NaCl stress is a major abiotic stress limiting the productivity and the geographical distribution of many plant species. Roots are the primary site of salinity perception. To understand better NaCl stress responses in Arabidopsis roots, a comparative proteomic analysis of roots that had been exposed to 150 mM NaCl for either 6 h or 48 h was conducted. Changes in the abundance of protein species within roots were examined using two-dimensional electrophoresis. Among the >1000 protein spots reproducibly detected on each gel, the abundance of 112 protein spots decreased and 103 increased, at one or both time points, in response to NaCl treatment. Through liquid-chromatography-tandem mass spectrometry, identity was assigned to 86 of the differentially abundant spots. The proteins identified included many previously characterized stress-responsive proteins and others related to processes including scavenging for reactive oxygen species; signal transduction; translation, cell wall biosynthesis, protein translation, processing and degradation; and metabolism of energy, amino acids, and hormones. At the resolution of individual genes and proteins, poor statistical correlation (6 h, r= -0.13; 48 h, r=0.11) of these protein expression data with previous microarray results was detected, supporting the concept that post-transcriptional regulation plays an important role in stress-responsive gene expression, and highlighting the need for combined transcriptomic and proteomic analyses.

Previous microarray analyses of Arabidopsis roots identified two closely related WRKY transcription factors (WRKY25 and WRKY33) among the transcripts that increased in abundance following treatment with NaCl. Here, we report further characterization of these genes, which we found to be inducible by a variety of abiotic stresses in an SOS-pathway independent manner, although WRKY33 induction was dependent on ABA signaling. Transcripts of both genes were detected in roots and leaves, while specific patterns of enrichment were observed in stems and floral buds for WRKY25 and WRKY33, respectively. We also identified upstream intergenic regions from each gene that were sufficient to confer stress-inducible expression on a reporter gene. However, the stress sensitivity of wrky25 null mutants did not differ from wild-type under any assay condition, while wrky33 null mutants and wrky25wrky33 double mutants showed only a moderate increase in NaCl-sensitivity, suggesting functional redundancy with other transcription factors. Nevertheless, overexpression of WRKY25 or WRKY33 was sufficient to increase Arabidopsis NaCl tolerance, while increasing sensitivity to ABA. Through microarray analyses of relevant genotypes, we identified 31 and 208 potential downstream targets of WRKY25 and WRKY33, respectively, most of which contained a W-box in their upstream regions.

Background: Members of plant WRKY transcription factor families are widely implicated in defense responses and various other physiological processes. For canola (Brassica napus L.), no WRKY genes have been described in detail. Because of the economic importance of this crop, and its evolutionary relationship to Arabidopsis thaliana, we sought to characterize a subset of canola WRKY genes in the context of pathogen and hormone responses.

Arabidopsis K+ transporter 1 (AKT1) participates in K+ uptake in roots, especially under low-K conditions. We recently identified a Ca²⁺ signaling pathway consisting of multiple calcineurin B-like calcium sensors (CBLs) and multiple target kinases (CBL-interacting protein kinases or CIPKs) that phosphorylate and activate AKT1, whereas a specific PP2C-type phosphatase inactivates CIPK-dependent AKT1 activity. In this study, we analyzed the interactions between PP2Cs and the CBL-CIPK pathway and found previously unsuspected mechanisms underlying the CBL-CIPK-PP2C signaling processes. The interaction between the CIPKs and PP2Cs involves the kinase domain of the CIPK component, in addition to the protein phosphatase interacting motif (PPI) in the regulatory domain. Furthermore, specific CBLs physically interact with and inactivate PP2C phosphatases to recover the CIPK-dependent AKT1 channel activity. These findings provide further insights into the signaling network consisting of CBL-CIPK-PP2C interactions in the activation of the AKT1 channel.

Background: Roots are an attractive system for genomic and post-genomic studies of NaCl responses, due to their primary importance to agriculture, and because of their relative structural and biochemical simplicity. Excellent genomic resources have been established for the study of Arabidopsis roots, however, a comprehensive microarray analysis of the root transcriptome following NaCl exposure is required to further understand plant responses to abiotic stress and facilitate future, systems-based analyses of the underlying regulatory networks.

BackgroundCanola (Brassica napus L.) is one of the most important oil-producing crops in China and worldwide. The yield and quality of canola is frequently threatened by environmental stresses including drought, cold and high salinity. Calcium is a ubiquitous intracellular secondary messenger in plants. Calcineurin B-like proteins (CBLs) are Ca2+ sensors and regulate a group of Ser/Thr protein kinases called CBL-interacting protein kinases (CIPKs). Although the CBL-CIPK network has been demonstrated to play crucial roles in plant development and responses to various environmental stresses in Arabidopsis, little is known about their function in canola.ResultsIn the present study, we identified seven CBL and 23 CIPK genes from canola by database mining and cloning of cDNA sequences of six CBLs and 17 CIPKs. Phylogenetic analysis of CBL and CIPK gene families across a variety of species suggested genome duplication and diversification. The subcellular localization of three BnaCBLs and two BnaCIPKs were determined using green fluorescence protein (GFP) as the reporter. We also demonstrated interactions between six BnaCBLs and 17 BnaCIPKs using yeast two-hybrid assay, and a subset of interactions were further confirmed by bimolecular fluorescence complementation (BiFC). Furthermore, the expression levels of six selected BnaCBL and 12 BnaCIPK genes in response to salt, drought, cold, heat, ABA, methyl viologen (MV) and low potassium were examined by quantitative RT-PCR and these CBL or CIPK genes were found to respond to multiple stimuli, suggesting that the canola CBL-CIPK network may be a point of convergence for several different signaling pathways. We also performed a comparison of interaction patterns and expression profiles of CBL and CIPK in Arabidospsis, canola and rice, to examine the differences between orthologs, highlighting the importance of studying CBL-CIPK in canola as a prerequisite for improvement of this crop.ConclusionsOur findings indicate that CBL and CIPK family members may form a dynamic complex to respond to different abiotic or hormone signaling. Our comparative analyses of the CBL-CIPK network between canola, Arabidopsis and rice highlight functional differences and the necessity to study CBL-CIPK gene functions in canola. Our data constitute a valuable resource for CBL and CPK genomics.

In our previous microarray analysis of NaCl-treated Arabidopsis roots, we identified a basic-helix-loop-helix (bHLH) transcription factor, bHLH92 (At5g43650), as one of the transcripts showing the greatest fold-increase in abundance upon NaCl exposure. Here, we characterize the role of bHLH92 in the context of abiotic stress physiology and hormone responses. We observed that bHLH92 transcript abundance increases in response to NaCl, dehydration, mannitol, and cold treatments, and compared these responses to those of two closely related genes: bHLH41 and bHLH42. The NaCl-inducibility of bHLH92 was only partially dependent on abscisic acid (ABA) biosynthesis and SALT OVERLY SENSITIVE2 (SOS2) pathways. As compared to WT, root elongation of bhlh92 mutants was more sensitive to mannitol, and these mutants also showed increased electrolyte leakage following NaCl treatments. Overexpression of bHLH92 moderately increased the tolerance to NaCl and osmotic stresses. Finally, we identified at least 19 putative downstream target genes of bHLH92 under NaCl treatment using an oligonucleotide microarray. Together these data show that bHLH92 functions in plant responses to osmotic stresses, although the net contribution of bHLH92-regulated genes to stress tolerance appears relatively limited in proportion to what might be expected from its transcript expression pattern.

BackgroundEukaryotic mitogen-activated protein kinase (MAPK/MPK) signaling cascades transduce and amplify environmental signals via three types of reversibly phosphorylated kinases to activate defense gene expression. Canola (oilseed rape, Brassica napus) is a major crop in temperate regions. Identification and characterization of MAPK and MAPK kinases (MAPKK/MKK) of canola will help to elucidate their role in responses to abiotic and biotic stresses.ResultsWe describe the identification and analysis of seven MKK (BnaMKK) and 12 MPK (BnaMPK) members from canola. Sequence alignments and phylogenetic analyses of the predicted amino acid sequences of BnaMKKs and BnaMPKs classified them into four different groups. We also examined the subcellular localization of four and two members of BnaMKK and BnaMPK gene families, respectively, using green fluorescent protein (GFP) and, found GFP signals in both nuclei and cytoplasm. Furthermore, we identified several interesting interaction pairs through yeast two-hybrid (Y2H) analysis of interactions between BnaMKKs and BnaMPKs, as well as BnaMPK and BnaWRKYs. We defined contiguous signaling modules including BnaMKK9-BnaMPK1/2-BnaWRKY53, BnaMKK2/4/5-BnaMPK3/6-BnaWRKY20/26 and BnaMKK9-BnaMPK5/9/19/20. Of these, several interactions had not been previously described in any species. Selected interactions were validated in vivo by a bimolecular fluorescence complementation (BiFC) assay. Transcriptional responses of a subset of canola MKK and MPK genes to stimuli including fungal pathogens, hormones and abiotic stress treatments were analyzed through real-time RT-PCR and we identified a few of BnaMKKs and BnaMPKs responding to salicylic acid (SA), oxalic acid (OA), Sclerotinia sclerotiorum or other stress conditions. Comparisons of expression patterns of putative orthologs in canola and Arabidopsis showed that transcript expression patterns were generally conserved, with some differences suggestive of sub-functionalization.ConclusionsWe identified seven MKK and 12 MPK genes from canola and examined their phylogenetic relationships, transcript expression patterns, subcellular localization, and protein-protein interactions. Not all expression patterns and interactions were conserved between canola and Arabidopsis, highlighting the limitations of drawing inferences about crops from model species. The data presented here provide the first systematic description of MKK-MPK-WRKY signaling modules in canola and will further improve our understanding of defense responses in general and provide a basis for future crop improvement.