Wenqiang Tang

Integration of Brassinosteroid Signal Transduction with the Transcription Network for Plant Growth Regulation in Arabidopsis

Brassinosteroids (BRs) regulate a wide range of developmental and physiological processes in plants through a receptor-kinase signaling pathway that controls the BZR transcription factors. Here, we use transcript profiling and chromatin-immunoprecipitation microarray (ChIP-chip) experiments to identify 953 BR-regulated BZR1 target (BRBT) genes. Functional studies of selected BRBTs further demonstrate roles in BR promotion of cell elongation. The BRBT genes reveal numerous molecular links between the BR-signaling pathway and downstream components involved in developmental and physiological processes. Furthermore, the results reveal extensive crosstalk between BR and other hormonal and light-signaling pathways at multiple levels. For example, BZR1 not only controls the expression of many signaling components of other hormonal and light pathways but also coregulates common target genes with light-signaling transcription factors. Our results provide a genomic map of steroid hormone actions in plants that reveals a regulatory network that integrates hormonal and light-signaling pathways for plant growth regulation.

BSKs mediate signal transduction from the receptor kinase BRI1 in Arabidopsis

Brassinosteroids (BRs) bind to the extracellular domain of the receptor kinase BRI1 to activate a signal transduction cascade that regulates nuclear gene expression and plant development. Many components of the BR signaling pathway have been identified and studied in detail. However, the substrate of BRI1 kinase that transduces the signal to downstream components remains unknown. Proteomic studies of plasma membrane proteins lead to the identification of three homologous BR-signaling kinases (BSK1, BSK2, and BSK3). The BSKs are phosphorylated by BRI1 in vitro and interact with BRI1 in vivo. Genetic and transgenic studies demonstrate that the BSKs represent a small family of kinases that activate BR signaling downstream of BRI1. These results demonstrate that BSKs are the substrates of BRI1 kinase that activate downstream BR signal transduction.

Brassinosteroid signal transduction from cell-surface receptor kinases to nuclear transcription factors

Brassinosteroid (BR) regulates gene expression and plant development through a receptor kinase-mediated signal transduction pathway. Despite the identification of many components of this pathway, it remains unclear how the BR signal is transduced from the cell surface to the nucleus. Here we describe a complete BR signalling pathway by elucidating key missing steps. We show that phosphorylation of BSK1 (BR-signalling kinase 1) by the BR receptor kinase BRI1 (BR-insensitive 1) promotes BSK1 binding to the BSU1 (BRI1 suppressor 1) phosphatase, and BSU1 inactivates the GSK3-like kinase BIN2 (BR-insensitive 2) by dephosphorylating a conserved phospho-tyrosine residue (pTyr 200). Mutations that affect phosphorylation/dephosphorylation of BIN2 pTyr200 (bin2-1, bin2-Y200F and quadruple loss-of-function of BSU1-related phosphatases) support an essential role for BSU1-mediated BIN2 dephosphorylation in BR-dependent plant growth. These results demonstrate direct sequential BR activation of BRI1, BSK1 and BSU1, and inactivation of BIN2, leading to accumulation of unphosphorylated BZR (brassinazole resistant) transcription factors in the nucleus. This study establishes a fully connected BR signalling pathway and provides new insights into the mechanism of GSK3 regulation.

PP2A activates brassinosteroid-responsive gene expression and plant growth by dephosphorylating BZR1

When brassinosteroid levels are low, the GSK3-like kinase BIN2 phosphorylates and inactivates the BZR1 transcription factor to inhibit growth in plants. Brassinosteroid promotes growth by inducing dephosphorylation of BZR1, but the phosphatase that dephosphorylates BZR1 has remained unknown. Here, using tandem affinity purification, we identified protein phosphatase 2A (PP2A) as a BZR1-interacting protein. Genetic analyses demonstrated a positive role for PP2A in brassinosteroid signalling and BZR1 dephosphorylation. Members of the B’ regulatory subunits of PP2A directly interact with BZR1’s putative PEST domain containing the site of the bzr1-1D mutation. Interaction with and dephosphorylation by PP2A are enhanced by the bzr1-1D mutation, reduced by two intragenic bzr1-1D suppressor mutations, and abolished by deletion of the PEST domain. This study reveals a crucial function for PP2A in dephosphorylating and activating BZR1 and completes the set of core components of the brassinosteroid-signalling cascade from cell surface receptor kinase to gene regulation in the nucleus.

Evidence of a Novel Cell Signaling Role for Extracellular Adenosine Triphosphates and Diphosphates in Arabidopsis

Extracellular ATP is a known receptor agonist in animals and was previously shown to alter plant growth, and so we investigated whether ATP derivatives could function outside plant cells as signaling agents. Signaling responses induced by exogenous nucleotides in animal cells typically include increases in free cytoplasmic calcium concentration ([Ca2+]cyt). We have evaluated the ability of exogenously applied adenosine 5′-[γ-thio]triphosphate (ATPγS), adenosine 5′-[β-thio]diphosphate (ADPβS), and adenosine 5′-O-thiomonophosphate to alter [Ca2+]cyt in intact apoaequorin transgenic Arabidopsis thaliana seedlings. ATPγS and ADPβS increase [Ca2+]cyt, and this increase is enhanced further when the nucleotides are added with the elicitor oligogalacturonic acid. Exogenous treatment with ATP also increases the level of transcripts encoding mitogen-activated protein kinases and proteins involved in ethylene biosynthesis and signal transduction. The increase in [Ca2+]cyt induced by nucleotide derivatives can be ablated by Ca2+-channel blocking agents and by the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), and the changes in gene expression can be partially blocked by these agents. These observations suggest that extracellular ATP can activate calcium-mediated cell-signaling pathways in plants, potentially playing a physiological role in transducing stress and wound responses.

A Proteomics Study of Brassinosteroid Response in Arabidopsis

The plant steroid hormones brassinosteroids (BRs) play an important role in a wide range of developmental and physiological processes. How BR signaling regulates diverse processes remains unclear. To understand the molecular details of BR responses, we performed a proteomics study of BR-regulated proteins in Arabidopsis using two-dimensional DIGE coupled with LC-MS/MS. We identified 42 BR-regulated proteins, which are predicted to play potential roles in BR regulation of specific cellular processes, such as signaling, cytoskeleton rearrangement, vesicle trafficking, and biosynthesis of hormones and vitamins. Analyses of the BR-insensitive mutant bri1-116 and BR-hypersensitive mutant bzr1-1D identified five proteins (PATL1, PATL2, THI1, AtMDAR3, and NADP-ME2) affected both by BR treatment and in the mutants, suggesting their importance in BR action. Selected proteins were further studied using insertion knock-out mutants or immunoblotting. Interestingly about 80% of the BR-responsive proteins were not identified in previous microarray studies, and direct comparison between protein and RNA changes in BR mutants revealed a very weak correlation. RT-PCR analysis of selected genes revealed gene-specific kinetic relationships between RNA and protein responses. Furthermore BR-regulated posttranslational modification of BiP2 protein was detected as spot shifts in two-dimensional DIGE. This study provides novel insights into the molecular networks that link BR signaling to specific cellular and physiological responses.

Extracellular ATP Inhibits Root Gravitropism at Concentrations That Inhibit Polar Auxin Transport

Raising the level of extracellular ATP to mmconcentrations similar to those found inside cells can block gravitropism of Arabidopsis roots. When plants are grown in Murashige and Skoog medium supplied with 1 mm ATP, their roots grow horizontally instead of growing straight down. Medium with 2 mm ATP induces root curling, and 3 mm ATP stimulates lateral root growth. When plants are transferred to medium containing exogenous ATP, the gravity response is reduced or in some cases completely blocked by ATP. Equivalent concentrations of ADP or inorganic phosphate have slight but usually statistically insignificant effects, suggesting the specificity of ATP in these responses. The ATP effects may be attributable to the disturbance of auxin distribution in roots by exogenously applied ATP, because extracellular ATP can alter the pattern of auxin-induced gene expression in DR5-β-glucuronidase transgenic plants and increase the response sensitivity of plant roots to exogenously added auxin. The presence of extracellular ATP also decreases basipetal auxin transport in a dose-dependent fashion in both maize (Zea mays) and Arabidopsis roots and increases the retention of [3H]indole-3-acetic acid in root tips of maize. Taken together, these results suggest that the inhibitory effects of extracellular ATP on auxin distribution may happen at the level of auxin export. The potential role of the trans-plasma membrane ATP gradient in auxin export and plant root gravitropism is discussed.

Proteomics shed light on the brassinosteroid signaling mechanisms

Large numbers of receptor-like kinases (RLKs) play key roles in plant development and defense by perceiving extracellular signals. The mechanisms of ligand-induced kinase activation and downstream signal transduction have been studied for only a few RLK pathways, among which the brassinosteroid (BR) pathway is the best characterized. Recently, proteomics studies identified new components that bridge the last gap in the genetically defined BR-signaling pathway, establishing the first complete pathway from an RLK to transcription factors in plants. Furthermore, analyses of phosphorylation events, mostly by mass spectrometry, provided insights into the mechanistic details of receptor kinase activation and regulation of downstream components by phosphorylation. This review focuses on recent progress in understanding BR signal transduction made by proteomics studies.

Extracellular Nucleotides and Apyrases Regulate Stomatal Aperture in Arabidopsis

This study investigates the role of extracellular nucleotides and apyrase enzymes in regulating stomatal aperture. Prior data indicate that the expression of two apyrases in Arabidopsis (Arabidopsis thaliana), APY1 and APY2, is strongly correlated with cell growth and secretory activity. Both are expressed strongly in guard cell protoplasts, as determined by reverse transcription-polymerase chain reaction and immunoblot analyses. Promoter activity assays for APY1 and APY2 show that expression of both apyrases correlates with conditions that favor stomatal opening. Correspondingly, immunoblot data indicate that APY expression in guard cell protoplasts rises quickly when these cells are moved from darkness into light. Both short-term inhibition of ectoapyrase activity by polyclonal antibodies and long-term suppression of APY1 and APY2 transcript levels significantly disrupt normal stomatal behavior in light. Stomatal aperture shows a biphasic response to applied adenosine 5′-[γ-thio]triphosphate (ATPγS) or adenosine 5′-[β-thio] diphosphate, with lower concentrations inducing stomatal opening and higher concentrations inducing closure. Equivalent concentrations of adenosine 5′-O-thiomonophosphate have no effect on aperture. Two mammalian purinoceptor inhibitors block ATPγS- and adenosine 5′-[β-thio] diphosphate-induced opening and closing and also partially block the ability of abscisic acid to induce stomatal closure and of light to induce stomatal opening. Treatment of epidermal peels with ATPγS induces increased levels of nitric oxide and reactive oxygen species, and genetically suppressing the synthesis of these agents blocks the effects of nucleotides on stomatal aperture. A luciferase assay indicates that treatments that induce either the closing or opening of stomates also induce the release of ATP from guard cells. These data favor the novel conclusion that ectoapyrases and extracellular nucleotides play key roles in regulating stomatal functions.

Proteomic Analysis of Brassica Stigmatic Proteins Following the Self-incompatibility Reaction Reveals a Role for Microtubule Dynamics During Pollen Responses

Mate selection and maintenance of genetic diversity is crucial to successful reproduction and species survival. Plants utilize self-incompatibility system as a genetic barrier to prevent self pollen from developing on the pistil, leading to hybrid vigor and diversity. In Brassica (canola, kale, and broccoli), an allele-specific interaction between the pollen SCR/SP11 (S-locus cysteine rich protein/S locus protein 11) and the pistil S Receptor Kinase, results in the activation of SRK which recruits the Arm Repeat Containing 1 (ARC1) E3 ligase to the proteasome. The targets of Arm Repeat Containing 1 are proposed to be compatibility factors, which when targeted for degradation by Arm Repeat Containing 1 results in pollen rejection. Despite the fact that protein degradation is predicted to be important for successful self-pollen rejection, the identity of the various proteins whose abundance is altered by the SI pathway has remained unknown. To identify potential candidate proteins regulated by the SI response, we have used the two-dimensional difference gel electrophoresis analysis, coupled with matrix-assisted laser desorption ionization/time of flight/MS. We identified 56 differential protein spots with 19 unique candidate proteins whose abundance is down-regulated following self-incompatible pollinations. The identified differentials are predicted to function in various pathways including biosynthetic pathways, signaling, cytoskeletal organization, and exocytosis. From the 19 unique proteins identified, we investigated the role of tubulin and the microtubule network during both self-incompatible and compatible pollen responses. Moderate changes in the microtubule network were observed with self-incompatible pollinations; however, a more distinct localized break-down of the microtubule network was observed during compatible pollinations, that is likely mediated by EXO70A1, leading to successful pollination.