Abstract: Maize PBF (prolamin-box binding factor) belongs to the Dof class of plant specific transcription factors containing one highly conserved zinc finger DNA-binding domain, called Dof (DNA binding with one finger) domain. Maize PBF trans-activates the gamma-zein gene (gammaZ) promoter in developing maize seeds as shown by transient expression in maize endosperms. Co-transfection of a gammaZ:GUS construct with 35S:PBF resulted in a sevenfold increase in GUS expression, however, PBF mutation in Cys residues within the Dof domain abolishes both, binding to DNA and the capacity to activate gammaZ promoter. We present two pieces of evidence that PBF transactivates gammaZ promoter by binding to the Pb3 motif (TGTAAAG). First, recombinant Dof domain of PBF (bdPBF) specifically recognized Pb3 site as shown by gel mobility shift assays and second, co-expression of PBF with gammaZ promoter mutated in Pb3 motif suppressed PBF trans-activation capacity. Immunocytochemical analysis on developing endosperm sections shows that PBF is localized in the nuclei of the peripheral layer cells of starchy endosperm, the tissue in which the initial accumulation of gamma-zein protein occurs. By contrast, PBF is detected in the cytosol of the starchy endosperm cells newly differentiated from aleurone daughter cells, where gamma-zein was absent. Taken together these data indicate that maize PBF plays an essential role in the regulation of the temporal and spatial expression of gammaZ gene.

Abstract: Accumulation of storage compounds in the embryo and endosperm of developing seeds is a highly regulated process that allows seedling growth upon germination until photosynthetic capacity is acquired. A critical regulatory element in the promoters of seed storage protein (SSP) genes from dicotyledonous species is the RY box, a target of B3-type transcription factors. However, the functionality of this motif in the transcriptional regulation of SSP genes from cereals has not been fully established. We report here the identification and molecular characterization of barley FUSCA3, a B3-type transcription factor as yet uncharacterized in monocotyledonous plants. Our results show that both the barley and Arabidopsis FUS3 genes maintain a conserved functionality for the regulation of SSP genes and anthocyanin biosynthesis in these two distantly related phylogenetic groups. Complementation of the loss-of-function mutant fus3 in Arabidopsis by the barley HvFus3 gene resulted in restored transcription from the At2S3 gene promoter and normal accumulation of anthocyanins in the seed. In barley, HvFUS3 participates in transcriptional activation of the endosperm-specific genes Hor2 and Itr1. HvFUS3, which specifically binds to RY boxes in EMSA experiments, trans-activates Hor2 and Itr1 promoters containing intact RY boxes in transient expression assays in developing endosperms. Mutations in the RY boxes abolished the HvFUS3-mediated trans-activation. HvFus3 transcripts accumulate in the endosperm and in the embryo of developing seeds, peaking at mid maturation phase. Remarkably, HvFUS3 interacts with the Opaque2-like bZIP factor BLZ2 in yeast, and this interaction is essential for full trans-activation of the seed-specific genes in planta.

Abstract: This article deals with the origin and evolution of the DOF transcription factor family through a phylogenetic analysis of those DOF sequences identified from a variety of representative organisms from different taxonomic groups: the green unicellular alga Chlamydomonas reinhardtii, the moss Physcomitrella patens, the fern Selaginella moellendorffii, the gymnosperm Pinus taeda, the dicotyledoneous Arabidopsis thaliana and the monocotyledoneous angiosperms Oryza sativa and Hordeum vulgare. In barley, we have identified 26 different DOF genes by sequence analyses of clones isolated from the screening of genomic libraries and of ESTs, whereas a single DOF gene was identified by bioinformatics searches in the Chlamydomonas genome. The phylogenetic analysis groups all these genes into six major clusters of orthologs originated from a primary basal grade. Our results suggest that duplications of an ancestral DOF, probably formed in the photosynthetic eukaryotic ancestor, followed by subsequent neo-, sub-functionalization and pseudogenization processes would have triggered the expansion of the DOF family. Loss, acquisition and shuffling of conserved motifs among the new DOFs likely underlie the mechanism of formation of the distinct subfamilies.

Abstract: In vivo protein-protein interactions are frequently studied by means of yeast two-hybrid analysis. However, interactions detected in yeast might differ considerably in the plant system. Based on GAL4 DNA-binding (BD) and activation domains (AD) we established an Arabidopsis protoplast two-hybrid (P2H) system. The use of Gateway-compatible vectors enables the high-throughput screening of protein-protein interactions in plant cells. The efficiency of the system was tested by examining the homo- and heterodimerization properties of basic leucine zipper (bZIP) transcription factors. A comprehensive heterodimerization matrix of Arabidopsis thaliana group C and group S bZIP transcription factors was generated by comparing the results of yeast and protoplast two-hybrid experiments. Surprisingly, almost no homodimerization but rather specific and selective heterodimerization was detected. Heterodimers were preferentially formed between group C members (AtbZIP9, -10, -25, -63) and members of group S1 (AtbZIP1, -2, -11, -44, -53). In addition, significant but low-affinity interactions were detected inside group S1, S2 or C AtbZIPs, respectively. As a quantitative approach, P2H identified weak heterodimerization events which were not detected in the yeast system. Thus, in addition to cell biological techniques, P2H is a valuable tool for studying protein-protein interaction in living plant cells.

Abstract: Proline metabolism has been implicated in plant responses to abiotic stresses. The Arabidopsis thaliana proline dehydrogenase (ProDH) is catalysing the first step in proline degradation. Transcriptional activation of ProDH by hypo-osmolarity is mediated by an ACTCAT cis element, a typical binding site of basic leucine zipper (bZIP) transcription factors. In this study, we demonstrate by gain-of-function and loss-of-function approaches, as well as chromatin immunoprecipitation (ChIP), that ProDH is a direct target gene of the group-S bZIP factor AtbZIP53. Dimerisation studies making use of yeast and Arabidopsis protoplast-based two-hybrid systems, as well as bimolecular fluorescence complementation (BiFC) reveal that AtbZIP53 does not preferentially form dimers with group-S bZIPs but strongly interacts with members of group-C. In particular, a synergistic interplay of AtbZIP53 and group-C AtbZIP10 was demonstrated by colocalisation studies, strong enhancement of ACTCAT-mediated transcription as well as complementation studies in atbzip53 atbzip10 T-DNA insertion lines. Heterodimer mediated activation of transcription has been found to operate independent of the DNA-binding properties and is described as a crucial mechanism to modulate transcription factor activity and function.

Abstract: Plants, as sessile life forms, have evolved diverse mechanisms to circumvent unfavourable growth conditions, among them interruption of the life cycle is one of the most successful strategies. During seed formation, embryo development can proceed through a maturation phase that allows the entry into a quiescent state and represents an evolutionary advantage, since it facilitates dispersal and resuming of growth under optimal environmental conditions. In the maturation phase different gene expression programmes devoted to the accumulation of storage compounds, acquisition of desiccation tolerance and entry into quiescence occur, which are highly coordinated and under fine regulatory control. This paper is focused on recent findings related to central transcription factors which regulate gene expression during maturation. The structure of established regulatory networks is presented based on extensive gene promoter analyses and the characterisation of mutants affecting seed development and maturation. Different aspects of gene regulation both in the embryo and endosperm are covered and comparisons between monocot and dicot species on the basis of current knowledge of the system are also presented.

Abstract: The expression of many seed storage protein genes in cereals relies on transcription factors of the bZIP class, belonging to the maize OPAQUE2 family. Here, we describe a survey of such factors in the genome of Arabidopsis thaliana, and the characterization of two of them, AtbZIP10 and AtbZIP25. Expression analysis by in situ hybridization shows that the occurrence of their mRNAs in the seed starts from early stages of development, peaks at maturation, and declines later in seed development, matching temporally and spatially those of the seed storage protein genes encoding 2S albumins and cruciferins. Gel mobility shift assays showed that AtbZIP10 and AtbZIP25 bind the ACGT boxes present in At2S and CRU3 promoters. Moreover, using the yeast two-hybrid system we show that AtbZIP10 and AtbZIP25 can interact in vivo with ABI3, an important regulator of gene expression in the seed of Arabidopsis. Transient expression analyses of a reporter gene under the control of the At2S1 promoter in transgenic plants overexpressing ectopically AtbZIP10, AtbZIP25, and ABI3 reveal that none of these factors could activate significantly the reporter gene when expressed individually. However, co-expression of AtbZIP10/25 with ABI3 resulted in a remarkable increase in the activation capacity over the At2S1 promoter, suggesting that they are part of a regulatory complex involved in seed-specific expression. This study shows a common mechanism of ABI3 in regulating different seed-specific genes through combinatorial interactions with particular bZIP proteins and a conserved role of O2-like bZIPs in monocot and dicot species.

Abstract: BACKGROUND: Dof proteins are a family of plant-specific transcription factors that contain a particular class of zinc-finger DNA-binding domain. Members of this family have been found to play diverse roles in gene regulation of processes restricted to the plants. The completed genome sequences of rice and Arabidopsis constitute a valuable resource for comparative genomic analyses, since they are representatives of the two major evolutionary lineages within the angiosperms. In this framework, the identification of phylogenetic relationships among Dof proteins in these species is a fundamental step to unravel functionality of new and yet uncharacterised genes belonging to this group. RESULTS: We identified 30 different Dof genes in the rice Oryza sativa genome and performed a phylogenetic analysis of a complete collection of the 36-reported Arabidopsis thaliana and the rice Dof transcription factors identified herein. This analysis led to a classification into four major clusters of orthologous genes and showed gene loss and duplication events in Arabidopsis and rice, that occurred before and after the last common ancestor of the two species. CONCLUSIONS: According to our analysis, the Dof gene family in angiosperms is organized in four major clusters of orthologous genes or subfamilies. The proposed clusters of orthology and their further analysis suggest the existence of monocot specific genes and invite to explore their functionality in relation to the distinct physiological characteristics of these evolutionary groups.

Abstract: HvGAMYB, a MYB transcription factor previously shown to be expressed in barley aleurone cells in response to gibberellin during germination, also has an important role in gene regulation during endosperm development. The mRNA was detected early (10 DAF) in the seeds where it accumulates, not only in the aleurone layer, starchy endosperm, nucellar projection and vascular tissue, but also in the immature embryo as shown by in situ hybridization analysis. The HvGAMYB protein, expressed in bacteria, binds to oligonucleotides containing the 5'-TAACAAC-3' or 5'-CAACTAAC-3' sequences, derived from the promoter regions of the endosperm-specific genes Hor2 and Itr1, encoding a B-hordein and trypsin-inhibitor BTI-CMe, respectively. Binding is prevented when these motifs are mutated to 5'-TgACAAg-3' and 5'-CgACTgAC-3'. Transient expression experiments in co-bombarded developing endosperms demonstrate that HvGAMYB trans-activates transcription from native Hor2 and Itr1 promoters through binding to the intact motifs described above. Trans-activation of the Hor2 promoter also requires an intact prolamine box (PB). This suggests that HvGAMYB interacts in developing barley endosperms with the PB-binding factor BPBF, an endosperm-specific DOF transcriptional activator of the Hor2 gene. The in vivo interaction experiment between HvGAMYB and BPBF was done in the yeast two-hybrid system, where HvGAMYB potentiates the BPBF trans-activation capacity through interaction with its C-terminal domain.

Abstract: In plants, basic region/leucine zipper motif (bZIP) transcription factors regulate processes including pathogen defence, light and stress signalling, seed maturation and flower development. The Arabidopsis genome sequence contains 75 distinct members of the bZIP family, of which approximately 50 are not described in the literature. Using common domains, the AtbZIP family can be subdivided into ten groups. Here, we review the available data on bZIP functions in the context of subgroup membership and discuss the interacting proteins. This integration is essential for a complete functional characterization of bZIP transcription factors in plants, and to identify functional redundancies among AtbZIP factors.

Abstract: 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).

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: The prolamin box (P-box) is a highly conserved 7-bp sequence element (5'-TGTAAAG-3') found in the promoters of many cereal seed storage protein genes. Nuclear factors from maize endosperm specifically interact with the P-box present in maize prolamin genes (zeins). The presence of the P-box in all zein gene promoters suggests that interactions between endosperm DNA binding proteins and the P-box may play an important role in the coordinate activation of zein gene expression during endosperm development. We have cloned an endosperm-specific maize cDNA, named prolamin-box binding factor (PBF), that encodes a member of the recently described Dof class of plant Cys2-Cys2 zinc-finger DNA binding proteins. When tested in gel shift assays, PBF exhibits the same sequence-specific binding to the P-box as factors present in maize endosperm nuclei. Additionally, PBF interacts in vitro with the basic leucine zipper protein Opaque2, a known transcriptional activator of zein gene expression whose target site lies 20 bp downstream of the P-box in the 22-kDa zein gene promoter. The isolation of the PBF gene provides an essential tool to further investigate the functional role of the highly conserved P-box in regulating cereal storage protein gene expression.

Abstract: We have amplified and cloned DNA sequences derived from a gene encoding a SNF1 (sucrose-non-fermenting 1)-related protein kinase which differs from that previously reported from barley. Northern blot and polymerase chain reaction (PCR) analysis of RNA populations, using specific probes and oligonucleotide primers, indicated that the two SNF1-related genes are differentially regulated. One is expressed in all tissues, whereas the other is expressed at high levels in the seed endosperm and aleurone, but at levels undetectable by northern blot analysis in other tissues. Comparisons with other plant SNF1-related protein kinase genes suggest that the form which is expressed at greatly enhanced levels in the seed is less similar to the other plant homologues which have been reported and may be unique to cereals.

Abstract: 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.

Abstract: This work presents new information on the structure and organization of B hordein genes in the Hor 2 locus of barley. Data obtained by Southern blot analysis and cloning and sequencing of different members of this multigene family are discussed.

Abstract: 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.

Abstract: Genomic sequences homologous to the yeast gene SNF1 have been isolated from barley (Hordeum vulgare) cv. Sunbar. SNF1 encodes a protein serine/threonine kinase required for the derepression of a number of genes, including SUC2 (invertase) in response to glucose deprivation. Southern blotting showed the presence of a family of related genes in barley and full-length sequences have been determined for two members of the family, one of which lacks an exon and is almost certainly non-functional. A partial sequence has been obtained for a third member of the family. The transcription start site of one of the genes has been determined by S1 nuclease protection. A transcript almost identical in sequence to the exons of one of the genes has been amplified from barley endosperm mRNA using the polymerase chain reaction. One of the full-length genomic sequences contains nine introns and 10 exons and the number and position of the introns in the second full-length sequence is identical except that it lacks exon 2. However, the length and sequence of the introns vary. Northern blot analyses indicated that related transcripts are present in aleurones, coleoptiles, endosperms, internodes, leaves, ovules, roots and root tips, with highest levels of expression in the aleurones and endosperms.