Pilar Carbonero

Genome-wide comparative phylogenetic analysis of the rice and Arabidopsis Dof gene families

BackgroundDof 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.ResultsWe 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.ConclusionsAccording 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.

A role for the DOF transcription factor BPBF in the regulation of gibberellin-responsive genes in barley aleurone.

Functional analyses of a number of hydrolase gene promoters, induced by gibberellin (GA) in aleurone cells following germination, have identified a GA-responsive complex as a tripartite element containing a pyrimidine box motif 5′-CCTTTT-3′. We describe here that BPBF, a barley (Hordeum vulgare) transcription factor of the DOF (DNA-Binding with One Finger) class, previously shown to be an activator of reserve protein encoding genes during development, also has a role in the control of hydrolase genes following seed germination. Northern-blot, reverse transcriptase-polymerase chain reaction, and in situ hybridization analyses evidenced that the transcripts of the BPBF-encoding gene (Pbf), besides being present during endosperm development, are also expressed in aleurone cells of germinated seeds where they are induced by GA, an effect counteracted by abscisic acid. Electrophoretic mobility shift assays have shown that the BPBF protein binds specifically to the pyrimidine box motif in vitro within the different sequence contexts that naturally occur in the promoters of genes encoding a cathepsin B-like protease (Al21) and a low-isoelectric point α-amylase (Amy2/32b), both induced in the aleurone layers in response to GA. In transient expression experiments, BPBF repressed transcription of theAl21 promoter in GA-treated barley aleurone layers and reverted the GAMYB-mediated activation of this protease promoter.

Increased insect resistance in transgenic wheat stably expressing trypsin inhibitor CMe

Proteinase inhibitors have been proposed to function as plant defence agents against herbivorous pests. We have introduced the barley trypsin inhibitor CMe (BTI-CMe) into wheat (Triticum aestivum L.) by biolistic bombardment of cultured immature embryos. Of the 30 independent transgenic wheat lines selected, 16 expressed BTI-CMe. BTI-CMe was properly transcribed and translated as indicated by northern and western blot, with a level of expression in transgenic wheat seeds up to 1.1% of total extracted protein. No expression was detected in untransformed wheat seeds. Functional integrity of BTI-CMe was confirmed by trypsin inhibitor activity assay. The significant reduction of the survival rate of the Angoumois grain moth (Sitotroga cerealella, Lepidoptera: Gelechiidae), reared on transgenic wheat seeds expressing the trypsin inhibitor BTI-CMe, compared to the untransformed control confirmed the potential of BTI-CMe for the increase of insect resistance. However, only early-instar larvae were inhibited in transgenic seeds and expression of BTI-CMe protein in transgenic leaves did not have a significant protective effect against leaf-feeding insects.

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.

The family of DOF transcription factors: from green unicellular algae to vascular plants

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.

Comparative phylogenetic analysis of cystatin gene families from arabidopsis, rice and barley

The plant cystatins or phytocystatins comprise a family of specific inhibitors of cysteine proteinases. Such inhibitors are thought to be involved in the regulation of several endogenous processes and in defence against pests and pathogens. Extensive searches in the complete rice and Arabidopsis genomes and in barley EST collections have allowed us to predict the presence of twelve different cystatin genes in rice, seven in Arabidopsis, and at least seven in barley. Structural comparisons based on alignments of all the protein sequences using the CLUSTALW program and searches for conserved motifs using the MEME program have revealed broad conservation of the main motifs characteristic of the plant cystatins. Phylogenetic analyses based on their deduced amino acid sequences have allowed us to identify groups of orthologous cystatins, and to establish homologies and define examples of gene duplications mainly among the rice and barley cystatin genes. Moreover, the absence of a counterpart between the two monocots, as well as strong variations in the motifs that interact with the cysteine proteinases, may be related to a species-specific evolutionary process. This cystatin classification should facilitate the assignment of proteinase specificities and functions to other cystatins as new information is obtained.

Differential expression of two types of sucrose synthase-encoding genes in wheat in response to anaerobiosis, cold shock and light

The expression of two types of sucrose synthase-encoding genes, Ss1 and Ss2, in hexaploid wheat (Triticum aestivum, L.), has been investigated using type-specific probes, corresponding to the 250-270 bp C-terminal portions of the respective cDNA clones. Both types of genes are highly expressed in developing endosperm, where the expression of the Ss2 type slightly precedes in time that of the Ss1 type. Expression of Ss genes is lower in etiolated leaves and in roots than in endosperm. In the first two tissues, the Ss1 mRNA is much more abundant than the Ss2 mRNA, and the Ss1 mRNA level sharply increases in response to anaerobiosis and to cold shock (6 degrees C), while the level of Ss2 mRNA is not significantly affected. Upon illumination of etiolated leaves, the Ss1 level mRNA decreases significantly and the Ss2 mRNA level increases.

Transgenic Expression of Trypsin Inhibitor CMe from Barley in Indica and Japonica Rice, Confers Resistance to the Rice Weevil Sitophilus Oryzae

Indica and japonica rice (Oryza sativa L.) plants were transformed by particle bombardment with the Itr1 gene encoding the barley trypsin inhibitor BTI-CMe, under the control of its own promoter that confers endosperm specificity, and the maize ubiquitin promoter. From 38 independent transgenic lines of indica (breeding line IR58) and 15 of the japonica (cv Senia) selected, 22 and 11, respectively, expressed the barley inhibitor at detectable levels. The transgene was correctly translated as indicated by western blot analysis with a level of expression in R3 seeds up to 0.31% (IR58) and 0.43% (Senia) of the total extracted protein. The functional integrity of BTI-CMe was confirmed by trypsin activity assays in liquid media and by activity staining gels, performed with seed extracts. The significant reduction of the survival rate of the rice weevil (Sitophilus oryzae, Coleoptera: Curculionidae) reared on homozygous transgenic indica and japonica rice seeds expressing the BTI-CMe, compared to non-transformed controls, and the decrease in the trypsin-like activity of insect crude midgut extracts, confirmed the utility of this proteinase inhibitor gene for the control of important storage pests.

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.

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

The barley genesHvLtp4.2 andHvLtp4.3 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 reportedLtp4 cDNA (nowLtp4.1). Southern blot analysis indicated the existence of three or moreLtp4 genes per haploid genome and showed considerable polymorphism among barley cultivars. We have investigated the transient expression of genesHvLtp4.2 andHvLtp4.3 following transformation by particle bombardment, using promoter fusions to theβ-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 thatLtp4.2 was more active thanLtp4.3 in endosperm, andLtp4.3 was active in roots, whileLtp4.2 was not. The promoters of both genes were induced by low temperature, both in winter and spring barley cultivars. Northern blot analysis, using theLtp4-specific probe, indicated thatXanthomonas campestris pv.translucens induced an increase over basal levels ofLtp4 mRNA, whilePseudomonas syringae pv.japonica caused a decrease. TheLtp4.3-Gus promoter fusion also responded in opposite ways to these two compatible bacterial pathogens, whereas theLtp4.2-Gus construction did not respond to infection.