Methyl jasmonate and bean leaf abscission

The effect of rac-methyl jasmonate, both in solution and as a vapour, on the separation of pulvinar and petiolar tissues in explants containing the distal abscission zone of primary leaves of Phaseolus vulgaris var. Contender was investigated. The effects of rac-methyl jasmonate were compared to those of (±)-abscisic acid, -naphthalene acetic acid, ethylene and 2-chloroethylphosphonic acid. Abscission times were determined in explants prepared from 14-day-old control plants and in explants prepared from plants that had been pretreated for 24h with the ethylene-action inhibitor, silver thiosulphate. While silver-pretreatment, or treatment with -naphthalene acetic acid delayed abscission, treatment with ethylene or 2-chloroethylphosphonic acid accelerated tissue separation. However, (±)-abscisic acid delayed abscission under these conditions. In all instances, treatment with rac-methyl jasmonate had no apparent effect on abscission. The loss of chlorophyll from bean leaf discs incubated in the dark was enhanced by treatment with 2-chloroethylphosphonic acid or (±)-abscisic acid and was retarded in discs incubated in benzyl adenine. While incubation in -naphthalene acetic acid was without effect, incubation in solution of rac-methyl jasmonate also retarded chlorophyll loss when compared to water controls.     

The expression of a chimeric Phaseolus vulgaris nodulin 30-GUS gene is restricted to the rhizobially infected cells in transgenic Lotus corniculatus nodules

In Phaseolus vulgaris there is a nodulin family, Npv30, of ca. 30 kDa, as detected in an in vitro translation assay [2]. We isolated a gene (npv30-1) for one of the members of this family. The nucleotide sequence of the promoter of npv30-1 contains nodule-specific motifs common to other late nodulin genes. The promoter was fused to the GUS reporter gene; this chimeric fusion was introduced into Lotus corniculatus via Agrobacterium rhizogenes transformation. GUS activity was only detected in the infected cells of the nodules of transgenic plants. By contrast, the expression of a 35S-GUS construct was restricted to the uninfected cells and the vascular tissue.     

Jasmonates promote abscission in bean petiole expiants: Its relationship to the metabolism of cell wall polysaccharides and cellulase activity

Jasmonic acid (JA) and its methyl ester (JA-Me) promoted the abscission of bean petiole expiants in the dark and light, and the activity of these compounds was almost same. JA and JA-Me did not enhance ethylene production in bean petiole expiants in the light, indicating that the abscission-promoting effects of these compounds are not the result of ethylene. Cells in the petiole adjacent to the abscission zone expanded during abscission but not in the pulvinus, and JA-Me promoted cell expansion in the petiole and the pulvinus. JA-Me had no effect on the total amounts of pectic and hemicellulosic polysaccharides in 2-mm segments of the abscission region, which included 1 mm of pulvinus and 1 mm of petiole from the abscission zone. On the other hand, the total amounts of cellulosic polysaccharides in this region were reduced significantly by the addition of JA-Me in the light. JA-Me had no effect on the neutral sugar composition of hemicellulosic polysaccharides during abscission. The decrease in the endogenous levels of UDP-sugars in the petiole adjacent to the abscission zone was accelerated during abscission by the addition of JA-Me in the light. Cellulase activities of pulvinus and petiole in 10-day-old seedlings were enhanced by the addition of JA. These results suggest that the promoting effect of JA or JA-Me on the abscission of bean petiole explants is due to the change of sugar metabolism in the abscission zone, in which the increase in cellulase activity involves the degradation of cell wall polysaccharides. Jasmonic acid (JA) and its methyl ester (JA-Me) are considered to be putative plant hormones for a number of reasons, including their wide occurrence in the plant kingdom, biologic, activities in multiple aspects at low concentrations, and their interaction with other plant hormones (for reviews see Parthier 1991, Hamberg and Gardner 1992, Sembdner and Parthier 1993, Ueda et al. 1994a). We have already reported that JA and JA-Me and C₁₈-unsaturated fatty acids, which are considered to be the substrates of the biosynthesis of jasmonates, are powerful senescence-promoting substances (Ueda et al. 1982b, 1991a). Senescence symptoms induced by these compounds are identical to those of natural senescence. Recently we have also found that JA inhibited indole-3-acetic acid (IAA)-induced elongation of oat (Avena sativa L. cv. Victory) coleoptile segments by inhibiting the synthesis of cell wall polysaccharides (Ueda et al. 1994b, 1995). These facts led us to study the mode of actions of JA and JA-Me on promoting abscission, which is considered the last dramatic phenomenon of senescence. In this paper we report that JA and JA-Me promote abscission in bean (Phaseolus vulgaris L. cv. Masterpiece) petiole expiants and that the changes in the metabolism of cell wall polysaccharides in the petiole and the pulvinus adjacent to the abscission zone are involved in the promotive effects of these compounds.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - DCB 2,6-dichlorobenzonitrile - HPLC high performance liquid chromatography - IAA indole-3-acetic acid - JA jasmonic acid - JA-Me methyl jasmonate - MES 2-(N-morpholino)ethane-sulfonic acid, monohydrate - TCA trichloroacetic acid - Tris 2-amino-2-hydroxymethy-1,3-propanediole     

Polygalacturonase expression during leaf abscission of normal and transgenic tomato plants

Polygalacturonase (PG, EC 3.2.1.15), an enzyme commonly found in ripening fruit, has also been shown to be associated with abscission. A zone-specific rise in PG activity accompanies the abscission of both leaves and flowers of tomato (Lycopersicon esculentum Mill.) plants. Studies of transgenic plants expressing an antisense RNA for fruit PG indicate that although the enzyme activity in transgenic fruit is < 1 % of that in untransformed fruit, the PG activity in the leaf abscission zone increases during separation to a similar value to that in untransformed plants. The timing and rate of leaf abscission in transgenic plants are unaffected by the introduction of the antisense gene. A polyclonal antibody raised against tomato fruit PG does not recognise the leaf abscission protein. Furthermore a complementary DNA (cDNA) clone (pTOM6), which has been demonstrated to code for fruit PG, does not hybridise to mRNA isolated from the abscission-zone region of tomato leaves. These results indicate that the PG protein in abscission zones of tomato is different from that in the fruit, and that the gene coding for this protein may also be different.Abbreviation PG polygalacturonase The authors of this paper are grateful to David Jackson of the John Innes Institute, Norwich, UK for his assistance with the in-situ hybridisation work. This research was supported by an Agricultural and Food Research Council Post-Doctoral award to J.E.T., and by a grant to D.G. from the Science and Engineering Research Council Biotechnology Directorate in association with ICI seeds. The work was carried out under Ministry of Agriculture, Food and Fisheries licences.     

Ethylene-promoted tomato flower abscission and the possible involvement of an inhibitor

The abscission zone in tomato (Lycopersicon esculentum (L.) Mill. flower pedicels is morphologically distinguishable prior to separation and is delineated by an indentation of the epidermis. Exposure of excised pedicels with the flower attached to ethylene results in abscission within 12 h and this can be accelerated by flower removal. Abscission of excised pedicels with the flower removed takes place in the absence of exogenous ethylene but this is delayed by pretreatment with aminoethoxyvinyl glycine, an inhibitor of ethylene biosynthesis. The data presented support the hypothesis that flower tissue is the source of an abscission inhibitor.Abbreviations AVG aminoethoxyvinyl glycine - IAA indole-3-acetic acid     

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 -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.     

Expression of a cellulase gene, celA, from the rumen fungus Neocallimastix patriciarum in Streptococcus bovis by means of promoter fusions

The aerotolerant rumen bacterium, Streptococcus bovis, has been used as a host for expression of genes of eukaryotic origin. The coding regions of the celA cellulase gene from the rumen fungus, Neocallimastix patriciarum, were fused with bacterial promoter/signal peptide regions from the Ruminococcus flavefaciens xynD and S. bovis -(1,3-1,4)-glucanase genes. Fusion cassettes were built into shuttle vector constructs based on pIL253 or pTRW10 and constructs carrying celA were transformed into S. bovis JB1. Active N. patriciarum cellulase was produced in S. bovis with either promoter, although better expression levels were obtained with the native S. bovis -glucanase promoter fragment.     

The use of immunological methods to study the activity of cellulase isozymes (B 1:4 glucan 4-glucan hydrolase) in bean leaf abscission

Abstract The changes in the levels of two different isozymes of cellulase (EC 3.2.1.4) have been followed during the abscission of the primary leaves of bean (Phaseolus vulgaris c.v. Red Kidney), using antibodies raised against the 9.5 form of the enzyme. Data from both radioimmune and direct assay show that the 9.5 form of cellulase is undetectable prior to the induction of abscission. After a 12 h lag this isozyme increases in activity, the increase preceding a decrease in integrity of the abscission zone cell walls. The results are consistent not only with the view that this specific isozyme is involved in wall hydrolysis but also with previous data which showed that cellulase is synthesized ‘de novo’. The 4.5 isozyme of cellulase is more widely spread throughout the plant, being most active in young tissues. During abscission the activity of this isozyme in the abscission layer falls and consequently it is not thought to be involved directly in the abscission process.     

Ozone- and ethylene-induced regulation of a grapevine resveratrol synthase promoter in transgenic tobacco

Stilbene synthases (STSs) are enzymes that play a critical role in the biosynthesis of stilbene, phytoalexins in a small number of unrelated plant species, and are induced by various biotic and abiotic stressors like pathogen attack, UV-irradiation or ozone exposure. To investigate the molecular basis for ozone-induced plant stress responses, we have examined the promoter of the grapevine resveratrol synthase (Vst1). In this report we summarize the influence of ozone on gene regulation. In transgenic tobacco a chimeric gene construct, containing the Vst1 promoter combined with the β-glucuronidase (GUS) reporter gene, is rapidly induced by ozone (0.1 μl·l⁻¹, 12 h). The same construct is also strongly induced by ethylene (20 μl·l⁻¹, 12 h). Promoter deletion analysis of the 5′ flanking sequence identified a positive regulatory element between −430 bp and −280 bp. This region contains ethylene-responsive enhancer elements, as well as an elicitor-responsive sequence in inverse orientation.     

Effects of ethylene and inhibitors of ethylene synthesis and action on nodulation in common bean (Phaseolus vulgaris L.)

The ethylene releasing compound, 2-chloroethylphosphonic acid (ethephon) inhibited nodule development in common bean (Phaseolus vulgaris L.) plants. In contrast, inhibitors of ethylene synthesis or its physiological activity enhanced nodulation. In a co-culture of bean seeds and rhizobia, ethephon inhibited rhizobial growth while inhibitors of ethylene synthesis or action did not influence the growth and proliferation of rhizobia. These data emphasize the role of ethylene as a regulator of nodulation in determinate nodulators and indicate that the ethylene signaling pathway involved in the nodulation process is not limited to the plant host but also involves the bacterial symbiont.     

The sequence of a pea vicilin gene and its expression in transgenic tobacco plants

A 5.5 kb Eco RI fragment containing a vicilin gene was selected from a Pisum sativum genomic library, and the protein-coding region and adjacent 5 and 3 regions were sequenced. A DNA construction comprising this 5.5 kb fragment together with a gene for neomycin phosphotransferase II was stably introduced into tobacco using an Agrobacterium tumefaciens binary vector, and the fidelity of expression of the pea vicilin gene in its new host was studied. The seeds of eight transgenic tobacco plants showed a sixteen-fold range in the level of accumulated pea vicilin. The level of accumulation of vicilin protein and mRNA correlated with the number of integrated copies of the vicilin gene. Pea vicilin was confined to the seeds of transgenic tobacco. Using immunogold labelling, vicilin was detected in protein bodies of eight out of ten embryos (axes plus cotyledons) and, at a much lower level, in two out of eleven endosperms. Pea vicilin was synthesized early in tobacco seed development; some molecules were cleaved as is the case in pea seeds, yielding a major parental component of M _r50000 together with a range of smaller polypeptides.     

Utilizing the potential for increased nitrogen fixation in common bean

Many variables affect the amount of N₂ fixation that occurs in field-grown common beans (Phaseolus vulgaris L.). When environmental conditions are optimized, genetically superior plants that are nodulated with efficient rhizobia are able to fix enough N₂ to support grain yields of up to 2000 kg ha^–1. All of the required components are available for common bean to fulfill its potential as a nitrogen-fixing food crop. However, these components must be assembled and presented to growers in a usable package that is economically attractive.     

Cloning and expression analysis of SKn-type dehydrin gene from bean in response to heavy metals

A heavy metal responsive gene PvSR3 (GenBank accession number U54703) encoding an acid dehydrin was isolated from a mercuric chloride-treated bean (Phaseolus vulgaris L.) leaf cDNA library by differential screening using cDNAs derived from treated and untreated plants. The PvSR3 cDNA is 981-bp long and has a 606-bp open-reading frame with a 202-residue-deduced amino acid sequence. The PvSR3 sequence contains two conserved repeats of the characteristic lysine-rich K segment (EKKGIMDKIKEKLPG) preceded by an 8-serine residue stretch, whereas the Y segment (DEYGNP) conserved motif is absent. The deduced protein has a calculated molecular weight of 23 kDa and an isoelectric point of 5.2. Sequence similarity and comparative analysis showed that PvSR3 shares 70 and 73% similarity with the dehydrin of poplar and pepper, respectively. Southern hybridizations indicated that PvSR3 was a low copy-number gene. Northern blot analysis revealed that PvSR3 mRNA was weakly detected in seedling leaves. However, the gene expression was strongly stimulated by heavy metals, such as mercury, cadmium, arsenic, and coppper, whereas virus infection and salt had little effect on it. In contrast, PvSR3 was not responsive to drought or abscisic acid (ABA), and was downregulated by UV radiation. Furthermore, PvSR3 was upregulated by the exogenous signaling molecules, including salicylic acid (SA) and hydrogen peroxide (H₂O₂). It is suggested that PvSR3 is extremely related to heavy metal stress, and might play an important role in metal detoxification and resistance to the damage caused by heavy metals.     

Inheritance of foreign genes in transgenic bean (Phaseolus vulgaris L.) co-transformed via particle bombardment

Exploiting the biolistic process we have generated stable transgenic bean (Phaseolus vulgaris L.) plants with unlinked and linked foreign genes. Co-transformation was conducted using plasmid constructions containing a fusion of the gus and neo genes, which were co-introduced with the methionine-rich 2S albumin gene isolated from the Brazil nut and the antisense sequence of AC1, AC2, AC3 and BC1 genes from the bean golden mosaic geminivirus. The results revealed a co-transformation frequency ranging from 40% to 50% when using unlinked genes and 100% for linked genes. The introduced foreign genes were inherited in a Mendelian fashion in most of the transgenic bean lines. PCR and Southern blot hybridization confirmed the integration of the foreign genes in the plant genome.     

Meristem-specific gene expression directed by the promoter of the S-phase-specific gene,cyc07, in transgenic Arabidopsis

A genomic clone for the cyc07 gene, which is expressed specifically at the S phase during the cell cycle in synchronous cultures of periwinkle (Catharanthus roseus) cells, was isolated. Determination of the nucleotide sequence of the clone revealed that the cyc07 gene consists of seven exons separated by six introns. Genomic Southern analysis indicated that the cyc07 gene is present as a single copy per haploid genome in periwinkle. Expression of related genes was detected in a wide range of other plants. Transgenic Arabidopsis plants were generated that expressed the gene for -glucuronidase (GUS) under the control of the promoter of the cyc07 gene. The tissue-specific pattern of expression directed by the promoter was investigated by analysis of GUS activity. Histochemical tests demonstrated that 589 bp of the 5-upstream sequence of the cyc07 gene could direct specifical expression of the GUS reporter gene in meristematic tissues in transgenic plants. The spatial pattern of expression directed by the promoter was closely correlated with meristematic activity and cell proliferation, suggesting an association between the function of the cyc07 gene and cell proliferation.