraction and chromosome walking--either rely on or are greatly facilitated by the availability of these types of mutations. This article gives a detailed characterization of ionizing radiation-induced mutations in plants. The Arabidopsis genes encoding chalcone flavanone isomerase (CHI) and dihydroflavonol 4-reductase (DFR) were cloned and found to correspond to two transparent testa loci. A CHI allele, generated by fast-neutron irradiation, consisted of an inversion within the gene. A 272-bp fragment from 38 centimorgans away on the same chromosome was transferred to one end of this inversion. A DFR allele, induced by x-irradiation, contained two deletions and an inversion of the 2.8-centimorgan intervening region. Sequence analysis of the break points in both mutants indicate that repair of radiation-induced damage involves mechanisms similar or identical to those that mediate the integration of foreign sequences into the genome. The chromosome rearrangements found in these mutants have important implications for the use of ionizing radiation-induced alleles in classical and molecular genetic experiments in plants.

Alcohol Oxidoreductases↗
PubMed1990

A potential role for RNA turnover in the light regulation of plant gene expression: ribulose-1,5-bisphosphate carboxylase small subunit in soybean.

Post-transcriptional regulation of the genes encoding the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase was examined in soybean seedlings. Substantial discrepancies were detected between relative in vitro transcription rates and steady-state RNA levels in light- and dark-grown seedling leaves, indicating that rbcS RNA may be degraded more rapidly in light than in darkness. Additional data imply that the turnover mechanism is rapidly induced by light, maintained for some time in darkness, and that it may be negatively controlled by far-red light. The proposed RNA turnover system does not affect all RNAs equally since a soybean actin gene showed equivalent in vitro transcription rates and RNA levels in light and darkness. Soybean rbcS genes may be subject to a novel mode of control in which light-induced expression is accompanied by an increased rate of RNA degradation. Models for the specific regulation of rbcS RNA stability in response to light are presented.

Blotting, Western↗
PubMed1990

Comparison of the expression of two highly homologous members of the soybean ribulose-1,5-bisphosphate carboxylase small subunit gene family.

Two soybean ribulose-1,5-bisphosphate carboxylase small subunit (SSU) genes, SRS1 and SRS4, are highly homologous over a region that includes 4 kb of 5' and 1 kb of 3' flanking sequences. The expression of these genes was compared using synthetic oligonucleotide probes. Analysis of a soybean leaf cDNA library indicates that SRS1 and SRS4 are the most highly expressed members of the soybean SSU gene family. Similar changes were observed in the RNA levels for these genes in response to white light, far-red light and darkness, although SRS1 was expressed at a four-fold higher level in total RNA than SRS4 under all conditions. However, nuclear run-on assays indicate that SRS1 is transcribed at a lower rate than SRS4, which suggests that SRS1 RNA is more stable. S1 nuclease analysis and oligonucleotide directed RNase H cleavage indicate that transcripts from both genes are polyadenylated within two principle regions separated by 35 nt. Sequence analysis of 16 independent cDNA clones identified seven different polyadenylation sites, and six of these sites lie within these two regions. Although SRS1 RNA was poorly recovered during poly(A)+ fractionation, RNase H cleavage experiments showed that transcripts from SRS1 and SRS4 had similar poly (A) tail lengths ranging from 0 to 220 nt. In addition, and despite differences in the untranslated leader sequences, SRS1 and SRS4 RNAs are assembled into polysomes with equal efficiencies. The overall similarity in expression patterns for these two genes further illustrates the coordinate evolution of individual members of a SSU gene family and is consistent with the proposal that gene conversion homogenizes both the coding and regulatory regions of these genes.

Base Sequence↗
PubMed1987

5' proximal sequences of a soybean ribulose-1,5-bisphosphate carboxylase small subunit gene direct light and phytochrome controlled transcription.

Two closely related ribulose-1,5-bisphosphate carboxylase small subunit (SSU) genes, SRS1 and SRS4, are transcribed at high levels in soybean plants in response to light. Transgenic petunia plants containing 5' sequences from SRS1 or SRS4 fused to the polypeptide encoding region of a neomycin phosphotransferase (NPTII) gene exhibit selectable kanamycin resistance. Deletion of three ATG codons from the region preceding the normal NPTII translation start site has little effect on the levels of kanamycin resistance in transformed plants. Run-on transcription assays in isolated nuclei demonstrate that transcription of the SRS1/NPTII chimera and the native petunia SSU11A gene subfamily is light regulated and under phytochrome control in leaves of transgenic plants. In young expanding leaves of fully light grown plants, transcription of these genes is markedly reduced within minutes of far-red treatment, while ribosomal DNA and actin gene transcription remains unchanged. This is analogous to the transcriptional response we observed for SRS1 and SRS4 in soybean seedlings. These data suggest (1) that transcription of SSU genes in both soybean and petunia require the continued presence or synthesis of phytochrome in the Pfr form and (2) that 5' sequences are sufficient to direct the phytochrome controlled transcriptional response of the SRS1 gene. In fully expanded mature leaves we found the transcription rates of the native SSU11A gene subfamily, the chimeric SRS1/NPTII gene, the rDNA genes, and several other control genes to be reduced markedly after far-red treatment or after extended periods of darkness. The contrast between results in young and mature leaves is discussed.

Chlorophyll↗