Identification and characterization of CBL and CIPK gene families in canola (Brassica napus L.)

BACKGROUND: Canola (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 Ca(2+) 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. RESULTS: In 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. CONCLUSIONS: Our 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

Comprehensive transcriptional profiling of NaCl-stressed Arabidopsis roots reveals novel classes of responsive genes

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. RESULTS: 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. CONCLUSION: 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

Separable whorl-specific expression and negative regulation by enhancer elements within the AGAMOUS second intron

We analyzed the 4-kb intragenic control region of the AGAMOUS (AG) gene to gain insight into the mechanisms controlling its expression during early flower development. We identified three major expression patterns conferred by 19 AG::reporter gene constructs: the normal AG pattern, a stamen-specific pattern, and a predominantly carpel pattern. To determine whether these three expression patterns were under negative control by APETALA2 (AP2) or LEUNIG (LUG), we analyzed b-glucuronidase staining patterns in Arabidopsis plants homozygous for strong ap2 and lug mutations. Our results indicated that the stamen-specific pattern was independent of AP2 but dependent on LUG; conversely, the carpel-specific pattern was independent of LUG but dependent on AP2. These results lead to a model of control of AG expression such that expression in each of the two inner whorls is under independent positive and negative control

Soil and seed both influence bacterial diversity in the microbiome of the Cannabis sativa seedling endosphere

IntroductionPhytobiomes have a significant impact on plant health. The microbiome of Cannabis sativa is particularly interesting both because of renewed interest in this crop and because it is commercially propagated in two different ways (i.e. clonally and by seed). Angiosperms obtain a founding population of seed-borne endophytes from their seed-bearing parent. This study examines the influence of both seed and soil-derived bacteria on the endospheres of cannabis seedlings of both hemp- and drug-types.MethodsA multi-factorial metagenomic study was conducted with three cannabis genotypes and two soil sources, which were tested both before and after autoclave sterilization. Seedlings were grown on soil, then rinsed and surface-sterilized, and 16S rDNA amplicons from seedling endophytes were sequenced, taxonomically classified, and used to estimate alpha- and beta-diversity in Qiime2. The statistical significance of differences in seedling microbiomes across treatments was tested, and PiCRUST2 was used to infer the functional relevance of these differences.ResultsSoil was found to have a profound effect on the alpha-diversity, beta-diversity, relative abundance, and functional genes of endophytic bacteria in germinating cannabis seedlings. Additionally, there was a significant effect of cannabis genotype on beta diversity, especially when genotypes were grown in sterilized soil. Gammaproteobacteria and Bacilli were the two most abundant taxa and were found in all genotypes and soil types, including sterilized soil.DiscussionThe results indicated that a component of cannabis seedling endosphere microbiomes is seed-derived and conserved across the environments tested. Functional prediction of seedling endophytes using piCRUST suggested a number of important functions of seed-borne endophytes in cannabis including nutrient and amino acid cycling, hormone regulation, and as precursors to antibiotics. This study suggested both seed and soil play a critical role in shaping the microbiome of germinating cannabis seedlings

Transcriptional profiling of pea ABR17 mediated changes in gene expression in Arabidopsis thaliana

<p>Abstract</p> <p>Background</p> <p>Pathogenesis-related proteins belonging to group 10 (PR10) are elevated in response to biotic and abiotic stresses in plants. Previously, we have shown a drastic salinity-induced increase in the levels of ABR17, a member of the PR10 family, in pea. Furthermore, we have also demonstrated that the constitutive expression of pea <it>ABR17 </it>cDNA in <it>Arabidopsis thaliana </it>and <it>Brassica napus </it>enhances their germination and early seedling growth under stress. Although it has been reported that several members of the PR10 family including ABR17 possess RNase activity, the exact mechanism by which the aforementioned characteristics are conferred by ABR17 is unknown at this time. We hypothesized that a study of differences in transcriptome between wild type (WT) and <it>ABR17 </it>transgenic <it>A. thaliana </it>may shed light on this process.</p> <p>Results</p> <p>The molecular changes brought about by the expression of pea <it>ABR17 </it>cDNA in <it>A. thaliana </it>in the presence or absence of salt stress were investigated using microarrays consisting of 70-mer oligonucleotide probes representing 23,686 <it>Arabidopsis </it>genes. Statistical analysis identified number of genes which were over represented among up- or down-regulated transcripts in the transgenic line. Our results highlight the important roles of many abscisic acid (ABA) and cytokinin (CK) responsive genes in <it>ABR17 </it>transgenic lines. Although the transcriptional changes followed a general salt response theme in both WT and transgenic seedlings under salt stress, many genes exhibited differential expression patterns when the transgenic and WT lines were compared. These genes include plant defensins, heat shock proteins, other defense related genes, and several transcriptional factors. Our microarray results for selected genes were validated using quantitative real-time PCR.</p> <p>Conclusion</p> <p>Transcriptional analysis in <it>ABR17 </it>transgenic <it>Arabidopsis </it>plants, both under normal and saline conditions, revealed significant changes in abundance of transcripts for many stress responsive genes, as well as those related to plant growth and development. Our results also suggest that <it>ABR17 </it>may mediate stress tolerance through the modulation of many ABA- and CK-responsive genes and may further our understanding of the role of ABR17 in mediating plant stress responses.</p

Auxin transport-feedback models of patterning in plants

Many patterning events in plants are regulated by the phytohormone auxin. In fact, so many things are under the influence of auxin that it seems difficult to understand how a single hormone can do so much. Auxin moves throughout the plant via a network of specialized membrane-bound import and export proteins, which are often differentially expressed and polarized depending on tissue type. Here, we review simulation models of pattern formation that are based on the control of these transporters by auxin itself. In these transport-feedback models, diversity in patterning comes not from the addition of more morphogens, but rather by varying the mechanism that regulates the transporters

APETALA2 control of barley internode elongation

Many plants dramatically elongate their stems during flowering, yet how this response is coordinated with the reproductive phase is unclear. We demonstrate that microRNA (miRNA) control of APETALA2 (AP2) is required for rapid, complete elongation of stem internodes in barley, especially of the final 'peduncle' internode directly underneath the inflorescence. Disrupted miR172 targeting of AP2 in the Zeo1.b barley mutant caused lower mitotic activity, delayed growth dynamics and premature lignification in the peduncle leading to fewer and shorter cells. Stage- and tissue-specific comparative transcriptomics between Zeo1.b and its parent cultivar showed reduced expression of proliferation-associated genes, ectopic expression of maturation-related genes and persistent, elevated expression of genes associated with jasmonate and stress responses. We further show that applying methyl jasmonate (MeJA) phenocopied the stem elongation of Zeo1.b, and that Zeo1.b itself was hypersensitive to inhibition by MeJA but less responsive to promotion by gibberellin. Taken together, we propose that miR172-mediated restriction of AP2 may modulate the jasmonate pathway to facilitate gibberellin-promoted stem growth during flowering

Transcriptional profiling of pea ABR17 mediated changes in gene expression in Arabidopsis thaliana

Background: Pathogenesis-related proteins belonging to group 10 (PR10) are elevated in response to biotic and abiotic stresses in plants. Previously, we have shown a drastic salinityinduced increase in the levels of ABR17, a member of the PR10 family, in pea. Furthermore, we have also demonstrated that the constitutive expression of pea ABR17 cDNA in Arabidopsis thaliana and Brassica napus enhances their germination and early seedling growth under stress. Although it has been reported that several members of the PR10 family including ABR17 possess RNase activity, the exact mechanism by which the aforementioned characteristics are conferred by ABR17 is unknown at this time. We hypothesized that a study of differences in transcriptome between wild type (WT) and ABR17 transgenic A. thaliana may shed light on this process. Results: The molecular changes brought about by the expression of pea ABR17 cDNA in A. thaliana in the presence or absence of salt stress were investigated using microarrays consisting of 70-mer oligonucleotide probes representing 23,686 Arabidopsis genes. Statistical analysis identified number of genes which were over represented among up- or down-regulated transcripts in the transgenic line. Our results highlight the important roles of many abscisic acid (ABA) and cytokinin (CK) responsive genes in ABR17 transgenic lines. Although the transcriptional changes followed a general salt response theme in both WT and transgenic seedlings under salt stress, many genes exhibited differential expression patterns when the transgenic and WT lines were compared. These genes include plant defensins, heat shock proteins, other defense related genes, and several transcriptional factors. Our microarray results for selected genes were validated using quantitative real-time PCR. Conclusion: Transcriptional analysis in ABR17 transgenic Arabidopsis plants, both under normal and saline conditions, revealed significant changes in abundance of transcripts for many stress responsive genes, as well as those related to plant growth and development. Our results also suggest that ABR17 may mediate stress tolerance through the modulation of many ABA- and CKresponsive genes and may further our understanding of the role of ABR17 in mediating plant stress responses