Synthesis of the peptide-transport carrier of barley scutellum during the early stages of germination

Development of peptide-transport activity in the scutella of isolated barley (Hordeum vulgare l. cv. Maris Otter, Winter) embryos is shown to increase rapidly after about 15 h of imbibition, with the bulk of the transport activity being expressed by 24 h. This development is prevented by treatment of 15 h embryos with cycloheximide. Protein synthesis is found to increase in a closely related manner and also to be abolished by cycloheximide. Measurement of the incorporation of bound [³⁵S]methionine by 15 to 21-h embryos indicates that de-novo protein synthesis during this period is greater in the scutellum than in the embryonic axis. Previous studies have shown that the peptide-transport system possesses essential dithiol groups, probably located at the substrate-binding site (Walker-Smith and Payne 1983 b, 1984b). Treatment of 15-h embryos with the non-penetrant thiol reagent p-chloromercuribenzene sulphonic acid did not affect development of peptide-transport activity during the following 6 h, whereas with 3-d embryos identical treatment inhibited uptake almost completely during a subsequent 6-h period. Radioautography revealed that amongst the proteins synthesised during this early phase of germination and labelled in vitro with [³⁵S]methionine some are found within the epithelial plasmalemmae of the scutellum, which is the location of the peptide-transport carrier identified previously by externally labelling with a radioactive thiol reagent. The results provide evidence that protein(s) of the peptide-transport system are synthesised and inserted into the scutellum during early germination, allowing the system to play a major role in the nitrogen nutrition of the embryo.Abbreviations Gly Glycine - Phe phenylalanine     

Analysis of the peptide carrier in the scutellum of barley embryos by photoaffinity labelling

The preparation of a phenylalanine analogue containing an azido group and its incorporation into dipeptides is described. Peptides modified in this way are taken up into barley (Hordeum vulgare L.) scutella via the previously characterized peptide-transport system. Photoactivation of modified peptides in the presence of isolated scutella resulted in irreversible inhibition of peptide uptake in a concentration-dependent manner. Transport of other solutes which share a common mechanism of energy coupling, but which are transported via distinct carriers, was not inhibited after photo-derivatization of scutella with the modified peptides. Derivatization of isolated scutellar tissue with a ¹⁴C-labelled peptide analogue, resulted in incorporation of label into two proteins of M_r = 54000 and 41000. Scutellar tissue from early-germinating seeds, which do not show active peptide uptake, did not incorporate label into these polypeptides. It is concluded that these proteins are components of the barley peptide-transport system.Abbreviations Ala alanine - Gly glycine - PAGE polyacrylamide gel electrophoresis - Phe phenylalanine - Pro proline - SDS sodium dodecyl sulphate This work was supported by a grant from the Agricultural and Food Research Council.     

Isolation and identification of proteins from the peptide-transport carrier in the scutellum of germinating barley (Hordeum vulgare L.) embryos

Peptide-transport proteins, intrinsic to the epithelial plasmalemmae of the scutella of germinating barley (Hordeum vulgare L.) embryos, have been selectively labelled with p-chloro-[²⁰³Hg]mercuribenzenesulphonate using both a substrate-screening technique and a procedure developed to label exclusively vicinal dithiol groups, which were shown previously (Walker-Smith and Payne, 1983, FEBS Lett. 160, 25–30) to be essential components of the peptide-transport system. After radioactive labelling, proteins from the scutellar membranes have been solubilised with lithium diiodosalicylate plus sodium dodecyl sulphate and separated by using polyacrylamide gel electrophoresis. Fluorography and silver staining of these gels has for the first time allowed identification of two presumptive components of the peptide-transport system. These components only become detectable in an extract of the scutellar epithelia after 15 h imbibition, concomitant with a dramatic increase in peptide-transport activity, and they remain present at least 3 d after the onset of germination. [³⁵] Methionine was shown to be incorporated into these proteins between 15–20 h after imbibition, but its incorporation during a similar 5 h period into scutella isolated after 3 d was undetectable, implying a slow turnover of these proteins during the later stages of germination.Abbreviations Ala₂, Ala₃ dialanine, trialanine - CHAPS 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulphonate - p-CMBS p-chloromercuribenzenesulphonic acid - NEM N-ethylmaleimide - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Characteristics of the active transport of peptides and amino acids by germinating barley embryos

Use of two different assays involving either radioactively labelled substrates or a fluorescent-labelling procedure, gave good agreement for the rates of transport of peptides and amino acids into the scutellum of germinating grains of barley (Hordeum vulgare cv. Maris Otter, Winter). However, evidence was obtained for the enzymic decarboxylation of transpored substrate, which can cause underestimates of transport rates when using radioactively labelled substrates. The peptide Gly-Phe, was shown to be rapidly hydrolysed after uptake, and autoradiography of transported Gly-[U-¹⁴C]Phe indicated a rapid distribution of tracer, i.e. [U-¹⁴C] phenylalanine into the epithelium and sub-epithelial layers of the scutellum. The developmental patterns of transport activity indicate that peptide transport is more important nutritionally during the early stages of germination (1–3 d) whereas amino acids become relatively more important later (4–6 d). A range of amino acids is shown to be actively transported and several compete for uptake. At physiological concentrations, e.g. 2mM, transport of peptides and amino acids is inhibited about 80% by protonophore uncouplers, but at higher concentrations (10–100 mM) passive uptake predominates.Abbreviations Gly glycine - Leu leucine - Phe phenylalanine - Pro proline     

Stereospecificity of peptide transport by germinating barley embryos

The stereospecific requirements for peptide transport in the scutellum of germinating barley (Hordeum vulgare) embryos are described. Replacement of an L-amino acid residue in a peptide by its D-stereoisomer decreases the affinity of the peptide for the transport site, leading to a reduction in transport. Substitution of a second D-residue reduces affinity still further. The extent to which transport is inhibited depends upon the position of the D-residue in the primary sequence, with D-residues at the C-terminus of the peptide having the greatest effect. Competition between D- and L-peptides indicates that they both enter via the same transport system. Although D-amino acids can be accumulated when presented as a peptide, these same D-residues are not transported when supplied as the free amino acids. L-Leu-D-leu is accumulated intact against a concentration gradient, indicating the operation of an active transport mechanism that can function without the involvement of peptidase activity.     

Peptide transport by germinating barley embryos

Glycylsarcosine, a dipeptide which is resistant to peptidase activity, was accumulated intact against a concentration gradient by germinating embryos of Hordeum vulgare L., var. Maris Otter, Winter. This is the first clear evidence for the presence of a dipeptide transport system involved in the movement of protein reserves across the scutellum from the endosperm to the embryo during germination.Abbreviations gly-sar glycylsarcosine - TCA trichloroacetic acid     

Peptide transport by germinating barley embryos: Uptake of physiological di- and oligopeptides

The uptake of a variety of physiological di- and oligopeptides by germinating barley (Hordeum vulgare L.) embryos is described. Peptides as large as pentaalanine can be absorbed. Evidence is presented suggesting the peptides are absorbed intact and subsequently undergo rapid intracellular hydrolysis. Uptake shows stereospecificity. The transport of peptides is generally faster than the transport of amino acids, making it likely that the former could play an important role in the mobilization of the protein storage reserves during germination. The peptide transport system in barley is compared with similar systems from other groups of organisms.Abbreviations Gly-sar glycylsarcosine - Gly-sar-sar glycylsarcosylsarcosine - Gly-sar-sar-sar Glycylsarcosylsarcosylsarcosine     

Peptide transport by germinating barley embryos: Evidence for a single common carrier for di- and oligopeptides

Competition for uptake of a range of amino acids and peptides by germinating barley (Hordeum vulgare L.) embryos was studied. Peptides and amino acids show no competition and are apparently absorbed by independent transport systems. However, peptides of widely different structures do compete and it seems that only a single peptide transport system is present in barley embryos, capable of handling both di- and oligopeptides. The ability of physiological peptides to totally inhibit the uptake of glycylsarcosine indicates they share a common uptake system which previously has been shown to have the properties of an active transport process. The characteristics of the barley peptide transport system are compared with those found in other organisms.     

Mobilization of proline in the starchy endosperm of germinating barley grain

In germinating grains of barley, Hordeum vulgare L. cv. Himalaya, free proline accumulated in the starchy endosperm during the period of rapid mobilization of reserve proteins. When starchy endosperms were separated from germinating grains and homogenized in a dilute buffer of pH 5 (the pH of the starchy endosperm), the liberation of proline continued in these suspensions. The process was completely inhibited by diisopropylfluorophosphate, indicating that it was totally dependent on serine carboxy-peptidases. The carboxypeptidases present in the starchy endosperms of germinating grains were fractionated by chromatography on DEAE-cellulose. Four peaks were obtained, all with different activity spectra on the seven carbobenzoxydipeptides (Z-dipeptides) tested. Two of the peaks corresponded to previously known barley carboxypeptidases; these as well as a third peak hydrolyzed substrates of the types Z-X-Y and Z-X-Pro (X and Y denote any amino acid residue except proline). The fourth peak corresponded to a proline carboxypeptidase specific for substrates of the Z-Pro-X type. Apparently, in the hydrolysis of longer proline-containing peptides there must be sequential cooperation between the two carboxypeptidase types. The carboxypeptidases in extracts of starchy endosperms also liberated proline from the peptides Ala-Ala-Ala-Pro and Ala-Ala-Pro while Ala-Pro and Pro-Ala were not attacked. The dipeptides, however, were rapidly hydrolyzed around pH 7 by extracts prepared from the scutella of germinating grains. It is concluded that one part of the proline residues of the reserve proteins is liberated in situ in the starchy endosperm through the combined action of acid proteinases and carboxypeptidases, while another part is taken up in the form of small peptides by the scutellum, where proline is liberated by amino- and/or dipeptidases in some neutral compartment.Abbreviations DFP diisopropylfluorophosphate - DTT dithiothreitol - TNBS 2,4,6-trinitrobenzenesulphonic acid - Z N-carbobenzoxy - TLC thin layer chromatography A preliminary account of these results was given at the Meeting of the Federation of European Plant Physiological Societies in Edinburgh in July 1978. Abstract No. 181     

Intracellular levels of polyadenylated RNA and loss of vigour in germinating wheat embryos

Polyadenylated-RNA (Poly(A)⁺RNA) levels have been studied during the germination of wheat embryos of high viability but differing vigour. In high-vigour embryos imbibed at 20°C the level of poly(A)⁺RNA falls dramatically over the first hour of imbibition, then remains constant up to 3 h of imbibition before increasing rapidly to a level similar to that found in the quiescent state by 7 h of imbibition. Median-vigour embryos imbibed at 20°C show similar changes in poly(A)⁺RNA content but the initial decrease and subsequent increase in poly(A)⁺RNA levels are less marked. On imbibition at 10°C, the poly(A)⁺RNA content in high-vigour embryos decreases to a lesser extent during the first hour than at 20°C and the level increases more slowly over the next 6 h than during the same time period at 20°C. The level of poly(A)⁺RNA in medianvigour embryos remains constant over the first 4 h of germination and then falls to a level of about half that found in quiescent high-vigour embryos. Polyacrylamide gel electrophoresis of total-RNA samples shows that the polyadenylic acid (poly(A)) sequences occur in RNA species ranging in size from 35-7S. Polyacrylamide gel electrophoresis of isolated poly(A) sequences demonstrates the presence of two size classes of poly(A) in quiescent embryos, but at 20°C a more heterodisperse pattern appears by 2 h of imbibition. At 10°C, two size classes of poly(A) persist throughout the period studied in both high- and median-vigour embryos, although in median-vigour embryos the ratio of larger: smaller poly(A)-tail sizes decreases more rapidly than in high-vigour embryos.Abbreviations Poly(A) polyadenylic acid - poly(U) polyuridylic acid - poly(A)⁺RNA polyadenylated RNA     

Contrasting responses of sulphate and phosphate transport in barley (Hordeum vulgare L.) roots to protein-modifying reagents and inhibition of protein synthesis

The uptake of sulphate into roots of barley seedlings is highly sensitive to phenylglyoxal (PhG), an arginine-binding reagent. Uptake was inhibited by >80% by a 1-h pre-treatment of roots with 0.45 mol · m^–3 PhG. Inhibition was maximal in pre-treatment solutions buffered between pH 4.5 and 6.5. Phosphate uptake, measured simultaneously by double-labelling uptake solutions with ³²P and ³⁵S, was less susceptible to inhibition by PhG, particularly at pH <6.5, and was completely insensitive to the less permeant reagent p-hydroxyphenylglyoxal (OH-PhG) administered at 1 mol · m^–3 at pH at 5.0 or 8.2; sulphate uptake was inhibited in -S plants by 90% by OH-PhG-treatment. Root respiration in young root segments was unaffected by OH-PhG pre-treatment for 1 h and inhibited by only 17% after 90 min pre-treatment. The uptake of both ions was inhibited by the dithiol-specific reagent, phenylarsine oxide even after short exposures (0.5–5.0 min). Sulphate uptake was more severely inhibited than that of phosphate, but in both cases inhibition could be substantially reversed by 5 min washing of treated roots by 5 mol · m^–3 dithioerythritol. After longer pre-treatment (50 min) with phenylarsine oxide, inhibition of the ion fluxes was not relieved by washing with dithioerythritol. Inhibition of sulphate influx by PhG was completely reversed by washing the roots for 24 h with culture solution lacking the inhibitor. The reversal was dependent on protein synthesis; less than 20% recovery was seen in the presence of 50 mmol · m^–3 cycloheximide. Sulphate uptake declined rapidly when -S roots were treated with cycloheximide. In the same roots the phosphate influx was little affected, small significant inhibitions being seen only after 4 h of treatment. Respiration was depressed by only 20% in apical and by 31% in basal root segments by cycloheximide pre-treatment for 2 h. Similar rates of collapse of the sulphate uptake and insensitivity of phosphate uptake were seen when protein synthesis was inhibited by azetidine carboxylic acid, p-fluorophenylalanine and puromycin. Considering the effects of all of the protein-synthesis inhibitors together leads to the conclusion that the sulphate transporter itself, or some essential sub-component of the uptake system, turns over rapidly with a half-time of about 2.5 h. The turnover of the phosphate transporter is evidently much slower. The results are discussed in relation to strategies for identifying the transport proteins and to the regulation of transporter activity during nutrient stress.Abbreviations CAP chloramphenicol - CHM cycloheximide - DTE dithioerythritol - OH-PhG p-hydroxyphenylglyoxal - PhAsO phenylarsine - PhG phenylglyoxal Paper dedicated to the memory of the late Ken Treharne who did much to encourage this collaboration.D.T.C. gratefully acknowledges a fellowship provided by Le Ministére des Etrangers during his stay in Montpellier.     

Preformed and newly synthesized messenger RNA in germinating wheat embryos

In 6 h germinated wheat (Triticum aestivum L. cv. Cama) embryos, more than half of the messenger RNAs are actively involved in translation. Neither preformed nor newly synthesized poly A⁺-RNA is translated preferentially. Germination in the presence of cordycepin showed that the half-life of the templates is about 2 h and that the newly synthesized messengers are essential to support protein synthesis in the embryo from the first hours of germination. Most of the messenger RNAs in 6 h germinated embryos are newly synthesized. The polypeptides coded for by either the endogenous messenger ribonucleoproteins or purified poly A⁺-RNA from both dry and germinated embryos are qualitatively identical; minor quantitative differences can however be observed.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate - TCA trichloroacetic acid - mRNP messenger ribonucleoprotein - poly A⁺-RNA polyadenylic acid containing RNA - PB polysome buffer - GM germination medium     

Gibberellin biosynthesis from gibberellin A12-aldehyde in a cell-free system from germinating barley (Hordeum vulgare L., cv. Himalaya) embryos

Gibberellin (GA) metabolism from GA₁₂-aldehyde was studied in cell-free systems from 2-d-old germinating embryos of barley. [¹⁴C]- or [17-²H₂]Gibberellins were used as substrates and all products were identified by combined gas chromatography-mass spectrometry. Stepwise analysis demonstrated the conversion of GA₁₂-aldehyde via the 13-deoxy pathway to GA₅₁ and via the 13-hydroxylation pathway to GA₂₉, GA₁ and GA₈. In addition, GA₃ was formed from GA₂₀ via GA₅. We conclude that the embryo is capable of producing gibberellins that can induce -amylase production in the aleurone layer. There was no evidence for 12- or 18-hydroxylation and GA₄ was neither synthesised nor metabolised by the system. All metabolically obtained GAs, with the exception of GA₃, were also found as endogenous components of the cell-free system in spite of ammonium-sulfate precipitation and desalting steps.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography We thank Mrs. G. Bodtke and Mrs. B. Schattenberg for preparing the barley embryos and the Deutsche Forschungsgemeinschaft for supporting this work.     

Precociously germinating rapeseed embryos retain characteristics of embryogeny

We compared the germination of Brassica napus L. embryos at three stages of development-mid-cotyledon, maturation and mature dry-to determine at which stage they acquired the capacity for normal germination and seedling development. Embryos were removed from the seed and cultured on hormone-free medium, allowing them to germinate. The transition from embryogeny to germination was monitored both morphologically and biochemically, using synthesis of 12 S storage protein as a marker of embryogeny. The mature embryos (dry seeds) set the standard for normal seedling development: radicle emergence, hypocotyl extension and cotyledon expansion occurred within 2 d and true leaves were formed within a week of germination. Rocket immunoelectrophoresis indicated that the storage proteins in seedlings from mature dry embryos were completely degraded within a week. In contrast, the midcotyledon-stage embryos appeared to germinate abnormally, retaining many embryonic characteristics. Although the roots emerged, the hypocotyls did not elongate and secondary cotyledons instead of leaves were formed at the shoot apex. Also, the seedlings continued to synthesize and accumulate storage proteins. The maturation-stage embryos did develop into normal-looking seedlings, but complete degradation of storage proteins required several weeks, presumably reflecting continued synthesis and turnover. We conclude that embryogenic and germination-specific processes can occur concurrently and that the capacity to develop as normal seedlings is acquired gradually during the maturation process.Abbreviations dpa days post anthesis - EDTA ethylenedi-aminetetraacetic acid - FW fresh weight     

Nucleic acid and protein synthesis and loss of vigour in germinating wheat embryos

A study has been made of the RNA and protein synthesising systems of wheat embryos isolated from seed lots having high viability but differing in vigour. The rate of RNA and protein synthesis in wheat embryos during the early hours of germination is related to the vigour of the seed lot. The imposition of a stress factor, in the nature of a sub-optimal germination temperature, during germination of isolated wheat embryos magnifies the differences in rates of protein and RNA synthesis between high and low vigour seed. Using cell-free protein synthesising systems it has been demonstrated that an important difference between high and low vigour embryos lies in the relative levels of messenger RNA in the embryo. High vigour embryos contain relatively higher levels of poly A⁺-RNA (i.e. potential mRNA species) than lower vigour embryos and furthermore the level of poly A⁺-RNA in high vigour embryos increases during early germination whilst in lower vigour embryos the level decreases. The difference in poly A⁺-RNA levels accounts, at least partially, for the differences in rates of protein synthesis observed between embryos from high and low vigour wheat seed during early germination at both optimal and sub-optimal germination temperatures.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - poly A⁺-RNA polyadenylated RNA - GM germination medium - PMS post-mitochondrial supernatant fraction     

Reconstitution of the tonoplast amino-acid carrier into liposomes

The tonoplast amino-acid transporter of barley (Hordeum vulgare L.) mesophyll cells was functionally reconstituted by incorporating solubilized tonoplast membranes, vacuoplast membranes or tonoplast-enriched microsomal vesicles into phosphatidylcholine liposomes. (i) Time-, concentration- and ATP-dependence of amino-acid uptake were similar to results with isolated vacuoles. Although the orientation of incorporation could not be controlled, the results indicate that the transporter functions as a uniport system which allows regulated equilibration by diffusion between the cytosolic and vacuolar amino-acid pools. (ii) The ATP-modulated amino-acid carrier was also successfully reconstituted from barley epidermal protoplasts and Valerianella or Tulipa vacuoplasts, indicating its general occurrence. (iii) Fractionation of solubilized tonoplasts by size-exclusion chromatography followed by reconstitution of the fractions for glutamine transport gave two activity peaks: the first eluted in the region of high-molecular-mass vesicles and the second at a size of 300 kDa for the Triton-protein micelle.Abbreviation SDS-PAGE sodium dodecyl sulfate-polyacryl-amide gel electrophoresis This work was part of our research efforts within the Sonderforschungsbereich 176 of the University. We gratefully acknowledge experimental support by Marion Betz and valuable discussions with Professors U. Heber and U.-I. Flügge and Dr. Armin Gross (University of Würzburg) and Dr. E. Martinoia (ETH, Zürich, Switzerland).     

Lectin synthesis in developing and germinating wheat and rye embryos

Wheat (Triticum aestivum L.) and rye (Secale cereale L.) lectins are specifically synthesized during seed formation. They accumulate exponentially in the primary axes in a period coinciding with the development of this complex organ. Since the specific lectin content also increases dramatically, there is apparently an outburst of lectin synthesis during the development of the primary axes. Germinating embryos also synthesize some lectin. The fortunate availability of a highly specific procedure for the isolation of cereal lectins enabled us to follow the kinetics of their synthesis during early germination. Stored mRNAs appear to be involved in this residual lectin synthesis.     

Increase of protein synthesis after isolation in dormant and afterripened Agrostemma githago L. embryos

Isolated embryos are more suitable for in vivo study of protein synthesis than non-isolated embryos because, after isolation, the uptake of labeled amino acids is about 1000 times higher. However, isolation also stimulates protein synthesis: Up to 4 h after isolation, the capacity to incorporate labeled amino acids increased 7 times. Therefore, data on incorporation obtained with isolated embryos cannot be extended to non-isolated embryos. The increase of protein synthesis was not due to synthesis of specific proteins, but was a general increase. Furthermore, ripening, dormant, and afterripened embryos showed the same degree of increase. Isolation therefore stimulates protein synthesis nonspecifically. When embryos were kept under anaerobic conditions after isolation, protein synthesis did not increase. Therefore, higher oxygen consumption after removal of the seedcoat is probably the cause of the higher incorporation capacity. Furthermore, the activation of protein synthesis lagged several hours behind the increase of oxygen consumption.Abbreviations A afterripened - D dormant - pre-rRNA precursor of ribosomal RNA     

Tissue-specific localization of aspartic proteinase in developing and germinating barley grains

Resting seeds of several plant species, including barley grains, have been reported to contain aspartic proteinase (EC 3.4.23) activity. Here, the expression of the Hordeum vulgare L. aspartic proteinase (HvAP) was studied in developing and germinating grains by activity measurements as well as by immunocytochemical and in-situ hybridization techniques. Southern blotting suggests the presence of one to two HvAP-encoding genes in the barley genome, while Northern analysis reveals a single 2.1-kb mRNA in grains and vegetative tissues. Western blotting with antibodies to HvAP shows the same subunit structure in different grain parts. In developing grains, HvAP is produced in the embryo, aleurone layer, testa and pericarp, but in the starchy endosperm HvAP is present only in the crushed and depleted area adjacent to the scutellum. During seed maturation, HvAP-encoding mRNA remains in the aleurone layer and in the embryo, but the enzyme disappears from the aleurone cells. The enzyme, however, remains in the degenerating tissues of the testa and pericarp as well as in resting embryo and scutellum. During the first three days of germination, the enzyme reappears in the aleurone layer cells but is not secreted into the starchy endosperm. The HvAP is also expressed in the flowers, stem, leaves, and roots of barley. The wide localization of HvAP in diverse tissues suggests that it may have several functions appropriate to the needs of different tissues.Abbreviations DAA days after anthesis - DTT dithiothreitol - HvAP Hordeum vulgare aspartic proteinase Both authors have contributed equally to this workWe thank Mart Saarma, Pia Runeberg-Roos, Alan Schulman and Yrjö Helariutta for helpful discussions during the study, Tiina Arna and Sari Makkonen for their help in proteinase activity experiments as well as Jaana Korhonen (Department of Pathology, University of Helsinki), Salla Marttila and Ilkka Porali (Department of Biology, University of Jyväskylä, Jyväskylä, Finland) for their advice on microscopical techniques. We also thank Liisa Pyhälä and Leena Liesirova for the production of the antibodies to HvAP at the National Public Health Institute, Helsinki. This study was supported by grants from the Ministry of Agriculture and Forestry and the Academy of Finland.     

Lipid transfer protein genes specifically expressed in barley leaves and coleoptiles

Genes/cDNAs encoding so-called lipid-transfer proteins (LTPs) have been isolated from a variety of tissues from different plants, but the in-vivo function of the LTP proteins is not yet known. In barley (Hordeum vulgare L.), the LTP1 gene (encoding a probable amylase/ protease inhibitor, Mundy and Rogers 1986, Planta 169, 51–63) is active in aleurone tissue, and in this paper two LTP-encoding cDNAs isolated from green leaves are described. The encoded proteins start with signal sequences, they are 75% homologous to each other, 60–63% homologous to rice aleurone LTP and maize seed/ coleoptile LTP, but only 48% homologous to barley aleurone LTP. Northern hybridization experiments established that the two seedling-specific genes are both highly expressed in leaves and coleoptiles whereas the LTP1 gene is inactive in seedlings. No LTP gene expression was detected in roots using either seedling or aleurone cDNA clones as probes. Tissue-print hybridization indicates that the LTP genes are first expressed in young epidermal cells in leaves and coleoptiles, and subsequently expressed in the vascular strands. Genomic Southern analysis indicates that the barley LTP gene family has four to six members.Abbreviation LTP lipid transfer protein I thank Dr. J. Mundy, Carlsberg Research Laboratory, Copenhagen, Denmark for the PAPI cDNA clone and R. Barkardottir, Department of Molceular Biology, University of Aarhus, Denmark for providing RNA for some of the Northern analyses. I also thank I. Bjørndal and L. Kjeldbjerg for excellent technical assistance. This work was supported by the The Danish Biotechnology Programme.