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.

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. We used microarrays of 70-mer oligonucleotide probes representing 23,686 Arabidopsis genes to identify root transcripts that changed in relative abundance following 6 h, 24 h, or 48 h of hydroponic exposure to 150 mM NaCl. Enrichment analysis identified groups of structurally or functionally related genes whose members were statistically over-represented among up- or down-regulated transcripts. Our results are consistent with generally observed stress response themes, and highlight potentially important roles for underappreciated gene families, including: several groups of transporters (e.g. MATE, LeOPT1-like); signalling molecules (e.g. PERK kinases, MLO-like receptors), carbohydrate active enzymes (e.g. XTH18), transcription factors (e.g. members of ZIM, WRKY, NAC), and other proteins (e.g. 4CL-like, COMT-like, LOB-Class 1). We verified the NaCl-inducible expression of selected transcription factors and other genes by qRT-PCR. Micorarray profiling of NaCl-treated Arabidopsis roots revealed dynamic changes in transcript abundance for at least 20% of the genome, including hundreds of transcription factors, kinases/phosphatases, hormone-related genes, and effectors of homeostasis, all of which highlight the complexity of this stress response. Our identification of these transcriptional responses, and groups of evolutionarily related genes with either similar or divergent transcriptional responses to stress, will facilitate mapping of regulatory networks and extend our ability to improve salt tolerance in plants.

Background: Members of plant WRKY transcription factor families are widely implicated in defense responses and various other physiological processes. For canola (Brassica napusL.), no WRKY genes have been described in detail. Because of the economic importance of this crop, and its evolutionary relationship toArabidopsis thaliana, we sought to characterize a subset of canolaWRKYgenes in the context of pathogen and hormone responses.Results: In this study, we identified 46WRKYgenes from canola by mining the expressed sequence tag (EST) database and cloned cDNA sequences of 38BnWRKYs. A phylogenetic tree was constructed using the conserved WRKY domain amino acid sequences, which demonstrated that BnWRKYs can be divided into three major groups. We further comparedBnWRKYsto the 72WRKYgenes fromArabidopsisand 91WRKYfrom rice, and we identified 46 presumptive orthologs ofAtWRKYgenes. We examined the subcellular localization of four BnWRKY proteins using green fluorescent protein (GFP) and we observed the fluorescent green signals in the nucleus only.The responses of 16 selectedBnWRKYgenes to two fungal pathogens,Sclerotinia sclerotiorumandAlternaria brassicae, were analyzed by quantitative real time-PCR (qRT-PCR). Transcript abundance of 13BnWRKYgenes changed significantly following pathogen challenge: transcripts of 10WRKYs increased in abundance, twoWRKYtranscripts decreased after infection, and one decreased at 12 h post-infection but increased later on (72 h). We also observed that transcript abundance of 13/16BnWRKYgenes was responsive to one or more hormones, including abscisic acid (ABA), and cytokinin (6-benzylaminopurine, BAP) and the defense signaling molecules jasmonic acid (JA), salicylic acid (SA), and ethylene (ET). We compared these transcript expression patterns to those previously described for presumptive orthologs of these genes inArabidopsisand rice, and observed both similarities and differences in expression patterns.Conclusion: We identified a set of 13BnWRKYgenes from among 16BnWRKYgenes assayed, that are responsive to both fungal pathogens and hormone treatments, suggesting shared signaling mechanisms for these responses. This study suggests that a large number of BnWRKY proteins are involved in the transcriptional regulation of defense-related genes in response to fungal pathogens and hormone stimuli.

Calcium, as a ubiquitous second messenger, plays essential roles in tip-growing cells, such as animal neurons, plant pollen tubes, and root hairs. However, little is known concerning the regulatory mechanisms that code and decode Ca(2+) signals in plants. The evidence presented here indicates that a calcium-dependent protein kinase, CPK32, controls polar growth of pollen tubes. Overexpression of CPK32 disrupted the polar growth along with excessive Ca(2+) accumulation in the tip. A search of downstream effector molecules for CPK32 led to identification of a cyclic nucleotide-gated channel, CNGC18, as an interacting partner for CPK32. Co-expression of CPK32 and CNGC18 resulted in activation of CNGC18 in Xenopus oocytes where expression of CNGC18 alone did not exhibit significant calcium channel activity. Overexpression of CNGC18 produced a growth arrest phenotype coupled with accumulation of calcium in the tip, similar to that induced by CPK32 overexpression. Co-expression of CPK32 and CNGC18 had a synergistic effect leading to more severe depolarization of pollen tube growth. These results provide a potential feed-forward mechanism in which calcium-activated CPK32 activates CNGC18, further promoting calcium entry during the elevation phase of Ca(2+) oscillations in the polar growth of pollen tubes.