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.     

Amitrole treatment of etiolated barley seedlings leads to deregulation of tetrapyrrole synthesis and to reduced expression of Lhc and RbcS genes

The effect of amitrole, known as an inhibitor of carotenoid biosynthesis, upon tetrapyrrole biosynthesis and its regulation has been studied. Etiolated barley (Hordeum vulgare L.) seedlings, grown in 125 μM amitrole, accumulated high levels of 5-aminolevulinate, Mg-protoporphyrin, Mg-protoporphyrin monomethyl ester, and protochlorophyllide. The amitrole-treated seedlings did not form paracrystalline prolamellar bodies, and the induction of Lhc and RbcS gene expression was reduced by non-photooxidative, low-intensity light. None of these events was observed upon treatment of the seedlings with 100 μM norflurazon, another inhibitor of carotenoid biosynthesis. The effect of amitrole cannot be explained solely by interaction with a presumed feedback inhibition of 5-aminolevulinate synthesis since incubation with amitrole and 5-aminolevulinate indicated that deregulation also occurs at later steps of tetrapyrrole biosynthesis. A possible relationship between this deregulation and ultrastructural changes is discussed. In connection with previously published data, we discuss Mg-protoporphyrin and its monomethyl ester as possible candidates for a “plastid signal” that operates as a negative factor, reducing the expression of Lhc and RbcS genes in this higher plant. Received: 27 June 2000 / Accepted: 12 October 2000     

Control by phytochrome of the synthesis of protein related to senescence in chloroplasts of barley (Hordeum vulgare)

Protein synthesis has been measured in chloroplast isolated from detached leaves of barley ( Hordeum vulgare L. cv. Hassan). The effects of hormone and light treatments of the leaves on chloroplast protein synthesis have been compared with effects on other senescence symptoms. Interruption of the dark with red light retards senescence and increases chloroplast protein synthesis. The effect of red light was reversed by far-red light. Red light did not act additively with kinetin, and it competed with ethylene and abscisic acid, accelerators of senescence, which decreased protein synthesis. In contrast to the interruption of the dark with red light, continuous light decreased chloroplast protein synthesis. It may be concluded effects on chloroplast protein synthesis. The retardation of senescence by continuous light is not necessarily related to P_u Instead, energy provided by photosynthesis may be an important factor.     

Solution structure of barley lipid transfer protein complexed with palmitate. Two different binding modes of palmitate in the homologous maize and barley nonspecific lipid transfer proteins.

The structure of a nonspecific lipid transfer protein from barley (ns-LTPbarley) in complex with palmitate has been determined by NMR spectroscopy. The structure has been compared to the structure of ns-LTPbarley in the absence of palmitate, to the structure of ns-LTPbarley in complex with palmitoyl coenzyme A, to the structure of ns-LTPmaize in its free form, and to the maize protein complexed with palmitate. Binding of palmitate only affects the structure of ns-LTPbarley moderately in contrast to the binding of palmitoyl coenzyme A, which leads to a considerable expansion of the protein. The modes of binding palmitate to the maize and barley protein are different. Although in neither case there are major conformational changes in the protein, the orientation of the palmitate in the two proteins is exactly opposite.     

Multiple binding modes for palmitate to barley lipid transfer protein facilitated by the presence of proline 12

Molecular dynamics simulations have been used to characterise the binding of the fatty acid ligand palmitate in the barley lipid transfer protein 1 (LTP) internal cavity. Two different palmitate binding modes (1 and 2), with similar protein–ligand interaction energies, have been identified using a variety of simulation strategies. These strategies include applying experimental protein–ligand atom–atom distance restraints during the simulation, or protonating the palmitate ligand, or using the vacuum GROMOS 54B7 force‐field parameter set for the ligand during the initial stages of the simulations. In both the binding modes identified the palmitate carboxylate head group hydrogen bonds with main chain amide groups in helix A, residues 4 to 19, of the protein. In binding mode 1 the hydrogen bonds are to Lys 11, Cys 13, and Leu 14 and in binding mode 2 to Thr 15, Tyr 16, Val 17, Ser 24 and also to the OH of Thr 15. In both cases palmitate binding exploits irregularity of the intrahelical hydrogen‐bonding pattern in helix A of barley LTP due to the presence of Pro 12. Simulations of two variants of barley LTP, namely the single mutant Pro12Val and the double mutant Pro12Val Pro70Val, show that Pro 12 is required for persistent palmitate binding in the LTP cavity. Overall, the work identifies key MD simulation approaches for characterizing the details of protein–ligand interactions in complexes where NMR data provide insufficient restraints.     

Changes in tubulin protein expression accompany reorganization of microtubular arrays during cell shaping in barley leaves

Barley (Hordeum vulgare L.) leaves grow from the base and thus exhibit a smooth developmental gradient. Developing mesophyll cells acquire their typical lobed shape synchronously along this gradient. Successive changes in the patterns of cortical microtubules are involved in the shaping process. The changes include formation and dispersal of band-like structures, the establishment of a random network and a dramatic loss of microtubules after completion of cell shaping. When the relative tubulin contents were determined in consecutive segments taken along the leaf, two tubulin maxima were found. They coincided with the establishment of the microtubular bands and the random network, respectively. Concomitantly, tubulin populations differing in the abundance of individual isotypes were found. The data indicate that the changes in the patterns of the cortical microtubules were not simply effected by shifting whole, stable microtubules, as may be inferred from immunofluorescence microscopy. Rather it appears that the formation of these patterns were accompanied by, or perhaps a result of, not only microtubule assembly and diassembly cycles, but also tubulin turnover. Received: 15 March 1997 / Accepted: 1 July 1997     

Cloning and expression analysis of candidate genes involved in wax deposition along the growing barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) leaf

The aim of the present study was to isolate clones of genes which are likely to be involved in wax deposition on barley leaves. Of particular interest were those genes which encode proteins that take part in the synthesis and further modification of very long chain fatty acids (VLCFAs), the precursors of waxes. Previously, it had been shown that wax deposition commences within a spatially well-defined developmental zone along the growing barley leaf (Richardson et al. in Planta 222:472–483, 2005). In the present study, a barley microarray approach was used to screen for candidate contig-sequences (www.barleybase.org) that are expressed particularly in those leaf zones where wax deposition occurs and which are expressed specifically within the epidermis, the site of wax synthesis. Candidate contigs were used to screen an established in-house cDNA library of barley. Six full-length coding sequences clones were isolated. Based on sequence homologies, three clones were related to Arabidopsis CER6/CUT1, and these clones were termed HvCUT1;1, HvCUT1;2 and HvCUT1;3. A fourth clone, which was related to Arabidopsis Fiddlehead (FDH), was termed HvFDH1;1. These clones are likely to be involved in synthesis of VLCFAs. A fifth and sixth clone were related to Arabidopsis CER1, and were termed HvCER1;1 and HvCER1;2. These clones are likely to be involved in the decarbonylation pathway of VLCFAs. Semi-quantitative RT-PCR confirmed microarray expression data. In addition, expression analyses at 10-mm resolution along the blade suggest that HvCUT1;1 (and possibly HvCUT1;2) and HvCER1;1 are involved in commencement of wax deposition during barley leaf epidermal cell development. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Selective expression of a probable amylase/protease inhibitor in barley aleurone cells: Comparison to the barley amylase/subtilisin inhibitor

We have cloned and sequenced a 650-nucleotide cDNA from barley (Hordeum vulgare L.) aleurone layers encoding a protein that is closely related to a known -amylase inhibitor from Indian finger millet (Eleusine coracana Gaertn.), and that has homologies to certain plant trypsin inhibitors. mRNA for this probable amylase/protease inhibitor (PAPI) is expressed primarily in aleurone tissue during late development of the grain, as compared to that for the amylase/subtilisin inhibitor, which is expressed in endosperm during the peak of storage-protein synthesis. PAPI mRNA is present at high levels in aleurone tissue of desiccated, mature grain, and in incubated aleurone layers prepared from rehydrated mature seeds. Its expression in those layers is not affected by either abscisic acid or gibberellic acid, hormones that, respectively, increase and decrease the abundance of mRNA for the amylase/subtilisin inhibitor. PAPI mRNA is almost as abundant in gibberellic acid-treated aleurone layers as that for -amylase, and PAPI protein is synthesized in that tissue at levels that are comparable to -amylase. PAPI protein is secreted from aleurone layers into the incubation medium.Abbreviations ABA abscisic acid - ASI barley amylase/subtilisin inhibitor - bp nucleotide base pairs - Da dalton - dpa days post anthesis - GA₃ gibberellic acid - PAPI probable amylase/protease inhibitor - poly(A)RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Expression of XET-related genes and its relation to elongation in leaves of barley (Hordeum vulgare L.)

Five cDNA clones were isolated from barley (Hordeum vulgare L.) that encoded mRNAs related to xyloglucan endotransglycosylase (XET). One of the clones encoded a protein with XET activity in vitro. Sequence comparisons revealed five families of XET-related sequences, one of which (containing two of the barley genes) was novel. Hybridization studies using clone-specific probes indicated that the corresponding genes were represented once, or possibly twice, in the barley genome. Treatment of dwarf mutants with gibberellic acid (GA₃), or homozygosity at the ‘slender’ (sln1) locus, resulted in a 2.5-fold (approximately) stimulation of blade elongation rate. Three of the five clones detected mRNAs that were maximally expressed towards the base of the blade, and present in greater quantities in GA₃-treated or slender seedlings. The remaining two clones detected mRNAs that were maximally expressed in the middle of the blade. Relative elemental growth rate (REGR) profiles of leaves growing with or without GA₃ treatment revealed similar maximal REGR values despite a 2.5-fold difference in leaf elongation rate. Segments of GA₃-treated leaves attained their maximal REGR values more rapidly, this being associated with enhanced expression of the three ‘basal’ XET-related mRNAs. Highest XET activities were detected in the base of the elongation zone, and in GA₃-treated seedlings a second activity peak was observed near the distal end of the elongation zone. We conclude that there are likely to be several XET isoenzymes with different expression patterns, and identify those XET-related proteins potentially involved in leaf elongation.     

Isolation, characterization and expression of a cDNA coding for a jasmonate-inducible protein of 37 kDa in barley leaves

In barley leaves, there is a dramatic alteration of gene expression upon treatment with jasmonates leading to the accumulation of newly formed proteins, designated as jasmonate-inducible proteins (JIPs). In the present study, a new jasmonate-inducible cDNA, designated pHvJS37, has been isolated by differential screening of a γgt10 cDNA library constructed from mRNA of jasmonate-treated barley leaf segments. The open reading frame (ORF) encodes a 39-9 kDa polypeptide which cross-reacts with antibodies raised against the in vivo JIP-37. The hydropathic plot suggests that the protein is mainly hydrophilic, containing two hydrophilic domains near the C-terminus. Database searches did not show any sequence homology of pHv.JS37 to known sequences. Southern analysis revealed at least two genes coding for JIP-37 which map to the distal portion of the long arm of chromosome 3 and are closely related to genes coding for JIP-23. The expression pattern of the JIP-37 genes over time shows differential responses to jasmonate, abscisic acid (ABA), osmotic stress (such as sorbitol treatment) and desiccation stress. No expression was found under salt stress. From experiments using an inhibitor and intermediates of jasmonate synthesis such as α-linolenic acid and 12-oxophytodienoic acid, we hypothesize that there is a stress-induced lipid-based signalling pathway in which an endogenous rise of jasmonate switches on JIP-37 gene expression. Using immunocytochemical techniques, JIP-37 was found to be simultaneously located in the nucleus, the cytoplasm and the vacuoles.     

A bacterial expression system revisited for the recombinant production of cystine-rich plant lipid transfer proteins

Non-specific lipid transfer proteins (nsLTPs) are abundant and ubiquitous cystine-rich proteins that are capable, in vitro, of binding lipids and hydrophobic molecules. In view to probe the lipid binding properties of the wheat nsLTP1, mutant variants may represent a powerful tool. To this end, a synthetic gene, encoding a mature wheat nsLTP1 polypeptide, was designed to ensure high level expression in Escherichia coli. The bacterial expression host strain, a translational fusion strategy, and convenient cleavage and purification procedures were optimized to produce in standard fermentation conditions, a significant amount (15 mg/L final yield), of a soluble and correctly folded recombinant nsLTP1. This highly amenable expression system is helpful in order to investigate structure-activity relationships of plant nsLTP.     

Magnetization transfer from laser-polarized xenon to protons located in the hydrophobic cavity of the wheat nonspecific lipid transfer protein

Nonspecific lipid transfer protein from wheat is studied by liquid-state NMR in the presence of xenon. The gas-protein interaction is indicated by the dependence of the protein proton chemical shifts on the xenon pressure and formally confirmed by the first observation of magnetization transfer from laser-polarized xenon to the protein protons. Twenty-six heteronuclear nOes have allowed the characterization of four interaction sites inside the wheat ns-LTP cavity. Their locations are in agreement with the variations of the chemical shifts under xenon pressure and with solvation simulations. The richness of the information obtained by the noble gas with a nuclear polarization multiplied by approximately 12,000 makes this approach based on dipolar cross-relaxation with laser-polarized xenon promising for probing protein hydrophobic pockets at ambient pressure.