BSKs are partially redundant positive regulators of brassinosteroid signaling in Arabidopsis

Arabidopsis thaliana brassinosteroid signaling kinases (BSKs) constitute a receptor‐like cytoplasmic kinase sub‐family (RLCK‐XII) with 12 members. Previous analysis demonstrated a positive role for BSK1 and BSK3 in the initial steps of brassinosteroid (BR) signal transduction. To investigate the function of BSKs in plant growth and BR signaling, we characterized T‐DNA insertion lines for eight BSK genes (BSK1–BSK8) and multiple mutant combinations. Simultaneous elimination of three BSK genes caused alterations in growth and the BR response, and the most severe phenotypes were observed in the bsk3,4,7,8 quadruple and bsk3,4,6,7,8 pentuple mutants, which displayed reduced rosette size, leaf curling and enhanced leaf inclination. In addition, upon treatment with 24‐epibrassinolide, these mutants showed reduced hypocotyl elongation, enhanced root growth and alteration in the expression of BR‐responsive genes. Some mutant combinations also showed antagonistic interactions. In support of a redundant function in BR signaling, multiple BSKs interacted in vivo with the BR receptor BRI1, and served as its phosphorylation substrates in vitro. The BIN2 and BIL2 GSK3‐like kinases, which are negative regulators of BR signaling, interacted in vivo with BSKs and phosphorylated them in vitro, probably at different sites to BRI1. This study demonstrates redundant biological functions for BSKs, and suggests the existence of a regulatory link between BSKs and GSK3‐like kinases.     

Two substrates are better than one: dual specificities for Dnmt2 methyltransferases

Dnmt2 enzymes have been widely conserved during evolution and contain all of the signature motifs of DNA (cytosine-5)-methyltransferases; however, the DNA methyltransferase activity of these proteins is comparatively weak and their biochemical and functional properties remain enigmatic. Recent evidence now shows that Dnmt2 has a novel tRNA methyltransferase activity, raising the possibility that the biological roles of these proteins might be broader than previously thought. This finding has important implications for understanding the evolutionary relationships among these enzymes.     

OsCPL3 is involved in brassinosteroid signaling by regulating OsGSK2 stability

Glycogen synthase kinase 3(GSK3) proteins play key roles in brassinosteroid(BR) signaling during plant growth and development by phosphorylating various substrates. However,how GSK3 protein stability and activity are themselves modulated is not well understood.Here, we demonstrate in vitro and in vivo that C-TERMINAL DOMAIN PHOSPHATASELIKE 3(Os CPL3), a member of the RNA Pol II CTD phosphatase-like family, physically interacts with Os GSK2 in rice(Oryza sativa). Os CPL3 expression was widely det...     

Interdependency of brassinosteroid and auxin signaling in Arabidopsis

How growth regulators provoke context-specific signals is a fundamental question in developmental biology. In plants, both auxin and brassinosteroids (BRs) promote cell expansion, and it was thought that they activated this process through independent mechanisms. In this work, we describe a shared auxin:BR pathway required for seedling growth. Genetic, physiological, and genomic analyses demonstrate that response from one pathway requires the function of the other, and that this interdependence does not act at the level of hormone biosynthetic control. Increased auxin levels saturate the BR-stimulated growth response and greatly reduce BR effects on gene expression. Integration of these two pathways is downstream from BES1 and Aux/IAA proteins, the last known regulatory factors acting downstream of each hormone, and is likely to occur directly on the promoters of auxin:BR target genes. We have developed a new approach to identify potential regulatory elements acting in each hormone pathway, as well as in the shared auxin:BR pathway. We show that one element highly overrepresented in the promoters of auxin- and BR-induced genes is responsive to both hormones and requires BR biosynthesis for normal expression. This work fundamentally alters our view of BR and auxin signaling and describes a powerful new approach to identify regulatory elements required for response to specific stimuli.     

Two lipoproteins extracted from Escherichia coli K-12 LCD25 lipopolysaccharide are the major components responsible for Toll-like receptor 2-mediated signaling

Toll-like receptor 2 (TLR2)-mediated cell activation induced by commercial preparations of LPS was recently shown to arise from impurities whose identities are not known. In this work, we determined the molecules responsible for TLR2-mediated cell activation in LPS derived from Escherichia coli K-12 strain LCD25. When LCD25 LPS was phenol extracted, two proteins capable of TLR2-mediated cell activation were purified and identified as E. coli lipoproteins. We cloned, expressed, and purified these two lipoproteins, Lip19 and Lip12. Lip19 or Lip12 activated TNF-alpha production from RAW264.7 and THP-1 cells in a TLR2-dependent manner. However, neither Lip19 nor Lip12 activated HUVECs, which lack endogenous TLR2. Additionally, IkappaB kinase beta and c-Jun N-terminal kinase 1 activation in THP-1 cells induced by Lip19 or Lip12 was observed. TLR2 activation by Lip19 and Lip12 in HEK293 cells was blocked by inhibitory anti-TLR2 mAbs. The unlipidated mutants, Lip19-C19S and Lip12-C21S, in which the NH(2)-terminal cysteine was substituted by serine, lost their ability to activate TLR2-transfected HEK 293 cells. Taken together, these results demonstrate that two lipoproteins constitute the major contaminants responsible for TLR2-mediated cell activation in E. coli LCD25 LPS.     

Determination of substrate specificity and putative substrates of Chk2 kinase

Chk2/hCds1, the human homolog of Saccharomyces cerevisiae Rad53p and Schizosaccharomyces pombe Cds1p, plays a critical role in the DNA damage checkpoint pathway. While several in vivo targets of Chk2 have been identified, the other target proteins of Chk2 responsible for multiple functions, such as cell cycle arrest, DNA repair, and apoptosis, remain to be elucidated. We utilized the GST-peptide approach to identify physiological substrates for Chk2. Mutational analyses using GST-linked Cdc25A containing serine 123 revealed that residues at positions -5 and -3 are critical determinants for the recognition of the Chk2 substrate. We determined the general phosphorylation consensus sequence and identified in vitro targets of Chk2 using GST peptides as substrates. The newly identified in vitro target proteins include Abl1, Bub1R, Bub1, Bub3, Psk-H1, Smc3, Plk1, Cdc25B, Dcamkl1, Mre11, Pms1, and Xrcc9.     

Two membrane juxtaposed signaling modules in ANF-RGC are interlocked

Atrial natriuretic factor (ANF) receptor guanylate cyclase ANF-RGC is a single transmembrane spanning modular protein. Juxtaposed to each side of the transmembrane module is a Cys423-Cys432 disulfide ANF signaling module motif and the ATP-regulated transduction module (ARM) motif. The signaling module motif is conserved in nearly all membrane guanylate cyclases and is believed to be critical in the signaling activities of all membrane guanylate cyclases. The present study with the model system of the olfactory membrane guanylate cyclase shows that this concept is not valid. Furthermore, the study shows that in ANF-GC the signaling motif works through the ARM domain. A new signaling model is proposed where in its natural state the disulfide structural motif represses the ARM domain activity, which, in turn, represses the catalytic module activity of ANF-RGC. ANF signaling relieves the disulfide structural motif restraint on the ARM inhibition and stimulates the catalytic module of the cyclase.     

Attenuation of brassinosteroid signaling enhances FLC expression and delays flowering

A main developmental switch in the life cycle of a flowering plant is the transition from vegetative to reproductive growth. In Arabidopsis thaliana, distinct genetic pathways regulate the timing of this transition. We report here that brassinosteroid (BR) signaling establishes an unexpected and previously unidentified genetic pathway in the floral-regulating network. We isolated two alleles of brassinosteroid-insensitive 1 (bri1) as enhancers of the late-flowering autonomous-pathway mutant luminidependens (ld). bri1 was found to predominantly function as a flowering-time enhancer. Further analyses of double mutants between bri1 and known flowering-time mutants revealed that bri1 also enhances the phenotype of the autonomous mutant fca and of the dominant FRI line. Moreover, all of these double mutants exhibited elevated expression of the potent floral repressor FLOWERING LOCUS C (FLC). This molecular response could be efficiently suppressed by vernalization, leading to accelerated flowering. Additionally, specific reduction of the expression of FLC via RNA interference accelerated flowering in bri1 ld double mutants. Importantly, combining the BR-deficient mutant cpd with ld also resulted in delayed flowering and led to elevated FLC expression. Finally, we found increased histone H3 acetylation at FLC chromatin in bri1 ld mutants, as compared with ld single mutants. In conclusion, we propose that BR signaling acts to repress FLC expression, particularly in genetic situations, with, for example, dominant FRI alleles or autonomous-pathway mutants, in which FLC is activated.     

Genetic evidence that cholera toxin substrates are regulatory components of adenylate cyclase.

Cholera toxin, using [32P]NAD+ as substrate, specifically radiolabels at least two proteins in plasma membranes of wild type S49 mouse lymphoma cells. The toxin-specific substrates are detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as bands corresponding to molecular weights of 45,000 and a doublet of 52,000 to 53,000. Membranes of two other cell types exhibit similar patterns of radiolabeled bands specifically produced by incubation with cholera toxin: the "uncoupled" variant S49 cell, which possesses adenylate cyclase activity unresponsive to hormones, and the HTC4 rat hepatoma cell, which lacks detectable catalytic adenylate cyclase activity but contains components of the cyclase system necessary for regulation by guanyl nucleotides and NaF. Little or no toxin-specific radiolabeling is observed in membranes of a fourth cell type, the adenylate cyclase activity-deficient S49 variant, which functionally lacks components of the cyclase system involved in cholera toxin action and regulation by guanyl nucleotides and NaF. The toxin-specific labeling pattern is not observed in membranes prepared from wild type S49 cells previously treated with cholera toxin in culture. One or both of the toxin substrates thus appears to be involved in regulation of adenylate cyclase by guanyl nucleotides and fluoride ion.     

ROPGAP-dependent interaction between brassinosteroid and ROP2-GTPase signaling controls pavement cell shape in Arabidopsis

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Major components of the KARRIKIN INSENSITIVE2-dependent signaling pathway are conserved in the liverwort Marchantia polymorpha

KARRIKIN INSENSITIVE2 (KAI2) was first identified as a receptor of karrikins, smoke-derived germination stimulants. KAI2 is also considered a receptor of an unidentified endogenous molecule called the KAI2 ligand. Upon KAI2 activation, signals are transmitted through the degradation of D53/SMXL proteins via MAX2-dependent ubiquitination. Although components in the KAI2-dependent signaling pathway, namely MpKAI2A and MpKAI2B, MpMAX2, and MpSMXL, exist in the genome of the liverwort Marchantia polymorpha, their functions remain unknown. Here, we show that early thallus growth is retarded and gemma dormancy in the dark is suppressed in Mpkai2a and Mpmax2 loss-of-function mutants. These defects are counteracted in Mpkai2a Mpsmxl and Mpmax2 Mpsmxl double mutants indicating that MpKAI2A, MpMAX2, and MpSMXL act in the same genetic pathway. Introduction of MpSMXL^d53, in which a domain required for degradation is mutated, into wild-type plants mimicks Mpkai2a and Mpmax2 plants. In addition, the detection of citrine fluorescence in Nicotiana benthamiana cells transiently expressing a SMXL-Citrine fusion protein requires treatment with MG132, a proteasome inhibitor. These findings imply that MpSMXL is subjected to degradation, and that the degradation of MpSMXL is crucial for MpKAI2A-dependent signaling in M. polymorpha. Therefore, we claim that the basic mechanisms in the KAI2-dependent signaling pathway are conserved in M. polymorpha.

Functions of genes in the KARRIKIN INSENSITIVE2-dependent signaling pathway are conserved in the liverwort Marchantia polymorpha and control early development of the thallus.