SummaryHvGAMYB, 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-speci®c 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-speci®c DOF transcriptional activator of the Hor2 gene. The in vivo interaction experiment between HvGAMYB and BPBF was done in the yeast twohybrid system, where HvGAMYB potentiates the BPBF trans-activation capacity through interaction with its C-terminal domain.

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.During seed development, storage reserves accumulate mostly in the form of carbohydrates and proteins, whose degradation upon germination will provide nutrients to the growing seedling before the photosynthetic capacity is fully acquired. Seed storage proteins (SSP) 1 are specifically synthesized in developing seeds, both in the endosperm and in the embryo. In the seed of monocotyledoneous species, the former is the predominant reserve tissue, whereas in dicotyledoneous plants the endosperm is commonly re-absorbed as maturation proceeds, and storage proteins are preferentially accumulated in the embryo. Because SSP-encoding genes are specifically induced and tightly regulated during seed development, they represent an interesting model system for studying the mechanisms of temporal and tissue-specific gene regulation.In the developing seed, different programs of gene expression have been defined that comprise distinct classes of genes that are coordinately regulated (1, 2). The MAT (maturation) class includes major SSP genes (like 2S albumins and 12S globulins) expressed at early and mid-maturation phases, whereas the LEA (late embryogenesis abundant) class includes primarily genes involved in the acquisition of desiccation tolerance expressed at later stages of maturation (3, 4). Unraveling the molecular basis of seed-specific gene expression has been mainly focused on the identification of cis-acting promoter el...

Arabidopsis thaliana S-Phase Kinase-Associated Protein 2A (SKP2A) is an F-box protein that regulates the proteolysis of cell cycle transcription factors. The plant hormone auxin regulates multiple aspects of plant growth and development, including cell division. We found that auxin induces the ubiquitin-dependent degradation of SKP2A both in vivo and in vitro, suggesting that this hormone acts as a signal to trigger SKP2A proteolysis. In this article, we show that auxin binds directly and specifically to SKP2A. By TIR1-based superposition and docking analyzes, we identified an auxin binding site in SKP2A. Mutations in this binding site reduce the ability of SKP2A to bind to auxin and generate nondegradable SKP2A forms. In addition, these non-auxin binding proteins are unable to promote E2FC/DPB degradation in vivo or to induce cell division in the root meristem. Auxin binds to TIR1 to promote its interaction with the auxin/indole-3-acetic acid target proteins. Here, we show that auxin also enhanced the interaction between SKP2A and DPB. Finally, a mutation in SKP2A leads to auxinresistant root growth, an effect that is additive with the tir1-1 phenotype. Thus, our data indicate that SKP2A is an auxin binding protein that connects auxin signaling with cell division.