Jan Sadowski

Exogenously induced expression of ethylene biosynthesis, ethylene perception, phospholipase D, and Rboh-oxidase genes in broccoli seedlings.

In higher plants, copper ions, hydrogen peroxide, and cycloheximide have been recognized as very effective inducers of the transcriptional activity of genes encoding the enzymes of the ethylene biosynthesis pathway. In this report, the transcriptional patterns of genes encoding the 1-aminocyclopropane-1-carboxylate synthases (ACSs), 1-aminocyclopropane-1-carboxylate oxidases (ACOs), ETR1, ETR2, and ERS1 ethylene receptors, phospholipase D (PLD)-α1, -α2, -γ1, and -δ, and respiratory burst oxidase homologue (Rboh)-NADPH oxidase-D and -F in response to these inducers in Brassica oleracea etiolated seedlings are shown. ACS1, ACO1, ETR2, PLD-γ1, and RbohD represent genes whose expression was considerably affected by all of the inducers used. The investigations were performed on the seedlings with (i) ethylene insensitivity and (ii) a reduced level of the PLD-derived phosphatidic acid (PA). The general conclusion is that the expression of ACS1, -3, -4, -5, -7, and -11, ACO1, ETR1, ERS1, and ETR2, PLD-γ 1, and RbohD and F genes is undoubtedly under the reciprocal cross-talk of the ethylene and PAPLD signalling routes; both signals affect it in concerted or opposite ways depending on the gene or the type of stimuli. The results of these studies on broccoli seedlings are in agreement with the hypothesis that PA may directly affect the ethylene signal transduction pathway via an inhibitory effect on CTR1 (constitutive triple response 1) activity.

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.

Dual role of the histone variant H2A.Z in transcriptional regulation of stress-response genes

H2A.Z is removed from nucleosomes localized in genes upon transcriptional activation in response to drought stress conditions in Arabidopsis thaliana. The influence of the histone variant H2A.Z on transcription remains a long-standing conundrum. Here, by analyzing the actin-related protein6 mutant, which is impaired in H2A.Z deposition, and by H2A.Z profiling in stress conditions, we investigated the impact of this histone variant on gene expression in Arabidopsis thaliana. We demonstrate that the arp6 mutant exhibits anomalies in response to osmotic stress. Indeed, stress-responsive genes are overrepresented among those hyperactive in arp6. In wild-type plants, these genes exhibit high levels of H2A.Z in the gene body. Furthermore, we observed that in drought-responsive genes, levels of H2A.Z in the gene body correlate with transcript levels. H2A.Z occupancy, but not distribution, changes in parallel with transcriptional changes. In particular, we observed H2A.Z loss upon transcriptional activation and H2A.Z gain upon repression. These data suggest that H2A.Z has a repressive role in transcription and counteracts unwanted expression in noninductive conditions. However, reduced activity of some genes in arp6 is associated with distinct behavior of H2A.Z at their +1 nucleosome, which exemplifies the requirement of this histone for transcription. Our data support a model where H2A.Z in gene bodies has a strong repressive effect on transcription, whereas in +1 nucleosomes, it is important for maintaining the activity of some genes.

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.

Comparative analysis of the Brassica oleracea genetic map and the Arabidopsis thaliana genome

We further investigated genome macrosynteny for Brassica species and Arabidopsis thaliana. This work aimed at comparative map construction for B. oleracea and A. thaliana chromosomes based on 160 known A. thaliana probes: 147 expressed sequence tags (ESTs) and 13 full-length cDNA clones. Based on an in silico study of the A. thaliana genome, most of the selected ESTs (83%) represented unique or low-copy genes. We identified conserved segments by the visual inspection of comparative data with a priori assumptions, and established their significance with the LineUp algorithm. Evaluation of the number of B. oleracea gene copies per A. thaliana EST revealed a fixed upward trend. We established a segregation distortion pattern for all genetic loci, with particular consideration of the type of selection (gametic or zygotic), and discuss its possible impact on genetic map construction. Consistent with previous reports, we found evidence for numerous chromosome rearrangements and the genome fragment replication of B. oleracea that have taken place since the divergence of the two species. Also, we found that over 54% of the B. oleracea genome is covered by 24 segments conserved with the A. thaliana genome. The average conserved segment is composed of 5 loci covering 19.3 cM in the B. oleracea genetic map and 2.42 Mb in the A. thaliana physical map. We have also attempted to use a unified system of conserved blocks (previously described) to verify our results and perform a comprehensive comparison with other Brassica species.

Genes involved in biosynthesis and signalisation of ethylene in Brassica oleracea and Arabidopsis thaliana: identification and genome comparative mapping of specific gene homologues

The study reported was aimed at the identification and determination of the chromosomal organisation of genes involved in the ethylene biosynthesis and signalling pathways in Brassica oleracea, on the basis of the Arabidopsisthaliana DNA probes and in silico genome analysis. Because of its polyploidal origin, the B. oleracea genome is characterised by extensive gene redundancy. Therefore, an important aspect of gene expression in B. oleracea response to environmental stimuli is to identify the specific gene copy involved. This aspect should also be taken into consideration while studying the genetic basis of biosynthesis and signal transduction in relation to basic phytohormones. Our present work concerns the identification of homologue genes involved in ethylene biosynthesis such as SAM, ACS and ACO, as well as those involved in the ethylene signalling pathway, mainly ETR1, CTR1, MKK4, MKK5, EIN2, EIN3, EREBP, ERF5 and ERF7 on the basis of the restriction fragment length polymorphism (RFLP) and PCR mapping. In the case of ACC synthases, (ACSs) the in silico analysis of gene variants in the genome of A. thaliana was followed by the identification of homologues to ACS2, ACS6 and ACS7 in the B. oleracea database. In total, 22 loci with sequence homology to the genes under analysis were included in the existing B. oleracea RFLP chromosomal map. Based on the stress responsiveness of most of the A. thaliana genes analysed in this study, we performed initial functional analysis of some gene homologues mapped. With the use of the RT-PCR approach the conservation of differential transcriptional induction of ACS homologues in the B. oleracea and A. thaliana was demonstrated during ozone stress.

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.

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.

1-Aminocyclopropane-1-carboxylate synthase — genes and expression

The immediate precursor of ethylene in higher plants is 1-aminocyclopropane-1-carboxylic acid (ACC), which is a product of the reaction controlled by 1-aminocyclopropane-1-carboxylate synthase catalytic activity (S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14). This review summarizes the up-to-date knowledge about organization and gene structure, and transcriptional expression of ACS genes from different plant species hitherto characterized. ACS is encoded by a highly divergent multigene family in each plant species. For example, the ACS families of tomato (LE-ACS) and Arabidopsis thaliana (AT-ACS) include numerous isoenzyme genes. Expression of selected members of the multigene ACS family in higher plants is induced by a diverse group of external and internal ethylene inducers such as: wounding, anaerobiosis, viral and fungal infection, chilling, drought, heavy metals, auxin, ripening and senescence processes. Enhanced transcription is the main regulatory step of this enzyme activity but the posttranscriptional regulation is operational as well.