Endogenous plant hormones of the broad bean,Vicia faba L. (-)-jasmonic acid,a plant growth inhibitor in pericarp

(-)-Jasmonic acid was identified as a plant growth inhibitor of the pericarp of Vicia faba by means of gas-liquid chromatography, high resolution mass spectrometry, ¹H-nuclear magnetic resonance (¹H-NMR), and ¹³C-NMR. Additionally, the pericarp contains very small amounts of abscisic acid (ABA) and 4-dihydrophaseic acid. The highest level of jasmonic acid was reached prior to full pericarp length. This amount (3 g g^-1 fresh weight) is similar to the maximal ABA content in the developing seed. Jasmonic acid is a plant growth inhibitor possessing a relative activity in the wheat seedling bioassay of 1–2.5%, compared to ABA. Contrary to ABA, jasmonic acid does not cause retardation of leaf emergence. The possible physiological role of jasmonic acid in the pericarp is discussed and compared with the assumed function of ABA in developing seeds.Abbreviations ABA abscisic acid - DPA 4-dihydrophaseic acid - DPAMeTMS methyl ester trimethylsilyl ether of DPA - EtOAc ethyl acetate - Et₂O ether - MS mass spectrometry - NMR nuclear magnetic resonance - GLC gas-liquid chromatography - TLC thin-layer chromatography - UV ultraviolet light     

Stimulation of adventitious root formation in non-woody stem cuttings by uridine

Uridine strongly stimulated adventitious root formation in stem cuttings of sunflower (Helianthus annuus L.), mung bean (Vigna radiata L.) and common bean (Phaseolus vulgaris L.). A dose response curve of uridine induced rooting showed that the optimum concentration of uridine was 0.1 µM. At all concentrations employed, uridine had no significant effect on root elongation. The rooting response of stem cuttings to the optimal concentration of indole-3-butyric acid (10 µM) in combination with 0.1 µM uridine did not significantly differ from their response to either of these compounds when applied alone. However, the rooting response of the cuttings to sub-optimal IBA (0.01 µM) was significantly stimulated by uridine. These findings suggested that uridine may have stimulated rooting by increasing the sensitivity of the rooting tissue to auxin.     

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.     

Promotion of adventitious root formation by 4-chlororesorcinol: A polyphenol oxidase inhibitor

The extent of rooting in cuttings of Phaseolus vulgaris L., and Vigna radiata Wilcz. was affected by 4-chlororesorcinol, a polyphenol oxidase inhibitor. More root primordia and more roots were formed after 4-chlororesorcinol treatment both with and without 10^-5M Indole butyric acid. Promotion of rooting was observed also in cuttings of Elaeagnus pungens, Gypsophilia elegans and Kalanchoe blossfeldiana. The enhancement in bean and mung bean was accompanied by a concomitant wider spatial distribution of the primordia and the resulting adventitious roots. The formation of primordia in the treated cuttings was delayed by 12–24 hours, compared to untreated cuttings. The treatment was effective only when given during the first hours after the preparation of the cutting of bean and mung bean, suggesting involvement in the initiation stage. Hypocotyl extracts of mung bean cuttings, pretreated with 4-chlororesorcinol, exhibited reduced polyphenol oxidase activity. The inhibition was not reversed by washing of the treated extract in 50% acetone or by an overnight dialysis, suggesting tight or maybe even irreversible binding of the inhibitor to the enzyme.Abbreviations 4-CR 4-chlororesorcinol - IBA Indole butyric acid - PPO polyphenol oxidase     

Isolation, Purification, and Characterization of an Endogenous Root-promoting Factor Obtained from Basal Sections of Pear Hardwood Cuttings

Basal segments taken from Old Home and Bartlett pear hardwood cuttings collected at intervals during the rooting period in September were extracted with ethanol and fractionated by paper chromatography in different solvent systems. Different zones on the chromatograms were bioassayed by the mung bean rooting test, which showed high levels of promotion in Old Home basal extracts when the cuttings were obtained during the period of maximum rooting. Extracts from Bartlett cuttings, however, showed considerably less promotion activity in the bioassay but did show high levels of inhibitory activity.

After the easily-rooted Old Home cuttings had been in the rooting medium for 10 days, a highly active endogenous root-promoting material was found in extracts from basal segments of cuttings having buds and which had been treated with indolebutyric acid. Similar extracts obtained from disbudded cuttings, or from cuttings with buds but not treated with indolebutyric acid, lacked this rooting-factor. Extracts obtained from all types of the difficult-to-root Bartlett cuttings also lacked this rooting-factor. The latter is believed to be produced by physiologically active Old Home buds, and is very effective in the mung bean bioassay, even at extremely low concentrations.

From paper chromatographic studies, tests with spray reagents, solubility determinations, biological tests, UV spectrum analysis, and infrared spectroscopy, it is believed that this rooting factor could be a condensation product between exogenous auxin (indolebutyric acid) and a phenolic compound produced by physiologically active Old Home pear buds.

     

Bruchid resistance of transgenic azuki bean expressing seed α-amylase inhibitor of common bean

Various species of bruchid beetles including Callosobruchus chinensis, C. maculatus and C. analis cause postharvest damage of azuki bean seeds, an important East Asian grain legume. The -amylase in the midguts of these insects is inhibited by the -amylase inhibitor (AI) present in common bean seeds. Transformation of azuki bean with the AI gene driven by the promoter of phytohemagglutinin results in high levels of AI in the seeds and the complete block of bruchid development on the seeds. Zabrotes subfasciatus, a South and Central American bruchid that is a storage pest of common bean, develops normally on the transgenic azuki bean.     

Adventitious rooting: examining the role of auxin in an easy- and a difficult-to-root plant

Therooting responses of cuttings of difficult-to-root lilac (Syringavulgaris) and easy-to-root forsythia(Forsythia×intermedia)were compared. The rooting ability of lilac cuttings declined over the growingseason (May–June). There was also a decline in the initial concentrationof free IAA at the base of the cuttings, but there was not a tight relationshipbetween basal IAA concentration and rooting ability. Polar auxin transportability was measured in lilac and forsythia during the period of maximum growthby [³H]IAA application to stem internodal tissue. Transport abilitydeclined in lilac over this time period, particularly in terms of transportintensity and percentage of [³H]IAA transported. In contrast thechanges in polar auxin transport ability in forsythia were less marked. Thisdifference between species was maintained in winter hardwood cuttings, withforsythia tissue showing greater polar auxin transport ability than lilac. Theimportance of polar auxin transport for adventitious rooting was demonstratedinboth lilac and forsythia softwood cuttings by use of the polar transportinhibitor 2,3,5-triiodobenzoic acid (TIBA). Overall the results indicate thatdifferences in polar auxin transport ability between lilac and forsythiacontribute to differences in rooting ability.     

Enhancement of adventitious root formation in mung bean cuttings by 3,5-dihalo-4-hydroxybenzoic acids and 2,4-dinitrophenol

3,5-Dihalo-4-hydroxybenzoic acids enhanced adventitious root formation in mung bean (Vigna radiata L.) cuttings. 3,5-Diiodo-4-hydroxybenzoic acid was more active than 3,5-dichloro-4-hydroxybenzoic acid, increasing the number of roots formed by about 4-fold. 2,4-Dinitrophenol also enhanced significantly adventitious root formation in mung bean cuttings. The phenolic compounds were active with or without indole-3-acetic acid. The possible mechanism by which these phenolic compounds enhance rooting is discussed.Abbreviations CCCP carbonyl cyanide 3-chlorophenylhydrazone - DIHB 3,5-diiodo-4-hydroxybenzoic acid - DNP 2,4-dinitrophenol     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.): gene integration,expression and inheritance

A reproducible method of Agrobacterium-mediated transformation was developed for Cicer arietinum (chickpea). Initial explants consisted of longitudinal slices from embryonic axes of imbibed, mature seed. The plasmid contained a bi-functional fusion gene conferring both -glucuronidase and neomycin phosphotransferase activities, under the control of a 35S35SAMV promoter. Using a series of tissue culture media for co-cultivation, shoot initiation and rooting, we recovered transgenic plants from approximately 1.3% of the sliced embryo axes. The addition of a shoot elongation medium to the protocol improved the success rate to 3.1% but increased the time in tissue culture. Inheritance of the gus gene was followed through four generations, both through expression and Southern hybridization assays, and showed the expected Mendelian inheritance pattern.NRCC Grant No. 46589.     

Identification and quantitation of adenosine-3′:5′-cyclic monophosphate in plants using gas chromatography-mass spectrometry and high-performance liquid chromatography

Direct evidence has been obtained for the presence of adenosine-3:5-cyclic monophosphate (cAMP) in tobacco (Nicotiana tabacum L.) callus tissue cultures, bean (Phaseolus vulgaris L.) seedlings and immature kernels of sweet corn (Zea mays L.) through the use of a highly specific and sensitive gas chromatography-mass spectrometric assay. Levels of endogenous cAMP ranged from 70 to 126 pmol/g fresh weight. Corresponding levels of cAMP determined for the same samples using radioimmunoassay were consistently three to four times higher. Contrary to previous reports for citrus plants, measurable levels of cAMP could not be detected in young lemon leaves within the limits of detection of the mass-spectrometric assay method. In the case of tobacco callus tissue, the coumarin glucoside, scopolin, which was present in large amounts and showed similar chromatographic behaviour to cAMP, interferred strongly with the mass-spectrometric measurements of cAMP in inadequately purified extracts. The use of high-performance liquid chromatography, in addition to standard chromatographic purification methods, produced highly purified plant extracts for quantitation of cAMP and also provided a method for the separation of cAMP from its 2:3-isomer.Abbreviations cAMP adenosine-3:5-cyclic monophosphate - 2:3-cAMP adenosine-2:3-cyclic monophosphate - GC-MS-MID combined gas chromatography-mass spectrometry with selected multiple-ion-detection - HPLC high-performance liquid chromatography - RIA radioimmunoassay - TLC thin-layer chromatography     

Activity of antioxidant enzymes in response to cadmium in Crotalaria juncea

The aromatic amine, -phenethylamine, was identified in various field-grown leguminous plants by analyses with HPLC, GC, GC-MS and ¹H-NMR. High concentration of -phenethylamine was generally detected only in mature root nodules, but not in other plant organs such as root, stem, leaf, pod and grain. Occurrence was specific to the root nodules formed by Bradyrhizobium infection. Ten of eleven legume crops including soybean [Glycine max (L.) Merr.], pigeon pea [Cajanus cajan (L.) Millsp.], adzuki bean (Vigna angularis), mung bean [V. radiata (L.) Wilczek] and cowpea (V. unguiculata) contained this aromatic amine, but groundnut (Arachis hypogaea L.) also nodulated by Bradyrhizobium sp. did not. Root nodules collected from garden pea (Pisum sativum L.), broad bean (Vicia fava L.), kidney bean (Phaseolus vulgaris L.) and various other herbaceous legumes nodulated by Rhizobium sp., Mesorhizobium sp., Sinorhizobium sp. or Azorhizobium caulinodans, and root-nodulated, woody non-legumes, nodulated by Frankia spp., contained little -phenethylamine.The amount of -phenethylamine in Bradyrhizobium-infected nodules varied with the legume species and their cultivars, and most significantly, with nodule age. In field-grown soybean plants, nodule -phenethylamine attained maximum concentration at the flowering stage and far exceeded that of the major polyamines of soybean nodules, putrescine and spermidine.     

Isolation and characterization of a cDNA from Cuphea lanceolata encoding a beta-ketoacyl-ACP reductase.

Summary A cDNA encoding -ketoacyl-ACP reductase (EC 1.1.1.100), an integral part of the fatty acid synthase type II, was cloned fromCuphea lanceolata. This cDNA of 1276 by codes for a polypeptide of 320 amino acids with 63 N-terminal residues presumably representing a transit peptide and 257 residues corresponding to the mature protein of 27 kDa. The encoded protein shows strong homology with the amino-terminal sequence and two tryptic peptides from avocado mesocarp -ketoacyl-ACP reductase, and its total amino acid composition is highly similar to those of the -ketoacyl-ACP reductases of avocado and spinach. Amino acid sequence homologies to polyketide synthase, -ketoreductases and short-chain alcohol dehydrogenases are discussed. An engineered fusion protein lacking most of the transit peptide, which was produced inEscherichia coli, was isolated and proved to possess -ketoacyl-ACP reductase activity. Hybridization studies revealed that inC. lanceolata -ketoacyl-ACP reductase is encoded by a small family of at least two genes and that members of this family are expressed in roots, leaves, flowers and seeds.     

Evolutionary relationships among proteins in the phytohemagglutinin-arcelin-α-amylase inhibitor family of the common bean and its relatives

The common bean, Phaseolus vulgaris, contains a family of defense proteins that comprises phytohemagglutinin (PHA), arcelin, and -amylase inhibitor (AI). Here we report eight new derived amino acid sequences of genes in this family obtained with either the polymerase chain reaction using genomic DNA, or by screening cDNA libraries made with RNA from developing beans. These new sequences are: two AI sequences and arcelin-4 obtained from a wild accession of P. vulgaris that is resistant to the Mexican bean weevil (Zabrotes subfasciatus) and the bean weevil (Acanthoscelides obtectus); an AI sequence from the related species P. acutifolius (tepary bean); a PHA and an arcelin-like sequence from P. acutifolius; an AI-like sequence from P. maculatus; and a PHA sequence from an arcelin-5 type P. vulgaris. A dendrogram of 16 sequences shows that they fall into the three identified groups: phytohemagglutinins, arcelins and AIs. A comparison of these derived amino acid sequences indicates that one of the four amino acid residues that is conserved in all legume lectins and is required for carbohydrate binding is absent from all the arcelins; two of the four conserved residues needed for carbohydrate binding are missing from all the AIs. Proteolytic processing at an Asn-Ser site is required for the activation of AI, and this site is present in all AI-like sequences; this processing site is also found at the same position in certain arcelins, which are not proteolytically processed. The presence of this site is therefore not sufficient for processing to occur.     

Enhancement of production of cloned α-amylase by lactic acid feeding from recombinant Saccharomyces cerevisiae using a SUC2 promoter

-Amylase production was higher (13 units ml^–1) when a recombinant Saccharomyces cerevisiae containing a SUC2 promoter was grown with 10 g lactic acid l^–1 than without addition (8 units ml^–1). With continuous lactic acid feeding in the inducing phase, -amylase increased to 79 units ml^–1 in a 1-l jar fermenter.     

The Weak Cytokinins N,N′-bis-(1-naphthyl)urea and N,N′-bis-(2-naphthyl)urea may Enhance Rooting in Apple and Mung Bean

The present research investigates the possibility that 2 weak urea-type cytokinins, the N,N′-bis-(1-naphthyl)urea and the N,N′-bis-(2-naphthyl)urea, enhance adventitious root formation. The rooting activity was assessed using the stem slice test, the mung bean rooting test and the rooting of apple microcuttings. The two compounds influenced the adventitious rooting process differently as regards the bioassay used. In the stem slice test, in the presence of exogenous auxin, both compounds enhanced the rooted slice percentage. In mung bean shoots, the N,N′-bis-(1-naphthyl)urea enhanced the root formation at the lowest concentration used (0.01 μM) while the N,N′-bis-(2-naphthyl)urea enhanced rooting at higher concentrations. In the rooting test of apple microcuttings the N,N′-bis-(1-naphthyl)urea and the N,N′-bis-(2-naphthyl)urea slightly enhanced only the mean root number per microcutting.     

The effect of paclobutrazol on in vitro rooting,transplant establishment and growth of fruit plants

The effect of paclobutrazol on in vitro rooting and growth of sour cherry (Prunus cerasus) rootstock CAB 11E clone, of S 749 × S 1490 (Prunus persica × Prunus kansuensis) hybrid rootstock, and of pear (Pyrus communis), cv. Abbé Fetel is reported.PP333 increased rooting of S 749 × S 1490 and of Abbé Fetel, particularly at a concentration of 0.5 mg/l (a.i.); moreover, it induced a rooting percentage as high as auxin in the former and hastened rooting of the latter. By contrast, paclobutrazol did not affect root production of 11 E.PP333-treated plants had shorter and thicker roots than controls but similar survival rates during acclimatization. Otherwise they grew less than controls during the first part of the acclimatization phase.Abbreviations used in text and tables BA = 6-benzyladenine - IBA = indole-3-butyric acid - PP333 = paclobutrazol = (2RS,3RS)-1-(-4-chlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl)pentan-3-ol Part of the results referring to S 749 × S 1490 (P. persica × P. kansuensis) rootstock were presented at the meeting on Controllo della fruttificazione delle piante da frutto, Bologna, Italy, June 1986, and were published in the Riv. Ortoflorofrutt. It. 70 (6)(1986). This research was funded in part by the Italian Ministry of Education (M.P.I. 60%).     

铁核桃根与叶水浸提液对果园间作绿肥植物的化感作用及其生理机制

以西南地区具有代表性的16种绿肥植物为受体材料,采用培养皿药膜法研究了铁核桃(Juglans sigillata)根水浸提液对受体种子发芽率及幼苗鲜重、干重的化感效应;并进一步研究了铁核桃根、叶水浸提液和胡桃醌对化感效应存在明显差异的4种绿肥植物(绿豆、红三叶、白三叶、花生)种子萌发与幼苗生长以及抗氧化酶特性的影响,以筛选适宜中国西南地区核桃园种植的绿肥植物,探讨核桃根和凋落物对绿肥作物的化感作用机制。结果表明:(1)铁核桃根水浸提液对绿豆的发芽率没有影响,但对绿豆幼苗鲜重和干重有显著抑制作用,而对其他15种绿肥的发芽率和鲜重、干重均有抑制作用。(2)胡桃醌显著抑制绿豆种子萌发,而铁核桃根或叶水浸提液对绿豆种子萌发没有影响。(3)铁核桃根或叶水浸提液以及胡桃醌对绿肥植物幼苗生长的化感效应趋势一致,但核桃根或叶水浸提液的化感效应强于胡桃醌。(4)绿豆幼苗在铁核桃根或叶水浸提液以及胡桃醌处理下,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)的活性均高于其他3种(红三叶、白三叶、花生)受体幼苗,表明绿豆清除活性氧能力高,细胞受损害程度较低,受化感作用影响最弱。研究认为,绿豆为适宜中国西南地区幼龄核桃园种植的间作绿肥植物。     

Transgenic chickpea seeds expressing high levels of a bean α-amylase inhibitor

We describe a robust and reproducible Agrobacterium-mediated chickpea transformation method based on kanamycin selection, and its use to introduce the bean AI1 gene into a desi type of chickpea. Bean AI1 was specifically expressed in the seeds, accumulated up to 4.2% of seed protein and was processed to low molecular weight polypeptides as occurs in bean seeds. The transgenic protein was active as an inhibitor of porcine -amylase in vitro. Transgenic chickpeas containing -AI1 strongly inhibited the development of Callosobruchus maculatus and C. chinensis (Col. : Bruchidae) in insect bioassays.     

Sea anemone collagen: Further evidence for the existence of only oneα-chain type

Summary Collagen from an inferior invertebrate, namely from the sea anemoneActinia equina L., appears to consist of three identical subunits (-chains). New evidence for this situation is reported on the basis of preparative-scale experiments.No fractionation of sea anemone collagen was obtained by CM-cellulose chromatography which is the method of choice for the isolation of vertebrate collagen subunits. On the other hand, the-component (mol. wt., 95.000) could be isolated by gel chromatography. It was homogenous when investigated by polyacrylamide gel electrophoresis (in the presence of sodium dodecyl sulphate) and by ion exchange chromatography on CM-cellulose. The amino acid composition of the-component was identical with that of unfractionated collagen.Divergent evolution of the collagen molecule appears likely since vertebrate collagen molecules are made up of different-chain types.