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 5Ј-CCTTTT-3Ј. 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 ␣-amylase (Amy2/32b), both induced in the aleurone layers in response to GA. In transient expression experiments, BPBF repressed transcription of the Al21 promoter in GA-treated barley aleurone layers and reverted the GAMYB-mediated activation of this protease promoter.
Summary Functional analysis of hydrolase gene promoters, induced by gibberellin (GA) in aleurone cells following germination, has identified a GA‐responsive complex (GARC) as a tripartite element containing a pyrimidine‐box motif 5′‐CCTTTT‐3′. We describe here the characterization of a new barley gene (Sad gene) encoding a transcription factor (SAD) of the DNA binding with One Finger (DOF) class that binds to the pyrimidine box in vitro and activates transcription of a GA‐induced protease promoter in bombarded aleurone layers. RT‐PCR and in situ hybridization analyses showed that the Sad transcripts accumulated in all tissues analysed, being especially abundant in the scutellum and aleurone cells upon seed germination. The SAD protein, expressed in bacteria, binds in a specific manner to two oligonucleotides containing the sequence 5′‐G/CCTTTT/C‐3′, derived from the promoter region of the Al21 gene encoding a cathepsin B‐like cysteine protease. Although the Sad transcript accumulation did not respond to external GA‐incubation in aleurone cells, in transient expression experiments in co‐bombarded aleurone layers, SAD trans‐activated transcription from the Al21 promoter in a similar manner as did GAMYB, a MYB protein previously shown to respond to GA and to activate several hydrolase gene promoters in barley aleurone cells. In vivo interaction between the GAMYB and SAD proteins was shown in the yeast two‐hybrid system, where GAMYB potentiates the SAD trans‐activation capacity through interaction with its C‐terminal domain.
SummaryThe DOF protein, SAD, previously shown to be a transcriptional activator in barley aleurone cells upon seed germination, also has an important role in gene regulation during endosperm development. mRNA was detected in early (10 days after flowering) developing barley seeds where it accumulated in the starchy endosperm, aleurone cells, nucellar projection, vascular tissues and the immature embryo, as shown by RT-PCR and in situ hybridization analyses. The SAD protein, expressed in bacteria, binds to oligonucleotides containing the prolamine box, 5¢-A/TAAAG-3¢sequence, derived from the promoter regions of the endospermspecific genes Hor2 and Itr1, encoding a B-hordein and trypsin-inhibitor BTI-CMe, respectively. SAD competed for the same binding sites with another endosperm-expressed DOF protein, BPBF. Transient expression experiments in co-bombarded developing endosperms demonstrated that SAD trans-activated transcription from Hor2 and Itr1 promoters through binding to the intact DOF motifs. When the two DOF factors are co-bombarded together an additive effect was observed upon the expression of the Itr1 gene. In-frame fusion of the Sad ORF to the reporter green fluorescent protein gene directs the fluorescence expression to the nucleus in transiently transformed onion epidermal layers. The visualization of fluorescence in the nucleus of onion cells, using the bimolecular fluorescent complex (BiFC) approach, has shown the in vivo interaction between SAD and the R2R3MYB protein GAMYB. The interaction in plant cells has also been documented for the DOF protein BPBF and GAMYB, but nuclear interaction could not be detected between BPBF and SAD by this procedure.
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).
A study was conducted to determine how the 5′-AACA/TA-3′ motif interacts with a transcription factor (TF) of the MYB class. Developing endosperms (10-22 days after flowering; DAF), mature embryos and 7-day-old leaves and roots were used for RNA extraction, while developing endosperms collected at 18 DAF were used for transient expression assays. Northern blot analysis revealed that the encoding gene GAMYB was already present in developing endosperms at 10 DAF and was detected throughout all stages. The transcripts of B-hordein (Hor2 gene) and trypsin inhibitor CMe (Itr1 gene) were detectable at 10 DAF and peaked at 22 DAF. Electrophoretic mobility shift assays (EMSA) showed in vitro binding of GAMYB protein to 5′-C/TAACT/AACC/A-3′ motif in the promoters of Hor2 and Itr1 genes. When a reporter gene under the control of the -343- or -560-bp promoter regions from the Hor2 and Itr1 genes, respectively, containing intact MYB and DOF binding sites were co-transfected with GAMYB or BPBF as effectors, a three- to ten-fold increase in β-glucuronidase (GUS) activity was observed. Mutations in the MYB binding motif that prevent in vitro binding by the GAMYB protein in EMSA assays abolished GUS transactivation. The results indicate that GAMYB mediates the transactivation of the Itr1 and Hor2 gene promoters in developing barley endosperm through binding to the MYB motif, and that an interaction with the DOF factor BPBF is necessary for the full transactivation of Hor2. Yeast cells co-transformed with full-length cDNA fragments of both proteins activated the expression of a Lac reporter gene, indicating an interaction between the two TFs in this system. This interaction took place between HvGAMYB and the C-terminal part of the BPBF. The results implicate GAMYB protein from barley as a TF involved in the combinatorial regulation of genes specifically expressed in the endosperm during development.
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