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As an alternative to soluble plant transcription systems, we examined the reinitiation capacity of isolated parsley nuclei. Nuclear chalcone synthase in vitro transcripts were affinity-labelled with gamma-thio-ATP, gamma-thio-GTP or beta-thio-ATP, and purified by chromatography on a mercury Sepharose affinity column. Primer extension and subsequent PCR amplification of these in vitro transcripts revealed gamma-thio-ATP-dependent, but no beta-thio-ATP-dependent, signals, although affinity labelling of overall in vitro transcripts still occurred with beta-thio-ATP. We conclude that the described plant nuclei reinitiated transcription non-specifically and that post-transcriptional transfer of the gamma-thio affinity label severely interfered with the detection of reinitiated transcripts.
Plant Mol Biol 1993 Mar
PMID:Post-transcriptional transfer of gamma-thio affinity label to RNA in isolated parsley nuclei. 846 88

We have identified five different full length chalcone synthase (CHS) cDNA clones from a cDNA library produced from transcripts isolated from an elicitor-treated alfalfa cell suspension culture. Nucleotide sequence similarity between the clones varied from 88-93%. Oligonucleotides based on divergent sequences in the 5'-untranslated regions of the clones could distinguish individual genes, or groups of genes, and their corresponding transcripts. Developmentally regulated expression of the CHS transcripts was predominantly in roots and root nodules; other unidentified members of the CHS gene family are expressed in stems, leaves and nodules. One of the CHS transcripts was strongly expressed in floral tissue. All the CHS transcripts studied were induced in elicitor-treated cell suspension cultures. Transcripts were also induced in roots in response to wounding or spraying with various elicitors, and in leaves infected with Phoma medicaginis (but not in wounded leaves). The induction kinetics of CHS2 transcripts were more rapid and/or transient than those of other members of the CHS family in CuCl2-treated roots and Phoma-infected leaves. The results are discussed in terms of the evolution and functions of the CHS gene family in legumes.
Plant Mol Biol 1993 May
PMID:Stress responses in alfalfa (Medicago sativa L.). 15. Characterization and expression patterns of members of a subset of the chalcone synthase multigene family. 850 27

Flavonoids are plant phenolic compounds involved in leguminous plant-microbe interactions. Genes implied in the central branch (chalcone synthase (CHS), chalcone isomerase (CHI)) or in the isoflavonoid branch of the flavonoid biosynthesis pathway have been characterized in Medicago sativa. No information is available to date, however, on genes whose products are involved in the synthesis of other types of flavonoids. In this paper we present the genomic organization as well as the nucleotide sequence of one flavanone-3-hydroxylase (F3H) encoding gene of M. sativa, containing two introns and exhibiting 82-89% similarity at the amino acid level to other F3H proteins. This is the first report on the genomic organization of a f3h gene so far. We present also the sequence of a partial dihydroflavonol-4-reductase (DFR) M. sativa cDNA clone. Southern blot experiments indicated that f3h and dfr genes are each represented by a single gene within the tetraploid genome of M. sativa. By a combination of Northern blot and RT-PCR analysis, we showed that both f3h and dfr genes are expressed in flowers, nodules and roots, with a pattern distinct from chs expression. Finally, we show that dfr is expressed in M. sativa leaves whereas f3h is not. The role played by these two genes in organs other than flowers remains to be determined.
Plant Mol Biol 1995 Nov
PMID:Molecular characterization and expression of alfalfa (Medicago sativa L.) flavanone-3-hydroxylase and dihydroflavonol-4-reductase encoding genes. 854 3

Nuclear transcript run-on analysis was used to investigate++ the relative transcription rates of genes encoding enzymes of isoflavonoid phytoalexin biosynthesis and related pathways in elicitor-treated alfalfa (Medicago sativa L.) cell suspension cultures. Genes encoding L-phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and chalcone reductase (CHR) were most rapidly activated, with increases in transcription measurable within 10-20 min after elicitation. Cinnamic acid 4-hydroxylase (C4H), chalcone isomerase (CHI), isoflavone reductase (IFR) and caffeic acid 3-0-methyltransferase (COMT) genes were also rapidly activated, but at a slower initial rate. Transcription of chalcone 2'-O-methyltransferase (CHOMT), and 1,3-beta-D-glucanase genes was less rapid, with lag periods of 60 and 30 min post-elicitation, respectively. Treatment of cells with a PAL inhibitor L-alpha-aminooxy-beta-phenylpropionic acid (AOPP) resulted in increased transcription of PAL, CHS and CHR, but reduced transcription of CHOMT, indicating a role for phenylpropanoid products as both negative and positive regulators of gene expression within the phenylpropanoid pathway.
Plant Mol Biol 1996 Feb
PMID:Stress responses in alfalfa (Medicago sativa L.). XX. Transcriptional activation of phenlpropanoid pathway genes in elicitor-induced cell suspension cultures. 860 96

The relationship between the length of anthers and the stage of development of microspores was examined in rice (Oryza sativa L. cv. Hayayuki). Anthers of < or = 2 mm and 2.1-2.2 mm in length and those ready to dehiscence were determined to be at the uninucleate, binucleate and trinucleate microspore stage, respectively. Two cDNAs (YY1 and YY2), representing genes that are specifically expressed in anthers at the uninucleate microspore stage, were isolated and characterized. YY1 cDNA encoded an open reading frame of 95 amino acids. Eight cysteine residues with the potential to form disulfide bridges were present in the amino acid sequence. There was a hydrophobic region at the N-terminus of the putative protein, suggesting that the YY1 protein might be secreted. This cysteine motif and the hydrophobic N-terminus are conserved among products of several anther-specific genes or cDNAs isolated from various plant species. These proteins are thought to form a superfamily of proteins that are confined to anthers. The YY1 transcript was localized in the tapetal cells and the peripheral cells of the vascular bundle. YY2 cDNA encoded an open reading frame of 389 amino acids and the deduced amino acid sequence exhibited substantial homology to that of chalcone synthase. Expression of YY2 mRNA was confined to the tapetal cells. The genes correspond to YY1 and YY2 cDNAs were shown to exist as single copies in the rice genome.
Plant Mol Biol 1996 Mar
PMID:Isolation and characterization of two cDNA clones for mRNAs that are abundantly expressed in immature anthers of rice (Oryza sativa L.). 870 28

Many eukaryotic DNA-binding proteins share a conserved amino acid sequence known as the basic region leucine zipper (bZIP) domain. bZIP proteins recognise DNA, upon dimerization, in a sequence-specific manner. The Common Plant Regulatory Factor 1 (CPRF1) is a bZIP transcription factor from parsley (Petroselinum crispum), which recognises defined elements containing ACGT cores. CPRF1 genomic DNA was cloned and the gene was sequenced. Analysis of the sequence data revealed the existence of 12 exons and 11 introns within a stretch of about 9 kb. A second RNA species hybridising to CPRF1 probes was identified as an alternatively spliced, additional CPRF1 transcript containing intron 8. This polyadenylated RNA species showed accumulation characteristics very similar to those of the CPRF1 mRNA. CPRF1 specifically binds an ACGT-containing element which is located within the composite regulatory unit that is necessary and sufficient for light activation of the parsley chalcone synthase (CHS) minimal promoter. Expression studies at the mRNA level demonstrated that CPRF1 mRNA is present in all organs of light-grown plants in which CHS mRNA expression is detectable, and light-dependent CHS mRNA accumulation was shown to be blocked by cycloheximide. Therefore, translation of a protein factor, possibly CPRF1, may be a prerequisite for CHS promoter activation.
Mol Gen Genet 1996 Jul 26
PMID:The transcriptional regulator CPRF1: expression analysis and gene structure. 875 92

Cis-regulatory elements involved in the activation of the plant defense-related gene encoding chalcone synthase 1 (PsChs1) in pea (Pisum sativum L.) were examined by transient transfection, gel mobility shift assay and in vitro DNase I-footprinting analysis. Transient transfection assay revealed that a 61 bp DNA fragment spanning from -242 to -182 of PsChs1 was required for the maximal promoter activity and possibly involved in the enhancement of elicitor-mediated activation. Nuclear isolate from elicitor-treated pea epicotyl tissues contained some factor(s) that specifically bound to this DNA fragment to form a complex with low mobility (LMC, low mobility complex) in gel mobility shift assay. DNase I-footprinting analysis of LMC revealed that among three protected regions detected in a 61 bp DNA fragment, two regions contained identical AT-rich sequence, TAAAATACT. Site directed mutation in either or both identical sequences, TAAAATACT to TGGAATACT, resulted in the reduction or loss in the ability to form LMC. Detailed analysis of 61 bp DNA fragment demonstrated that the region from -242 to -226 containing promoter-distal TAAAATACT motif was imperative for the maximal elicitor-mediated activation of PsChs1.
Plant Mol Biol 1996 Jun
PMID:Analysis of cis-regulatory elements involved in the activation of a member of chalcone synthase gene family (PsChs1) in pea. 879 Feb 82

We studied potential mechanisms of eosinophil accumulation in nonallergic chronic hyperplastic sinusitis with nasal polyposis (CHS/NP). We measured expression of endothelial vascular cell adhesion molecule-1 (VCAM-1), which mediates selective eosinophil transendothelial migration, the cytokines interleukin (IL)-1 beta, TNF-alpha and IL-13 which upregulate VCAM-1 expression, and the chemokine RANTES which mediates lymphocyte, monocyte, and eosinophil chemotaxis in chronic hyperplastic sinusitis with nasal polyposis (CHS/NP) nasal polyps (nonallergic versus allergic) and middle turbinate biopsies from normal controls. By immunohistochemical staining, the density of EG2+ eosinophils was increased in both the nonallergic and allergic CHS/NP subgroups compared to normal controls. VCAM-1 expression was significantly increased in CHS/NP subjects compared to normal controls (P = 0.0005), with the highest intensity seen in nonallergic CHS/NP. By in situ hybridization, the densities of IL-1 beta, TNF-alpha, IL-13, and RANTES mRNA+ cells were all increased in nonallergic CHS/NP compared to normal controls (P = 0.009, 0.0005, 0.0005, and 0.001, respectively). In comparison to allergic CHS/NP, nonallergic CHS/NP had a significantly higher tissue density of TNF-alpha (P = 0.04) and a lower density of IL-13 (P = 0.005) mRNA+ cells. In general, VCAM-1 expression correlated strongly in CHS/NP with the density of TNF-alpha (R = .91, P = 0.0005) but not the density of IL-1 beta, IL-13, or RANTES mRNA+ cells. We conclude that upregulation of VCAM-1 and elaboration of RANTES may contribute to the marked accumulation of eosinophils in nonallergic CHS/NP. TNF-alpha may play a critical role in VCAM-1 upregulation in this nonallergic eosinophilic disorder.
Am J Respir Cell Mol Biol 1996 Oct
PMID:Eosinophil infiltration in nonallergic chronic hyperplastic sinusitis with nasal polyposis (CHS/NP) is associated with endothelial VCAM-1 upregulation and expression of TNF-alpha. 887 77

The chalcone synthase is a key enzyme that catalyses the first dedicated reaction of the flavonoid pathway in higher plants. The chs gene and its protein product in rice has been investigated. The presence of a chalcone synthase (CHS) protein in rice seedlings and its developmental stage-specific expression has been demonstrated by western analysis. The chalcone synthase of rice was found to be immunologically similar to that of maize. A rice cDNA clone, Os-chs cDNA, encoding chalcone synthase, isolated from a leaf cDNA library of an indica rice variety Purpleputtu has been mapped to the centromeric region of chromosome 11 of rice. It was mapped between RFLP markers RG2 and RG103. RG2 is the nearest RFLP marker located at a genetic distance of 3.3 cM. Some segments of chromosome 11 of rice including chs locus are conserved on chromosome 4 of maize. The markers, including chs locus on chromosome 11 of rice are located, though not in the same order, on chromosome 4 of maize. Genetic analysis of purple pigmentation in two rice lines, Abhaya and Shyamala, used in the present mapping studies, indicated the involvement of three genes, one of which has been identified as a dominant inhibitor of leaf pigmentation. The Os-chs cDNA shows extensive sequence homology, both for DNA and protein (deduced), to that of maize, barley and also to different monocots and dicots.
Plant Mol Biol 1996 Nov
PMID:Chalcone synthase in rice (Oryza sativa L.): detection of the CHS protein in seedlings and molecular mapping of the chs locus. 898 May 25

Expression of chalcone synthase (CHS), the first enzyme in the flavonoid branch of the phenylpropanoid biosynthetic pathway in plants, is induced by developmental cues and environmental stimuli. We used plant transformation technology to delineate the functional structure of the French bean CHS15 gene promoter during plant development. In the absence of an efficient transformation procedure for bean, Nicotiana tabacum was used as the model plant. CHS15 promoter activity, evaluated by measurements of beta-D-glucuronidase (GUS) activity, revealed a tissue-specific pattern of expression similar to that reported for CHS genes in bean. GUS activity was observed in flowers and root tips. Floral expression was confined to the pigmented part of petals and was induced in a transient fashion. Fine mapping of promoter cis-elements was accomplished using a set of promoter mutants generated by unidirectional deletions or by site-directed mutagenesis. Maximal floral and root-specific expression was found to require sequence elements located on both sides of the TATA-box. Two adjacent sequence motifs, the G-box (CACGTG) and H-box (CCTACC(N)7CT) located near the TATA-box, were both essential for floral expression, and were also found to be important for root-specific expression. The CHS15 promoter is regulated by a complex interplay between different cis-elements and their cognate factors. The conservation of both the G-box and H-box in different CHS promoters emphasizes their importance as regulatory motifs.
Plant Mol Biol 1996 Dec
PMID:Functional dissection of a bean chalcone synthase gene promoter in transgenic tobacco plants reveals sequence motifs essential for floral expression. 898 May 36


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