Gérard Burkard

Bean cyclophilin gene expression during plant development and stress conditions

Cyclophilins (Cyp) are ubiquitous proteins with peptidyl-prolyl cis-trans isomerase activity that catalyses rotation of X-Pro peptide bonds and facilitates the folding of proteins; these enzymes are believed to play a role in in vivo protein folding. During development of normal bean plants, Cyp transcripts are first detected three days after beginning of germination and are present in all plant tissues examined. In a general way, higher amounts of Cyp mRNAs are found in developing tissues. Cyp mRNA accumulates in alfalfa mosaic virus-infected bean leaves and after ethephon and salicylic acid treatments. In response to a localized chemical treatment Cyp mRNA accumulation is observed in the untreated parts of the plants; however these changes in mRNA levels are restricted to the aerial part of the plant. A comparative study of Cyp mRNA accumulation in bean and maize in response to various external stimuli shows striking differences in profiles between the two plants. For instance, in response to heat shock, maize Cyp mRNA significantly accumulates, whereas no remaining mRNA is observed a few hours after the beginning of the heat stress in bean. Differences in mRNA accumulation profiles are also observed upon salt stress which induces the response earlier in maize than in bean, whereas the opposite situation is observed when plants are cold-stressed. All these findings further suggest that cyclophilin might be a stress-related protein.

Identification and characterization of maize pathogenesis-related proteins. Four maize PR proteins are chitinases.

Eight pathogenesis-related proteins extractable at pH 2.8 were found to accumulate in maize leaves after mercuric chloride treatment or brome mosaic virus infection. These proteins were called PRm (pathogenesis-related maize) proteins. Seven PRm proteins were purified to homogeneity by preparative polyacrylamide gel electrophoresis and their amino acid compositions determined. Estimated molecular weights in SDS-containing gels were: PRm 1 14.2 kDa; Prm 2 16.5 kDa; PRm 3 and PRm 4 25 kDa; PRm 6b 30.5 kDa; PRm 6a 32 kDa; PRm 7 34.5 kDa. Antisera raised against either PRm 3 or PRm 4 reacted specifically each with PRm 3 or PRm 4. Antisera raised against PRm 6b reacted with PRm 6b as well as with PRm 6a and antisera against PRm 7 reacted with PRm 7 and PRm 5. Tobacco anti-PR 1b antisera reacted with maize PRm 2.Chitinase (poly[1,4-(N-acetyl-β-D-glucosamide)]glycanhydrolase, EC 3.2.1.14) activity was found for PRm 3, PRm 4, PRm 5, and PRm 7.

Effects of abiotic stresses on cyclophilin gene expression in maize and bean and sequence analysis of bean cyclophilin cDNA

Abstract In a search for stress-related genes, a differential screening of a mercuric chloride-treated maize cDNA library has been performed. Among the positive clones identified, one, designated as CHEM 7, has been found to be expressed at a significantly higher level in stressed plants than in non-stressed plants. The sequence of CHEM 7 showed complete identity to maize ( Zea mays L.) cyclophilin (CyP) described earlier. CHEM 7 was used as a probe to screen a chemically-stressed bean cDNA library. DNA sequencing of the longest positive clone revealed a high homology with maize CyP cDNA. Time course studies of CyP mRNA accumulation upon chemical stress in maize and bean ( Phaseolus vulgaris L.) showed that in maize maximum amounts of mRNA are found 6–7 h after treatment, whereas in bean there is a rather long delay before mRNA synthesis is stimulated and the maximum accumulation is only reached 48 h after the onset of stress. Southern blot analysis suggests that maize CyP is encoded by a small family of genes (6–7) but there is only a single CyP gene in bean. CyP mRNA synthesis is not only stimulated by a mercuric chloride treatment but also by other abiotic stresses such as heat-shock, wounding, salt stress and low temperature.

Heavy-metal-responsive genes in maize: identification and comparison of their expression upon various forms of abiotic stress

To identify genes involved in defense against heavy-metal stresses, a cDNA library originating from mercuric chloride-treated maize (Zea mays L. cv. INRA 258) leaves was constructed and analysed by differential screening using cDNAs derived from treated and untreated plants. Transcriptionally activated cDNA clones, designated CHEM (chemically-activated), were isolated and characterized. They represent various known proteins, such as glycine-rich proteins, pathogenesis-related proteins, chaperones and membrane proteins. The expression of the genes encoding these proteins was studied in maize subjected to other forms of abiotic stress. Expression of glycine-rich proteins was greatly enhanced by heat stress, and also stimulated by NaCl, polluted rainwater, wounding and cold stress. Pathogenesis-related proteins were strongly induced by ultraviolet light and to a lesser extent by NaCl, polluted rainwater and wounding. Heat-shock protein was mainly induced by heat and cold, and ubiquitin by wounding. Expression of the membrane channel protein was stimulated by heat stress, NaCl, polluted rainwater and ultraviolet-light irradiation.

Maize pathogenesis-related proteins: characterization and cellular distribution of 1,3-β-glucanases and chitinases induced by brome mosaic virus infection or mercuric chloride treatment.

Abstract Ten pathogenesis-related (PR) proteins have been extracted at pH 5·2 and purified from maize leaves after bromegrass mosaic virus infection or mercuric chloride treatment. Among these PR proteins several have been found to be 1,3-β-glucanases and chitinases. Thus PRm6a and PRm6b proteins are two acidic glucanases and PRm Ba1 a basic glucanase; PRmBa2 is a basic chitinase found in addition to the four previously characterized acidic chitinases (PRm3, PRm4, PRm5 and PRm7 proteins). The basic 1,3-β-glucanase account for about 15% of the total 1,3-β-glucanase activity and the basic chitinase for about 20% of the total chitinase activity. The cellular distribution of several PR proteins have been investigated, by immunodetection, in the intercellular fluid and in the crude extracts from maize protoplasts. It has been found that the acidic 1,3-β-glucanases and acidic chitinases have an extracellular localization, whereas the corresponding basic enzymes are located inside the cell. PRm2 protein a tobacco PR1b-type protein, is also located in the intercellular spaces of maize leaves.

DNA sequence analysis of a cyclophilin gene from maize: developmental expression and regulation by salicylic acid

In plants, such as maize, cyclophilin (Cyp) genes are expressed at a basal level in all tissues. Amounts of Cyp mRNA above the basic level are observed in germinating seedlings, in growing tissues/organs such as roots and leaf meristematic tissue of young maize plants, nodes and embryonic female inflorescences of adult plants and also in non-proliferating tissues such as the internodes of adult plants. Salicylic acid (SA) enhances the transcription of maize Cyp genes. The possible involvement of SA in the pathway leading to defense responses induced by abiotic stresses such as mercuric chloride treatment is discussed. A maize Cyp genomic clone isolated using a maize Cyp cDNA probe contains 737 by of the 5′ upstream and the entire coding region. This Cyp gene is not interrupted by intervening sequences. In the 5′ upstream region, characteristic transcription signals as well as putative regulatory sequences were identified. Two TATA boxes are found at positions −56 by and −66 by with respect to the transcription start site. Two putative heat shock elements were identified in the promoter region; a metal regulatory element and a third heat shock element were localized in the 5′ untranslated leader. Several putative polyadenylation signals and (G)T-rich sequence motifs were identified in the 3′ untranslated region.

Analysis of acid extractable bean leaf proteins induced by mercuric chloride treatment and alfalfa mosaic virus infection. Partial purification and characterization

Abstract Mercuric chloride treatment of Phaseolus vulgaris (var. ‘Saxa’) leaves, induces the synthesis of four new soluble proteins extractable at pH 2.8. The molecular weights of these proteins were found to be 17 000 for pathogenesis related (PR) 1 and PR 2 proteins, 28 000 for PR 3 protein and 32 000 for PR 4 protein, when determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. In Alfalfa Mosaic Virus (AMV)-infected bean leaves only three new soluble proteins were found, corresponding to the mercuric chloride-induced PR 1, PR 3 and PR 4 proteins. The four mercuric chloride-induced proteins were purified by a technique including an ammonium sulfate fractionation and a preparative polyacrylamide gel electrophoresis. Some biochemical and serological properties of these proteins have been studied.

Stress responses in maize: sequence analysis of cDNAs encoding glycine-rich proteins.

A number of sequences for plant glycine-rich proteins (GRPs) have been obtained by cDNA or genomic cloning. All the proteins encoded by these DNAs are characterized by a high glycine content with repetitive glycine stretches. Their role is not clearly established yet, some of these proteins are thought to be components of the cell wall, but so far the bean GRP 1.8 is the only one for which direct evidence exists for its location in the cell wall [8]. The expression of some of the GRP genes seems to be organ-specific and developmentally regulated [ 11 ]. These genes are also influenced by environmental stimuli, thus wounding induces the petunia [ 1 ] and bean genes [9]. Salicylic acid and virus infection induce the tobacco gene [7]. In maize, abscisic acid (ABA) and desiccation induce a GRP in embryo and leaves respectively [6]. Here we report on the isolation and sequence analysis of two cDNA clones encoding GRPs in chemically treated maize leaves. One GRP shows a complete identity with that induced by ABA, the second is structurally distinct. Maize plants (Zea mays) were treated when 12 days old by spraying a 0.2?o mercuric chloride solution onto the leaves. Polyadenylated RNA was extracted 6 hours after onset of the chemical treatment; details have been described elsewhere [3]. Doubled-stranded cDNA was synthesized with the Pharmacia kit and cloned into the Eco RI site of 2gtl0 cloning vector. About 40 000 recombinant clones (10 ~o of the total library) have been screened using [ 32p ]_labelled cDNAs synthesized by using RNAs isolated from treated and control plants as probes. Among the various positive clones obtained, two have been selected, designated CHEM1 and CHEM2, subcloned into the Bluescript KS( + ) plasmid vector and sequenced. The nucleotide sequence of the cDNAs was determined by the dideoxy method [ 14]. DNA sequence analysis was performed on VAX computer using the GCG package [ 5 ]. Maize genomic DNA was isolated from germinated embryos according to Dellaporta et al. [4]. Total RNA was isolated as previously described [3]. Southern and northern transfers and hybridization were performed according to Sambrook et al. [13] using Hybond N membranes. The cDNA clones CHEM1 and CHEM2 were obtained from a cDNA library prepared from

Differential expression of bean chitinase genes by virus infection, chemical treatment and UV irradiation

Three chitinases have been shown previously to be induced upon various stresses of bean leaves. Time course studies of mRNA accumulation of two of them (P3- and P4-chitinases) have been studied upon virus infection, mercuric chloride treatment and UV irradiation. In alfalfa mosaic virus (A1MV)-infected plants both mRNAs, absent in uninfected bean leaves, become detectable 36 h after inoculation. A maximum level of mRNAs is reached 84 h after inoculation and, whereas the amount of P3-ch mRNA decreases soon after having reached the maximum, the amount of P4-ch mRNA remains at high levels for several days. In mercuric chloride-treated leaves P4-ch mRNA becomes detectable 1–1.5 h after onset of treatment and a maximum level is observed between 6 h and 24 h after treatment; P3-ch mRNA becomes detectable later than P4-ch mRNA in treated leaves and reaches a maximum as late as 18 h after treatment has been applied. UV light also induces the synthesis of both mRNAs but, here again, important differences are observed in the accumulation rate of the two transcripts. The relative amounts of each mRNA induced by the different stresses have been compared. The most effective inducer of P3-ch mRNA is A1MV. In contrast, mercuric chloride induces P4-ch mRNA more efficiently than A1MV or UV light. We have also determined the complete nucleotide sequence of the cDNA encoding P3-chitinase that has been isolated from a cDNA library by using the cucumber lysozyme-chitinase cDNA as a probe. The 1072 bp P3-ch cDNA encodes a mature protein of 268 amino acid residues and the 25 residue NH2-terminal signal peptide of the precursor. Because of its high structural homology to the cucumber and Arabidopsis acidic chitinases as well as to the N-terminal amino acid sequence of the bifunctional lysozyme-chitinase from P. quinquifolia, bean P3-chitinase can be considered to belong to the class III chitinases. Southern blot analysis of bean genomic DNA revealed that P3-chitinase is encoded by a single gene.

Construction of the physical map of the chloroplast DNA of Phaseolus vulgaris and localization of ribosomal and transfer RNA genes

Construction of a physical map of the chloroplast DNA from Phaseolus vulgaris showed that this circular molecule is segmentally organized into four regions. Unlike other chloroplast DNAs which have analogous organization, two single-copy regions that separate two inverted repeats have been demonstrated to exist in both relative orientations, giving rise to two populations of DNA molecules. Hybridization studies using individual rRNA and tRNA species revealed the location of a set of rRNA genes and at least seven tRNA genes in each inverted repeat region, a minimum of 17 tRNA genes in the large single-copy region and one tRNA gene in the small single-copy region. The tRNA genes code for 24 tRNA species corresponding to 16 amino acids. Comparison of this gene map with those of other chloroplast DNAs suggests that DNA sequence rearrangements, involving some tRNA genes, have occurred.