Functional analysis of the anaphase promoting complex activator CCS52A highlights the crucial role of endo‐reduplication for fruit growth in tomato

Tomato fruit growth is characterized by the occurrence of numerous rounds of DNA endo‐reduplication in connection with cell expansion and final fruit size determination. Endo‐reduplication is an impairment of mitosis that originates from the selective degradation of M phase‐specific cyclins via the ubiquitin‐mediated proteolytic pathway, requiring the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Two types of APC/C activators, namely CCS52 and CDC20 proteins, exist in plants. We report here the molecular characterization of such APC/C activators during fruit development, and provide an in planta functional analysis of SlCCS52A, a gene that is specifically associated with endo‐reduplication in tomato. Altering SlCCS52A expression in either a negative or positive manner had an impact on the extent of endo‐reduplication in fruit, and fruit size was reduced in both cases. In SlCCS52A over‐expressing fruits, endo‐reduplication was initially delayed, accounting for the altered final fruit size, but resumed and was even enhanced at 15 days post anthesis (dpa), leading to fruit growth recovery. This induction of growth mediated by endo‐reduplication had a considerable impact on nitrogen metabolism in developing fruits. Our data contribute to unravelling of the physiological role of endo‐reduplication in growth induction during tomato fruit development.     

Both FGF23 and extracellular phosphate activate Raf/MEK/ERK pathway via FGF receptors in HEK293 cells

Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone produced by bone and exerts its function in the target organs by binding the FGF receptor (FGFR) and Klotho. Since recent studies suggested that extracellular inorganic phosphate (Pi) itself triggers signal transduction and regulates gene expression in some cell types, we tested the notion that extracellular Pi induces signal transduction in the target cells of FGF23 also and influences its signaling, utilizing a human embryonic kidney cell line HEK293. HEK293 cells expressed low levels of klotho, and treatment with a recombinant FGF23[R179Q], a proteolysis‐resistant mutant of FGF23, resulted in phosphorylation of ERK1/2 and induction of early growth response‐1 (EGR1) expression. Interestingly, increased extracellular Pi resulted in activation of the Raf/MEK/ERK pathway and expression of EGR1, which involved type III sodium/phosphate (Na⁺/Pi) cotransporter PiT‐1. Since the effects of an inhibitor of Na⁺/Pi cotransporter on FGF23 signaling suggested that the signaling triggered by increased extracellular Pi shares the same downstream cascade as FGF23 signaling, we further investigated their convergence point. Increasing the extracellular Pi concentration resulted in the phosphorylation of FGF receptor substrate 2α (FRS2α), as did treatment with FGF23. Knockdown of FGFR1 expression diminished the phosphorylation of both FRS2α and ERK1/2 induced by the Pi. Moreover, overexpression of FGFR1 rescued the decrease in Pi‐induced phosphorylation of ERK1/2 in the cells where the expression of PiT‐1 was knocked down. These results suggest that increased extracellular Pi triggers signal transduction via PiT‐1 and FGFR and influences FGF23 signaling in HEK293 cells. J. Cell. Biochem. 111: 1210–1221, 2010. © 2010 Wiley‐Liss, Inc.     

Chemical genetic identification of a lectin receptor kinase that transduces immune responses and interferes with abscisic acid signaling

Insight into how plants simultaneously cope with multiple stresses, for example, when challenged with biotic stress from pathogen infection and abiotic stress from drought, is important both for understanding evolutionary trade‐offs and optimizing crop responses to these stresses. Mechanisms by which initial plant immune signaling antagonizes abscisic acid (ABA) signal transduction require further investigation. Using a chemical genetics approach, the small molecule [5‐(3,4‐dichlorophenyl)furan‐2‐yl]‐piperidine‐1‐ylmethanethione (DFPM) has previously been identified due to its ability to suppress ABA signaling via plant immune signaling components. Here, we have used forward chemical genetics screening to identify DFPM‐insensitive loci by monitoring the activity of ABA‐inducible pRAB18::GFP in the presence of DFPM and ABA. The ability of DFPM to attenuate ABA signaling was reduced in rda mutants (resistant to DFPM inhibition of ABA signaling). One of the mutants, rda2, was mapped and is defective in a gene encoding a lectin receptor kinase. RDA2 functions in DFPM‐mediated inhibition of ABA‐mediated reporter expression. RDA2 is required for DFPM‐mediated activation of immune signaling, including phosphorylation of mitogen‐activated protein kinase (MAPK) 3 (MPK3) and MPK6, and induction of immunity marker genes. Our study identifies a previously uncharacterized receptor kinase gene that is important for DFPM‐mediated immune signaling and inhibition of ABA signaling. We demonstrate that the lectin receptor kinase RDA2 is essential for perceiving the DFPM signal and activating MAPKs, and that MKK4 and MKK5 are required for DFPM interference with ABA signal transduction.     

Plant stress surveillance monitored by ABA and disease signaling interactions

Abiotic and biotic stresses are the major factors that negatively impact plant growth. In response to abiotic environmental stresses such as drought, plants generate resistance responses through abscisic acid (ABA) signal transduction. In addition to the major role of ABA in abiotic stress signaling, ABA signaling was reported to downregulate biotic stress signaling. Conversely recent findings provide evidence that initial activation of plant immune signaling inhibits subsequent ABA signal transduction. Stimulation of effector-triggered disease response can interfere with ABA signal transduction via modulation of internal calcium-dependent signaling pathways. This review overviews the interactions of abiotic and biotic stress signal transduction and the mechanism through which stress surveillance system operates to generate the most efficient resistant traits against various stress condition.     

OsCERK1 and OsRLCK176 play important roles in peptidoglycan and chitin signaling in rice innate immunity

Microbe‐associated molecular pattern (MAMP)‐triggered immunity plays critical roles in the basal resistance defense response in plants. Chitin and peptidoglycan (PGN) are major molecular patterns for fungi and bacteria, respectively. Two rice (Oryza sativa) lysin motif‐containing proteins, OsLYP4 and OsLYP6, function as receptors that sense bacterial PGN and fungal chitin. These membrane receptors, which lack intracellular kinase domains, likely contain another component for transmembrane immune signal transduction. Here, we demonstrate that the rice LysM receptor‐like kinase OsCERK1, a key component of the chitin elicitor signaling pathway, also plays an important role in PGN‐triggered immunity in rice. Silencing of OsCERK1 suppressed PGN‐induced (and chitin‐induced) immunity responses, including reactive oxygen species generation, defense gene expression, and callose deposition, indicating that OsCERK1 is essential for both PGN and chitin signaling initiated by OsLYP4 and OsLYP6. OsLYP4 associated with OsLYP6 and the rice chitin receptor chitin oligosaccharide elicitor‐binding protein (CEBiP) in the absence of PGN or chitin, and treatment with PGN or chitin led to their disassociation in vivo. OsCERK1 associated with OsLYP4 or OsLYP6 when induced by PGN but it associated with OsLYP4, OsLYP6, or CEBiP under chitin treatment, suggesting the presence of different patterns of ligand‐induced heterooligomeric receptor complexes. Furthermore, the receptor‐like cytoplasmic kinase OsRLCK176 functions downstream of OsCERK1 in the PGN and chitin signaling pathways, suggesting that these MAMPs share overlapping intracellular signaling components. Therefore, OsCERK1 plays dual roles in PGN and chitin signaling in rice innate immunity and as an adaptor involved in signal transduction at the plasma membrane in conjunction with OsLYP4 and OsLYP6.     

Role of the JAK/STAT signal transduction pathway in the regulation of gene expression in CNS

Over the last 20 years the JAK/STAT signal transduction pathway has been extensively studied. An enormous amount of data on different cell signal transduction pathways is now available. The JAK/STAT signal transduction pathway is one of the intracellular signaling pathways activated by cytokines and growth factors that was first studied in the hematopoietic system, but recent data demonstrate that this signal transduction is also greatly utilized by other systems. The JAK/STAT pathway is a signaling cascade that links the activation of specific cell membrane receptors to nuclear gene expression. This review is focused on the role of JAK/STAT signal transduction pathway activation in the central nervous system (CNS).     

Signal transduction during cold, salt, and drought stresses in plants

Abiotic stresses, especially cold, salinity and drought, are the primary causes of crop loss worldwide. Plant adaptation to environmental stresses is dependent upon the activation of cascades of molecular networks involved in stress perception, signal transduction, and the expression of specific stress-related genes and metabolites. Plants have stress-specific adaptive responses as well as responses which protect the plants from more than one environmental stress. There are multiple stress perception and signaling pathways, some of which are specific, but others may cross-talk at various steps. In this review article, we first expound the general stress signal transduction pathways, and then highlight various aspects of biotic stresses signal transduction networks. On the genetic analysis, many cold induced pathways are activated to protect plants from deleterious effects of cold stress, but till date, most studied pathway is ICE-CBF-COR signaling pathway. The Salt-Overly-Sensitive (SOS) pathway, identified through isolation and study of the sos1, sos2, and sos3 mutants, is essential for maintaining favorable ion ratios in the cytoplasm and for tolerance of salt stress. Both ABA-dependent and -independent signaling pathways appear to be involved in osmotic stress tolerance. ROS play a dual role in the response of plants to abiotic stresses functioning as toxic by-products of stress metabolism, as well as important signal transduction molecules and the ROS signaling networks can control growth, development, and stress response. Finally, we talk about the common regulatory system and cross-talk among biotic stresses, with particular emphasis on the MAPK cascades and the cross-talk between ABA signaling and biotic signaling.     

Wnt meets Warburg: another piece in the puzzle?

One of the most common abnormalities of cancer cells is their predilection to engage in a high rate of glycolysis despite the continued availability of oxygen. First described by Otto Warburg, this phenomenon of aerobic glycolysis (or the Warburg effect) has recently been proposed to result from cancer‐associated alterations in signal transduction pathways. In this issue of The EMBO Journal, Pate et al provide further support for this hypothesis by demonstrating that Wnt signaling plays an important role in establishing aerobic glycolysis as a mechanism to support in vivo cancer cell proliferation.     

Large‐scale proteomic analysis of tyrosine‐phosphorylation induced by T‐cell receptor or B‐cell receptor activation reveals new signaling pathways

Activation of the T‐cell receptor (TCR) and that of the B‐cell receptor (BCR) elicits tyrosine‐phosphorylation of proteins that belongs to similar functional categories, but result in distinct cellular responses. Large‐scale analyses providing an overview of the signaling pathways downstream of TCR or BCR have not been described, so it has been unclear what components of these pathways are shared and which are specific. We have now performed a systematic analysis and provide a comprehensive list of tyrosine‐phosphorylated proteins (PY proteome) with quantitative data on their abundance in T cell, B cell, and nonlymphoid cell lines. Our results led to the identification of novel tyrosine‐phosphorylated proteins and signaling pathways not previously implicated in immunoreceptor signal transduction, such as clathrin, zonula occludens 2, eukaryotic translation initiation factor 3, and RhoH, suggesting that TCR or BCR signaling may be linked to downstream processes such as endocytosis, cell adhesion, and translation. Thus comparative and quantitative studies of tyrosine‐phosphorylation have the potential to expand knowledge of signaling networks and to promote understanding of signal transduction at the system level.     

An Arabidopsis kinase cascade influences auxin‐responsive cell expansion

Mitogen‐activated protein kinase (MPK) cascades are conserved mechanisms of signal transduction across eukaryotes. Despite the importance of MPK proteins in signaling events, specific roles for many Arabidopsis MPK proteins remain unknown. Multiple studies have suggested roles for MPK signaling in a variety of auxin‐related processes. To identify MPK proteins with roles in auxin response, we screened mpk insertional alleles and identified mpk1‐1 as a mutant that displays hypersensitivity in auxin‐responsive cell expansion assays. Further, mutants defective in the upstream MAP kinase kinase MKK3 also display hypersensitivity in auxin‐responsive cell expansion assays, suggesting that this MPK cascade affects auxin‐influenced cell expansion. We found that MPK1 interacts with and phosphorylates ROP BINDING PROTEIN KINASE 1 (RBK1), a protein kinase that interacts with members of the Rho‐like GTPases from Plants (ROP) small GTPase family. Similar to mpk1‐1 and mkk3‐1 mutants, rbk1 insertional mutants display auxin hypersensitivity, consistent with a possible role for RBK1 downstream of MPK1 in influencing auxin‐responsive cell expansion. We found that RBK1 directly phosphorylates ROP4 and ROP6, supporting the possibility that RBK1 effects on auxin‐responsive cell expansion are mediated through phosphorylation‐dependent modulation of ROP activity. Our data suggest a MKK3 ? MPK1 ? RBK1 phosphorylation cascade that may provide a dynamic module for altering cell expansion.     

Distinct UV-B and UV-A/blue light signal transduction pathways induce chalcone synthase gene expression in Arabidopsis cells.

UV and blue light control the expression of flavonoid biosynthesis genes in a range of higher plants. To investigate the signal transduction processes involved in the induction of chalcone synthase (CHS) gene expression by UV-B and UV-A/blue light, we examined the effects of specific agonists and inhibitors of known signaling components in mammalian systems in a photomixotrophic Arabidopsis cell suspension culture. CHS expression is induced specifically by these wavelengths in the cell culture, in a manner similar to that in mature Arabidopsis leaf tissue. Both the UV-B and UV-A/blue phototransduction processes involve calcium, although the elevation of cytosolic calcium is insufficient on its own to stimulate CHS expression. The UV-A/blue light induction of CHS expression does not appear to involve calmodulin, whereas the UV-B response does; this difference indicates that the signal transduction pathways are, at least in part, distinct. We provide evidence that both pathways involve reversible protein phosphorylation and require protein synthesis. The UV-B and UV-A/blue light signaling pathways are therefore different from the phytochrome signal transduction pathway regulating CHS expression in other species.     

植物PP2C蛋白磷酸酶ABA信号转导及逆境胁迫调控机制研究进展

蛋白磷酸酶(protein phosphatase,PP)是蛋白质可逆磷酸化调节机制中的关键酶,而PP2C磷酸酶是一类丝氨酸/苏氨酸残基蛋白磷酸酶,是高等植物中最大的蛋白磷酸酶家族,包含76个家族成员,广泛存在于生物体中。迄今为止,在植物体内已经发现了4种PP2C蛋白磷酸酶。蛋白激酶和蛋白磷酸酶协同催化蛋白质可逆磷酸化,在植物体内信号转导和生理代谢中起着重要的调节作用,蛋白质的磷酸化几乎存在于所有的信号转导途径中。大量研究表明,PP2Cs参与多条信号转导途径,包括PP2C参与ABA调控,对干旱、低温、高盐等逆境胁迫的响应,参与植物创伤和种子休眠或萌发等信号途径,其调控机制不同,但酶催化活性都依赖于Mg2+或Mn2+的浓度。植物PP2C蛋白的C端催化结构域高度保守,而N端功能各异。文中还综述了高等植物PP2C的分类、结构、ABA受体与PP2Cs蛋白互作、PP2C基因参与ABA信号途径以及其他逆境信号转导途径的研究进展。     

Wnt/β‐catenin signaling in the mouse embryonic cranial mesenchyme is required to sustain the emerging differentiated meningeal layers

Cranial neural crest cells (CNCCs) give rise to cranial mesenchyme (CM) that differentiates into the forebrain meningeal progenitors in the basolateral and apical regions of the head. This occurs in close proximity to the other CNCC‐CM‐derivatives, such as calvarial bone and dermal progenitors. We found active Wnt signaling transduction in the forebrain meningeal progenitors in basolateral and apical populations and in the non‐meningeal CM preceding meningeal differentiation. Here, we dissect the source of Wnt ligand secretion and requirement of Wnt/β‐catenin signaling for the lineage selection and early differentiation of the forebrain meninges. We find persistent canonical Wnt/β‐catenin signal transduction in the meningeal progenitors in the absence of Wnt ligand secretion in the CM or surface ectoderm, suggesting additional sources of Wnts. Conditional mutants for Wntless and β‐catenin in the CM showed that Wnt ligand secretion and Wnt/β‐catenin signaling were dispensable for specification and proliferation of early meningeal progenitors. In the absence of β‐catenin in the CM, we found diminished laminin matrix and meningeal hypoplasia, indicating a structural and trophic role of mesenchymal β‐catenin signaling. This study shows that β‐catenin signaling is required in the CM for maintenance and organization of the differentiated meningeal layers in the basolateral and apical populations of embryonic meninges.     

A novel Axin2 knock‐in mouse model for visualization and lineage tracing of WNT/CTNNB1 responsive cells

Wnt signal transduction controls tissue morphogenesis, maintenance and regeneration in all multicellular animals. In mammals, the WNT/CTNNB1 (Wnt/β‐catenin) pathway controls cell proliferation and cell fate decisions before and after birth. It plays a critical role at multiple stages of embryonic development, but also governs stem cell maintenance and homeostasis in adult tissues. However, it remains challenging to monitor endogenous WNT/CTNNB1 signaling dynamics in vivo. Here, we report the generation and characterization of a new knock‐in mouse strain that doubles as a fluorescent reporter and lineage tracing driver for WNT/CTNNB1 responsive cells. We introduced a multi‐cistronic targeting cassette at the 3′ end of the universal WNT/CTNNB1 target gene Axin2. The resulting knock‐in allele expresses a bright fluorescent reporter (3xNLS‐SGFP2) and a doxycycline‐inducible driver for lineage tracing (rtTA3). We show that the Axin2^{P2A‐rtTA3‐T2A‐3xNLS‐SGFP2} strain labels WNT/CTNNB1 responsive cells at multiple anatomical sites during different stages of embryonic and postnatal development. It faithfully reports the subtle and dynamic changes in physiological WNT/CTNNB1 signaling activity that occur in vivo. We expect this mouse strain to be a useful resource for biologists who want to track and trace the location and developmental fate of WNT/CTNNB1 responsive stem cells in different contexts.     

High-Resolution Fractionation of Signaling Endosomes Containing Different Receptors

Receptor endocytosis is regulated by ligand binding, and receptors may signal after endocytosis in signaling endosomes. We hypothesized that signaling endosomes containing different types of receptors may be distinct from one another and have different physical characteristics. To test this hypothesis, we developed a high-resolution organelle fractionation method based on mass and density, optimized to resolve endosomes from other organelles. Three different types of receptors undergoing ligand-induced endocytosis were localized predominately in endosomes that were resolved from one another using this method. Endosomes containing activated receptor tyrosine kinases (RTKs), TrkA and EGFR, were similar to one another. Endosomes containing p75^NTR (in the tumor necrosis receptor superfamily) and PAC1 (a G-protein-coupled receptor) were distinct from each other and from RTK endosomes. Receptor-specific endosomes may direct the intracellular location and duration of signal transduction pathways to dictate response to signals and determine cell fate.