Developmental phenotypes of Arabidopsis plants expressing phosphovariants of AtMYB75.

The Arabidopsis transcription factor Myeloblastosis protein 75 (MYB75, AT1G56650) is a well-established transcriptional activator of genes required for anthocyanin and flavonoid production, and a repressor of lignin and other secondary cell wall biosynthesis genes. MYB75 is itself tightly regulated at the transcriptional, translational and post-translational levels, including protein phosphorylation by Arabidopsis MAP kinases Examination of the behavior of different phosphovariant versions of MYB75 in vitro and in vivo revealed that overexpression of the MYB75T131E phosphovariant had a particularly marked effect on global changes in gene expression suggesting that phosphorylated MYB75 could be involved in a broader range of functions than previously recognized. Here, we describe a range of distinct developmental phenotypes observed among Arabidopsis lines expressing various phosphovariant forms of MYB75. Expression of either MYB75T131E or MYB75T131A phosphovariants, from the endogenous MYB75 promoter, in Arabidopsis myb75- mutants (Nossen background), resulted in severely impaired germination rates, and developmental arrest at early seedling stages. Arabidopsis plants overexpressing MYB75T131E from a strong constitutive Cauliflower mosaic virus (CaMV35S) promoter displayed slower development, with delayed bolting, flowering and onset of senescence. Conversely, MYB75T131A -overexpressing lines flowered and set seed earlier than either Col-0 WT controls or other MYB75-overexpressors (MYB75WT and MYB75T131E ). Histochemical analysis of mature stems also revealed ectopic vessel development in plants overexpressing MYB75; this phenotype was particularly prominent in the MYB75T131E phosphovariant. These data suggest that MYB75 plays a significant role in plant development, and that this aspect of MYB75 function is influenced by its phosphorylation status.

Biological impacts of phosphomimic AtMYB75.

MAIN CONCLUSION: The phosphorylation status of MYB75 at T-131 affects protein stability, flavonoid profiles, and patterns of gene expression. The Arabidopsis transcription factor Myeloblastosis protein 75 (MYB75, AT1G56650) is known to act as a positive transcriptional regulator of genes required for flavonoid and anthocyanin biosynthesis. MYB75 was also shown to negatively regulate lignin and other secondary cell wall biosynthetic genes (Bhargava et al. in Plant Physiol 154(3):1428-1438, 2010). While transcriptional regulation of MYB75 has been described in numerous publications, little is known about post-translational control of MYB75 protein function. In a recent publication, light-induced activation of a MAP kinase (MPK4, AT4G01370) in Arabidopsis was reported to lead to MYB75 phosphorylation at two canonical MPK target sites, threonines, T-126 and T-131. This double phosphorylation event positively influenced MYB75 protein stability (Li et al. in Plant Cell 28(11):2866-2883, 2016). We have examined this phenomenon through use of phosphomutant forms of MYB75 and found that MYB75 is phosphorylated primarily at T-131, and that the phosphorylation of MYB75 recombinant protein in vitro can be catalyzed by multiple MAP kinases, including MPK3 (AT3G45640), MPK6 (AT2G43790), MPK4 and MPK11 (AT1G01560). We also demonstrate that MYB75 can bind to a large number of Arabidopsis MPK's in vitro, suggesting it could be a target of multiple signalling pathways. The impact of MYB75 phosphorylation at T-131 on the function of this transcription factor, in terms of localization, stability, and protein-protein interactions with known binding partners was examined in transgenic lines expressing phosphomimic and phosphonull versions of MYB75, to capture the behaviour of permanently phosphorylated and unphosphorylated MYB75 protein, respectively. In addition, we describe how ectopic over-expression of different phosphovariant forms of MYB75 (MYB75WT, MYB75T131A, and MYB75T131E) affects flavonoid biochemical profiles and global changes of gene expression in the corresponding transgenic Arabidopsis plants.

Analysis of MAPK and MAPKK gene families in wheat and related Triticeae species.

BACKGROUND: The mitogen-activated protein kinase (MAPK) family is involved in signal transduction networks that underpin many different biological processes in plants, ranging from development to biotic and abiotic stress responses. To date this class of enzymes has received little attention in Triticeae species, which include important cereal crops (wheat, barley, rye and triticale) that represent over 20% of the total protein food-source worldwide. RESULTS: The work presented here focuses on two subfamilies of Triticeae MAPKs, the MAP kinases (MPKs), and the MAPK kinases (MKKs) whose members phosphorylate the MPKs. In silico analysis of multiple Triticeae sequence databases led to the identification of 152 MAPKs belonging to these two sub-families. Some previously identified MAPKs were renamed to reflect the literature consensus on MAPK nomenclature. Two novel MPKs, MPK24 and MPK25, have been identified, including the first example of a plant MPK carrying the TGY activation loop sequence common to mammalian p38 MPKs. An EF-hand calcium-binding domain was found in members of the Triticeae MPK17 clade, a feature that appears to be specific to Triticeae species. New insights into the novel MEY activation loop identified in MPK11s are offered. When the exon-intron patterns for some MPKs and MKKs of wheat, barley and ancestors of wheat were assembled based on transcript data in GenBank, they showed deviations from the same sequence predicted in Ensembl. The functional relevance of MAPKs as derived from patterns of gene expression, MPK activation and MKK-MPK interaction is discussed. CONCLUSIONS: A comprehensive resource of accurately annotated and curated Triticeae MPK and MKK sequences has been created for wheat, barley, rye, triticale, and two ancestral wheat species, goat grass and red wild einkorn. The work we present here offers a central information resource that will resolve existing confusion in the literature and sustain expansion of MAPK research in the crucial Triticeae grains.

Identification and Characterization of Arabidopsis Seed Coat Mucilage Proteins.

Plant cell wall proteins are important regulators of cell wall architecture and function. However, because cell wall proteins are difficult to extract and analyze, they are generally poorly understood. Here, we describe the identification and characterization of proteins integral to the Arabidopsis (Arabidopsis thaliana) seed coat mucilage, a specialized layer of the extracellular matrix composed of plant cell wall carbohydrates that is used as a model for cell wall research. The proteins identified in mucilage include those previously identified by genetic analysis, and several mucilage proteins are reduced in mucilage-deficient mutant seeds, suggesting that these proteins are genuinely associated with the mucilage. Arabidopsis mucilage has both nonadherent and adherent layers. Both layers have similar protein profiles except for proteins involved in lipid metabolism, which are present exclusively in the adherent mucilage. The most abundant mucilage proteins include a family of proteins named TESTA ABUNDANT1 (TBA1) to TBA3; a less abundant fourth homolog was named TBA-LIKE (TBAL). TBA and TBAL transcripts and promoter activities were detected in developing seed coats, and their expression requires seed coat differentiation regulators. TBA proteins are secreted to the mucilage pocket during differentiation. Although reverse genetics failed to identify a function for TBAs/TBAL, the TBA promoters are highly expressed and cell type specific and so should be very useful tools for targeting proteins to the seed coat epidermis. Altogether, these results highlight the mucilage proteome as a model for cell walls in general, as it shares similarities with other cell wall proteomes while also containing mucilage-specific features.

Nucleocytoplasmic partitioning of tobacco N receptor is modulated by SGT1.

SGT1 (Suppressor of G2 allele of SKP1) is required to maintain plant disease Resistance (R) proteins with Nucleotide-Binding (NB) and Leucine-Rich Repeat (LRR) domains in an inactive but signaling-competent state. SGT1 is an integral component of a multi-protein network that includes RACK1, Rac1, RAR1, Rboh, HSP90 and HSP70, and in rice the Mitogen-Activated Protein Kinase (MAPK), OsMAPK6. Tobacco (Nicotiana tabacum) N protein, which belongs to the Toll-Interleukin Receptor (TIR)-NB-LRR class of R proteins, confers resistance to Tobacco Mosaic Virus (TMV). Following transient expression in planta, we analyzed the functional relationship between SGT1, SIPK - a tobacco MAPK6 ortholog - and N, using mass spectrometry, confocal microscopy and pathogen assays. Here, we show that tobacco SGT1 undergoes specific phosphorylation in a canonical MAPK target-motif by SIPK. Mutation of this motif to mimic SIPK phosphorylation leads to an increased proportion of cells displaying SGT1 nuclear accumulation and impairs N-mediated resistance to TMV, as does phospho-null substitution at the same residue. Forced nuclear localization of SGT1 causes N to be confined to nuclei. Our data suggest that one mode of regulating nucleocytoplasmic partitioning of R proteins is by maintaining appropriate levels of SGT1 phosphorylation catalyzed by plant MAPK.

Immunoprofiling reveals unique cell-specific patterns of wall epitopes in the expanding Arabidopsis stem.

The Arabidopsis inflorescence stem undergoes rapid directional growth, requiring massive axial cell-wall extension in all its tissues, but, at maturity, these tissues are composed of cell types that exhibit markedly different cell-wall structures. It is not clear whether the cell-wall compositions of these cell types diverge rapidly following axial growth cessation, or whether compositional divergence occurs at earlier stages in differentiation, despite the common requirement for cell-wall extensibility. To examine this question, seven cell types were assayed for the abundance and distribution of 18 major cell-wall glycan classes at three developmental stages along the developing inflorescence stem, using a high-throughput immunolabelling strategy. These stages represent a phase of juvenile growth, a phase displaying the maximum rate of stem extension, and a phase in which extension growth is ceasing. The immunolabelling patterns detected demonstrate that the cell-wall composition of most stem tissues undergoes pronounced changes both during and after rapid extension growth. Hierarchical clustering of the immunolabelling signals identified cell-specific binding patterns for some antibodies, including a sub-group of arabinogalactan side chain-directed antibodies whose epitope targets are specifically associated with the inter-fascicular fibre region during the rapid cell expansion phase. The data reveal dynamic, cell type-specific changes in cell-wall chemistry across diverse cell types during cell-wall expansion and maturation in the Arabidopsis inflorescence stem, and highlight the paradox between this structural diversity and the uniform anisotropic cell expansion taking place across all tissues during stem growth.

The interacting MYB75 and KNAT7 transcription factors modulate secondary cell wall deposition both in stems and seed coat in Arabidopsis.

The Arabidopsis thaliana KNAT7 (KNOX family) and MYB75 (MYB family) transcription factors were each shown earlier to interact in yeast two-hybrid assays, and to modulate secondary cell wall formation in inflorescence stems. We demonstrate here that their interaction also occurs in vivo, and that specific domains of each protein mediate this process. The participation of these interacting transcription factors in secondary cell wall formation was then extended to the developing seed coat through the use of targeted transcript analysis and SEM in single loss-of-function mutants. Novel genetic and protein-protein interactions of MYB75 and KNAT7 with other transcription factors known to be involved in seed coat regulation were also identified. We propose that a MYB75-associated protein complex is likely to be involved in modulating secondary cell wall biosynthesis in both the Arabidopsis inflorescence stem and seed coat, and that at least some parts of the transcriptional regulatory network in the two tissues are functionally conserved.

Postal code for a plant MAPK.

Plants contain hundreds of protein kinases that are believed to provide cellular signal transduction services, but the identities of the proteins they are targeting are largely unknown. Using an Arabidopsis MAPK (mitogen-activated protein kinase) (MPK6) as a model, Sörensson et al. describe in this issue of the Biochemical Journal how arrayed combinatorial peptide scanning offers an efficient route to discovery of new potential kinase substrates.

Mitogen-activated protein kinase signaling in plant-interacting fungi: distinct messages from conserved messengers.

Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved proteins that function as key signal transduction components in fungi, plants, and mammals. During interaction between phytopathogenic fungi and plants, fungal MAPKs help to promote mechanical and/or enzymatic penetration of host tissues, while plant MAPKs are required for activation of plant immunity. However, new insights suggest that MAPK cascades in both organisms do not operate independently but that they mutually contribute to a highly interconnected molecular dialogue between the plant and the fungus. As a result, some pathogenesis-related processes controlled by fungal MAPKs lead to the activation of plant signaling, including the recruitment of plant MAPK cascades. Conversely, plant MAPKs promote defense mechanisms that threaten the survival of fungal cells, leading to a stress response mediated in part by fungal MAPK cascades. In this review, we make use of the genomic data available following completion of whole-genome sequencing projects to analyze the structure of MAPK protein families in 24 fungal taxa, including both plant pathogens and mycorrhizal symbionts. Based on conserved patterns of sequence diversification, we also propose the adoption of a unified fungal MAPK nomenclature derived from that established for the model species Saccharomyces cerevisiae. Finally, we summarize current knowledge of the functions of MAPK cascades in phytopathogenic fungi and highlight the central role played by MAPK signaling during the molecular dialogue between plants and invading fungal pathogens.

The Class II KNOX gene KNAT7 negatively regulates secondary wall formation in Arabidopsis and is functionally conserved in Populus.

• The formation of secondary cell walls in cell types such as tracheary elements and fibers is a defining characteristic of vascular plants. The Arabidopsis transcription factor KNAT7 is a component of a transcription network that regulates secondary cell wall biosynthesis, but its function has remained unclear. • We conducted anatomical, biochemical and molecular phenotypic analyses of Arabidopsis knat7 loss-of-function alleles, KNAT7 over-expression lines and knat7 lines expressing poplar KNAT7. • KNAT7 was strongly expressed in concert with secondary wall formation in Arabidopsis and poplar. Arabidopsis knat7 loss-of-function alleles exhibited irregular xylem phenotypes, but also showed increased secondary cell wall thickness in fibers. Increased commitment to secondary cell wall biosynthesis was accompanied by increased lignin content and elevated expression of secondary cell wall biosynthetic genes. KNAT7 over-expression resulted in thinner interfascicular fiber cell walls. • Taken together with data demonstrating that KNAT7 is a transcriptional repressor, we hypothesize that KNAT7 is a negative regulator of secondary wall biosynthesis, and functions in a negative feedback loop that represses metabolically inappropriate commitment to secondary wall formation, thereby maintaining metabolic homeostasis. The conservation of the KNAT7 regulatory module in poplar suggests new ways to manipulate secondary cell wall deposition for improvement of bioenergy traits in this tree.

Arabidopsis ovate family proteins, a novel transcriptional repressor family, control multiple aspects of plant growth and development.

BACKGROUND: The Arabidopsis genome contains 18 genes that are predicted to encode Ovate Family Proteins (AtOFPs), a protein family characterized by a conserved OVATE domain, an approximately 70-amino acid domain that was originally found in tomato OVATE protein. Among AtOFP family members, AtOFP1 has been shown to suppress cell elongation, in part, by suppressing the expression of AtGA20ox1, AtOFP4 has been shown to regulate secondary cell wall formation by interact with KNOTTED1-LIKE HOMEODOMAIN PROTEIN 7 (KNAT7), and AtOFP5 has been shown to regulate the activity of a BEL1-LIKEHOMEODOMAIN 1(BLH1)-KNAT3 complex during early embryo sac development, but little is known about the function of other AtOFPs. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated here that AtOFP proteins could function as effective transcriptional repressors in the Arabidopsis protoplast transient expression system. The analysis of loss-of-function alleles of AtOFPs suggested AtOFP genes may have overlapping function in regulating plant growth and development, because none of the single mutants identified, including T-DNA insertion mutants in AtOFP1, AtOFP4, AtOFP8, AtOFP10, AtOFP15 and AtOFP16, displayed any apparent morphological defects. Further, Atofp1 Atofp4 and Atofp15 Atofp16 double mutants still did not differ significantly from wild-type. On the other hand, plants overexpressing AtOFP genes displayed a number of abnormal phenotypes, which could be categorized into three distinct classes, suggesting that AtOFP genes may also have diverse functions in regulating plant growth and development. Further analysis suggested that AtOFP1 regulates cotyledon development in a postembryonic manner, and global transcript profiling revealed that it suppress the expression of many other genes. CONCLUSIONS/SIGNIFICANCE: Our results showed that AtOFPs function as transcriptional repressors and they regulate multiple aspects of plant growth and development. These results provided the first overview of a previously unknown transcriptional repressor family, and revealed their possible roles in plant growth and development.

AtMKK6 and AtMPK13 are required for lateral root formation in Arabidopsis.

The mitogen-activated protein (MAP) kinase cascades are important signaling components that mediate various biological pathwaysin all eukaryotic cells. In our recent publication,1 we identified AtMPK4 as one of the downstream targets of AtMKK6 that is required for executing male-specific meiotic cytokinesis. Here we provide evidence that another target, AtMPK13, is developmentally co-expressed with AtMKK6 in Arabidopsis, and both AtMPK13 and AtMKK6 display high Promoter::GUS activity in the primary root tips and at the lateral root primordia. Partial suppression of either AtMKK6 or AtMPK13 expression significantly reduces the number of lateral roots in the transgenic lines, suggesting that the AtMKK6-AtMPK13 module positively regulates lateral root formation.

AtMPK4 is required for male-specific meiotic cytokinesis in Arabidopsis.

Mitogen-activated protein kinase (MAPK) cascades have been implicated in regulating various aspects of plant development, including somatic cytokinesis. The evolution of expanded plant MAPK gene families has enabled the diversification of potential MAPK cascades, but functionally overlapping components are also well documented. Here we report that Arabidopsis MPK4, an MAPK that was previously described as a regulator of disease resistance, can interact with and be phosphorylated by the cytokinesis-related MAP kinase kinase, AtMKK6. In mpk4 mutant plants, anthers can develop normal microspore mother cells (MMCs) and peripheral supporting tissues, but the MMCs fail to form a normal intersporal callose wall after male meiosis, and thus cannot complete meiotic cytokinesis. Nevertheless, the multinucleate mpk4 microspores subsequently proceed through mitotic cytokinesis, resulting in enlarged mature pollen grains that possess increased sets of the tricellular structure. This pollen development phenotype is reminiscent of those observed in both atnack2/tes/stud and anq1/mkk6 mutants, and protein-protein interaction analysis defines a putative signalling module linking AtNACK2/TES/STUD, AtANP3, AtMKK6 and AtMPK4 together as a cascade that facilitates male-specific meiotic cytokinesis in Arabidopsis.

Involvement of Arabidopsis RACK1 in protein translation and its regulation by abscisic acid.

Earlier studies have shown that RACK1 functions as a negative regulator of abscisic acid (ABA) responses in Arabidopsis (Arabidopsis thaliana), but the molecular mechanism of the action of RACK1 in these processes remains elusive. Global gene expression profiling revealed that approximately 40% of the genes affected by ABA treatment were affected in a similar manner by the rack1 mutation, supporting the view that RACK1 is an important regulator of ABA responses. On the other hand, coexpression analysis revealed that more than 80% of the genes coexpressed with RACK1 encode ribosome proteins, implying a close relationship between RACK1's function and the ribosome complex. These results implied that the regulatory role for RACK1 in ABA responses may be partially due to its putative function in protein translation, which is one of the major cellular processes that mammalian and Saccharomyces cerevisiae RACK1 is involved in. Consistently, all three Arabidopsis RACK1 homologous genes, namely RACK1A, RACK1B, and RACK1C, complemented the growth defects of the S. cerevisiae cross pathway control2/rack1 mutant. In addition, RACK1 physically interacts with Arabidopsis Eukaryotic Initiation Factor6 (eIF6), whose mammalian homolog is a key regulator of 80S ribosome assembly. Moreover, rack1 mutants displayed hypersensitivity to anisomycin, an inhibitor of protein translation, and displayed characteristics of impaired 80S functional ribosome assembly and 60S ribosomal subunit biogenesis in a ribosome profiling assay. Gene expression analysis revealed that ABA inhibits the expression of both RACK1 and eIF6. Taken together, these results suggest that RACK1 may be required for normal production of 60S and 80S ribosomes and that its action in these processes may be regulated by ABA.

MYB75 functions in regulation of secondary cell wall formation in the Arabidopsis inflorescence stem.

Deposition of lignified secondary cell walls in plants involves a major commitment of carbon skeletons in both the form of polysaccharides and phenylpropanoid constituents. This process is spatially and temporally regulated by transcription factors, including a number of MYB family transcription factors. MYB75, also called PRODUCTION OF ANTHOCYANIN PIGMENT1, is a known regulator of the anthocyanin branch of the phenylpropanoid pathway in Arabidopsis (Arabidopsis thaliana), but how this regulation might impact other aspects of carbon metabolism is unclear. We established that a loss-of-function mutation in MYB75 (myb75-1) results in increased cell wall thickness in xylary and interfascicular fibers within the inflorescence stem. The total lignin content and S/G ratio of the lignin monomers were also affected. Transcript profiles from the myb75-1 inflorescence stem revealed marked up-regulation in the expression of a suite of genes associated with lignin biosynthesis and cellulose deposition, as well as cell wall modifying proteins and genes involved in photosynthesis and carbon assimilation. These patterns suggest that MYB75 acts as a repressor of the lignin branch of the phenylpropanoid pathway. Since MYB75 physically interacts with another secondary cell wall regulator, the KNOX transcription factor KNAT7, these regulatory proteins may form functional complexes that contribute to the regulation of secondary cell wall deposition in the Arabidopsis inflorescence stem and that integrate the metabolic flux through the lignin, flavonoid, and polysaccharide pathways.

Novel and expanded roles for MAPK signaling in Arabidopsis stomatal cell fate revealed by cell type-specific manipulations.

Mitogen-activated protein kinase (MAPK) signaling networks regulate numerous eukaryotic biological processes. In Arabidopsis thaliana, signaling networks that contain MAPK kinases MKK4/5 and MAPKs MPK3/6 function in abiotic and biotic stress responses and regulate embryonic and stomatal development. However, how single MAPK modules direct specific output signals without cross-activating additional downstream processes is largely unknown. Studying relationships between MAPK components and downstream signaling outcomes is difficult because broad experimental manipulation of these networks is often lethal or associated with multiple phenotypes. Stomatal development in Arabidopsis follows a series of discrete, stereotyped divisions and cell state transitions. By expressing a panel of constitutively active MAPK kinase (MAPKK) variants in discrete stomatal lineage cell types, we identified a new inhibitory function of MKK4 and MKK5 in meristemoid self-renewal divisions. Furthermore, we established roles for MKK7 and MKK9 as both negative and (unexpectedly) positive regulators during the major stages of stomatal development. This has expanded the number of known MAPKKs that regulate stomatal development and allowed us to build plausible and testable subnetworks of signals. This in vivo cell type-specific assay can be adapted to study other protein families and thus may reveal insights into other complex signal transduction pathways in plants.

MAP kinases MPK9 and MPK12 are preferentially expressed in guard cells and positively regulate ROS-mediated ABA signaling.

Reactive oxygen species (ROS) mediate abscisic acid (ABA) signaling in guard cells. To dissect guard cell ABA-ROS signaling genetically, a cell type-specific functional genomics approach was used to identify 2 MAPK genes, MPK9 and MPK12, which are preferentially and highly expressed in guard cells. To provide genetic evidence for their function, Arabidopsis single and double TILLING mutants that carry deleterious point mutations in these genes were isolated. RNAi-based gene-silencing plant lines, in which both genes are silenced simultaneously, were generated also. Mutants carrying a mutation in only 1 of these genes did not show any altered phenotype, indicating functional redundancy in these genes. ABA-induced stomatal closure was strongly impaired in 2 independent RNAi lines in which both MPK9 and MPK12 transcripts were significantly silenced. Consistent with this result, mpk9-1/12-1 double mutants showed an enhanced transpirational water loss and ABA- and H(2)O(2)-insensitive stomatal response. Furthermore, ABA and calcium failed to activate anion channels in guard cells of mpk9-1/12-1, indicating that these 2 MPKs act upstream of anion channels in guard cell ABA signaling. An MPK12-YFP fusion construct rescued the ABA-insensitive stomatal response phenotype of mpk9-1/12-1, demonstrating that the phenotype was caused by the mutations. The MPK12 protein is localized in the cytosol and the nucleus, and ABA and H(2)O(2) treatments enhance the protein kinase activity of MPK12. Together, these results provide genetic evidence that MPK9 and MPK12 function downstream of ROS to regulate guard cell ABA signaling positively.

Quantitative proteomics identifies oxidant-induced, AtMPK6-dependent changes in Arabidopsis thaliana protein profiles.

In Arabidopsis thaliana, oxidant-induced signalling has been shown to utilize the mitogen-activated protein kinase (MAPK), AtMPK6. To identify proteins whose accumulation is altered by ozone in an AtMPK6-dependent manner we employed isotope-coded affinity tagging (ICAT) technology to investigate the impact of AtMPK6-suppression on the protein profiles in Arabidopsis both before (air control) and during continuous ozone (O(3)) fumigation (500 nL L(-1) for 8 h). Among the 150 proteins positively identified and quantified in the O(3)-treated plants, we identified thirteen proteins whose abundance was greater in the AtMPK6-suppressed genotype than in wild-type (WT). These include the antioxidant proteins, monodehydroascorbate reductase, peroxiredoxin Q, and glutathione reductase. A further eighteen proteins were identified whose abundance was lower in the ozone-treated AtMPK6-suppressed line relative to ozone-exposed WT plants. These predominantly comprised proteins involved in carbohydrate-, energy-, and amino acid metabolism, and tetrapyrrole biosynthesis. In control plants, five proteins increased, and nine proteins decreased in abundance in the AtMPK6-suppressed genotype compared to that of the WT, reflecting changes in the protein composition of plants that have AtMPK6 constitutively suppressed. Since a number of these proteins are part of the redox response pathway, and loss of AtMPK6 renders Arabidopsis more susceptible to oxidative stress, we propose that AtMPK6 plays a key role in the plant's overall ability to manage oxidative stress.

Suppression of MKK5 reduces ozone-induced signal transmission to both MPK3 and MPK6 and confers increased ozone sensitivity in Arabidopsis thaliana.

In Arabidopsis thaliana, ozone-induced signaling has been shown to involve the mitogen-activated protein kinases (MAPKs) MPK3 and MPK6. To identify a possible ozone-induced mitogen-activated protein kinase kinase (MAPKK) involved in the activation of these specific MAPKs, we employed RNA interference-(RNAi)-based suppression of MKK5, a known cognate MAPKK to both MPK3 and MPK6. When exposed to ozone, activation of both MPK3 and MPK6 was markedly reduced in the MKK5-suppressed plants compared to WT. Additionally, the MKK5-suppressed plants were found to be highly sensitive to ozone as determined by visible leaf damage concomitant with elevated levels of leaf-localised H(2)O(2). Taken together, our data suggest MKK5 functions both in ozone-induced activation of MPK3 and MPK6 and in integrating ROS homeostasis during ozone stress.