Specific TCP transcription factors interact with and stabilize PRR2 within different nuclear sub-domains.

Plants possess a large set of transcription factors both involved in the control of plant development or in plant stress responses coordination. We previously identified PRR2, a Pseudo-Response Regulator, as a plant-specific CML-interacting partner. We reported that PRR2 acts as a positive actor of plant defense by regulating the production of antimicrobial compounds. Here, we report new data on the interaction between PRR2 and transcription factors belonging to the Teosinte branched Cycloidea and PCF (TCP) family. TCPs have been described to be involved in plant development and immunity. We evaluated the ability of PRR2 to interact with seven TCPs representative of the different subclades of the family. PRR2 is able to interact with TCP13, TCP15, TCP19 and TCP20 in yeast two-hybrid system and in planta interactions were validated for TCP19 and TCP20. Transient expression in tobacco highlighted that PRR2 protein is more easily detected when co-expressed with TCP19 or TC20. This stabilization is associated with a specific sub-nuclear localization of the complex in Cajal bodies or in nuclear speckles according to the interaction of PRR2 with TCP19 or TCP20 respectively. The interaction between PRR2 and TCP19 or TCP20 would contribute to the biological function in specific nuclear compartments.

The involvement of calmodulin and protein kinases in the upstream of cytosolic and nucleic calcium signaling induced by hypoosmotic shock in tobacco cells.

Changes in Ca2+ concentrations in cytosol ([Ca2+]C) or nucleus ([Ca2+]N) may play some vital roles in plants under hypoosmotic shock (Hypo-OS). Here, we observed that Hypo-OS induces biphasic increases in [Ca2+]C and [Ca2+]N in two tobacco cell lines (BY-2) expressing apoaequorin either in the cytosol or in the nucleus. Both [Ca2+]C and [Ca2+]N were sensitively modulated by the inhibitors of calmodulin and protein kinases, supporting the view that calmodulin suppresses the 1st peaks and and protein kinases enhance 2nd peaks in [Ca2+]C and [Ca2+]N. Data also suggested that the 1st and 2nd events depend on the internal and extracellular Ca2+ sources, respectively.

PRR2, a pseudo-response regulator, promotes salicylic acid and camalexin accumulation during plant immunity.

Calcium signalling mediated by Calmodulin (CaM) and calmodulin-like (CML) proteins is critical to plant immunity. CaM and CML regulate a wide range of target proteins and cellular responses. While many CaM-binding proteins have been identified, few have been characterized for their specific role in plant immunity. Here, we report new data on the biological function of a CML-interacting partner, PRR2 (PSEUDO-RESPONSE REGULATOR 2), a plant specific transcription factor. Until now, the physiological relevance of PRR2 remained largely unknown. Using a reverse genetic strategy in A. thaliana, we identified PRR2 as a positive regulator of plant immunity. We propose that PRR2 contributes to salicylic acid (SA)-dependent responses when challenged with the phytopathogenic bacterium Pseudomonas syringae. PRR2 is transcriptionally upregulated by SA and P. syringae, enhances SA biosynthesis and SA signalling responses; e.g. in response to P. syringae, PRR2 induces the production of SA and the accumulation of the defence-related protein PR1. Moreover, PRR2 overexpressing lines exhibit an enhanced production of camalexin, a phytoalexin that confers enhanced resistance against pathogens. Together, these data reveal the importance of PRR2 in plant immune responses against P. syringae and suggest a novel function for this particular plant specific transcription factor in plant physiology.

Exploration of plant growth and development using the European Modular Cultivation System facility on the International Space Station.

Space experiments provide a unique opportunity to advance our knowledge of how plants respond to the space environment, and specifically to the absence of gravity. The European Modular Cultivation System (EMCS) has been designed as a dedicated facility to improve and standardise plant growth in the International Space Station (ISS). The EMCS is equipped with two centrifuges to perform experiments in microgravity and with variable gravity levels up to 2.0 g. Seven experiments have been performed since the EMCS was operational on the ISS. The objectives of these experiments aimed to elucidate phototropic responses (experiments TROPI-1 and -2), root gravitropic sensing (GRAVI-1), circumnutation (MULTIGEN-1), cell wall dynamics and gravity resistance (Cell wall/Resist wall), proteomic identification of signalling players (GENARA-A) and mechanism of InsP3 signalling (Plant signalling). The role of light in cell proliferation and plant development in the absence of gravity is being analysed in an on-going experiment (Seedling growth). Based on the lessons learned from the acquired experience, three preselected ISS experiments have been merged and implemented as a single project (Plant development) to study early phases of seedling development. A Topical Team initiated by European Space Agency (ESA), involving experienced scientists on Arabidopsis space research experiments, aims at establishing a coordinated, long-term scientific strategy to understand the role of gravity in Arabidopsis growth and development using already existing or planned new hardware.

14-3-3-regulated Ca(2+)-dependent protein kinase CPK3 is required for sphingolipid-induced cell death in Arabidopsis.

In eukaryotic cells, sphingoid long chain bases (LCBs) such as sphingosine or phytosphingosine (PHS) behave as second messengers involved in various processes including programmed cell death (PCD). In plants, induction of PCD by LCBs has now been described, but the signalling pathway is still enigmatic. Using Arabidopsis, we identify new key steps in this pathway. We demonstrate that PHS induces activation of the calcium-dependent kinase CPK3, which phosphorylates its binding partners, the 14-3-3 proteins. This phosphorylation leads to the disruption of the complex and to CPK3 degradation. Using cpk3 knockout lines, we demonstrate that CPK3 is a positive regulator of LCB-mediated PCD. These findings establish 14-3-3-regulated CPK3 as a key component of the LCB pathway leading to PCD in plants.

Changes in gravitational forces induce modifications of gene expression in A. thaliana seedlings.

By comparing the expression patterns of selected genes from Arabidopsis thaliana (L.) Heynh. grown either at 1 g or on a clinostat (horizontally or vertically inverted, 1 rpm), and either used directly or after hypergravity stimulation, we have shown that the pattern of expression did not proceed in a stereotypical manner. Rather, the selected genes fell into different classes. These classes include (i) those insensitive to the gravitational conditions, (ii) those that are regulated in an opposite manner by hypergravity and clinostat conditions, (iii) those that are desensitised to hypergravity by long-term culture on a clinostat, and (iv) those enhanced by such a treatment. Our data suggest that rapid reorientation of gene expression is likely to occur in response to changes in the gravitational conditions.

The nucleus together with the cytosol generates patterns of specific cellular calcium signatures in tobacco suspension culture cells.

Plant cell suspension cultures respond to osmotic changes by alterations in levels of free cellular calcium. Using the aequorin recombinant method, we have measured the spatial and temporal characteristics of calcium signatures in the nucleus and the cytosol of BY-2 tobacco suspension cells challenged with hypo- or hyper-osmotic shock. We show here that the nuclear compartment contributes together with the cytosol to produce calcium signal patterns that discriminate hypo- from hyper-osmotic treatments, i.e. turgor from tension. We also demonstrate that calcium responses in the nucleus and the cytosol are differentially modulated by the strength and the nature of hyper-osmotic treatments. We conclude that qualitative and quantitative changes in the parameters of an external stimulus such as osmotic changes are converted into calcium signatures, distinctive in their temporal and subcellular characteristics, involving both the nucleus and the cytosol. Our results illustrate the versatility of calcium signaling in plant cells. In addition to the physiological 'address' of the cell, the compartmentation of the calcium signal is probably an important parameter in encoding response specificity.

[Basis for the calcium specificity in the plant response to extracellular stimulation]. / Les supports de la spécificité calcique dans la réponse des plantes aux stimuli extracellulaires.

The Ca2+ cation is fully recognized as an important intracellular second messenger coupling a wide range of extracellular stimuli to characteristic responses in plant cells. Such a pleiotropic effect raises questions regarding the mechanisms by which the signalling pathways, all of then involving an increase in intracellular calcium concentration, can be specific to a given stimulus. Here, we present recent results which shed light into different concepts which may explain the response specificity in signalling processes, such as "the cross-talk between signalling pathways", "the Ca2+ signatures" and "the compartmentation of Ca(2+)-signalling".

Plant graviperception and gravitropism: a newcomer's view.

Gravitropism is an adaptable mechanism corresponding to the directed growth by which plants orient in response to the gravity vector. The overall process is generally divided into three distinct stages: graviperception, gravitransduction, and asymmetric growth response. The phenomenology of these different steps has been described by using refined cell biology approaches combined with formal and molecular genetics. To date, it clearly appears that the cellular organization plays crucial roles in gravisensing and that gravitropism is genetically different between organs. Moreover, while interfering with other physical or chemical stimuli and sharing probably some common intermediary steps in the transduction pathway, gravity has its own perception and transduction systems. The intimate mechanisms involved in these processes have to be unveiled at the molecular level and their biological relevance addressed at the cellular and whole plant levels under normal and microgravitational conditions. gravitropism: a newcomer's view.

Plasma membrane depolarization-activated calcium channels, stimulated by microtubule-depolymerizing drugs in wild-type Arabidopsis thaliana protoplasts, display constitutively large activities and a longer half-life in ton 2 mutant cells affected in the organization of cortical microtubules.

Depolarization-activated plasma membrane calcium channels have been suggested to play prominent roles in signal perception and transduction processes during growth and development of higher plants. The existence of such channels has recently been established in higher plant cells. However, patch-clamp experiments have shown that their activity is very low and decreases very rapidly after the establishment of the whole-cell configuration, due most probably to protein-protein interactions involving microtubules. The present study takes advantage of the existence of Arabidopsis thaliana mutants referred to as ton 2 mutants reported to be affected in their microtubule organization, to address the physiological relevance of such a hypothesis based on a pharmacological approach. Patch-clamp studies showed that depolarization-activated calcium channel activities in ton 2 protoplasts were 10-fold higher and their relative half-life three-times longer than in wild-type protoplasts. In addition, oryzalin and colchicine, which disrupt the microtubule organization, stimulated and stabilized calcium channel activities in wild-type but remained ineffective on ton 2 protoplasts. However, although the microtubules appeared important in the regulation of calcium channels in A. thaliana, immunocytological staining of tubulin demonstrated that there was no visible difference in the general organization of microtubule networks or in the amount of microtubules bound to the plasma membrane in ton 2 and wild-type protoplasts. It is suggested that the down-regulation of calcium channels implicating microtubules involves additional component(s) corresponding probably to gene product(s) defective in ton 2 mutant cells.

Organization of cytoskeleton controls the changes in cytosolic calcium of cold-shocked Nicotiana plumbaginifolia protoplasts.

Using Nicotiana plumbaginifolia constitutively expressing the recombinant bioluminescent calcium indicator, aequorin, it has been previously demonstrated that plant cells react to cold-shock by an immediate rise in cytosolic calcium. Such an opportune system has been exploited to address the regulatory pathway involved in the calcium response. For this purpose, we have used protoplasts derived from N. plumbaginifolia leaves that behave as the whole plant but with a better reproducibility. By both immunodetecting cytoskeletal components on membrane ghosts and measuring the relative change in cytosolic calcium, we demonstrate that the organization of the cytoskeleton has profound influences on the calcium response. The disruption of the microtubule meshwork by various active drugs, such as colchicin, oryzalin and vinblastin, leads to an important increase in the cytosolic calcium (up to 400 nM) in cold-shocked protoplasts over control. beta-Lumicolchicin, an inactive analogue of colchicin, is ineffective either on cytoplasmic calcium increase or on microtubule organization. A microfilament disrupting drug, cytochalasin D, exerts a slight stimulatory effect, whereas the simultaneous disruption of microtubule and microfilament meshworks results in a dramatic increase in the calcium response to cold-shock. The results described in the present paper illustrate the role of the intracellular organization and, more specifically, the role of cytoskeleton in controlling the intensity of calcium response to an extracellular stimulus.

Activation of plasma membrane voltage-dependent calcium-permeable channels by disruption of microtubules in carrot cells.

Plasma membrane-bound voltage-dependent calcium channels may couple the perception of an initial stimulus to a regulated pathway for calcium influx. The activities of these channels have been shown to be very low and highly unstable but may be recruited by large-predepolarizing pulses, according to a process referred to as recruitment. By combining pharmacological and electrophysiological approaches, we demonstrate in the present paper that the cytoskeleton plays an important role in the regulation of the activity and stability of voltage-dependent calcium channels during whole-cell patch-clamp experiments on carrot protoplasts. Whereas drugs affecting the organization of the microfilament network have no measurable effect, the manipulation of the microtubule network elicits important changes. Thus, the addition of colchicine or oryzalin, which are known to disrupt microtubule organization, leads to a 6-10-fold increase in calcium channel activities and half-life. In contrast, stabilization of the microtubules by taxol has no effect on any of these parameters. The data obtained suggest that interactions of microtubules and voltage-dependent calcium channels by either direct or indirect mechanisms inhibit channel activities and decrease their half-life. In contrast, the disruption of the network overcomes such an inhibitory effect and allows the activation of calcium channels. It is speculated that under normal physiological conditions these protein-protein interactions may work in a reversible manner and contribute to signal transduction in higher plants.

Expression of membrane targeted aequorin in Xenopus laevis oocytes.

We described here a system for high level of expression of the calcium activated photoprotein aequorin. This protein has been targeted to the plasma membrane of Xenopus oocyte by nuclear microinjection of a plasmid containing a construction of a chimeric cDNA encoding a fusion protein composed of the photoprotein aequorin and the 5-HT1A receptor. The expression of this fusion protein is placed under the control of RSV promoter. Functional photoprotein was reconstituted in the oocyte by incubation with coelenterazine. The amount of photoprotein 24 h after nuclear microinjection of the plasmid was sufficient to trigger a detectable light emission following calcium entry. The efficiency of the expression is correlated with the dose of plasmid injected. Intracytoplasmic injection of the plasmid always failed in photoprotein expression. Targeting of the apoprotein was demonstrated by immunolocalization under confocal microscopy. In our experimental conditions, the apoprotein was always localized at the animal pole above the nucleus. We never observed expression and targeting to the plasma membrane of the vegetal pole. WE suggest that such expression might be of great interest for the study of numerous problems of developmental biology, in which calcium-dependent pathways are involved.