Functional dissection of a bean chalcone synthase gene promoter in transgenic tobacco plants reveals sequence motifs essential for floral expression.

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.

Stress responses in alfalfa (Medicago sativa L.). XX. Transcriptional activation of phenlpropanoid pathway genes in elicitor-induced cell suspension cultures.

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.

Cis elements and potential trans-acting factors for the developmental regulation of the Phaseolus vulgaris CHS15 promoter.

A nuclear factor (SBF-1) has previously been identified in Phaseolus vulgaris L. (bean) suspension cell nuclear extracts that binds in vitro to three DNase I-footprinted elements (SBF-1 boxes I, II, and III, 5' to 3') in the 5' region of the bean CHS15 (chalcone synthase) gene promoter. To define the functional role of the three SBF-1 boxes in development, we examined transgenic tobacco plants carrying a series of nested CHS15 promoter-beta-glucuronidase (GUS) fusions for GUS activity by histochemical staining. We show that the CHS15 promoter deleted to position -173 and lacking all three SBF-1 boxes directs the same qualitative pattern of expression in initiating lateral roots and in developing seeds as the full length promoter (-326). Thus, activation of expression in these organs is mediated by sequence elements located downstream of the three SBF-1 boxes. However, specific deletions within the -326 to -173 region modulate expression. Thus, deletion of box II abolishes GUS activity in initiating lateral roots. Further deletion of box III fails to restore expression but subsequent deletion of an additional 43 bp to position -173 re-establishes expression. We show that sequence-specific DNA-binding activities consistent with these results are present in nuclear extracts of bean roots and seeds. These studies reveal cis elements within the CHS15 promoter, and potential trans factors, that permit organ- and tissue-specific developmental patterns of regulation to be combined with a flexible response to environmental cues.

Isolation of a monocot 3-hydroxy-3-methylglutaryl coenzyme A reductase gene that is elicitor-inducible.

The rice (Oryza sativa) phytoalexins, momilactones and oryzalexins, are synthesized by the isoprenoid pathway. An early step in this pathway, one that is rate-limiting in mammalian systems, is catalyzed by the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). A gene that encodes this enzyme has been isolated from rice, and found to contain an open reading frame of 1527 bases. The encoded protein sequence of the rice HMGR appears to be conserved with respect to other HMGR proteins, and 1 or 2 membrane-spanning domains characteristic of plant HMGRs are predicted by a hydropathy plot of the amino acid sequence. The protein is truncated at its 5' end, and shows reduced sequence conservation in this region as compared to other plant sequences. The rice genome contains a small family of HMGR genes. The isolated gene, HMGR I, is expressed at low levels in both vegetative and floral organs of rice plants. It is not induced in plants by wounding, but is strongly and rapidly induced in suspension cells by a fungal cell wall elicitor from the pathogen Magnaporthe grisea, causal agent of rice blast disease. This suggests that HMGR I may be important in the induction of rice phytoalexin biosynthesis in response to pathogen attack, and therefore may play a key role as a component of the inducible defense mechanism in rice.

Purification and biochemical characterization of proteins which bind to the H-box cis-element implicated in transcriptional activation of plant defense genes.

The H-box (CCTACC(N)7CT(N)4A), which occurs three times within the -154 to -42 region of the bean chalcone synthase chs15 promoter, is important for developmental regulation of chs15, and induction of chs15 and coordinately regulated defense genes by elicitors and other stress stimuli. Two protein factors, KAP-1 and KAP-2, which recognize conserved features in the H-box motif, were purified from bean cell suspension cultures by a combination of ion exchange chromatography and DNA affinity chromatography. KAP-1 is a 97 kDa polypeptide, whereas KAP-2 comprises two polypeptides of 76 and 56 kDa. KAP-1 and KAP-2 also differ in the sensitivity of their DNA-bound forms to trypsin. Dephosphorylation of KAP-1 or KAP-2 affects the mobility of the protein/H-box binding complex in gel shift assays but does not inhibit DNA binding. Elicitation of bean cell suspensions with glutathione does not affect the total cellular activities of KAP-1 or KAP-2, but causes a rapid increase in the specific activities of both factors in the nuclear fraction, consistent with a role for these factors in the signal pathway for elicitor induction of chs15 and related defense genes.

Dissection of the functional architecture of a plant defense gene promoter using a homologous in vitro transcription initiation system.

CHS15 is one of a family of bean genes encoding chalcone synthase, which catalyzes the first reaction in a branch pathway of phenylpropanoid biosynthesis for the production of flavonoid pigments and UV protectants and isoflavonoid-derived phytoalexins. The functional architecture of the CHS15 promoter was dissected by a novel homologous plant in vitro transcription initiation system in which whole-cell and nuclear extracts from suspension-cultured soybean cells direct accurate and efficient RNA polymerase II-mediated transcription from an immobilized promoter template. Authentic transcription from the CHS15 promoter template was also observed with whole-cell extracts from suspension-cultured cells of bean, tobacco, and the monocot rice, and the soybean whole-cell extract transcribed several other immobilized promoter templates. Hence, this procedure may be of general use in the study of plant gene regulation mechanisms in vitro. Assay of the effects of depletion of the soybean whole-cell extract by preincubation with small regions of the CHS15 promoter or defined cis elements showed that trans factors that bind to G-box (CACGTG, -74 to -69) and H-box (CCTACC, -61 to -56 and -121 to -126) cis elements, respectively, make major contributions to the transcription of the CHS15 promoter in vitro. Both cis element/trans factor interactions in combination are required for maximal activity. Delineation of these functional cis element/trans factor interactions in vitro provides the basis for study of the mechanisms underlying developmental expression of CHS15 in pigmented petal cells established by G-box and H-box combinatorial interactions, and for characterization of the terminal steps of the signal pathway for stress induction of the phytoalexin defense response.

Combination of H-box [CCTACC(N)7CT] and G-box (CACGTG) cis elements is necessary for feed-forward stimulation of a chalcone synthase promoter by the phenylpropanoid-pathway intermediate p-coumaric acid.

The phenylpropanoid pathway intermediate p-coumaric acid (4-CA) stimulates expression of the bean (Phaseolus vulgaris L.) chalcone synthase (malonyl-CoA:4-coumaroyl-CoA, EC 2.3.1.74) chs15 gene promoter in electroporated protoplasts of alfalfa (Medicago sativa L.). We have analyzed the effects of 5' deletions, mutations, and competition with promoter sequences in trans on the expression of a chs15 promoter-chloramphenicol acetyltransferase gene fusion in elicited alfalfa protoplasts. Two distinct sequence elements, the H-box (consensus CCTACC(N)7CT) and the G-box (CACGTG), are required for stimulation of the chs15 promoter by 4-CA. Furthermore, a 38-base-pair chs15 promoter sequence containing both cis elements conferred responsiveness to 4-CA on the cauliflower mosaic virus 35S minimal promoter. The H-box and G-box in combination establish the complex developmental pattern of chs15 expression and are also involved in stress induction. Hence, potential internal pathway regulation through feed-forward stimulation by 4-CA operates by modulation of the signal pathways for developmental and environmental regulation.

cis-element combinations determine phenylalanine ammonia-lyase gene tissue-specific expression patterns.

The bean phenylalanine ammonia-lyase gene 2 (PAL2) is expressed in the early stages of vascular development at the inception of xylem differentiation, associated with the synthesis of lignin precursors. This is part of a complex program of developmental expression regulating the synthesis of functionally diverse phenylpropanoid natural products. Analysis of the expression of PAL2 promoter-beta-glucuronidase gene fusions in transgenic tobacco plants showed that functionally redundant cis elements located between nucleotides -289 and -74 relative to the transcription start site were essential for xylem expression, but were not involved in expression in leaf primordia and stem nodes or in establishing tissue specificity in petals. The -135 to -119 region implicated in xylem expression contains a negative element that suppresses the activity of a cryptic cis element for phloem expression located between -480 and -289. The functional properties of each vascular element are conserved in stem, petiole, and root, even though the xylem and phloem are organized in different patterns in these organs. We conclude that the PAL2 promoter has a modular organization and that tissue-specific expression in the vascular system involves a negative combinatorial interaction, modulation of which may provide a flexible mechanism for modification of tissue specificity.

Characterization of a nuclear protein that binds to three elements within the silencer region of a bean chalcone synthase gene promoter.

The chalcone synthase (EC 2.3.1.74) gene promoter from the bean Phaseolus vulgaris L. contains a silencer element between positions -140 and -326 fro the transcription start site that is functional in electroporated soybean protoplasts. This element contains three binding sites for a bean nuclear factor (SBF-1) with DNA sequence recognition properties that are very similar to those of nuclear factor GT-1. By using a synthetic tetramer of one of the binding sites as probe, we have purified sequence-specific SBF-1 activity approximately 1750-fold from suspension-cell nuclei, by using a combination of ammonium sulfate precipitation, gel filtration, heparin-agarose chromatography, and sequence-specific DNA affinity chromatography. The factor exhibited an apparent molecular weight of 160,000-200,000 on the basis of gel filtration. A subunit molecular weight of approximately 95,000 was determined from SDS/polyacrylamide gel electrophoretic analysis of purified fractions, followed by Southwestern blot analysis (a protein blot probed with oligonucleotide probes), and from UV-cross-linking experiments. The factor lost DNA-binding activity on treatment with alkaline phosphatase. We discuss the properties of SBF-1 in relation to the functionality of GT-1 binding sequences in plant genes.

Silencer region of a chalcone synthase promoter contains multiple binding sites for a factor, SBF-1, closely related to GT-1.

Bean nuclear extracts were used in gel retardation assays and DNase I footprinting experiments to identify a protein factor, designated SBF-1, that specifically interacts with regulatory sequences in the promoter of the bean defense gene CHS15, which encodes the flavonoid biosynthetic enzyme chalcone synthase. SBF-1 binds to three short sequences designated boxes 1, 2 and 3 in the region -326 to - 173. This cis-element, which is involved in organ-specific expression in plant development, functions as a transcriptional silencer in electroporated protoplasts derived from undifferentiated suspension-cultured soybean cells. The silencer element activates in trans a co-electroporated CHS15-chloramphenicol acetyl-transferase gene fusion, indicating that the factor acts as a repressor in these cells. SBF-1 binding in vitro is rapid, reversible and sensitive to prior heat or protease treatment. Competitive binding assays show that boxes 1, 2 and 3 interact cooperatively, but that each box can bind the factor independently, with box 3 showing the strongest binding and box 2 the weakest binding. GGTTAA(A/T)(A/T)(A/T), which forms a consensus sequence common to all three boxes, resembles the binding site for the GT-1 factor in light-responsive elements of the pea rbcS-3A gene, which encodes the small subunit of ribulose bisphosphate carboxylase. Binding to the CHS15 -326 to -173 element, and to boxes 1, 2 or 3 individually, is competed by the GT-1 binding sequence of rbcS-3A, but not by a functionally inactive form, and likewise the CHS sequences can compete with authentic GT-1 sites from the rbcS-3A promoter for binding.(ABSTRACT TRUNCATED AT 250 WORDS)

cDNA cloning and characterization of a putative 1,3-beta-D-glucanase transcript induced by fungal elicitor in bean cell suspension cultures.

Synthetic oligonucleotides based on similarity between tobacco 1,3-beta-D-glucanase and barley 1,3-1,4-beta-D-glucanase were used to prime the synthesis and amplification of a 162 bp bean (Phaseolus vulgaris L.) beta-glucanase cDNA by the polymerase chain reaction (PCR). The PCR product was used to isolate a near full-length beta-glucanase cDNA corresponding to an approximately 1400 bp full-length transcript, from a library containing cDNA sequences complementary to mRNA from fungal elicitor-treated bean cells. At the amino acid level, the bean beta-glucanase cDNA was 59% similar to tobacco 1,3-beta-D-glucanase, 46% similar to barley 1,3-beta-D-glucanase and 46% similar to barley 1,3-1,4-beta-D-glucanase. At the nucleotide level, the similarities were 65, 50 and 53% respectively. The beta-glucanase appeared to be encoded by a single gene with similar genomic organization in bean cultivars Canadian Wonder, Imuna and Saxa. On the basis of predicted Mr, isoelectric point, sequence similarity, and comparisons of rate of transcript appearance with induced enzyme activity, it was concluded that the cDNA encodes the basic bean endo-1,3-beta-D-glucanase. Glucanase transcripts were induced, from very low basal levels, with similar kinetics to chitinase transcripts in elicitor-treated bean cell suspension cultures.

Bean pathogenesis-related (PR) proteins deduced from elicitor-induced transcripts are members of a ubiquitous new class of conserved PR proteins including pollen allergens.

We have searched for induced transcripts in a cDNA library derived from bean cell supension cultures treated with an elicitor from Colletotrichum lindemuthianum. Six independently isolated cDNAs corresponding to rapidly induced small mRNAs have been classified by their DNA sequence and slightly different induction behaviour into two groups. 5'- and 3'-untranslated regions exhibit little similarity, but the deduced small acidic proteins designated PvPR1 and PvPR2 are 89% identical. No relationship was found with the well-characterized PR1 proteins from tobacco. However, the PvPR proteins are closely related to pI49 in pea (64% identity), pSTH2 in potato (41% identity) and PcPR1-1 in parsley (39% identity), which are also induced in response to elicitor or microbial attack. Moreover, a major pollen allergen in birch (BetvI) has a 44% identity with PvPR1 proteins. These similarities establish a ubiquitous class of conserved defense-related proteins and suggest a common yet still unknown function. Southern blot analysis indicates that PvPR protein gene organization is highly complex with an estimated copy number of more than 12 genes.

Developmental and environmental regulation of a bean chalcone synthase promoter in transgenic tobacco.

Regulatory properties of a 1.4-kilobase promoter fragment of the bean chalcone synthase CHS8 gene were examined by analysis of glucuronidase (GUS) activity in transgenic tobacco containing a CHS8-GUS gene fusion. The promoter was highly active in the root apical meristem and in petals, exclusively in those cells of the inner epidermis that accumulate anthocyanins. The gene fusion was only weakly expressed in other floral organs, mature leaves, and stems. The early stages of seedling development were characterized by an apparent wound induction of the promoter in the endosperm and strong expression in the immature root, which became localized to the apical meristem and perivascular tissue at the root-hypocotyl junction. The promoter became active during lateral root formation in both the new root and damaged tissue of the main root. The gene fusion was also expressed in greening cotyledons and primary leaves but not in the shoot apical meristem. Light modulated expression in the cotyledons and root-shoot junction but had no effect on other aspects of the developmental program. Wounding or fungal elicitor treatment of mature leaves activated the promoter in a well-defined zone adjacent to the stress site. Stress induction occurred in mesophyll and vascular tissues as well as in the epidermis. We conclude that the CHS8 promoter contains cis-elements required to establish temporal and spatial control of flavonoid biosynthesis during development and in response to diverse environmental stimuli.

Differential regulation of phenylalanine ammonia-lyase genes during plant development and by environmental cues.

Phenylalanine ammonia-lyase (PAL) catalyzes the first reaction in the biosynthesis from phenylalanine of a wide variety of phenylpropanoid natural products including lignin, flavonoid pigments, and phytoalexins. In bean (Phaseolus vulgaris L.), PAL is encoded by a family of three genes. We show here by RNase protection with gene-specific probes that these genes are expressed differentially during development and in response to different environmental cues. While all three genes are expressed at high levels in roots, only PAL1 and PAL2 are expressed in shoots and only PAL1 is expressed in leaves. Strikingly, PAL2 is expressed at very high levels in petals, where PAL1 is only very weakly expressed and PAL3 is not expressed. All three genes are induced by mechanical wounding of hypocotyls, but fungal infection only activates PAL1 and PAL3. Illumination of etiolated hypocotyls activates PAL1 and PAL2 but not PAL3. Corresponding differential patterns of synthesis of specific PAL polypeptide isoforms were observed by two-dimensional gel electrophoretic analysis of in vitro translation products encoded by RNA isolated from hypocotyls stimulated by light, wounding, or infection. The specific isoforms encoded by transcripts of the three PAL genes were identified by inhibition of synthesis in vitro with gene-specific anti-sense transcripts followed by comparative two-dimensional gel electrophoretic analysis of the pattern of translation products. These data indicate that selective expression of PAL genes encoding functional variants is governed by a complex set of regulatory networks for developmental and environmental control of phenylpropanoid biosynthesis.

Molecular cloning of plant transcripts encoding protein kinase homologs.

Oligonucleotides, corresponding to conserved regions of animal protein-serine/threonine kinases, were used to isolate cDNAs encoding plant homologs in the dicot bean (Phaseolus vulgaris L.) and the monocot rice (Oryzae sativa L.). The C-terminal regions of the deduced polypeptides encoded by the bean (PVPK-1) and rice (G11A) cDNAs, prepared from mRNAs of suspension cultures and leaves, respectively, contain features characteristic of the catalytic domains of eukaryotic protein-serine/threonine kinases, indicating that these cDNAs encode plant protein kinases. The putative catalytic domains are most closely related to cyclic nucleotide-dependent protein kinases and the protein kinase C family, suggesting the plant homologs may likewise transduce extracellular signals. However, outside these domains, PVPK-1 and G11A exhibit no homology either to each other or to regulatory domains of other protein kinases, indicating the plant homologs are modulated by other signals. PVPK-1 corresponds to a 2.4-kb transcript in suspension cultured bean cells. Southern blots of genomic DNA indicate that PVPK-1 and G11A correspond to single copy genes that form part of a family of related plant sequences.

Glycine-rich cell wall proteins in bean: gene structure and association of the protein with the vascular system.

A single genomic clone (14 kb) isolated from bean (Phaseolus vulgaris L.) contains two genes that encode glycine-rich proteins. These genes are present as single copies in the genome, are separated by 2.85 kb and encode transcripts of 1.8 kb and 1.0 kb respectively. The encoded proteins contain 60% glycine and have amino-terminal signal peptides. The 1.8 kb transcript is present in young hypocotyls and in ovary tissue. Excision-wounding transiently induced this transcript in old, but not in young hypocotyl tissue. Antibodies raised against regions of the glycine-rich protein 1.8, expressed as a lacZ fusion protein in bacteria, react with a protein of 53 kd in a protein fraction extracted from cell walls of bean ovaries. Tissue imprints of bean ovaries treated with anti-glycine-rich protein antibodies showed that the glycine-rich protein was distributed in a regular pattern of small, highly localized discrete sites. The immunoreactive regions correspond to the pattern of vascular tissue in the pod. In young hypocotyls, glycine-rich protein is present at four pairs of discrete sites symmetrically arranged on the inner side of the vascular ring. These results suggest a close relationship between glycine-rich proteins and development of the vascular system.

Cinnamyl-alcohol dehydrogenase, a molecular marker specific for lignin synthesis: cDNA cloning and mRNA induction by fungal elicitor.

Cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) catalyzes the final step in a branch of phenylpropanoid synthesis specific for production of lignin monomers. We have isolated a full-length cDNA clone encoding CAD, as a molecular marker specific for lignification, by immunoscreening a lambda gt11 library containing cDNAs complementary to mRNA from elicitor-treated cell cultures of bean (Phaseolus vulgaris L.). The clone comprises a single long open reading frame of 1767 base pairs, 31 base pairs of 5' leader, and 152 base pairs of 3' untranslated sequence. The deduced 65-kDa CAD polypeptide has several features that are strongly conserved in alcohol dehydrogenases. Addition of fungal elicitor to cell cultures stimulates CAD transcription, which leads to a remarkably rapid, but transient, accumulation of CAD mRNA, with no detectable lag and maximal levels after 1.5 hr. Southern blot analysis of bean genomic DNA indicates that elicitor-induced CAD is encoded by a single gene. The regulatory significance of the rapid activation of this CAD gene and the possible existence of a second, divergent CAD gene involved in lignification during xylogenesis are discussed.

Differential regulation of a hydroxyproline-rich glycoprotein gene family in wounded and infected plants.

We have characterized three different transcripts induced by fungal elicitor, wounding, or infection which encode apoproteins of cell wall hydroxyproline-rich glycoproteins involved in plant defense against infection. The proteins encoded by two of these transcripts contain a proline-rich domain involving tandem repetition of the 16-amino-acid unit Tyr3-Lys-Ser-Pro4-Ser-Pro-Ser-Pro4. The third transcript encodes a protein with a proline-rich domain involving a variant of this 16-mer canonical repeat: Tyr3-His-Ser-Pro4-Lys-His-Ser-Pro4. Each transcript is encoded by a separate gene present at single or low copy number in the haploid genome. These transcripts exhibit markedly different patterns of accumulation in different stress conditions, indicating the operation of several distinct intercellular stress signal systems in higher plants.