Phytohormones have essential roles in coordinately regulating a large array of developmental processes. Studies have revealed that brassinosteroids (BRs) and abscisic acid (ABA) interact to regulate hundreds of expression in genes, governing many biological processes. However, whether their interaction is through modification or intersection of their primary signaling cascades, or by independent or parallel pathways remains a big mystery. Using biochemical and molecular markers of BR signaling and ABA biosynthetic mutants, we demonstrated that exogenous ABA rapidly inhibits BR signaling outputs as indicated by the phosphorylation status of BES1 and BR-responsive gene expression. Experiments using a bri1 null-allele, bri1-116, and analysis of subcellular localization of BKI1-YFP further revealed that the BR receptor complex is not required for ABA to act on BR signaling outputs. However, when the BR downstream signaling component BIN2 is inhibited by LiCl, ABA failed to inhibit BR signaling outputs. Also, using a set of ABA insensitive mutants, we found that regulation of ABA on the BR primary signaling pathway depends on the ABA early signaling components, ABI1 and ABI2. We propose that the signaling cascades of ABA and BR primarily cross-talk after BR perception, but before their transcriptional activation. This model provides a reasonable explanation for why a large proportion of BR-responsive genes are also regulated by ABA, and provides an insight into the molecular mechanisms by which BRs could interact with ABA.cross-talk ͉ gene expression ͉ phosphorylation ͉ seed germination ͉ signal transduction U nlike animals, plants are sessile and need to constantly regulate their developmental and physiological processes to respond to various internal and external stimuli. Studies have revealed that many biological processes result from integrating multiple hormonal signals, and extensive cross-talk among different hormonal signaling pathways is present in plants (1, 2). Recently, a large set of microarray data verified that many genes are coregulated by multiple hormones, suggesting the importance of hormones to coordinately regulate biological processes in plants (3,4). A few studies have also elucidated specific molecular mechanisms of hormonal cross-talk. They include the role of auxin and ethylene in regulating root meristem development (1), the antagonistic relationship between abscisic acid (ABA) and gibberellins (GAs) on seed dormancy and germination (5, 6), and integration of the primary signaling pathways of auxin and brassinosteroids (BRs) by auxin response factor 2 (ARF2) (7).Studies have indicated that BRs and ABA can coregulate the expression of hundreds of genes (4), and they interact physiologically in controlling many developmental processes (5,(8)(9)(10). It is also well known that ABA is required to establish seed dormancy during embryo maturation and to inhibit seed germination (5), whereas BRs promote seed germination, likely through enhancing the embryo growth potential to antagonize the effect...

Arabidopsis glycogen synthase kinase 3 (GSK3)-like kinases have versatile functions in plant development and in responding to abiotic stresses. Although physiological evidence suggested a potential role of GSK3-like kinases in abscisic acid (ABA) signaling, the underlying molecular mechanism was largely unknown. Here we identified members of Snf1-related kinase 2s (SnRK2s), SnRK2.2 and SnRK2.3, that can interact with and be phosphorylated by a GSK3-like kinase, brassinosteroid insensitive 2 (BIN2). bin2-3 bil1 bil2, a loss-offunction mutant of BIN2 and its two closest homologs, BIN2 like 1 (BIL1) and BIN2 like 2 (BIL2), was hyposensitive to ABA in primary root inhibition, ABA-responsive gene expression, and phosphorylating ABA Response Element Binding Factor (ABF) 2 fragment by in-gel kinase assays, whereas bin2-1, a gain-of-function mutation of BIN2, was hypersensitive to ABA, suggesting that these GSK3-like kinases function as positive regulators in ABA signaling. Furthermore, BIN2 phosphorylated SnRK2.3 on T180, and SnRK2.3 T180A had decreased kinase activity in both autophosphorylation and phosphorylating ABFs. Bikinin, a GSK3 kinase inhibitor, inhibited the SnRK2.3 kinase activity and its T180 phosphorylation in vivo. Our genetic analysis further demonstrated that BIN2 regulates ABA signaling downstream of the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS receptors and clade A protein phosphatase 2C but relies on SnRK2.2 and SnRK2.3. These findings provide significant insight into the modulation of ABA signaling by Arabidopsis GSK3-like kinases.signal transduction | phosphorylation cascades | kinase activation

The development of the TMTpro-16plex series expanded the breadth of commercial isobaric tagging reagents by nearly 50% over classic TMT-11plex. In addition to the described 16plex reagents, the proline-based TMTpro molecule can accommodate two additional combinations of heavy carbon and nitrogen isotopes. Here, we introduce the final two labeling reagents, TMTpro-134C and TMTpro-135N, which permit the simultaneous global protein profiling of 18 samples with essentially no missing values. For example, six conditions with three biological replicates can now be perfectly accommodated. We showcase the 18plex reagent set by profiling the proteome and phosphoproteome of a pair of isogenic mammary epithelial cell lines under three conditions in triplicate. We compare the depth and quantitative performance of this data set with a TMTpro-16plex experiment in which two samples were omitted. Our analysis revealed similar numbers of quantified peptides and proteins, with high quantitative correlation. We interrogated further the TMTpro-18plex data set by highlighting changes in protein abundance profiles under different conditions in the isogenic cell lines. We conclude that TMTpro-18plex further expands the sample multiplexing landscape, allowing for complex and innovative experimental designs.

SUMMARY The plant hormones brassinosteroids (BRs) participate in light-mediated regulation of plant growth, although the underlying mechanisms are far from being fully understood. In addition, the function of the core transcription factor in the BR signaling pathway, BRI1-EMS-SUPPRESSOR 1 (BES1), largely depends on its phosphorylation status and its protein stability, but the regulation of BES1 is not well understood. Here, we report that SINA of Arabidopsis thaliana (SINATs) specifically interact with dephosphorylated BES1 and mediate its ubiquitination and degradation. Our genetic data demonstrated that SINATs inhibit BR signaling in a BES1-dependent manner. Interestingly, we found that the protein levels of SINATs were decreased in the dark and increased in the light, which changed BES1 protein levels accordingly. Thus, our study not only uncovered a new mechanism of BES1 degradation but also provides significant insights into how light conditionally regulates plant growth through controlling accumulation of different forms of BES1.