Agnieszka Ludwików

Gene Networks in Plant Ozone Stress Response and Tolerance

For many plant species ozone stress has become much more severe in the last decade. The accumulating evidence for the significant effects of ozone pollutant on crop and forest yield situate ozone as one of the most important environmental stress factors that limits plant productivity worldwide. Today, transcriptomic approaches seem to give the best coverage of genome level responses. Therefore, microarray serves as an invaluable tool for global gene expression analyses, unravelling new information about gene pathways, in-species and cross-species gene expression comparison, and for the characterization of unknown relationships between genes. In this review we summarize the recent progress in the transcriptomics of ozone to demonstrate the benefits that can be harvested from the application of integrative and systematic analytical approaches to study ozone stress response. We focused our consideration on microarray analyses identifying gene networks responsible for response and tolerance to elevated ozone concentration. From these analyses it is now possible to notice how plant ozone defense responses depend on the interplay between many complex signaling pathways and metabolite signals.

Gene expression profiling of ozone-treated Arabidopsis abi1td insertional mutant: protein phosphatase 2C ABI1 modulates biosynthesis ratio of ABA and ethylene

We report on the characterization of the interaction between reactive oxygen species signalling and abscisic acid (ABA)-mediated gene network in ozone (O3) stress response. To identify the stress-related signalling pathways and possible cross-talk controlled by an ABA-negative regulator, the protein phosphatase 2C abscisic acid insensitive1 (ABI1), we performed a genome-wide transcription profiling of O3-treated wild-type and ABI1 knockout (abi1td) plants. In addition, to better understand ABA signalling and the interactions between stress response pathways, we performed a microarray analysis of drought-treated plants. Functional categorization of the identified genes showed that ABI1 is involved in the modulation of several cellular processes including metabolism, transport, development, information pathways and variant splicing. Comparisons with available transcriptome data sets revealed the extent of ABI1 involvement in both ABA-dependent and ABA-independent gene expression. Furthermore, in O3 stress the ABA hypersensitivity of abi1td resulted in a significant reduction of the ABA level, ethylene (ET) over-production and O3 tolerance. Moreover, the physical interaction of ABI1 with ACC synthase2 and ACC synthase6 was shown. We provide a model explaining how ABI1 can regulate both ABA and ET biosynthesis. Altogether, our findings indicate that ABI1 plays the role of a general signal transducer linking ABA and ET biosynthesis as well as signalling pathways to O3 stress tolerance.

Arabidopsis ABA-Activated Kinase MAPKKK18 is Regulated by Protein Phosphatase 2C ABI1 and the Ubiquitin–Proteasome Pathway

Phosphorylation and dephosphorylation events play an important role in the transmission of the ABA signal. Although SnRK2 [sucrose non-fermenting1-related kinase2] protein kinases and group A protein phosphatase type 2C (PP2C)-type phosphatases constitute the core ABA pathway, mitogen-activated protein kinase (MAPK) pathways are also involved in plant response to ABA. However, little is known about the interplay between MAPKs and PP2Cs or SnRK2 in the regulation of ABA pathways. In this study, an effort was made to elucidate the role of MAP kinase kinase kinase18 (MKKK18) in relation to ABA signaling and response. The MKKK18 knockout lines showed more vigorous root growth, decreased abaxial stomatal index and increased stomatal aperture under normal growth conditions, compared with the control wild-type Columbia line. In addition to transcriptional regulation of the MKKK18 promoter by ABA, we demonstrated using in vitro and in vivo kinase assays that the kinase activity of MKKK18 was regulated by ABA. Analysis of the cellular localization of MKKK18 showed that the active kinase was targeted specifically to the nucleus. Notably, we identified abscisic acid insensitive 1 (ABI1) PP2C as a MKKK18-interacting protein, and demonstrated that ABI1 inhibited its activity. Using a cell-free degradation assay, we also established that MKKK18 was unstable and was degraded by the proteasome pathway. The rate of MKKK18 degradation was delayed in the ABI1 knockout line. Overall, we provide evidence that ABI1 regulates the activity and promotes proteasomal degradation of MKKK18.

AtEAF1 is a potential platform protein for Arabidopsis NuA4 acetyltransferase complex

BackgroundHistone acetyltransferase complex NuA4 and histone variant exchanging complex SWR1 are two chromatin modifying complexes which act cooperatively in yeast and share some intriguing structural similarities. Protein subunits of NuA4 and SWR1-C are highly conserved across eukaryotes, but form different multiprotein arrangements. For example, the human TIP60-p400 complex consists of homologues of both yeast NuA4 and SWR1-C subunits, combining subunits necessary for histone acetylation and histone variant exchange. It is currently not known what protein complexes are formed by the plant homologues of NuA4 and SWR1-C subunits.ResultsWe report on the identification and molecular characterization of AtEAF1, a new subunit of Arabidopsis NuA4 complex which shows many similarities to the platform protein of the yeast NuA4 complex. AtEAF1 copurifies with Arabidopsis homologues of NuA4 and SWR1-C subunits ARP4 and SWC4 and interacts physically with AtYAF9A and AtYAF9B, homologues of the YAF9 subunit. Plants carrying a T-DNA insertion in one of the genes encoding AtEAF1 showed decreased FLC expression and early flowering, similarly to Atyaf9 mutants. Chromatin immunoprecipitation analyses of the single mutant Ateaf1b-2 and artificial miRNA knock-down Ateaf1 lines showed decreased levels of H4K5 acetylation in the promoter regions of major flowering regulator genes, further supporting the role of AtEAF1 as a subunit of the plant NuA4 complex.ConclusionsGrowing evidence suggests that the molecular functions of the NuA4 and SWR1 complexes are conserved in plants and contribute significantly to plant development and physiology. Our work provides evidence for the existence of a yeast-like EAF1 platform protein in A. thaliana, filling an important gap in the knowledge about the subunit organization of the plant NuA4 complex.

Targeting proteins for proteasomal degradation—a new function of Arabidopsis ABI1 protein phosphatase 2C

The ubiquitin/26S proteasome system (UPS) has been implicated in the regulation of many physiological processes including hormone signaling. The plant hormone abscisic acid (ABA) employs the UPS to control its own synthesis and signaling and to regulate stress response and tolerance. Among the known effectors of ABA signaling, the ABI1 (abscisic acid-insensitive 1) protein phosphatase, which belongs to group A of the type 2C protein phosphatases, is recognized as a key component of the pathway. Molecular and genetic evidence implicates this protein phosphatase in numerous plant responses. This mini-review discusses recent progress in understanding the role of ABI1 in ABA signaling, with particular emphasis on recent data that link ABI1 to protein degradation via the UPS.

Involvement of genes encoding ABI1 protein phosphatases in the response of Brassica napus L. to drought stress

In this report we characterized the ArabidopsisABI1 gene orthologue and Brassica napus gene paralogues encoding protein phosphatase 2C (PP2C, group A), which is known to be a negative regulator of the ABA signaling pathway. Six homologous B. napus sequences were identified and characterized as putative PP2C group A members. To gain insight into the conservation of ABI1 function in Brassicaceae, and understand better its regulatory effects in the drought stress response, we generated transgenic B. napus plants overexpressing A. thalianaABI1. Transgenic plants subjected to drought showed a decrease in relative water content, photosynthetic pigments content and expression level of RAB18- and RD19A-drought-responsive marker genes relative to WT plants. We present the characterization of the drought response of B. napus with the participation of ABI1-like paralogues. The expression pattern of two evolutionarily distant paralogues, BnaA01.ABI1.a and BnaC07.ABI1.b in B. napus and their promoter activity in A. thaliana showed differences in the induction of the paralogues under dehydration stress. Comparative sequence analysis of both BnaABI1 promoters showed variation in positions of cis-acting elements that are especially important for ABA- and stress-inducible expression. Together, these data reveal that subfunctionalization following gene duplication may be important in the maintenance and functional divergence of the BnaABI1 paralogues. Our results provide a framework for a better understanding of (1) the role of ABI1 as a hub protein regulator of the drought response, and (2) the differential involvement of the duplicated BnaABI1 genes in the response of B. napus to dehydration-related stresses.

Phosphatase ABI1 and okadaic acid-sensitive phosphoprotein phosphatases inhibit salt stress-activated SnRK2.4 kinase.

BackgroundSNF1-related protein kinases 2 (SnRK2s) are key regulators of the plant response to osmotic stress. They are transiently activated in response to drought and salinity. Based on a phylogenetic analysis SnRK2s are divided into three groups. The classification correlates with their response to abscisic acid (ABA); group 1 consists SnRK2s non-activated in response to ABA, group 2, kinases non-activated or weakly activated (depending on the plant species) by ABA treatment, and group 3, ABA-activated kinases. The activity of all SnRK2s is regulated by phosphorylation. It is well established that clade A phosphoprotein phosphatases 2C (PP2Cs) are negative regulators of ABA-activated SnRK2s, whereas regulators of SnRK2s from group 1 remain unidentified.ResultsHere, we show that ABI1, a PP2C clade A phosphatase, interacts with SnRK2.4, member of group 1 of the SnRK2 family, dephosphorylates Ser158, whose phosphorylation is needed for the kinase activity, and inhibits the kinase, both in vitro and in vivo. Our data indicate that ABI1 and the kinase regulate primary root growth in response to salinity; the phenotype of ABI1 knockout mutant (abi1td) exposed to salt stress is opposite to that of the snrk2.4 mutant. Moreover, we show that the activity of SnRK2s from group 1 is additionally regulated by okadaic acid-sensitive phosphatase(s) from the phosphoprotein phosphatase (PPP) family.ConclusionsPhosphatase ABI1 and okadaic acid-sensitive phosphatases of the PPP family are negative regulators of salt stress-activated SnRK2.4. The results show that ABI1 inhibits not only the ABA-activated SnRK2s but also at least one ABA-non-activated SnRK2, suggesting that the phosphatase is involved in the cross talk between ABA-dependent and ABA-independent stress signaling pathways in plants.

Regulation of Arabidopsis MAPKKK18 by ABI1 and SnRK2, components of the ABA signaling pathway.

ABSTRACT The plant hormone abscisic acid (ABA), a key regulator in many crucial developmental and physiological processes, recruits diverse components into precisely regulated signaling network. We recently discovered that MAPKKK18, an ABA-activated kinase, is regulated by the protein phosphatase type 2C (PP2C) ABI1 and the kinase SnRK2.6, both components of the ABA core signaling pathway. ABI1 acts to inhibit MAPKKK18 kinase activity, but also affects MAPKKK18 protein turnover via the ubiquitin-proteasome pathway. SnRK2.6 kinase also seems to be important for the regulation of MAPKKK18 function. In this review we summarize the mechanisms that are exclusively involved in MAPKKK18 kinase regulation and that ensure specificity in its activation.

A Role for Barley Calcium-Dependent Protein Kinase CPK2a in the Response to Drought.

Increasing the drought tolerance of crops is one of the most challenging goals in plant breeding. To improve crop productivity during periods of water deficit, it is essential to understand the complex regulatory pathways that adapt plant metabolism to environmental conditions. Among various plant hormones and second messengers, calcium ions are known to be involved in drought stress perception and signaling. Plants have developed specific calcium-dependent protein kinases that convert calcium signals into phosphorylation events. In this study we attempted to elucidate the role of a calcium-dependent protein kinase in the drought stress response of barley (Hordeum vulgare L.), one of the most economically important crops worldwide. The ongoing barley genome project has provided useful information about genes potentially involved in the drought stress response, but information on the role of calcium-dependent kinases is still limited. We found that the gene encoding the calcium-dependent protein kinase HvCPK2a was significantly upregulated in response to drought. To better understand the role of HvCPK2a in drought stress signaling, we generated transgenic Arabidopsis plants that overexpressed the corresponding coding sequence. Overexpressing lines displayed drought sensitivity, reduced nitrogen balance index (NBI), an increase in total chlorophyll content and decreased relative water content. In addition, in vitro kinase assay experiments combined with mass spectrometry allowed HvCPK2a autophosphorylation sites to be identified. Our results suggest that HvCPK2a is a dual-specificity calcium-dependent protein kinase that functions as a negative regulator of the drought stress response in barley.

Mitogen-activated protein kinase cascades in plant hormone signaling

Mitogen-activated protein kinase (MAPK) modules play key roles in the transduction of environmental and developmental signals through phosphorylation of downstream signaling targets, including other kinases, enzymes, cytoskeletal proteins or transcription factors, in all eukaryotic cells. A typical MAPK cascade consists of at least three sequentially acting serine/threonine kinases, a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK) and finally, the MAP kinase (MAPK) itself, with each phosphorylating, and hence activating, the next kinase in the cascade. Recent advances in our understanding of hormone signaling pathways have led to the discovery of new regulatory systems. In particular, this research has revealed the emerging role of crosstalk between the protein components of various signaling pathways and the involvement of this crosstalk in multiple cellular processes. Here we provide an overview of current models and mechanisms of hormone signaling with a special emphasis on the role of MAPKs in cell signaling networks. One-sentence summary: In this review we highlight the mechanisms of crosstalk between MAPK cascades and plant hormone signaling pathways and summarize recent findings on MAPK regulation and function in various cellular processes.