SUMMARY Light and brassinosteroid (BR) antagonistically regulate the developmental switch from etiolation in the dark to photomorphogenesis in the light in plants. Here we identify GATA2 as a key transcriptional regulator that mediates the crosstalk between BR- and light-signaling pathways. Overexpression of GATA2 causes constitutive photomorphogenesis in the dark, whereas suppression of GATA2 reduces photomorphogenesis caused by light, BR deficiency, or the constitutive photomorphogenesis mutant cop1. Genome profiling and chromatin immunoprecipitation experiments show that GATA2 directly regulates genes that respond to both light and BR. BR represses GATA2 transcription through the BR-activated transcription factor BZR1, whereas light causes accumulation of GATA2 protein and feedback inhibition of GATA2 transcription. Dark-induced proteasomal degradation of GATA2 is dependent on the COP1 E3 ubiquitin ligase, and COP1 can ubiquitinate GATA2 in vitro. This study illustrates a molecular framework for antagonistic regulation of gene expression and seedling photomorphogenesis by BR and light.

Seed development is important for agriculture productivity. We demonstrate that brassinosteroid (BR) plays crucial roles in determining the size, mass, and shape of Arabidopsis (Arabidopsis thaliana) seeds. The seeds of the BR-deficient mutant de-etiolated2 (det2) are smaller and less elongated than those of wild-type plants due to a decreased seed cavity, reduced endosperm volume, and integument cell length. The det2 mutant also showed delay in embryo development, with reduction in both the size and number of embryo cells. Pollination of det2 flowers with wild-type pollen yielded seeds of normal size but still shortened shape, indicating that the BR produced by the zygotic embryo and endosperm is sufficient for increasing seed volume but not for seed elongation, which apparently requires BR produced from maternal tissues. BR activates expression of SHORT HYPOCOTYL UNDER BLUE1, MINISEED3, and HAIKU2, which are known positive regulators of seed size, but represses APETALA2 and AUXIN RESPONSE FACTOR2, which are negative regulators of seed size. These genes are bound in vivo by the BR-activated transcription factor BRASSINAZOLE-RESISTANT1 (BZR1), and they are known to influence specific processes of integument, endosperm, and embryo development. Our results demonstrate that BR regulates seed size and seed shape by transcriptionally modulating specific seed developmental pathways.

Multiple repeats of membrane occupation and recognition nexus (MORN) motifs were detected in plant phosphatidylinositl monophosphate kinase (PIPK), a key enzyme in PI-signaling pathway. Structural analysis indicates that all the MORN motifs (with varied numbers at ranges of 7-9), which shared high homologies to those of animal ones, were located at N-terminus and sequentially arranged, except those of OsPIPK1 and AtPIPK7, in which the last MORN motif was separated others by an ~100 amino-acid "island" region, revealing the presence of two kinds of MORN arrangements in plant PIPKs. Through employing a yeast-based SMET (sequence of membrane-targeting) system, the MORN motifs were shown being able to target the fusion proteins to cell plasma membrane, which were further confirmed by expression of fused MORN-GFP proteins. Further detailed analysis via deletion studies indicated the MORN motifs in OsPIPK1, together with the 104 amino-acid "island" region are involved in the regulation of differential subcellular localization, i.e. plasma membrane or nucleus, of the fused proteins. Fat Western blot analysis of the recombinant MORN polypeptide, expressed in Escherichia coli, showed that MORN motifs could strongly bind to PA and relatively slightly to PI4P and PI(4,5)P 2 . These results provide informative hints on mechanisms of subcellular localization, as well as regulation of substrate binding, of plant PIPKs.

The phosphatidylinositol (PI) metabolic pathway is considered critical in plant responses to many environmental factors, and previous studies have indicated the involvement of multiple PI-related gene families during cellular responses. Through a detailed analysis of the Arabidopsis thaliana genome, 82 polypeptides were identified as being involved in PI signaling. These could be grouped into different families including PI synthases (PIS), PI-phosphate kinases (PIPK), phospholipases (PL), inositol polyphosphate phosphatases (IPPase), inositol polyphosphate kinases (IPK), PI transfer proteins and putative inositol polyphosphate receptors. The presence of more than 10 isoforms of PIPK, PLC, PLD and IPPase suggested that these genes might be differentially expressed during plant cellular responses or growth and development. Accordingly, DNA chip technology was employed to study the expression patterns of various isoforms. In total, 79 mRNA clones were amplified and used for DNA chip generation. Expression profile analysis was performed using samples that represented multiple tissues or cellular responses. Tested samples included normal leaf, stem and flower tissues, and leaves from plants treated with various hormones (auxin, cytokinin, gibberellin, abscisic acid and brassinosteroid) or environmental factors (temperature, calcium, sodium, drought, salicylic acid and jasmonic acid). Results showed that many PI pathway-related genes were differentially expressed under these experimental conditions. In particular, the different isoforms of each family were specifically expressed in many cases, suggesting their involvement in tissue specificity and cellular responses to environmental conditions. This work provides a starting point for functional studies of the relevant PI-related proteins and may help shed light onto the role of PI pathways in development and cellular responses.

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Insulin-like growth factor-binding protein-3 (IGFBP-3mRNA levels almost 80% lower in hepatoma tissues than in normal liver. The decreased IGFBP-3 expression has been associated with epigenetic changes such as DNA methylation and histone deacetylation (3, 4). IGFBP-3 is the most abundant insulin-like growth factor-binding protein in the human circulation, where it forms part of the IGF transport complex that stabilizes IGF-I and IGF-II and limits their access to tissues (5). In addition to its transport function, IGFBP-3 also has multiple biological roles at the cellular level, which depend on its interaction with a wide range of ligands (6, 7). By virtue of its ability to bind IGF-I and IGF-II with high affinity, IGFBP-3 can inhibit signaling through the type I IGF receptor. Other functions, independent of IGF binding, include effects on apoptosis, cell growth, differentiation, and migration, many of which appear dependent on the cell type and context (8 -10). For example, IGFBP-3 can induce apoptosis in various cancer cell models, including prostate and breast cancer (11, 12), although it can also have growth-stimulatory effects (13).Whereas in the rodent liver, IGFBP-3 gene expression is predominantly confined to Kupffer cells (14), in human liver, IGFBP-3 is synthesized by hepatocytes (15) and has been proposed to function as an inhibitor of cell proliferation in hepatocellular carcinoma (HCC) 4 (3, 16). MS-275 is a histone deacetylase inhibitor for which a significant anti-cancer role has been demonstrated in vitro and in vivo (17,18). In this study, we have investigated the involvement of endogenous IGFBP-3 in the anti-tumor effects of MS-275, by down-regulating its expression using siRNA. We describe roles for IGFBP-3 in hepatoma cell proliferation and migration, and we identify IGFBP-3-dependent proteins involved in mediating these effects. EXPERIMENTAL PROCEDURESMaterials-Cell culture reagents were from Invitrogen. MS-275, trypan blue, propidium iodide, and RNase were purchased from Sigma. Chemically synthesized siRNA against IGFBP-3, THBS2, and LYVE1 and an IGFBP-3 scrambled control were from Qiagen (Doncaster, Victoria, Australia). Recombinant human IGFBP-3 was produced in 911 human retinoblastoma cells (19). The following antibodies were purchased for Western analysis: histone H3, histone H3-acetyl-LYS 9/18, his-