Krisztina Ötvös

Arabidopsis PPR40 Connects Abiotic Stress Responses to Mitochondrial Electron Transport

Oxidative respiration produces adenosine triphosphate through the mitochondrial electron transport system controlling the energy supply of plant cells. Here we describe a mitochondrial pentatricopeptide repeat (PPR) domain protein, PPR40, which provides a signaling link between mitochondrial electron transport and regulation of stress and hormonal responses in Arabidopsis (Arabidopsis thaliana). Insertion mutations inactivating PPR40 result in semidwarf growth habit and enhanced sensitivity to salt, abscisic acid, and oxidative stress. Genetic complementation by overexpression of PPR40 complementary DNA restores the ppr40 mutant phenotype to wild type. The PPR40 protein is localized in the mitochondria and found in association with Complex III of the electron transport system. In the ppr40-1 mutant the electron transport through Complex III is strongly reduced, whereas Complex IV is functional, indicating that PPR40 is important for the ubiqinol-cytochrome c oxidoreductase activity of Complex III. Enhanced stress sensitivity of the ppr40-1 mutant is accompanied by accumulation of reactive oxygen species, enhanced lipid peroxidation, higher superoxide dismutase activity, and altered activation of several stress-responsive genes including the alternative oxidase AOX1d. These results suggest a close link between regulation of oxidative respiration and environmental adaptation in Arabidopsis.

The involvement of reactive oxygen species (ROS) in the cell cycle activation (G0-to-G1 transition) of plant cells

Reactive oxygen species (ROS) are involved in various cellular processes in plants. Among those, resistance to abiotic stress, defence mechanisms and cell expansion have been intensively studied during the last years. We recently demonstrated that ROS, in concert with auxin, have a role in cell cycle activation of differentiated leaf cells.1 In this addendum we provide further evidence to show that oxidative stress/ROS accelerate auxin-mediated cell cycle entry (G0-to-G1 Addendum to: Pasternak TP, Ötvös K, Domoki M, Fehér A. Linked activation of cell division and oxidative stress defense in alfalfa leaf protoplast-derived cells is dependent on exogenous auxin. Plant Growth Regul 2007; 51:109-17.

Plant Rho-type (Rop) GTPase-dependent activation of receptor-like cytoplasmic kinases in vitro.

Plants have evolved distinct mechanisms to link Rho‐type (Rop) GTPases to downstream signaling pathways as compared to other eukaryotes. Here, experimental data are provided that members of the Medicago, as well as Arabidopsis, receptor‐like cytoplasmic kinase family (RLCK Class VI) were strongly and specifically activated by GTP‐bound Rop GTPases in vitro. Deletion analysis indicated that the residues implicated in the interaction might be distributed on various parts of the kinases. Using a chimaeric Rop GTPase protein, the importance of the Rho‐insert region in kinase activation could also be verified. These data strengthen the possibility that RLCKs may serve as Rop GTPase effectors in planta.

The CRYPTOCHROME1-Dependent Response to Excess Light Is Mediated through the Transcriptional Activators ZINC FINGER PROTEIN EXPRESSED IN INFLORESCENCE MERISTEM LIKE1 and ZML2 in Arabidopsis

This work identifies ZML2 and its homolog ZML1 as key regulators of gene expression in the cry1-dependent response to excess light. ZML1/2 bind to the CryR1 cis-element in vitro and in vivo, and T-DNA insertion lines for ZML2 and ZML1 were sensitive to excess light, demonstrating misregulation of several cry1-dependent genes in response to excess light. Exposure of plants to light intensities that exceed the electron utilization capacity of the chloroplast has a dramatic impact on nuclear gene expression. The photoreceptor Cryptochrome 1 (cry1) is essential to the induction of genes encoding photoprotective components in Arabidopsis thaliana. Bioinformatic analysis of the cry1 regulon revealed the putative cis-element CryR1 (GnTCKAG), and here we demonstrate an interaction between CryR1 and the zinc finger GATA-type transcription factors ZINC FINGER PROTEIN EXPRESSED IN INFLORESCENCE MERISTEM LIKE1 (ZML1) and ZML2. The ZML proteins specifically bind to the CryR1 cis-element as demonstrated in vitro and in vivo, and TCTAG was shown to constitute the core sequence required for ZML2 binding. In addition, ZML2 activated transcription of the yellow fluorescent protein reporter gene driven by the CryR1 cis-element in Arabidopsis leaf protoplasts. T-DNA insertion lines for ZML2 and its homolog ZML1 demonstrated misregulation of several cry1-dependent genes in response to excess light. Furthermore, the zml1 and zml2 T-DNA insertion lines displayed a high irradiance-sensitive phenotype with significant photoinactivation of photosystem II (PSII), indicated by reduced maximum quantum efficiency of PSII, and severe photobleaching. Thus, we identified the ZML2 and ZML1 GATA transcription factors as two essential components of the cry1-mediated photoprotective response.

Use of the Foot-and-Mouth Disease Virus 2A Peptide Co-Expression System to Study Intracellular Protein Trafficking in Arabidopsis

Background A tool for stoichiometric co-expression of effector and target proteins to study intracellular protein trafficking processes has been provided by the so called 2A peptide technology. In this system, the 16–20 amino acid 2A peptide from RNA viruses allows synthesis of multiple gene products from single transcripts. However, so far the use of the 2A technology in plant systems has been limited. Methodology/Principal Findings The aim of this work was to assess the suitability of the 2A peptide technology to study the effects exerted by dominant mutant forms of three small GTPase proteins, RABD2a, SAR1, and ARF1 on intracellular protein trafficking in plant cells. Special emphasis was given to CAH1 protein from Arabidopsis, which is trafficking to the chloroplast via a poorly characterized endoplasmic reticulum-to-Golgi pathway. Dominant negative mutants for these GTPases were co-expressed with fluorescent marker proteins as polyproteins separated by a 20 residue self-cleaving 2A peptide. Cleavage efficiency analysis of the generated polyproteins showed that functionality of the 2A peptide was influenced by several factors. This enabled us to design constructs with greatly increased cleavage efficiency compared to previous studies. The dominant negative GTPase variants resulting from cleavage of these 2A peptide constructs were found to be stable and active, and were successfully used to study the inhibitory effect on trafficking of the N-glycosylated CAH1 protein through the endomembrane system. Conclusions/Significance We demonstrate that the 2A peptide is a suitable tool when studying plant intracellular protein trafficking and that transient protoplast and in planta expression of mutant forms of SAR1 and RABD2a disrupts CAH1 trafficking. Similarly, expression of dominant ARF1 mutants also caused inhibition of CAH1 trafficking to a different extent. These results indicate that early trafficking of the plastid glycoprotein CAH1 depends on canonical vesicular transport mechanisms operating between the endoplasmic reticulum and Golgi apparatus.

Immunodetection of retinoblastoma-related protein and its phosphorylated form in interphase and mitotic alfalfa cells

Plant retinoblastoma-related (RBR) proteins are primarily considered as key regulators of G1/S phase transition, with functional roles in a variety of cellular events during plant growth and organ development. Polyclonal antibody against the C-terminal region of the Arabidopsis RBR1 protein also specifically recognizes the alfalfa 115 kDa MsRBR protein, as shown by the antigen competition assay. The MsRBR protein was detected in all cell cycle phases, with a moderate increase in samples representing G2/M cells. Antibody against the human phospho-pRb peptide (Ser807/811) cross-reacted with the same 115 kDa MsRBR protein and with the in vitro phosphorylated MsRBR protein C-terminal fragment. Phospho-MsRBR protein was low in G1 cells. Its amount increased upon entry into the S phase and remained high during the G2/M phases. Roscovitine treatment abolished the activity of alfalfa MsCDKA1;1 and MsCDKB2;1, and the phospho-MsRBR protein level was significantly decreased in the treated cells. Colchicine block increased the detected levels of both forms of MsRBR protein. Reduced levels of the MsRBR protein in cells at stationary phase or grown in hormone-free medium can be a sign of the division-dependent presence of plant RBR proteins. Immunolocalization of the phospho-MsRBR protein indicated spots of variable number and size in the labelled interphase nuclei and high signal intensity of nuclear granules in prophase. Structures similar to phospho-MsRBR proteins cannot be recognized in later mitotic phases. Based on the presented western blot and immunolocalization data, the possible involvement of RBR proteins in G2/M phase regulation in plant cells is discussed.

The phosphomimetic mutation of an evolutionarily conserved serine residue affects the signaling properties of Rho of plants (ROPs)

Plant ROP (Rho of plants) proteins form a unique subgroup within the family of Rho-type small G-proteins of eukaryotes. In this paper we demonstrate that the phosphomimetic mutation of a serine residue conserved in all Rho proteins affects the signaling properties of plant ROPs. We found that the S74E mutation in Medicago ROP6 and Arabidopsis ROP4 prevented the binding of these proteins to their plant-specific upstream activator the plant-specific ROP nucleotide exchanger (PRONE)-domain-containing RopGEF (guanine nucleotide exchange factor) protein and abolished the PRONE-mediated nucleotide exchange reaction in vitro. Structural modeling supported the hypothesis that potential phosphorylation of the S74 residue interferes with the binding of the PRONE-domain to the adjacent plant-specific R76 residue which plays an important role in functional ROP-PRONE interaction. Moreover, we show that while the binding of constitutively active MsROP6 to the effector protein RIC (ROP-interactive CRIB-motif-containing protein) was not affected by the S74E mutation, the capability of this mutated protein to bind and activate the RRK1 kinase in vitro was reduced. These observations are in agreement with the morphology of tobacco pollen tubes expressing mutant forms of yellow fluorescent protein (YFP):MsROP6. The S74E mutation in MsROP6 had no influence on pollen tube morphology and attenuated the phenotype of a constitutively active form of MsROP6. The presented Medicago and Arabidopsis data support the notion that the phosphorylation of the serine residue in ROPs corresponding to S74 in Medicago ROP6 could be a general principle for regulating ROP activation and signaling in plants.

Nitric oxide, a signaling molecule in plant cell reactivation

Nitric oxide is known to act as a biological messenger in divers signal transduction pathways in animal organisms. Initial investigations suggest that plants use nitric oxide as a signaling molecule via pathways remarkably similar to those found in mammals. Especially, the siganiling role of NO during plant defense reactions is well established. However, mounting evidences support the hypothesis that NO is a more general effector of plant growth and development. In our laboratory, alfalfa cell cultures were used to investigate the possible involvement of NO in the regulation of cell division and differentiation in plant cells. The homogenous population of leaf protoplasts were cultured in the presence of a NO donor, sodium nitroprusside (SNP) and/or an inhibitor, NG-monomethyl-L-arginine (L-NMMA). BrdU incorporation frequency into the nuclei of the protoplast-derived cells indicated that the entry into the S-phase of the cell cycle is enhanced by SNP and inhibited by L-NMMA treatments, respectively (see Figure ​Figure1).1). Experiments have also been carried out with continuously dividing cell suspension cultures. The obtained data indicated that these type of cells are insensitive to similar treatments shown to affect protoplast-derived cell division (see Figure ​Figure22). Figure 1 BrdU incorporation frequency into the nuclei of leaf protplast-derived alfalfa cells during the third day of culture. The cells were treated by the indicated drugs affecting endogenous NO formation. For cell culture details see [2]. Figure 2 BrdU incorporation frequency into the nuclei of cell suspension-cultured alfalfa cells during the third day after subculture. The cells were treated by the indicated drugs affecting endogenous NO formation. For cell culture details see [2]. In addition to cell cycle progression, the effect of the above drugs on the auxin-induced formation of embryogenic competent cells from leaf protoplasts (for more details see [1,2]) have been followed. The promotive effect of SNP and the inhibitory effect of L-NMMA have been observed on the process, especially at low exogenous auxin (0.02 μM 2,4-dichlorophenoxyacetic acid) supplementation.

Specific features of RHO GTPase-dependent signaling in plants

Rho GTPases constitute a major branch of the Ras superfamily of small GTPases. To date, at least 18 mammalian Rho GTPases have been identified, with RHOA, RAC1, and CDC42 being the most intensely studied (Mackay and Hall, 1998). In animal cells, RHO GTPases function as regulated GDP/GTP switches that are activated by diverse extracellular stimuli that stimulate G protein-coupled receptors, receptor tyrosine kinases, integrins, and other cell surface receptors (Kjoller and Hall, 1999). Once activated, each RHO GTPase interacts with a wide spectrum of functionally diverse downstream effectors to initiate cytoplasmic signaling pathways that regulate both cytoplasmic and nuclear events (Aspenstrom, 1999). RHO family GTPases are involved in cellular processes where cell shape is changed due to division, morphogenesis or motility, and require the reorganization of the cytoskeleton (Ridley, 2001). In plants, a specific class of RHO GTPases exists forming the ROP subfamily (Valster et al., 2000). Their roles in polarized growth of pollen tubes (Fu et al., 2001) as well as polarization of Fucus embryos (Belanger and Quatrano, 2000) were hypothesized to be linked to cytoskeletal functions and polarized secretion. RAC/ ROP proteins also induce the activation of the NADPH oxidase enzyme complex leading to superoxide production in mammalian (e.g. macrophages) as well as in plant cells (e.g. Diebold and Bokoch, 2001; Park et al., 2000). However, only limited information has accumulated up to now on the signaling cascades activated by RHO-type GTPases in plants. In our laboratory, we have isolated four different cDNA clones coding for RHO-type GTPases from an alfalfa root-nodule cDNA library. Different mutant forms of these proteins have been created by site-specific in vitro mutagenesis. Yeast two-hybrid screens were performed to reveal protein–protein interactions. Screens with the dominant negative form (‘GDP-bound state’) of one of these proteins did not show any specific interaction. In contrast, using the dominant positive form (GTP-bound state) six ‘strong’ interacting partners could be identified including myosin-like proteins, a WD40 protein homologue, and two receptor-like serine/ threonin kinases (RLKs). Further, two-hybrid screens using one of the ROP-interacting RLK protein as bait revealed potential members of a receptor complex, several transcription factors, and proteins implicated in membrane trafficking (endocytosis, Golgi-functions). These results indicate that plant RHO(ROP)-related signaling can, in parallel, reach nuclear as well as cytoplasmic targets such as the cytoskeleton and membranes. Based on these and other published data, one can observe several obvious differences between animal and plant cells concerning RHO GTPase signaling. Plant ROP GTPases form a distinct class and do not belong to the CDC42/RAC/RHO subfamilies: their sequences are divergent from those of their animal counterparts in spite of the high degree of evolutionary conservation of the main functional elements. Interestingly, however, one of the alfalfa RHO proteins identified in our laboratory is distinct from plant RHO(ROP) proteins and is more closely related to animal RAC and CDC42 proteins (Fig. 1). Detailed characterization of this specific plant RHO GTPase is in progress. Concerning regulators and effectors of RHO GTPases, there are characteristic differences between plants and animals: no guanidine nucleotide exchange factors (GEFs) have been identified in plants; plant GTPase activating proteins (GAPs) carry the ‘CRIB’ interaction * Corresponding author. Tel.: +36-62-432-232; fax: +36-62-433-434. E-mail address: fehera@nucleus.szbk.u-szeged.hu (A. Fehér). Cell Biology International 27 (2003) 191–192 Cell Biology International