A role for inositol 1,4,5-trisphosphate in gravitropic signaling and the retention of cold-perceived gravistimulation of oat shoot pulvini

Plants sense positional changes relative to the gravity vector. To date, the signaling processes by which the perception of a gravistimulus is linked to the initiation of differential growth are poorly defined. We have investigated the role of inositol 1,4,5-trisphosphate (InsP(3)) in the gravitropic response of oat (Avena sativa) shoot pulvini. Within 15 s of gravistimulation, InsP(3) levels increased 3-fold over vertical controls in upper and lower pulvinus halves and fluctuated in both pulvinus halves over the first minutes. Between 10 and 30 min of gravistimulation, InsP(3) levels in the lower pulvinus half increased 3-fold over the upper. Changes in InsP(3) were confined to the pulvinus and were not detected in internodal tissue, highlighting the importance of the pulvinus for both graviperception and response. Inhibition of phospholipase C blocked the long-term increase in InsP(3), and reduced gravitropic bending by 65%. Short-term changes in InsP(3) were unimpaired by the inhibitor. Gravitropic bending of oat plants is inhibited at 4 degrees C; however, the plants retain the information of a positional change and respond at room temperature. Both short- and long-term changes in InsP(3) were present at 4 degrees C. We propose a role for InsP(3) in the establishment of tissue polarity during the gravitropic response of oat pulvini. InsP(3) may be involved in the retention of cold-perceived gravistimulation by providing positional information in the pulvini prior to the redistribution of auxin.     

Increased levels of reactive oxygen species and expression of a cytoplasmic aconitase/iron regulatory protein 1 homolog during the early response of maize pulvini to gravistimulation

The maize (Zea mays L.) stem pulvinus is a disc of tissue located apical to each node that functions to return a tipped stem to a more upright position via increased cell elongation on its lower side. We investigated the possibility that reactive oxygen species (ROS) and hydrogen peroxide (H2O2), in particular, are involved in the gravitropic response of the pulvinus prior to initiation of the growth response by employing the cytochemical stain 3,3'-diaminobenzidine (DAB). DAB polymers were found in the bundle sheath cells of gravistimulated pulvini in association with amyloplasts after 1 min of gravistimulation, and the signal spread throughout the cytosol of these cells by 30 min. Furthermore, treatment of maize stem explants containing pulvini with 1 mm H2O2 on their upper sides caused reversal of bending polarity. Similar, though less dramatic, results were obtained via application of 1 mm ascorbic acid to the lower side of the explants. In addition, we determined that a maize cytoplasmic aconitase/iron regulatory protein 1 (IRP1) homolog is up-regulated in the pulvinus bundle sheath cells after gravistimulation using suppressive subtractive hybridization PCR (SSH PCR), real-time RT-PCR and in situ hybridization. Although we do not yet know the role of the IRP1 homolog in the pulvinus, the protein is known to be a redox sensor in other systems. Collectively, our results point to an increase in ROS quite early in the gravitropic signalling pathway and its possible role in determining the direction of bending of the pulvini. We speculate that an ROS burst may serve to link the physical phenomenon of amyloplast sedimentation to the changes in cellular biochemistry and gene expression that facilitate directional growth.     

TpMRK regulates cell division of Tetrahymena in response to oxidative stress

TpMRK was identified as a stress‐responsive mitogen activated protein kinase (MAPK)‐related kinase and has been shown to play a critical role in the stress signalling in Tetrahymena cells. Here, we found that the mRNA expression of TpMRK was correlated with cell division of Tetrahymena with decreased expression occurring in cells prior to entering synchronous cell division induced by heat treatment. Notably, cell division was delayed with a lower division index of 40% after exposure to hydrogen peroxide while 85% of cells underwent cell division synchronously at 75 min after heat treatment without the oxidative exposure. Furthermore, inactivation of TpMRK signalling by p38 MAPK inhibitor SB203580 or MEK inhibitor PD 98059 partially derepressed cell division induced by hydrogen peroxide. Our data suggest that oxidative stimuli might cause aberration of synchronous cell division of Tetrahymena through activating the TpMRK cascade. Copyright © 2009 John Wiley & Sons, Ltd.     

Growth dynamics and cytoskeleton organization during stem maturation and gravity-induced stem bending in Zea mays L.

Characterization of gravitropic bending in the maize stem pulvinus, a tissue that functions specifically in gravity responses, demonstrates that the pulvinus is an ideal system for studying gravitropism. Gravistimulation during the second of three developmental phases of the pulvinus induces a gradient of cell elongation across the non-growing cells of the pulvinus, with the most elongation occurring on the lower side. This cell elongation is spatially and temporally separated from normal internodal cell elongation. The three characterized growth phases in the pulvinus correspond closely to a specialized developmental sequence in which structural features typical of cells not fully matured are retained while cell maturation occurs in surrounding internodal and nodal tissue. For example, the lignification of supporting tissue and rearrangement of transverse microtubules to oblique that occur in the internode when cell elongation ceases are delayed for up to 10 d in the adjacent cells of the pulvinus, and only occurs as a pulvinus loses its capacity to respond to gravistimulation. Gravistimulation does not modify this developmental sequence. Neither wall lignification nor rearrangement of transverse microtubules occurs in the rapidly elongating lower side or non-responsive upper side of the pulvinus until the pulvinus loses the capacity to bend further. Gravistimulation does, however, lead to the formation of putative pit fields within the expanding cells of the pulvinus. Received: 18 April 1998 / Accepted: 2 July 1998     

Gravistimulation leads to asymmetry of both auxin and gibberellin levels in barley pulvini

The auxin indole-3-acetic acid (IAA) is known to promote the biosynthesis of active gibberellins (GAs) in barley ( Hordeum vulgare ). We therefore investigated the possibility that this interaction might contribute to the gravitropic response of barley leaf sheath pulvini. Barley plants at the inflorescence stage were gravistimulated for varying times, and the pulvini were then separated into upper and lower halves for quantification of IAA and GAs by GC-MS. Consistent with the Cholodny–Went theory, the lower portion contained more IAA than did the upper portion. This difference was detected as early as 2.5 h after the start of gravistimulation, and bending was also observed at this stage. At later time points tested (6 h and 24 h), but not at 2.5 h or 3 h, the higher auxin content of the lower half was associated with a higher level of GA₁, the main bioactive GA in barley. Consistent with that result, the expression of Hv3ox2 , which encodes a key enzyme for the conversion of GA₂₀ to GA₁, was higher in the lower side than in the upper, after 6 h. It is suggested that in gravistimulated leaf sheath pulvini, auxin accumulates in the lower side, leading to a higher level of GA₁, which contributes to the bending response. Further evidence that GAs play a role in the gravitropic response was obtained from GA-related mutants, including the elongated sln1c mutant, in which GA signalling is constitutive. Pulvinar bending in the sln1c mutant was greater than in the wild-type. This result indicates that in the lower side of the gravistimulated pulvinus, the relatively high level of bioactive GA facilitates, but does not mediate, the bending response.     

Molecular mechanisms involved in the regulation of cytokine production by muramyl dipeptide

MDP (muramyl dipeptide), a component of peptidoglycan, interacts with NOD2 (nucleotide-binding oligomerization domain 2) stimulating the NOD2-RIP2 (receptor-interacting protein 2) complex to activate signalling pathways important for antibacterial defence. Here we demonstrate that the protein kinase activity of RIP2 has two functions, namely to limit the strength of downstream signalling and to stabilize the active enzyme. Thus pharmacological inhibition of RIP2 kinase with either SB 203580 [a p38 MAPK (mitogen-activated protein kinase) inhibitor] or the Src family kinase inhibitor PP2 induces a rapid and drastic decrease in the level of the RIP2 protein, which may explain why these RIP2 inhibitors block MDP-stimulated downstream signalling and the production of IL-1beta (interleukin-1beta) and TNFalpha (tumour necrosis factor-alpha). We also show that RIP2 induces the activation of the protein kinase TAK1 (transforming-growth-factor-beta-activated kinase-1), that a dominant-negative mutant of TAK1 inhibits RIP2-induced activation of JNK (c-Jun N-terminal kinase) and p38alpha MAPK, and that signalling downstream of NOD2 or RIP2 is reduced by the TAK1 inhibitor (5Z)-7-oxozeaenol or in TAK1-deficient cells. We also show that MDP activates ERK1 (extracellular-signal-regulated kinase 1)/ERK2 and p38alpha MAPK in human peripheral-blood mononuclear cells and that the activity of both MAPKs and TAK1 are required for MDP-induced signalling and production of IL-1beta and TNFalpha in these cells. Taken together, our results indicate that the MDP-NOD2/RIP2 and LPS (lipopolysaccharide)-TLR4 (Toll-like receptor 4) signalling pathways converge at the level of TAK1 and that many subsequent events that lead to the production of pro-inflammatory cytokines are common to both pathways.     

Parathyroid hormone synergizes with non‐cyclic AMP pathways to activate the cyclic AMP response element

Parathyroid hormone (PTH) activates multiple signaling pathways following binding to the PTH1 receptor in osteoblasts. Previous work revealed a discrepancy between cAMP stimulation and CRE reporter activation of truncated PTH peptides, suggesting that additional signaling pathways contribute to activation of the CRE. Using a CRE‐Luciferase reporter containing multiple copies of the CRE stably transfected into the osteoblastic cell line Saos‐2, we tested the ability of modulators of alternative pathways to activate the CRE or block the PTH‐induced activation of the CRE. Activators of non‐cyclic AMP pathways, that is, EGF (Akt, MAPK, JAK/STAT pathways); thapsigargin (intracellular calcium pathway); phorbol myristate acetate (protein kinase C, PKC pathway) induced minor increases in CRE‐luciferase activity alone but induced dramatic synergistic effects in combination with PTH. The protein kinase A (PKA) inhibitor H‐89 (10 µM) almost completely blocked PTH‐induced activation of the CRE‐reporter. Adenylate cyclase inhibitors SQ 22536 and DDA had profound and time‐dependent biphasic effects on the CRE response. The MAPK inhibitor PD 98059 partially inhibited basal and PTH‐induced CRE activity to the same degree, while the PKC inhibitor bisindolylmaleimide (BIS) had variable effects. The calmodulin kinase II inhibitor KN‐93 had no significant effect on the response to PTH. We conclude that non‐cAMP pathways (EGF pathway, calcium pathway, PKC pathway) converge on, and have synergistic effects on, the response of a CRE reporter to PTH. J. Cell. Biochem. 106: 887–895, 2009. © 2009 Wiley‐Liss, Inc.     

cAMP blocks MAPK activation and sclerotial development via Rap-1 in a PKA-independent manner in Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a filamentous ascomycete phytopathogen able to infect an extremely wide range of cultivated plants. Our previous studies have shown that increases in cAMP levels result in the impairment of the development of the sclerotium, a highly differentiated structure important in the disease cycle of this fungus. cAMP also inhibits the activation of a S. sclerotiorum mitogen-activated protein kinase (MAPK), which we have previously shown to be required for sclerotial maturation; thus cAMP-mediated sclerotial inhibition is modulated through MAPK. However, the mechanism(s) by which cAMP inhibits MAPK remains unclear. Here we demonstrate that a protein kinase A (PKA)-independent signalling pathway probably mediates MAPK inhibition by cAMP. Expression of a dominant negative form of Ras, an upstream activator of the MAPK pathway, also inhibited sclerotial development and MAPK activation, suggesting that a conserved Ras/MAPK pathway is required for sclerotial development. Evidence from bacterial toxins that specifically inhibit the activity of small GTPases, suggested that Rap-1 or Ras is involved in cAMP action. The Rap-1 inhibitor, GGTI-298, restored MAPK activation in the presence of cAMP, further suggesting that Rap-1 is responsible for cAMP-dependent MAPK inhibition. Importantly, inhibition of Rap-1 is able to restore sclerotial development blocked by cAMP. Our results suggest a novel mechanism involving the requirement of Ras/MAPK pathway for sclerotial development that is negatively regulated by a PKA-independent cAMP signalling pathway. Cross-talk between these two pathways is mediated by Rap-1.     

Resting and action potentials of excitabl cells in the main pulvinus of Mimosa pudica

Resting and action potentials of excitable cells in the motortissue of the main pulvinus of Mimosa pudica, recorded withan intracellular microelectrode, were –150 and 110 mV,respectively. The excitable cells were found in both upper andlower halves of the main pulvinus. (Received May 20, 1976; )     

Change in potassium distribution in a Phaseolus pulvinus during circadian movement of the leaf

Phaseolus moves its leaves upward and downward with circadianperiod. This movement of the leaf results from the differentialchange in the turgor on opposite sides of the pulvinus. Concentrations of K⁺, Na⁺, Mg⁺⁺, and Ca⁺⁺ in the upper and lowerhalves of the pulvinus and the water content of cells on bothsides of it were analyzed in relation to the deformation ofthe pulvinus. The results showed that (1) the pulvinus was deformedby expansion and contraction of the cells on its opposite sides;(2) among the four cations, the K⁺ concentration was markedlyhigh in both halves of the pulvinus; (3) the osmotic pressureof the upper and lower halves were nearly equal during the rhythmicdeformation of the pulvinus; (4) the expansion and contractionof the cells on the opposite sides of the pulvinus have a positivecorrelation only with a change in the K+ concentration expressedin terms of µmoles per mg protein; (5) the concentrationsof other cations such as Na⁺, Mg⁺⁺, Ca⁺⁺, expressed in termsof µmoles per mg protein, did not change during the circadiandeformation of the pulvinus. Thus, the rhythmic K⁺ movementseems to be the basis for pulvinar turgor movements. With respectto the mechanism of K⁺ movement, three possibilities are discussed. (Received November 7, 1975; )     

Gibberellins and Gravitropism in Maize Shoots : Endogenous Gibberellin-Like Substances and Movement and Metabolism of [3H]Gibberellin A20

[³H]Gibberellin A₂₀ (GA₂₀) of high specific radioactivity (49.9 gigabecquerel per millimole) was applied equilaterally in a ring of microdrops to the internodal pulvinus of shoots of 3-week-old gravistimulated and vertical normal maize (Zea mays L.), and to a pleiogravitropic (prostrate) maize mutant, lazy (la). All plants converted the [³H]GA₂₀ to [³H]GA₁⁻ and [³H]GA₂₉-like metabolites as well as to several metabolites with the partitioning and chromatographic behavior of glucosyl conjugates of [³H]GA₁, [³H]GA₂₉, and [³H]GA₈. The tentative identification of these putative [³H]GA glucosyl conjugates was further supported by the release of the free [³H]GA moiety after cleavage with cellulase. Within 12 hours of the [³H]GA₂₀ feed, there was a significantly higher proportion of total radioactivity in lower than in upper halves of internode and leaf sheath pulvini in gravistimulated normal maize. Further, there was a significantly higher proportion of putative free GA metabolites of [³H]GA₂₀, especially [³H]GA₁, in the lower halves of normal maize relative to upper halves. The differential localization of the metabolites between upper and lower halves was not apparent in the pleiogravitropic mutant, la. Endogenous GA-like substances were also examined in gravistimulated maize shoots. Forty-eight hours after gravistimulation of 3-week-old maize seedlings, endogenous free GA-like substances in upper and lower leaf sheath and internode pulvini halves were extracted, chromatographed, and bioassayed using the `Tanginbozu' dwarf rice microdrop assay. Lower halves contained consistently higher total levels of GA-like activity. The qualitative elution profile of GA-like substances differed consistently, upper halves containing principally a GA₂₀-like substance and lower halves containing mainly GA₁-like and GA₁₉-like substances. Gibberellins A₁ (10 nanograms per gram) and A₂₀ (5 nanograms per gram) were identified from these lower leaf sheath pulvini by capillary gas chromatography-selected ion monitoring. Results from all of these experiments are consistent with a role for GAs in the differential shoot growth that follows gravitropism, although the results do not eliminate the possibility that the redistribution of GAs results from the gravitropic response.     

FAK contributes to proteinuria in hypercholesterolaemic rats and modulates podocyte F‐actin re‐organization via activating p38 in response to ox‐LDL

Focal adhesion kinase (FAK) is a non‐receptor protein tyrosine kinase that regulates cell adhesion, proliferation and differentiation. In the present study, a rat model of high fat diet‐induced hypercholesterolaemia was established to investigate the involvement of FAK in lipid disorder‐related kidney diseases. We showed focal fusion of podocyte foot process that occurred at as early as 4 weeks in rats consuming high fat diet, preceding the onset of proteinuria when aberrant phosphorylation of FAK was found. These abnormalities were ameliorated by dietary intervention of TAE226, a reported inhibitor of FAK. FAK is also an adaptor protein initiating cascades of intracellular signals including c‐Src, Rho GTPase and mitogen‐activated protein kinase (MAPK). P38 MAPK belongs to the latter and is centrally involved in kidney diseases. Our cell culture data revealed oxidized low‐density lipoprotein (ox‐LDL) triggered hyper‐phosphorylation of FAK and p38, ectopic expression of cellular markers (manifested as decreased WT1, podocin and NEPH1, and increased vimentin and mmp9), and re‐arrangement of F‐actin filaments with enhanced cell motility; these mutations were significantly rectified by FAK shRNA. Notably, pre‐treatment of p38 inhibitor did not alter FAK activation, albeit its deletion of p38 hyper‐activity and attenuation of cellular abnormalities, demonstrating that p38 acted as a downstream effector of FAK signalling and ox‐LDL damaged podocytes in a FAK/p38‐dependent manner. This was further identified by animal data that p38 activation was also abrogated by TAE226 treatment in hypercholesterolaemic rats, suggesting that FAK/p38 axis might also be involved in in vivo events. These findings provided a potential early mechanism of hypercholesterolaemia‐related podocyte damage and proteinuria.