Basic peroxidases in isolated vacuoles of nicotiana tabacum L.

Vacuoles of tobacco mesophyll and of suspension-cultured cells were isolated in order to study the localization of peroxidase isoenzymes. Only basic peroxidases were detectable by electrophoretic separation of the vacuolar sap. Some of the basic peroxidases have formerly been described as an ionically bound cell-wall fraction. This fraction, however, was found to be an artifact produced by incomplete cell breakage. Reinvestigation of isolated cell walls confirmed that mainly acidic peroxidases are localized in the cell walls where they move freely or are bound. As a consequence of former and present results we think it probable that all of the peroxidase isoenzymes are secretory proteins because they have to be transported from the sites of synthesis in the cytoplasm to the sites of function, the extracytoplasmic spaces, cell wall (acidic peroxidases), and vacuole (basic peroxidases).Abbreviation ER endoplasmic reticulum - PAGE polyacrylamide gel electrophoresis     

Lateral diffusion of polarly transported indoleacetic acid and its role in the growth of Lupinus albus L. hypocotyls

The transport and metabolism of indole-3-acetic acid (IAA) was studied in etiolated lupin (Lupinus albus L, cv. Multolupa) hypocotyls, following application of dual-isotope-labelled indole-3-acetic acid, [5-³H]IAA plus [1-¹⁴C]IAA, to decapitated plants. To study the radial distribution of the transported and metabolized IAA, experiments were carried out with plants in which the stele was separated from the cortex by a glass capillary. After local application of labelled IAA to the cortex, radioactivity remained immobilized in the cortex, near the application point, showing that polar transport cannot occur in the outer tissues. However, following application of IAA to the stele, radioactivity appeared in the cortex in those hypocotyl sections below the first 1 cm (in which the capillary was inserted), and the basipetal IAA movement was similar to that observed after application of IAA to the complete cut surface. In both assays, longitudinal distribution of ¹⁴C and ³H in the stele outside the first 1 cm was positively correlated with that of cortex, indicating that there was a lateral migration of IAA from the transport pathway (in the stele) to the outer tissues and that this migration depended on the amount of IAA in the stele. Both tissues (stele and cortex) exhibited intensive IAA metabolism, decarboxylation being higher in the stele than in the cortex while IAA conjugation was the opposite. Decapitation of the seedlings caused a drastic reduction of hypocotyl growth in the 24 h following decapitation, unless the hypocotyls were treated apically with IAA. Thus, exogenous IAA, polarly transported, was able to substitute the endogenous source of auxin (cotyledons plus meristem) to permit hypocotyl growth. It is proposed that IAA escapes from the transporting cells (in the stele) to the outer tissues in order to reach the growth-responsive cells. The IAA metabolism in the outer tissues could generate the IAA gradient necessary for the maintenance of its lateral flow, and consequently the auxin-induced cell elongation.     

Characterisation of a developmentally related polypeptide with glutelin solubility characteristics from Lupinus albus L.

Proteins from Lupinus albus L. cv. Rio Maior seeds were fractionated according to solubility criteria. Patterns of concanavalin A (ConA)-binding polypeptides from the different classes, albumins, globulins, glutelins and prolamins, were established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands of apparent molecular masses of 29 and 23.5 kDa with glutelin solubility characteristics bound the lectin. The 23.5-kDa band was separated by two-dimensional electrophoresis into two components: one glycosylated and heterogeneous with an isoelectric point of approx. 10 (designated as G23) and another, not detected with ConA, precipitating in the first dimension. The amino acid and hexosamine analysis of G23 showed that it is particularly rich in Gly (11.2%), Glx (10.0%), Ser (9.0%), Leu (8.2%), Asx (7.5%), and Pro (6.7%) and that it has a considerable content of the sulphur-containing amino acids Met (2.0%) and Cys (5.8%) and contains glucosamine. The determined N-terminal amino acid sequence of G23 was: ¹KG(R)V⁵KGTGD¹⁰(T)PXXV¹⁵XLY(N)R²⁰T, and this had no significant similarity to any of the amino acid sequences contained in the data bank SWISS-PROT 26. The glycoprotein G23 was completely deglycosylated with peptide-N-glycosidase F, yielding a homogeneous 21-kDa polypeptide composed of approximately 191 amino acids. The structures of the major N-linked neutral oligosaccharides of G23, determined by exoglycosidase sequencing, were as follows: Man2Man6(Man3) Man6(Man2Man2Man3)Man4GlcNAc4GlcNAc (13%); ± Man2Man6(Man3)Man6(± Man2 Man2 Man3)Man4GlcNAc4GlcNAc (29%); Man6(Man3) Man6(Man2Man3)Man4GlcNAc4GlcNAc (13%); Man6(Man3)Man6(Man3)Man4GlcNAc4GlcNAc (16%); Man6(Man3)(Xyl2)Man4GlcNAc 4GlcNAc (28%). Changes in G23 abundance during seed development, germination and seedling growth were monitored with a specific antibody. The glycoprotein G23 started to accumulate appreciably during seed formation between the 40th and the 50th days after anthesis and was detected following seed imbibition, until the 9th day in cotyledons, the 2nd day in roots and the 4th day in hypocotyls and leaves.Abbreviations ConA concanavalin A - Endo H endo-N-acetyl--d-glucosaminidase H - GlcNAc N-acetylglucosamine - gu glucose unit - IEF isoelectric focusing - Man mannose - NEPHGE non-equilibrium pH gradient electrophoresis - PNGase F peptide-N-glycosidase F - PVDF polyvinylidenedifluoride - Xyl xylose We thank Geoffrey Guile (Oxford Glycobiology Institute, Oxford, UK) for help with HPLC separations and amino acid and hexosamine analysis, Terry Butters (Oxford Glycobiology Institute) for providing the exoglycosidases and advice in their use, Manuela Regala (Instituto de Tecnologia Química e Biológica Oeiras, Portugal) and Paula Veríssimo (University of Coimbra, Portugal) for determining the N-terminal amino acid sequence of G20 and G23 and Dr. Jorge Lampreia (Universidade Nova de Lisboa, Lisbon, Portugal) for the computerised search of the SWISS-PROT data bank. Lupinus albus seeds were provided by Dr. João Neves Martins (Instituto Superior de Agronomia Lisbon, Portugal). We also thank J. Romão (Instituto Gulbenkian de Ciência, Oeiras, Portugal) for technical assistance in antibody production. This work was supported by Junta National de Investigação Científica e Tecnológica, Portugal.     

De-novo synthesis and release of peroxidases in cell suspension cultures of Nicotiana tabacum L.

De-novo synthesis of acid and basic peroxidases has been studied in cell suspension cultures of tobacco by incorporation of ³H- and ¹⁴C-amino acids. Incorporation rates were found to be high for acid peroxidases and low for basic peroxidases. Synthesis of all peroxidases was inhibited by cycloheximide and actinomycin D. Subculturing of the cells increased the rates of radioactive amino-acid incorporation into all peroxidases within the first 24 h. This rise in peroxidase synthesis was correlated with the age of the transferred cells. The older the cells were the more pronounced was the effect. During the culture cycle the high rates of peroxidase synthesis at the second day dropped back to initial values. Peroxidase synthesis was thus inversely related to peroxidase accumulation which was very low at the beginning and increased continuously. By pulse-chase experiments it has been shown that newly synthesized acid peroxidases accumulated in the medium. This process was inhibited by monensin. Only the acid peroxidases were secreted into the cell wall and from there released. The basic peroxidases were not detectable in the medium.Abbreviations AA^* radioactive amino-acid mixture - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Indole-3-methanol is the main product of the oxidation of indole-3-acetic acid catalyzed by two cytosolic basic isoperoxidases from Lupinus

The nature of the products of the auxin catabolism mediated by both basic and acidic isoperoxidases has been studied. While indole-3-methanol is only a minor product of the oxidation of indole-3-acetic acid catalyzed by extracellular acidic isoperoxidases, it is the only product of the oxidation of indole-3-acetic acid catalyzed by two cytosolic basic isoperoxidases (EC 1.11.1.7) from lupin (Lupinus albus L.) hypocotyls. The putative indole-3-methanol formed by these latter isoperoxidases was isolated and then characterized by mass spectrometry and ¹H-nuclear magnetic resonance spectrometry. These results are discussed with respect to the diversity and compartmentation of the catabolism of indole-3-acetic acid in plant tissues.Abbreviations DCP 2,4-dichlorophenol - IAA indole-3-acetic acid - IM indole-3-methanol     

Turnover and transport of quinolizidine alkaloids. Diurnal fluctuations of lupanine in the phloem sap,leaves and fruits of Lupinus albus L.

Quinolizidine alkaloids formed in the leaves of Lupinus albus L. are translocated via the phloem to the other plant organs, especially the maturing fruits. Compared with amino-acid transport in the phloem, the alkaloids contribute about 8% to the overall nitrogen being exported from the leaf. Since it is likely that the alkaloids are subsequently degraded in the target tissues a minor role of quinolizidine alkaloids might be nitrogen transport. A marked diurnal fluctuation of alkaloids was observed in the leaves, the phloem sap, the roots and the fruits with an increase during the day and an amplitude of several hundred percent thus providing evidence for a rapid turnover of endogenous alkaloids.Abbreviations QA quinolizidine alkaloids - GLC gas-liquid chromatography     

Cell-wall synthesis and elongation growth in hypocotyls of Helianthus annuus L.

The relationship between growth and increase in cell-wall material (wall synthesis) was investigated in hypocotyls of sunflower seedlings (Helianthus annuus L.) that were either grown in the dark or irradiated with continuous white light (WL). The peripheral three to four cell layers comprised 30–50% of the entire wall material of the hypocotyl. The increase in wall material during growth in the dark and WL, respectively, was larger in the inner tissues than in the peripheral cell layers. The wall mass per length decreased continuously, indicating that wall thinning occurs during growth of the hypocotyl. When dark-grown seedlings were transfered to WL, a 70% inhibition of growth was observed, but the increase in wall mass was unaffected. Likewise, the composition of the cell walls (cellulose, hemicellulose, pectic substances) was not affected by WL irradiation. Upon transfer of dark-grown seedlings into WL a drastic increase in wall thickness and a concomitant decrease in cell-wall plasticity was measured. The results indicate that cell-wall synthesis and cell elongation are independent processes and that, as a result, WL irradiation of etiolated hypocotyls leads to a thickening and mechanical stiffening of the cell walls.     

Osmotic relations during elongation growth in hypocotyls of Helianthus annum L.

The relationship between growth, change in cell osmotic pressure and accumulation of osmotic solutes was investigated in hypocotyls of sunflower (Helianthus annum L.) seedlings. During growth in darkness the osmotic pressure decreased by 50% between days 2 and 6 after sowing. After irradiation of dark-grown seedlings with continuous white light (WL) an inhibition of hypocotyl growth was measured, but the osmotic pressure of the growing cells was not lower than in the dark-grown control. Growth in darkness and after WL irradiation was accompanied by an increase in the amount of osmotic substances (soluble sugars) which was proportional to the increase in length of the organ. During growth in continuous WL the cell osmotic pressure decreased by 45 % between days 2 and 6 after sowing. The transfer of WL-grown seedlings to darkness (“re-etiolation”) resulted in a rapid acceleration of hypocotyl growth, but the cell osmotic pressure was the same as that of the WL grown control. Growth in continuous WL was accompanied by a corresponding accumulation of osmotic substances (soluble sugars). The transition from WL to darkness resulted in an enhanced accumulation of osmotica and an increase in cell-wall extensibility. The results indicate that the relative maintenance of cell osmotic pressure during rapid hypocotyl growth in darkness is caused by an enhanced accumulation of soluble sugars into the growing cells of the organ.     

Somatic embryogenesis of agriculturally important lupin species (Lupinus angustifolius,L. albus,L. mutabilis)

Somatic embryos were obtained from immature cotyledons of Lupinus angustifolius, L. albus and L. mutabilis but not from L. luteus. Different kinds of basal media and plant growth regulators in primary and secondary culture were tested. The best induction media were based on B5 and were supplemented with 5 mg I^-1 2,4-D alone or with 0.25 mg I^-1 kinetin. Mature stage somatic embryos were obtained on media containing ABA (0.1–0.5 mg I^-1) and a high NH₄/NO₃ ratio. Embryo germination and plantlet development occurred on MS media supplemented with glutamine or GA₃.     

The relationship between diffusible,extractable and conjugated (base-labile) forms of indole-3-acetic acid in isolated coleoptile tips of Zea mays L.

Isolated, 2.5-mm-long coleoptile tips of Zea mays L. cv. Anjou 210 were analyzed for diffusible and tissue-extractable indole-3-acetic acid (IAA) in comparison with the level of base-labile conjugates at various times after excision. The results indicate that base-labile conjugates of IAA do not serve as major sources of free IAA in maize coleoptile tips.Abbreviations IAA indole-3-acetic acid - TLC thin-layer chromatography     

Enzymatic synthesis of quinolizidine alkaloid esters: a tigloyl-CoA: 13-hydroxylupanine O-tigloyltransferase from Lupinus albus L.

A tigloyl-CoA: 13-hydroxylupanine O-tigloyl-transferase could be demonstrated in crude enzyme preparations from Lupinus albus seedlings. The enzyme activity increases concomitantly with for formation of 13-tigloyloxylupanine in developing lupin seedlings. The transferase catalyzes specifically the transfer of an acyl group to 13-hydroxylupanine. The apparent Km-values are 140 M for tigloyl-CoA and 18 M for 13-hydroxylupanine. Other hydroxylated compounds, e.g., lupinine, 4-hydroxylupanine, and cholesterol are not acylated. The transferase shows optimal activity at pH 7–8 and at 30°C. It is activated by dithioerythritol and inhibited by thiol reagents. Tigloyl-CoA can be replaced as acyl donor by benzoyl-CoA and to a lesser extent by valeroyl-CoA, 3-methylbutyryl-CoA, butyryl CoA and propionyl-CoA but not acetyl-CoA. Preliminary evidence indicates that the transfer of the tigloyl and benzoyl moieties is catalyzed by different enzymes. The transferase activity could only be demonstrated in plants which produce quinolizidine alkaloids.Abbreviations DIECA diethyldithiocarbamate - DTE dithioerythritol - GLC gas-liquid chromatography     

Molecular cloning of the gene for indolepyruvate decarboxylase from Enterobacter cloacae

Summary Although indole-3-acetic acid (IAA) is a well-known plant hormone, the main IAA biosynthetic pathway from l-tryptophan (Trp) via indole-3-pyruvic acid (IPyA) has yet to be elucidated. Previous studies have suggested that IAA is produced by Enterobacter cloacae isolated from the rhizosphere of cucumbers and its biosynthetic pathway may possibly be the same as that in plants. To elucidate this pathway, the IAA biosynthetic gene was isolated from a genomic library of E. cloacae by assaying for the ability to convert Trp to IAA. DNA sequence analysis showed that this gene codes for only one enzyme and its predicted protein sequence has extensive homology with pyruvate decarboxylase in yeast and Zymomonas mobilis. Cell-free extracts prepared from Escherichia coli harboring this gene could convert IPyA to indole-3-acetaldehyde (IAAld). These results clearly show that this pathway is mediated only by indolepyruvate decarboxylase, which catalyzes the conversion of IPyA to IAAld.     

A high amount of satellite DNA in the genome of Lupinus angustifolius L.

Embryo DNA, isolated from ungerminated seeds of Lupinus angustifolius L., contains an exceptionally high amount of guanine-cytosine-rich satellite DNA. The thermal denaturation curve of total embryo DNA is biphasic with an inflexion point at 62% denaturation, indicating the presence of satellite DNA. The satellite fraction could be separated from the mainband DNA by three successive preparative CsCl-gradient centrifugations. The densities of the DNA fractions are 1.7045 g cm^-3 and 1.6925 g cm^-3, respectively. The percentages of guanine-cytosine calculated from these densities are comparable to the percentages of GC calculated from the melting temperatures. Finally, ressociation studies prove that foldback DNA and highly repeated sequences are much more frequent in the satellite DNA fraction than in the mainband DNA.Abbreviation C _o t the product of the DNA concentration (mol nucleotides l^-1) and the time (s) of incubation in a DNA reassociation reaction - GC guanine-cytosine - np nucleotide parirs - T temperature interval between 16 and 84% denaturation     

Effect of water stress on abscisic acid levels in white lupin (Lupinus albus L.) fruit,leaves and phloem exudate

Abscisic acid (ABA) was identified by combined gas liquid chromatography-mass spectrometry in sieve-tube exudate collected from the cut stylar ends of white lupin fruit. Water stress caused an increase in ABA levels in leaf, seed and pod tissues and phloem exudate. When compared with levels in extracts of these tissues, the concentration of ABA in sieve-tube sap was very high. It is suggested that ABA is actively transported out of mature leaves in the phloem and this finding is discussed in terms of the ABA balance of the plant.Abbreviations ABA abscisic acid - GLC gas liquid chromatography     

Characterization of long-term extension of isolated cell walls from growing cucumber hypocotyls

Walls from frozen-thawed cucumber (Cucumis sativus L.) hypocotyls extend for many hours when placed in tension under acidic conditions. This study examined whether such creep is a purely physical process dependent on wall viscoelasticity alone or whether enzymatic activities are needed to maintain wall extension. Chemical denaturants inhibited wall creep, some acting reversibly and others irreversibly. Brief (15 s) boiling in water irreversibly inhibited creep, as did pre-incubation with proteases. Creep exhibited a high Q₁₀ (3.8) between 20° and 30°C, with slow inactivation at higher temperatures, whereas the viscous flow of pectin solutions exhibited a much lower Q₁₀ (1.35). On the basis of its temperature sensitivity, involvement of pectic gel-sol transitions was judged to be of little importance in creep. Pre-incubation of walls in neutral pH irreversibly inactivated their ability to creep, with a half-time of about 40 min. At 1 mM, Cu²⁺, Hg²⁺ and Al³⁺ were strongly inhibitory whereas most other cations, including Ca²⁺, had little effect. Sulfhydryl-reducing agents strongly stimulated creep, apparently by stabilizing wall enzyme(s). The physical effects of these treatments on polymer interactions were examined by Instron and stress-relaxation analyses. Some treatments, such as pH and Cu²⁺, had significant effects on wall viscoelasticity, but others had little or no apparent effect, thus implicating an enzymatic creep mechanism. The results indicate that creep depends on relatively rugged enzymes that are firmly attached to or entangled in the wall. The sensitivity of creep to SH-reducing agents indicates that thiol reduction of wall enzymes might provide a control mechanism for endogenous cell growth.Abbreviations DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetraacetic acid - Hepes N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid     

Changes in cell-wall polysaccharides in relation to seedling development and the mobilisation of reserves in the cotyledons of Lupinus angustifolius cv. Unicrop

Some 22% of the dry weight of the cotyledons of resting seeds of Lupinus angustifolius cv. Unicrop has been shown to be non-starch polysaccharide material comprising the massively thickened walls of the storage mesophyll cells. On hydrolysis this material released galactose (76%), arabinose (13%), xylose (4%), uronic acid (7%): only traces of glucose were detected indicating the virtual absence of cellulose from the walls. Changes in the amount and composition of this material following germination have been studied in relation to parameters of seedling development and the mobilisation of protein, lipid and oligosaccharide reserves. Starch, which was not present in the resting seed, appeared transitorily following germination: under conditions of continuous darkness starch levels were reduced. During the period of bulk-reserve mobilisation, 92% of the non-starch polysaccharide material disappeared from the cotyledons. The residual cell-wall material released galactose (14%), arabinose (19%), xylose (24%) and uronic acid (43%). The galactose and arabinose residues of the cotyledonary cell walls clearly constitute a major storage material, quantitatively as important as protein. The overall role of the wall polysaccharides in seedling development is discussed.     

Endogenous indoles and the biosynthesis and metabolism of indole-3-acetic acid in cultures of Rhizobium phaseoli

Gas chromatography-mass spectrometric analyses of purified extracts from cultures of Rhizobium phaseoli wild-type strain 8002, grown in a non-tryptophan-supplemented liquid medium, demonstrated the presence of indole-3-acetic acid (IAA), indole-3-ethanol (IEt), indole-3-aldehyde and indole-3-methanol (IM). In metabolism studies with ³H-, ¹⁴C- and ²H-labelled substrates the bacterium was shown to convert tryptophan to IEt, IAA and IM; IEt to IAA and IM; and IAA to IM. Indole-3-acetamide (IAAm) could not be detected as either an endogenous constituent or a metabolite of [³H]tryptophan nor did cultures convert [¹⁴C]IAAm to IAA. Biosynthesis of IAA in R. phaseoli, thus, involves a different pathway from that operating in Pseudomonas savastanio and Agrobacterium tumefaciens-induced crown-gall tumours.Abbreviations IAA indole-3-acetic acid - IAld indole-3-aldehyde - IAAm indole-3-acetamide - IEt indole-3-ethanol - IM indole-3-methanol - HPLC-RC high-performance liquid chromatography-radio counting - GC-MS gas chromatography-mass spectrometry     

L- and D-tryptophan aminotransferases from maize coleoptiles

Two forms of L-tryptophan aminotransferases (L-TAT-1 and L-TAT-2) and one D-tryptophan aminotransferase (D-TAT) were separated from maize coleoptiles by using L- and D-tryptophan as amino group donors. The enzymes were partially purlfied by hydrophobic and gel filtration column chromatographies. L-TAT-1 and L-TAT-2 had similar properties, showing optimum pH at 8–9 and a high optimum temperature of 50–60 C for catalytic activity. As the amino group acceptor for these two enzymes, α-keto glutaric acid was more effective than pyruvic, oxaloacetic and glyoxylic acids. The molecular masses of L-TAT-1 and L-TAT-2 estimated by gel filtration were approximately 80 kDa and 45 kDa, respectively. D-TAT had an optimum pH similar to those of L-TATs, but the optimum temperature was conslderably lower (30 C). Pyruvic acid was an effective amino group acceptor for D-TAT, whereas oxaloacetic and α-keto glutaric acids were not. D-Cycloserine completely inhibited the activity. The molecular mass of D-TAT was approximately 55 kDa. These three TATs required pyridoxal-5-phosphate for their catalytic activities.     

A simple system for determining the phloem mobility of compounds using excised pods of lupin (Lupinus albus L.)

Lupin pods were isolated from the plant and the pedicels placed in solutions of either radiolabeled sucrose, glucose, abscisic acid, or gibberellin A₉ (GA₉). Phloem exudate was collected from the cut stylar ends of the pods and both quantitative and qualitative data were obtained on the activity in the sap. Fed sucrose, abscisic acid, and GA₉ were found in phloem exudate, but labeled glucose was immobile as the monosaccharide and activity in exudate appeared in sucrose.Abbreviations GA gibberellic acid - ABA abscisic acid - TLC thin layer chromatography     

An unusually high number of direct repeats detected by sequence analysis of the dispersed EcoRI-family fragments in Lupinus luteus L.

The Lupinus luteus genome contains a highly repetitive fraction of sequences named the EcoRI family. Two EcoRI molecules, 1071 and 1079 base pairs in length, were cloned, sequenced and compared. Analysis of the internal-sequence organization revealed a number of short direct repeats. Their involvement in the formation of the EcoRI-family fragments is postulated. Evidence is presented for the dispersed type of genomic organization of the EcoRI-family fragments.Abbreviations AluI, BspRI, EcoRI, Mbo, PstI restriction nucleases - bp base pair - G, A, T, C deoxynucleotides: dGMP, dAMP, dTMP and dCMP - pBR322 and pUC18 plasmids used as cloning vehicles