Ternary complex formation between HvMYBS3 and other factors involved in transcriptional control in barley seeds.

The SHAQKYF R1MYB transcription factor (TF) HvMYBS3 from barley is an activator of gene expression both during endosperm development and in aleurone cells upon seed germination. Its mRNA was detected as early as 10 days after flowering in developing barley endosperm, with a peak at 18 days, and in aleurone cells at 8 h after water imbibition, as shown by Northern blot and in situ hybridization analyses. The HvMYBS3 protein expressed in bacteria binds to oligonucleotides containing a GATA core derived from the promoters of: (i) the developing endosperm gene Itr1 (5'-GATAAGATA-3') encoding trypsin inhibitor BTI-CMe, and (ii) the post-germinating aleurone gene Amy6.4 (5'-TATCCAC-3'/5'-GTGGATA-3') encoding a high-pI alpha-amylase. Transient expression experiments in co-bombarded developing endosperms and in barley aleurone layers demonstrated that HvMYBS3 trans-activated transcription both from Itr1 and Amy6.4 promoters, in contrast with a previously reported seed-expressed R1MYB, HvMCB1, which was an activator of Itr1 and a transcriptional repressor of the Amy6.4 gene. In the yeast three-hybrid system, the HvMYBS3 protein formed a ternary complex with BPBF and BLZ2, two important seed TFs. However, no binary interactions could be detected between HvMYBS3 and BLZ2 or between HvMYBS3 and BPBF.

Inhibition of plant-pathogenic fungi by the barley cystatin Hv-CPI (gene Icy) is not associated with its cysteine-proteinase inhibitory properties.

The recombinant barley cystatin Hv-CPI inhibited the growth of three phytopathogenic fungi (Botrytis cinerea, Colletotrichum graminicola, and Plectosphaerella cucumerina) and the saprotrophic fungus Trichoderma viride. Several mutants of barley cystatin were generated by polymerase chain reaction approaches and both their antifungal and their cysteine-proteinase inhibitory properties investigated. Point mutants R38-->G, Q63-->L, and Q63-->P diminished their capacity for inhibiting papain and cathepsin B, retaining their antifungal properties. However, mutant C68-->G was more active for papain and cathepsin B than the wild type. These results indicate that in addition to the consensus cystatin-reactive site, Q63-V64-V65-A66-G67, the A37-R38-F39-A40-V41 region, common to all cereal cystatins, and the C68 residue are important for barley cystatin activity. On the other hand, the K92-->P mutant is inactive as a fungicide, but still retains measurable inhibitory activity for papain and cathepsin B. Against B. cinerea, the antifungal effect of Hv-CPI and of its derived mutants does not always correlate with their activities as proteinase inhibitors, because the Q63-->P mutant is inactive as a cystatin, while still inhibiting fungal growth, and the K92-->P mutant shows the reciprocal effects. These data indicate that inhibition of plant-pathogenic fungi by barley cystatin is not associated with its cysteine-proteinase inhibitory activity. Moreover, these results are corroborated by the absence of inhibition of intra- and extramycelia-proteinase activities by barley cystatin and by other well-known inhibitors of cysteine-proteinase activity in the fungal zymograms of B. cinerea.

A constitutive cystatin-encoding gene from barley (Icy) responds differentially to abiotic stimuli.

A barley cDNA clone encoding a cysteine proteinase inhibitor was characterized. The deduced amino acid sequence of this barley cystatin (Hv-CPI) contains the motif QXVXG conserved among members of the cystatin superfamily. The gene (Icy), located on chromosome 2, was expressed in embryos, developing endosperms, leaves and roots as assessed by northern blot analysis. Western blot analysis detected a slightly retarded band in leaves that was not present in roots or seeds. In these two organs a more precise location of Hv-CPI was done by immuno-histochemical analysis, with polyclonal antibodies raised against the recombinant CPI protein expressed in Escherichia coli. This protein efficiently inhibited papain (Ki 2.0 x 10(-8) M) and ficin (Ki 2.2 x 10(-8) M) and, to a lesser extent, chymopapain (Ki 1.6 x 10(-7) M) and was inactive against bromelain. The Icy mRNA expression in vegetative tissues increased in response to anaerobiosis, dark and cold shock (6 degrees C).

Adaptation of Spodoptera exigua (Lepidoptera: Noctuidae) to barley trypsin inhibitor BTI-CMe expressed in transgenic tobacco.

Nicotiana tabacum plants were transformed with the cDNA of barley trypsin inhibitor BTI-CMe under the control of the 35S CaMV promoter. Although the transgene was expressed and the protein was active in the homozygous lines selected, growth of Spodoptera exigua (Lepidoptera: Noctuidae) larvae reared on transgenic plants was not affected. The protease activity in larval midgut extracts after 2 days feeding on transformed tobacco leaves from the highest expressing plant showed a reduction of 25% in the trypsin-like activity compared to that from insects fed on non-transformed controls. The susceptibility of digestive serine-proteases to inhibition by BTI-CMe was confirmed by activity staining gels. This decrease was compensated with a significant induction of leucine aminopeptidase-like and carboxipeptidase A-like activities, while chymotrypsin-, elastase-, and carboxipeptidase B-like proteases were not affected.

Barley BLZ2, a seed-specific bZIP protein that interacts with BLZ1 in vivo and activates transcription from the GCN4-like motif of B-hordein promoters in barley endosperm.

A barley endosperm cDNA, encoding a DNA-binding protein of the bZIP class of transcription factors, BLZ2, has been characterized. The Blz2 mRNA expression is restricted to the endosperm, where it precedes that of the hordein genes. BLZ2, expressed in bacteria, binds specifically to the GCN4-like motif (GLM; 5'-GTGAGTCAT-3') in a 43-base pair oligonucleotide derived from the promoter region of a Hor-2 gene (B1-hordein). This oligonucleotide also includes the prolamin box (PB; 5'-TGTAAAG-3'). Binding by BLZ2 is prevented when the GLM is mutated to 5'-GTGctTCtc-3' but not when mutations affect the PB. The BLZ2 protein is a potent transcriptional activator in a yeast two-hybrid system where it dimerizes with BLZ1, a barley bZIP protein encoded by the ubiquitously expressed Blz1 gene. Transient expression experiments in co-bombarded developing barley endosperms demonstrate that BLZ2 transactivates transcription from the GLM of the Hor-2 gene promoter and that this activation is also partially dependent on the presence of an intact PB. A drastic decrease in GUS activity is observed in co-bombarded barley endosperms when using as effectors equimolar mixtures of Blz2 and Blz1 in antisense constructs. These results strongly implicate the endosperm-specific BLZ2 protein from barley, either as a homodimer or as a heterodimer with BLZ1, as an important transcriptional activator of seed storage protein genes containing the GLM in their promoters.

An endosperm-specific DOF protein from barley, highly conserved in wheat, binds to and activates transcription from the prolamin-box of a native B-hordein promoter in barley endosperm.

A cDNA encoding a DNA-binding protein of the DOF class of transcription factors was isolated from a barley endosperm library. The deduced amino acid sequence for the corresponding protein is 94% identical through the DOF domain to the prolamin-box (P-box) binding factor PBF from maize. The gene encoding the barley PBF (BPBF) maps to chromosome 7H, and its expression is restricted to the endosperm where it precedes that of the hordein genes. The BPBF expressed in bacteria as a GST-fusion binds a P-box 5'-TGTAAAG-3' containing oligonucleotide derived from the promoter region of an Hor2 gene. Binding was prevented when the P-box motif was mutated to 5'-TGTAgAc-3'. A P-box binding activity, present in barley and wheat endosperm nuclei, interacted similarly to BPBF with this synthetic oligonucleotide, and the binding was abolished by 1,10-phenanthroline. Transient expression experiments in developing barley endosperms demonstrate that BPBF transactivates transcription from the P-box element of a native Hor2 promoter and that direct binding of BPBF to its target site is essential for transactivation since mutations in the DOF DNA-binding domain or in the P-box motif of this promoter abolished both binding and transactivation. Evidence was also obtained for the presence in wheat of a Pbf homologue having similar DNA-binding properties to that of BPBF. These results strongly implicate this endosperm-specific DOF protein from barley as an important activator of hordein gene expression and suggest the evolutionary conservation of the Pbf gene function among small grain cereals.

Barley BLZ1: a bZIP transcriptional activator that interacts with endosperm-specific gene promoters.

A cDNA encoding a bZIP transcription factor was obtained from barley endosperm and used to identify the corresponding gene from a genomic library. The gene, designated Blz1, contained six exons and five introns, plus a 442 nt-long 5'-untranslated leader sequence, and was located on chromosome 5H. The Blz1 mRNA was detected early in endosperm development and was also expressed in roots and leaves. The BLZ1 protein was a potent transcriptional activator in a yeast system; 85% of its activity was associated with the first 203 amino acid residues at the N-terminus, which included two acidic regions. Presumptive involvement of Blz1 in the regulation of gene expression in endosperm was ascertained by the DNA-binding properties of BLZ1 in electrophoretic mobility shift assays (EMSA) and by transient expression in barley developing endosperms, using, as effectors, Blz1 in both sense and anti-sense orientations. In the co-bombardment experiments, the beta-glucuronidase (GUS) reported gene responded to Blz1 if under the control of the endosperm-specific ltr1 promoter or under a synthetic promoter containing the endosperm box of gene Hor2. Sucrose synthase promoters Ss1 and Ss2 and synthetic promoters containing mutated sequences of Hor2 were unaffected in trans by Blz1.

The Spatial Distribution of Sucrose Synthase Isozymes in Barley.

The sucrose (Suc) synthase enzyme purified from barley (Hordeum vulgare L.) roots is a homotetramer that is composed of 90-kD type 1 Suc synthase (SS1) subunits. Km values for Suc and UDP were 30 mM and 5 [mu]M, respectively. This enzyme can also utilize ADP at 25% of the UDP rate. Anti-SS1 polyclonal antibodies, which recognized both SS1 and type 2 Suc synthase (SS2) (88-kD) subunits, and antibodies raised against a synthetic peptide, LANGSTDNNFV, which were specific for SS2, were used to study the spatial distribution of these subunits by immunoblot analysis and immunolocalization. Both SS1 and SS2 were abundantly expressed in endosperm, where they polymerize to form the five possible homo- and heterotetramers. Only SS1 homotetramers were detected in young leaves, where they appeared exclusively in phloem cells, and in roots, where expression was associated with cap cells and the vascular bundle. In the seed both SS1 and SS2 were present in endosperm, but only SS1 was apparent in the chalazal region, the nucellar projection, and the vascular bundle. The physiological implications for the difference in expression patterns observed are discussed with respect to the maize (Zea mays L.) model.

Differential expression of pathogen-responsive genes encoding two types of glycine-rich proteins in barley.

Gene-specific probes (3' ends of cDNAs) were obtained from barley cDNAs encoding two types of glycine-rich proteins: HvGRP2, characterized by a cytokeratin-like and a cysteine-rich domain, and HvGRP3, whose main feature was an RNA-binding domain. Expression of genes Hvgrp2 and Hvgrp3, which are present at one (or two) copies per haploid genome, was ubiquitous and gene Hvgrp3 was under light/darkness modulation. Cold treatment increased Hvgrp2 and Hvgrp3 mRNA levels. Methyl jasmonate (10 microM) switched off the two genes. Expression of Hvgrp2, but not that of Hvgrp3, was induced by ethylene treatment (100 ppm). Fungal pathogens Erysiphe graminis and Rhynchosporium secalis increased the mRNAs levels of the two genes, both in compatible and in incompatible interactions, while bacterial pathogens did not.

Two cold-inducible genes encoding lipid transfer protein LTP4 from barley show differential responses to bacterial pathogens.

The barley genes HvLtp4.2 and HvLtp4.3 both encode the lipid transfer protein LTP4 and are less than 1 kb apart in tail-to-tail orientation. They differ in their non-coding regions from each other and from the gene corresponding to a previously reported Ltp4 cDNA (now Ltp4.1). Southern blot analysis indicated the existence of three or more Ltp4 genes per haploid genome and showed considerable polymorphism among barley cultivars. We have investigated the transient expression of genes HvLtp4.2 and HvLtp4.3 following transformation by particle bombardment, using promoter fusions to the beta-glucuronidase reporter sequence. In leaves, activities of the two promoters were of the same order as those of the sucrose synthase (Ss1) and cauliflower mosaic virus 35S promoters used as controls. Their expression patterns were similar, except that Ltp4.2 was more active than Ltp4.3 in endosperm, and Ltp4.3 was active in roots, while Ltp4.2 was not. The promoters of both genes were induced by low temperature, both in winter and spring barley cultivars. Northern blot analysis, using the Ltp4-specific probe, indicated that Xanthomonas campestris pv. translucens induced an increase over basal levels of Ltp4 mRNA, while Pseudomonas syringae pv. japonica caused a decrease. The Ltp4.3-Gus promoter fusion also responded in opposite ways to these two compatible bacterial pathogens, whereas the Ltp4.2-Gus construction did not respond to infection.

Isolation and promoter characterization of barley gene Itr1 encoding trypsin inhibitor BTI-CMe: differential activity in wild-type and mutant lys3a endosperm.

The gene Itr1, encoding trypsin inhibitor BTI-CMe, has been obtained from a genomic library of Hordeum vulgare L. The gene has no introns and presents in its 5'-upstream region 605 bp that are homologous to the long terminal repeats (LTR) of the 'copia-like' retro-transposon Bare-1. Functional analysis of the Itr1 promoter by transient expression in protoplasts derived from different barley tissues, has shown that in this system the Itr1 promoter retains its endosperm specifity and the trans-regulation mediated by the Lys3a gene. The proximal promoter extending 343 bp upstream of the translation initiation ATG codon is sufficient to confer full GUS expression and for endosperm specifity. In protoplasts derived from the lys3a mutant, Risø 1508, GUS activity was less than 5% of that obtained with the same constructs in the protoplasts of wild-type Bomi from which it derives. Gel retardation experiments, after incubation with proteins obtained from both types of endosperm nuclei, also show differential patterns. Possible reasons for these differences are discussed.

The promoter of the gene Itr1 from barley confers a different tissue specificity in transgenic tobacco.

Tissue-specific expression of the gene coding for trypsin inhibitor BTI-CMe in barley (Itr1) occurs during the first half of endosperm development. In transgenic tobacco, the Itr1 promoter drives expression of the beta-glucuronidase reporter gene not only in developing endosperm but also in embryo, cotyledons and the meristematic intercotyledonary zone of germinating seedlings. A promoter fragment extending 343 bp upstream of the translation initiation ATG codon was sufficient for full transgene expression, whereas, the proximal 83 bp segment of the promoter was inactive. Possible reasons for the differences in expression patterns are discussed.

Sharp divergence between wheat and barley at loci encoding novel members of the trypsin/alpha-amylase inhibitors family.

Amino acid sequences for three members (CMx1, CMx2, and CMx3) of a new subfamily of trypsin/alpha-amylase inhibitors in wheat have been deduced from the nucleotide sequences of the corresponding cDNAs. A cDNA clone encoding CMx1 was selected from a wheat developing endosperm library using a probe that encoded barley trypsin inhibitor BTI-CMe at low stringency. Sequences corresponding to CMx2 and CMx3 were obtained from cDNA amplified by the polymerase chain reaction. The three CMx sequences contain a premature stop codon after 363 nt, as well as a second stop codon at the same position as in BTI-CMe (nt 439-441). Southern analysis of DNAs from diploid, tetraploid, and hexaploid wheats, as well as from aneuploid lines, indicate that there is a single CMx locus in each of the three genomes of hexaploid wheat, respectively associated with chromosomal arms 4AS, 4BS, and 4DL. These genes are expressed early during endosperm development and not expressed at detectable levels in other tissues. Evolutionary implications are discussed.

Cloning of cDNA, expression, and chromosomal location of genes encoding the three types of subunits of the barley tetrameric inhibitor of insect alpha-amylase.

Three cDNA clones from barley developing endosperm, corresponding to proteins BTAI-CMa, BTAI-CMb and BTAI-CMd, which are the three types of subunits of the tetrameric inhibitor of insect alpha-amylases, have been identified and sequenced. The deduced amino acid sequence of BTAI-CMb corresponds to the CM16/CM17 type of subunit in wheat (92/90% identical residues) and has one putative N-glycosylation site (NLT) and a possible kinase-C phosphorylation site (SCR). The BTAI-CMa sequence differs at four amino acid residues from a previously reported one from cv. Bomi and the sequence deduced for BTAI-CMd completes (11 N-terminal residues) and confirms previously available data. The gene for BTAI-CMa (Iat1) is located in the beta arm of barley chromosome 7H (syn.1), while genes for both BTAI-CMb (Iat2) and BTAI-CMd (Iat3) are in the long arm of chromosome 4H. The three genes are expressed in endosperm and their mRNAs are not detected in the other tissues tested, except Iat1, which seems to be expressed at a low level in coleoptile and roots, where it is switched off by 50 microM methyl jasmonate.

Sucrose synthase genes in barley. cDNA cloning of the Ss2 type and tissue-specific expression of Ss1 and Ss2.

A cDNA of 2,708 bp encoding type 2 sucrose synthase (Ss2) from barley has been sequenced. Similarity of this cDNA with respect to that of type 1 (Ss1) is high in the coding region (75% identical positions), but low (41% identical residues) in the 3' non-coding region. Type-specific non cross-hybridizing probes for Northern blot analysis have been obtained from the 3' ends. The Ss1 type is highly expressed in developing endosperm and in roots and, at lower levels, in coleoptiles and aleurone. The Ss2 mRNA is abundant in endosperm, low in aleurone, and undetected in coleoptiles and roots.

The promoter of barley trypsin-inhibitor BTI-CMe, discriminates between wheat and barley endosperm protoplasts in transient expression assays.

Several promoter fragments from the barley gene coding for trypsin inhibitor, BTI-CMe, have been fused to the ß-glucuronidase (GUS) reporter gene and these chimeric constructs used for transient expression in protoplasts. Transfection of developing endosperm protoplasts from barley (cv Bomi) show a maximum GUS expression of about 50% of that driven by the cauliflower mosaic virus 35S promoter, while in wheat endosperm protoplasts expression is less than 10%. No significant expression is found in transfected leaf protoplasts from barley, wheat or tobacco (<2% of the 35S control). All the information required for endosperm and barley specificity is present in the 343 bp proximal to the translation initiation site.

A major barley allergen associated with baker's asthma disease is a glycosylated monomeric inhibitor of insect alpha-amylase: cDNA cloning and chromosomal location of the gene.

A 14.5 kDa barley endosperm protein that is a major allergen in baker's asthma disease, as previously shown by both in vitro (IgE binding) and in vivo tests, has been identified as a glycosylated monomeric member of the multigene family of inhibitors of alpha-amylase/trypsin from cereals. A cDNA encoding this allergen (renamed BMAI-1) has been isolated and characterized. The deduced sequence for the mature protein, which is 132 residues long, is identical in its N-terminal end to the 20 amino acid partial sequence previously determined from the purified allergen, and fully confirms that it is a member of the multigene family of cereal inhibitors. Southern-blot analysis of wheat/barley addition lines using the insert in the BMAI-1 cDNA clone as a probe, has led to the location of the allergen gene (Iam1) in barley chromosome 2, while another related member of this protein family, the barley dimeric alpha-amylase inhibitor BDAI-1 gene (Iad1) has been located in chromosome 6. Iam1 is the first member of this inhibitor family in cereals to be assigned to chromosome group 2, thus extending the dispersion of genes in the family to five out of the seven homology groups of chromosomes in wheat and barley (chromosomes 2, 3, 4, 6 and 7).

Homologous sucrose synthase genes in barley (Hordeum vulgare) are located in chromosomes 7H (syn. 1) and 2H. Evidence for a gene translocation?

The chromosomal location of the two types of sucrose synthase genes, Ss1 and Ss2, has been investigated in barley by Southern blot analysis of wheat-barley addition lines using non-cross-hybridizing-specific probes corresponding to the C-terminal regions of their respective cDNA clones (congruent to 250 bp). The Ss1 gene, whose cDNA of 2,667 bp has been entirely sequenced, is located in the beta-arm of chromosome 7H (syn. 1), while that corresponding to the homologous Ss2 is in the short arm of 2H, suggesting the existence of a translocation event between these two chromosomes in cultivated barley after an initial gene duplication and divergent evolution.