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Primary metabolites and genes corresponding to important metabolic pathways that may be potentially involved in mediating resistance to clubroot disease in canola were identified. Arrows indicate increase in metabolite levels and gene expression.

Pathogenic fungi cause significant yield losses and quality reductions to many crops including canola, wheat, and barley. Toxic metabolites produced by fungal pathogens, along with excessive application of synthetic fungicides, can also pose risks to human and livestock health. Hydroxy unsaturated fatty acids (HUFAs) are novel alternatives to commonly used fungicides. Here, the antifungal activities of two HUFAs, coriolic acid (CA) and ricinoleic acid (RA), were assessed in vitro and in planta for their activity against the important phytopathogens Fusarium graminearum Schwabe, Pyrenophora tritici-repentis (Died.) Drechsler, Pyrenophora teres f. teres Drechsler, Sclerotinia sclerotiorum (Lib.) de Bary, Leptosphaeria maculans Ces. & De Not., and Aspergillus niger Tiegh. on selected media, monocots or dicots. The results in vitro indicated that both CA and RA showed the strongest inhibitory activity against L. maculans and A. niger, but their activities varied with different fungi. On wheat and barley, CA but not RA reduced disease severity caused by Pyrenophora spp.; on canola, treatment with high levels of CA and RA led to oxidative damage of the plant tissues, and treatment with low concentrations of CA and RA did not reduce disease severity caused by L. maculans or S. sclerotiorum on canola. Our findings suggest that the utility of HUFAs in reducing disease severity caused by pathogenic fungi depends on the dosage and the plant and fungus targeted. High concentrations of HUFA can be phytotoxic on certain plants. In addition to their direct antifungal properties, additional mechanisms may be involved in the disease reduction of CA, suggesting the need for further evaluation of its potential use in crop protection.

Clubroot resistance in spring canola has been introgressed from different Brassica sources; however, molecular mechanism underlying this resistance, especially the involvement of long non-coding RNAs (lncRNAs), is yet to be understood. We identified 464 differentially expressed (DE) lncRNAs from the roots of clubroot-resistant canola, carrying resistance on chromosome BnaA03, and susceptible canola lines challenged with Plasmodiophora brassicae pathotype 3. Pathway enrichment analysis showed that most of the target genes regulated by these DE lncRNAs belonged to plant–pathogen interaction and hormone signaling, as well as primary and secondary metabolic pathways. Comparative analysis of these lncRNAs with 530 previously reported DE lncRNAs, identified using resistance located on BnaA08, detected 12 lncRNAs that showed a similar trend of upregulation in both types of resistant lines; these lncRNAs probably play a fundamental role in clubroot resistance. We identified SSR markers within 196 DE lncRNAs. Genotyping of two DH populations carrying resistance on BnaA03 identified a marker capable of detecting the resistance in 98% of the DH lines. To our knowledge, this is the first report of the identification of SSRs within lncRNAs responsive to P. brassicae infection, demonstrating the potential use of lncRNAs in the breeding of Brassica crops.

Clubroot disease, caused by Plasmodiophora brassicae Woronin, is a major threat to the production of Brassica’ crops. Resistance to different P. brassicae pathotypes has been reported in the A genome, chromosome A08; however, the molecular mechanism of this resistance, especially the involvement of long noncoding RNAs (lncRNAs), is not understood. We have used a strand-specific lncRNA-Seq approach to catalog lncRNAs from the roots of clubroot-susceptible and -resistant Brassica napus lines. In total, 530 differentially expressed (DE) lncRNAs were identified, including 88% of long intergenic RNAs and 11% natural antisense transcripts. Sixteen lncRNAs were identified as target mimics of the microRNAs (miRNAs) and eight were identified as the precursors of miRNAs. KEGG pathway analysis of the DE lncRNAs showed that the cis-regulated target genes mostly belong to the phenylpropanoid biosynthetic pathway (15%) and plant–pathogen interactions (15%) while the transregulated target genes mostly belong to carbon (18%) and amino acid biosynthesis pathway (19%). In all, 24 DE lncRNAs were identified from chromosome A08, which is known to harbor a quantitative trait locus conferring resistance to different P. brassicae pathotypes; however, eight of these lncRNAs showed expression only in the resistant plants. These results could form the basis for future studies aimed at delineating the roles of lncRNAs in plant–microbe interactions.

Clubroot resistance in spring canola has been introgressed from different Brassica sources; however, molecular mechanism underlying this resistance, especially the involvement of long non-coding RNAs (lncRNAs), is yet to be understood. We identified 464 differentially expressed (DE) lncRNAs from the roots of clubroot-resistant canola, carrying resistance on chromosome BnaA03, and susceptible canola lines challenged with

Salicylic acid (SA) and reactive oxygen species (ROS) are two well-defined inducers of leaf senescence. Here, we identified a novel WRKY transcription factor gene WSR1 (WRKY regulating SA and ROS 1) in Brassica napus (rapeseed) in promoting SA and ROS production, which eventually led to leaf senescence thereafter. Its expression increased in senescing leaves. Ca2+-dependent protein kinase (CPK) 5 and -6 interacted with and phosphorylated BnaWSR1. Overexpression of phosphomimic BnaWSR1 (BnaWSR1ca) in rapeseed protoplasts elicited ROS production and cell death while its ectopic expression in Arabidopsis enhanced SA and ROS levels and, hence, accelerated leaf senescence. Furthermore, BnaWSR1ca activated the expression of Isochorismate Synthase 1 (ICS1), Respiratory Burst Oxidase Homologue (Rboh) D, and Senescence-Associated Gene 14 (SAG14). Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays demonstrated that BnaWSR1ca directly bound to promoter regions of ICS1, RbohD, and SAG14. These data have identified a CPK-WSR1 module that integrates SA and ROS to control cell death and leaf senescence.

Sclerotinia sclerotiorum is a model necrotrophic pathogen causing great economic losses worldwide. Sclerotia are dormant structures that play significant biological and ecological roles in the life and disease cycles of S. sclerotiorum and other species of sclerotia-forming fungi. microRNA-like RNAs (milRNAs) as non-coding small RNAs play regulatory roles in fungal development and pathogenicity. Therefore, milRNAs associated with sclerotial development in S. sclerotiorum were investigated in this study. A total of 275 milRNAs with induced expression during sclerotia development were identified, in which 51 were differentially expressed. The target genes of all milRNAs were predicted. The putative functions of the targets regulated by milRNAs were annotated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The expression levels of six selected milRNAs that coordinated with their corresponding targets were validated by qRT-PCR. Among these six milRNAs, Ssc-milR-240 was potentially associated with sclerotial development by epigenetic regulation of its target histone acetyltransferase. This study will facilitate the better understanding of the milRNA regulation associated with sclerotial development in S. sclerotiorum and even other sclerotia-forming fungi. This work will provide novel insights into the molecular regulations of fungal morphogenesis and the candidate targets of milRNAs used for the sustainable management of plant diseases caused by S. sclerotiorum.

Low temperature is one of the most common environmental stresses that seriously affect the growth and development of plants. However, plants have the plasticity in their defence mechanisms enabling them to tolerate and, sometimes, even survive adverse environmental conditions. MicroRNAs (miRNAs) are small non-coding RNAs, approximately 18-24 nucleotides in length, and are being increasingly recognized as regulators of gene expression at the post-transcriptional level and have the ability to influence a broad range of biological processes. There is growing evidence in the literature that reprogramming of gene expression mediated through miRNAs is a major defence mechanism in plants enabling them to respond to stresses. To date, numerous studies have established the importance of miRNA-based regulation of gene expression under low temperature stress. Individual miRNAs can modulate the expression of multiple mRNA targets, and, therefore, the manipulation of a single miRNA has the potential to affect multiple biological processes. Numerous functional studies have attempted to identify the miRNA-target interactions and have elaborated the role of several miRNAs in cold-stress regulation. This review summarizes the current understanding of miRNA-mediated modulation of the expression of key genes as well as genetic and regulatory pathways, involved in low temperature stress responses in plants.

Gene expression profiles are increasingly applied to investigate molecular mechanism for which, normalization with suitable reference genes is critical. Previously we have reported several suitable reference genes for laticifer samples from rubber tree, however, little is known in leaf. The main objective of this current study was to identify some stable expression reference genes at various developmental stages of leaf, as well as during abiotic (high and low temperature extremes) and biotic stresses (pathogen stress). Gene expression profilings identified the ubiquitin-proteasome system as excellent potential as reference genes for rubber tree leaf. Among a total of 30 tested genes investigated, 24 new candidate (including 11 genes involved in the ubiquitin-proteasome system), 4 previously identified and 2 specific genes, were further evaluated using quantitative real-time PCR. Our results indicated that the new candidate genes had better expression stability comparing with others. For instance, an ubiquitin conjugating enzyme (RG0099) and three ubiquitin-protein ligases (RG0928, RG2190 and RG0118) expressed stably in all samples, and were confirmed to be suitable reference genes for rubber tree leaf under four different conditions. Finally, we suggest that using more than one reference gene may be appropriate in gene expression studies when employing different software to normalize gene expression data. Our findings have significant implications for the reliability of data obtained from genomics studies in rubber tree and perhaps in other species.

Backgroud Gene expression profiling is increasingly applied to investigate molecular mechanisms for which, normalization with suitable reference genes is critical. Previously we have reported several suitable reference genes for laticifer samples from rubber, however, little is known about reference genes in leaf. Results The main objective of this current study was to identify some reference genes with stable expression patterns in leaf at various developmental stages, as well as during abiotic (temperature extremes) and biotic stresses. Gene expression profiling experiments in rubber tree leaf identified the ubiquitin-proteasome system as having excellent potential as reference genes. Among a total of 30 tested genes investigated, 24 new (including 11 genes involved in the ubiquitin-proteasome system), 4 previously identified and 2 specific genes, were further evaluated using quantitative real-time PCR. Our results indicated that the new genes had better stability of expression when compared with others. For instance, an ubiquitin conjugating enzyme (RG0099) and three ubiquitin-protein ligases (RG0928, RG2190 and RG0118) expressed stably in all samples, and were confirmed to be suitable reference genes in rubber tree leaf in four different conditions. Finally, we suggest that using more than one reference gene may be appropriate in gene expression studies when employing different software to normalize gene expression data. Conclusion Our findings have significant implications for the reliability of data obtained from genomics studies in rubber tree and perhaps in other species.

Clubroot resistance has been introgressed into spring canola from various sources; however, the molecular basis of this using multiple resistances has not yet been investigated. We conducted a comparative transcriptomic study using resistance introgressed from rutabaga (RtR) and winter canola cv. ‘Mendel’ (MdR) and identified ~3500 upregulated genes of which 8–9% genes were involved in primary and secondary metabolic pathways. Treatment of the susceptible plants with trehalose suggested that the modulation of primary metabolism can increase resistance to clubroot. While comparing the DEGs from BnaA08 (RtR) and BnaA03 (MdR), we found that the gene CRF4 could be essential for mediating resistance. We also developed SSR markers from the upregulated genes for use in breeding.

In higher plants, hexokinase (HXK) is a crucial enzyme that phosphorylates glucose, because there is no evidence for the existence of glucokinase (GLK). In the rubber tree laticifers, the phosphorylation of glucose initiates glycolysis and pentose phosphate pathways, which provide energy and other compounds required for latex regeneration. However, the mechanism of how HXKs participate in the latex regeneration process in laticifers is currently unknown. In this study, four HXK cDNAs (HbHXK1-4) were isolated from the rubber tree (Hevea brasiliensis) and sequenced. Nucleotide-derived amino acid sequences revealed the presence of conserved amino acids that are required for catalytic activity and subcellular targeting. Among these genes, HbHXK2 and 4 were found to be the predominant isoforms in latex (cytoplasm of laticifers) when compared with others. Phylogenetic and subcellular localization analysis revealed that HbHXK2 was presumably targeted to the chloroplast and HbHXK4 to the mitochondria. Both HbHXK2 and 4 were confirmed to have catalytic activities towards glucose and fructose with a significantly higher affinity for glucose. In yield-stimulating experiments, HbHXK4 was strongly induced by tapping and ethephon treatments; whereas, HbHXK2 had the opposite expression pattern. The abundance of HbHXK4 transcripts was consistent with the observed catalytic activity and latex yield. Furthermore, in laticifers, HbHXK4 was more efficient in phosphorylating glucose compared with HbHXK2. Based on these results, it is concluded that HbHXK2 and 4 are the key enzymes for glucose metabolism during latex regeneration and may contribute to the productivity of H. brasiliensis (para rubber tree), especially HbHXK4.

Phoma macdonaldii causes black stem of sunflower, which severely affects sunflower yield and quality. There is currently little molecular information available for this pathogenic fungus. In this study, a global proteomic analysis of P. macdonaldii was performed to determine the biological characteristics and pathogenicity of this pathogen. A total of 1498 proteins were identified by LC-MS/MS in all biological replicates. Among the identified proteins, 1420 proteins were classified into the three main GO categories (biological process, cellular component, and molecular function) while 806 proteins were annotated into the five major KEGG database (metabolism, genetic information processing, environmental information processing, cellular processes, and organismal systems). The regulated expression levels of eight genes encoding selected identified proteins were investigated to assess their potential effects on fungal development and pathogenesis. To the best of our knowledge, this is the first study to characterize the proteome of the necrotrophic fungus P. macdonaldii. The presented results provide novel insights into the development and pathogenesis of P. macdonaldii and possibly other Phoma species. SIGNIFICANCE: Black stem of sunflower is a devastating disease caused by the necrotrophic fungus Phoma macdonaldii. Relatively little is known regarding the molecular characteristics of this pathogen, and no proteomic investigation has been reported. Thus, we conducted a global proteomic analysis of P. macdonaldii. Many proteins were found to be differentially regulated during fungal development and pathogenesis, suggesting they may be important for these two processes. This is the first proteomic study of P. macdonaldii, and the data presented herein will be useful for elucidating the molecular characteristics of this fungus as well as other Phoma species.

Temperature extremes, including cold, adversely impact plant growth and development. Plant responses to cold stress (CS) are regulated at both transcriptional and post-transcriptional levels. MicroRNAs (miRNAs), small non-coding RNAs, are known to be involved in post-transcriptional regulation of various developmental processes and metal stress in Brassica napus L. (canola), however, their role in response to CS is largely unknown. In this study, changes in various physiological parameters and endogenous abundance of miRNAs were characterized in spring canola seedlings (DH12075) exposed to 4 °C for 0–48 h. Cold stress induced electrolyte leakage, increased the levels of malondialdheyde and antioxidant enzymes and reduced photosynthetic efficiency. Using small RNA sequencing, 70 known and 126 novel miRNAs were identified in CS leaf tissues and among these, 25 known and 104 novel miRNAs were differentially expressed. Quantitative real-time (qRT) PCR analysis of eight selected miRNAs confirmed their CS responsiveness. Furthermore, the expression of six out of eight miRNAs exhibited an opposite trend in a winter variety of canola, ‘Mendel’, when compared to ‘DH12075’. This first study on the B. napus miRNAome provides a framework for further functional analysis of these miRNAs and their targets in response to CS which may contribute towards the future development of cold resilient crops.

Proteins of animal origin can represent a portion of the overall nitrogen (N) pool in the soil environment and there is a possibility that plants may utilize animal proteins as a N source. Using wheat (Triticum aestivum L.) we investigated if the model protein, ovalbumin was taken up into the roots and transported within the plant. In roots, ovalbumin was associated with the epidermis when no root damage was evident, but with minor root damage, it was present in intercellular spaces throughout the cortex and at the endodermis. Ovalbumin was only found in the stem when minor damage to the root system was evident. Suspension cultures of wheat protoplasts revealed that ovalbumin was not assimilated into individual plant cells. Our results suggest that ovalbumin uptake and subsequent movement in wheat is possible only after root damage has occurred. Apoplastic movement may enable animal protein to enter plant tissues above the soil level where they could be consumed by grazers.

Rasmussen, J., Gilroyed, B. H., Reuter, T., Badea, A., Eudes, F., Graf, R., Laroche, A., Kav, N. N. V. and McAllister, T. A. 2014. Efficiency of protein as a nitrogen source for wheat and morphological changes in roots exposed to high protein concentrations. Can. J. Plant Sci. 94: 603–613. Proteins of animal origin can enter the environment through application of agricultural by-products to arable or pastured land. In this study, wheat (Triticum aestivum cv. AC Andrew) was exposed to treatments with nitrogen (N) supplied as animal protein (bovine serum albumin; BSA), inorganic N or a combination of these sources at different iso-nitrogenous concentrations. Plant growth was assessed by monitoring both wet and dry mass of shoots and data showed that protein treatments did not differ (P>0.05) from controls lacking N. Analysis of N also showed that plants supplied with protein N displayed lower N (1.2–2.4%) concentration as compared with those supplied with inorganic N (up to 12.4%) with N remaining 71 mM, with the development of knob-like outgrowths with unknown function or significance. This study provides evidence that wheat plantlets grown under sterile conditions are unable to utilize BSA as efficiently as NH4NO3 as a N source, but their roots exhibit a morphological response to protein.

MicroRNAs (miRNAs) are a class of small endogenous RNAs conserved in eukaryotic organisms including plants. They suppress gene expression posttranscriptionally in many different biological processes. Previously, we reported salinity-induced changes in gene expression in transgenic Arabidopsis thaliana plants that constitutively expressed a pea abscisic acid-responsive (ABR17) gene. In the current study, we used microarrays to investigate the role of miRNA-mediated posttranscriptional gene regulation in these same transgenic plants in the presence and absence of salinity stress. We identified nine miRNAs that were significantly modulated due to ABR17 gene expression and seven miRNAs that were modulated in response to salt stress. The target genes regulated by these miRNAs were identified using sRNA target Base (starBase) degradome analysis and through 5′-RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). Our findings revealed miRNA–mRNA interactions comprising regulatory networks of auxin response factor (ARF), argonaute 1 (AGO1), dicer-like proteins 1 (DCL1), SQUAMOSA promoter binding (SPB), NAC, APETALA 2 (AP2), nuclear factor Y (NFY), RNA-binding proteins, A. thaliana vacuolar phyrophosphate 1 (AVP1), and pentatricopeptide repeat (PPR) in ABR17-transgenic A. thaliana, which control physiological, biochemical, and stress signaling cascades due to the imposition of salt stress. Our results are discussed within the context of the effect of the transgene, ABR17, and the roles of miRNA expression may play in mediating plant responses to salinity.

Low temperature stress is one of the major abiotic stress challenging the growth and productivity of economically important crops. Both chilling and freezing temperatures have severe effects on growth of plants and have resulted in temperate plants, such as perennial rye grass and wheat to evolve mechanisms to avoid or, at the very least, minimize this damage. Accumulating osmoprotectants including glycine betaine, sugars (trehalose and fructans), polyamines, changes in lipid membrane profile, photosynthetic acclimation along with extensive reprogramming at molecular level help temperate plants acquire tolerance to low temperatures. In this review, we have focused mainly on metabolic engineering of plants by introduction of biosynthetic genes involved in various metabolic pathways. Availability of genomic, transcriptomic sequences combined with post-transcriptional data is beginning to link the gene function, regulatory networks and epigenetic states to different phenotypes. Generation of this knowledge together with our ability to manipulate genes involved in mediating tolerance to various stressors including low temperature will lead to the development of cold-resistant genotypes.

Prion diseases are progressive, infectious neurodegenerative disorders caused primarily by the misfolding of the cellular prion protein (PrPc) into an insoluble, protease-resistant, aggregated isoform termed PrPsc. In native conditions, PrPc has a structured C-terminal domain and a highly flexible N-terminal domain. A part of this N-terminal domain consists of 4–5 repeats of an unusual glycine-rich, eight amino acids long peptide known as the octapeptide repeat (OR) domain. In this article, we successfully report the first crystal structure of an OR of PrPc bound to the Fab fragment of the POM2 antibody. The structure was solved at a resolution of 2.3 A by molecular replacement. Although several studies have previously predicted a β-turn-like structure of the unbound ORs, our structure shows an extended conformation of the OR when bound to a molecule of the POM2 Fab indicating that the bound Fab disrupts any putative native β turn conformation of the ORs. Encouraging results from several recent studies have shown that administering small molecule ligands or antibodies targeting the OR domain of PrP result in arresting the progress of peripheral prion infections both in ex vivo and in in vivo models. This makes the structural study of the interactions of POM2 Fab with the OR domain very important as it would help us to design smaller and tighter binding OR ligands.

The phytopathogen Sclerotinia sclerotiorum has been extensively studied, but factors contributing to the pathogenicity of this fungus are not comprehensively understood. With the annotation of the S. sclerotiorum genome and its characterization at the transcriptome and proteome levels, hypothetical and predicted proteins can be targeted for more detailed studies, helping to elucidate fungal infection and virulence mechanisms. Accordingly, a gene (ssv263) encoding a hypothetical, novel protein from S. sclerotiorum that is orthologous to a secreted protein from Botrytis cinerea, and which appears to be unique to these pathogens, was targeted for further analysis. Mutant strains of S. sclerotiorum were generated by disruption of the ssv263 gene and characterized for virulence on a susceptible canola (Brassica napus) genotype. Based on the extent of symptom development, the virulence of the ssv263-disrupted mutants was significantly reduced relative to the wild-type. To our knowledge, this is the fir...

Sclerotinia stem rot caused by Sclerotinia sclerotiorum affects canola production worldwide. Emerging evidence suggests that long non-coding RNAs (lncRNAs) play important roles in the regulation of gene expression in plants, in response to both abiotic and biotic stress. So far, identification of lncRNAs has been limited to a few model plant species, and their roles in mediating responses to biotic stresses are yet to be characterized in Brassica napus. The present study reports the identification of novel lncRNAs responsive to S. sclerotiorum infection in B. napus at two time points after infection (24 hpi and 48 hpi) using a stranded RNA-Sequencing technique and a detection pipeline for lncRNAs. Of the total 3,181 lncRNA candidates, 2,821 lncRNAs were intergenic, 111 were natural antisense transcripts, 76 possessed exonic overlap with the reference coding transcripts while the remaining 173 represented novel lnc- isoforms. Forty one lncRNAs were identified as the precursors for microRNAs (miRNAs) including miR156, miR169 and miR394, with significant roles in mediating plant responses to fungal phytopathogens. A total of 931 differentially expressed lncRNAs were identified in response to S. sclerotiorum infection and the expression of 12 such lncRNAs was further validated using qRT-PCR. B. napus antisense lncRNA, TCONS_00000966, having 90% overlap with a plant defensin gene, showed significant induction at both infection stages, suggesting its involvement in the transcriptional regulation of defense responsive genes under S. sclerotiorum infection. Additionally, nine lncRNAs showed overlap with cis-regulatory regions of differentially expressed genes of B. napus. Quantitative RT-PCR verification of a set of S. sclerotiorum responsive sense/antisense transcript pairs revealed contrasting expression patterns, supporting the hypothesis that steric clashes of transcriptional machinery may lead to inactivation of sense promoter. Our findings highlight the potential contributions of lncRNAs in regulating expression of plant genes that respond to biotic stress.

The necrotrophic phytopathogen, Sclerotinia sclerotiorum, causes Sclerotinia stem rot, which is a serious constraint to canola (Brassica napus L.) production worldwide. To understand the detailed molecular mechanisms underlying host response to Sclerotinia infection, we analyzed the transcript level changes in canola post-infection with S. sclerotiorum in a time course of a compatible interaction using strand specific whole transcriptome sequencing. Following infection, 161 and 52 genes (P≤0.001) were induced while 24 and 23 genes were repressed at 24h post-inoculation (hpi) and 48hpi, respectively. This suggests that, a gradual increase in host cell lyses and increase virulence of the pathogen led to the expression of only a fewer host specific genes at the later stage of infection. We observed rapid induction of key pathogen responsive genes, including glucanases, chitinases, peroxidases and WRKY Transcription factors (TFs) within 24hpi, indicating early detection of the pathogen by the host. Only 16 genes were significantly induced at both the time points suggesting a coordinated suppression of host responses by the pathogen. In addition to genes involved in plant-pathogen interactions, many novel disease responsive genes, including various TF sand those associated with jasmonate (JA) and ethylene (ET) signalling were identified. This suggests that canola adopts multiple strategies in mediating plant responses to the pathogen attack. Quantitative real time PCR (qRT-PCR) validation of a selected set of genes demonstrated a similar trend as observed by RNA-Seq analysis and highlighted the potential involvement of these genes by the host to defend itself from pathogen attack. Overall, this work presents an in-depth analysis of the interaction between host susceptibility and pathogen virulence in the agriculturally important B. napus-S. sclerotiorum pathosystem.

The aleurone layer of mature Arabidopsis thaliana seed plays important roles in seed germination and dormancy. However, the proteomic profile of this cell layer is unknown partly because it is difficult to separate this thin cell layer from the mature seeds. In this study, we have used a simple technique to separate the aleurone layer along with the seed coat following germination of seeds and determined for the first time the putative protein composition of this cell layer. By subjecting the total proteins extracted from the seed coat to 2D gel electrophoresis followed by liquid chromatography/tandem mass spectrometry, we identified four AGI loci, AT4G28520, AT5G44120, AT1G03880, and AT1G03890; all of which belong to the seed storage family of proteins. Because in Arabidopsis the diploid aleurone cells of the seed coat perform protein storage functions similar to that of triploid endosperm of other plant species, it is assumed that the above AGI loci are associated with the aleurone layer of the seed coat.

Canola (Brassica napus), an agriculturally important oilseed crop, can be significantly affected by diseases such as sclerotinia stem rot, blackleg, and alternaria black spot resulting in significant loss of crop productivity and quality. Cysteine-rich antimicrobial peptides isolated from plants have emerged as a potential resource for protection of plants against phytopathogens. Here we report the significance of an antimicrobial peptide, PmAMP1, isolated from western white pine (Pinus monticola), in providing canola with resistance against multiple phytopathogenic fungi. The cDNA encoding PmAMP1 was successfully incorporated into the genome of B. napus, and it's in planta expression conferred greater protection against Alternaria brassicae, Leptosphaeria maculans and Sclerotinia sclerotiorum. In vitro experiments with proteins extracted from transgenic canola expressing Pm-AMP1 demonstrated its inhibitory activity by reducing growth of fungal hyphae. In addition, the in vitro synthesized peptide also inhibited the growth of the fungi. These results demonstrate that generating transgenic crops expressing PmAMP1 may be an effective and versatile method to protect susceptible crops against multiple phytopathogens.

Pea abscisic acid responsive (ABR17) protein is a member of the pathogenesis-related 10 (PR10) family of proteins and its ribonuclease (RNase) activity has been reported previously. In order to investigate the amino acids important for the demonstrated ribonuclease activity of ABR17, site-directed mutants H69L and E148A were generated, expressed in Escherichia coli and purified to homogeneity. These mutations affected RNase activity differently; the H69L mutant exhibited a decreased RNase activity whereas E148A exhibited an elevated activity. A structural model for pea ABR17 has been generated using the three dimensional structure of Lupinus luteus PR10 protein in order to explain the possible effects of the H69L and the E148A mutations on substrate binding and catalysis.

PR-10 genes encode small, acidic, intracellular proteins that respond to abiotic and biotic stimuli. Transgenic expression of PR-10 genes has been shown to enhance early seedling growth of dicots in saline environments. To identify candidate PR-10 genes in cereals for increasing stress tolerance, we conducted phylogenetic analyses and real-time polymerase chain reaction of representatives of the two major clades of putative PR-10 genes in wheat. We observed that the abundance of BQ752893 was generally greater than the abundance of CV778999, particularly when measured in roots across four wheat genotypes. However, CV778999 transcripts were more abundant than BQ752893 in flag leaves. These data suggest that the transcripts define two functionally divergent groups of PR-10 type genes in wheat, both of which may be suitable targets for biotechnological manipulation under different circumstances.

Egg storage causes egg white to lose its viscous nature to form a thin liquid, commonly referred to as egg white thinning. To understand the mechanisms underlying egg white thinning, white-shell eggs were used in the present study to determine the proteome-level changes of egg white proteins occurred during storage. Egg white thinning was observed visually after 20 days of storage at ambient temperature (22 ± 2 °C) when the maximum number of proteome-level changes occurred. The proteins that showed significant changes in abundance during storage included ovalbumin, clusterin, ovoinhibitor, ovotransferrin, and prostaglandin D2 synthase. Among these, only the abundance of clusterin was observed to change continuously during the storage period. Hence, it is expected that the increase in the concentrations of clusterin and ovoinhibitor along with the change of ovalbumin content during storage might contribute to egg white thinning. Degradation of ovalbumin/clusterin during egg storage may be due to the combined effect of proteolysis and increase in pH; this may also be partly responsible for egg white thinning phenomenon.

Blackleg and blackspot are economically important diseases of canola caused by Leptosphaeria maculans and Alternaria brassicae, respectively, and can lead to significant crop losses. In this study, we investigated the effects of cytokinins on the symptoms caused by these two pathogens. We observed that cytokinin, especially 6-benzyl amino purine, was able to significantly reduce disease symptoms and mycelial growth within plant tissues. This cytokinin was also able to inhibit the in vitro growth of both fungi. Other cytokinins such as kinetin or adenine hemisulfate were unable to inhibit fungal growth in vitro, suggesting that the presence of a benzene ring structure is required for the inhibitory effects observed. Our findings are discussed within the context of plant–pathogen interactions.

Brassica napus (canola) is an important oilseed crop in many countries throughout the world including Canada. One of the major constraints on canola productivity is blackspot disease caused by the necrotrophic phytopathogenic fungus Alternaria brassicae. Two isolates of A. brassicae with significant differences in virulence have been characterized at the proteomelevel. The morphological observations indicated the Ontario isolate to be more virulent by virtue of increased disease severity score as compared to the UAMH7476 isolate. This was further confirmed through histological observations that indicated extensive colonization of the host tissue by the highly virulent isolate. Mycelial protein profiles of two differentially virulent A. brassicae isolates were compared using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) in order to identify proteins that may be responsible for the observed differences. The investigation suggested several differences in the mycelial proteomes of the two isolates. The proteins that were significantly abundant in the more virulent isolate included a protein with conserved actin related protein 2/3 domain, enolase, malate dehydrogenase and serine protease. These results suggest that the differential protein expression pattern could be exploited to identify putative virulence and pathogenicity factors in A. brassicae.

Canola (Brassica napus L.) is an agriculturally and economically important crop in Canada, and its growth and yield are frequently influenced by fungal pathogens. Sclerotinia sclerotiorum is among those fungal pathogens and causes stem rot disease in B. napus whereas it has been reported that Brassica carinata is moderately tolerant to S. sclerotiorum. Jasmonic acid/ethylene (JA/ET) and salicylic acid (SA) are phytohormones that are known to be involved in plant disease responses. To investigate the defense signaling cascades involved in the interaction of B. napus and B. carinata with S. sclerotiorum, we examined the expression of five orthologs of B. napus genes involved in JA/ET or SA signaling pathways using quantitative RT-PCR. Our results indicated that there are differences in the timing of JA/ET and SA signaling pathways between B. napus and B. carinata. Our results in these two Brassica species also support previous observations that necrotrophic pathogens trigger JA/ET signaling in response to infection. Finally, we observed that transgenic canola expressing 1-aminocyclopropane-1-carboxylate-deaminase producing low levels of ET was relatively more susceptible to S. sclerotiorum than its wild-type counterpart, suggesting that ET inhibits S. sclerotiorum-induced symptom development.

Oxalic acid (OA) occurs extensively in nature and plays diverse roles, especially in pathogenic processes involving various plant pathogens. However, proteome changes and modifications of signaling and oxidative network of plants in response to OA are not well understood. In order to investigate the responses of Brassica napus toward OA, a proteome analysis was conducted employing 2-DE with MS/MS. A total of 37 proteins were identified as responding to OA stress, of which 13 were up-regulated and 24 were down-regulated. These proteins were categorized into several functional groups including protein processing, RNA processing, photosynthesis, signal transduction, stress response, and redox homeostasis. Investigation of the effect of OA on phytohormone signaling and oxidative responses revealed that jasmonic acid-, ethylene-, and abscisic acid-mediated signaling pathways appear to increase at later time points, whereas those pathways mediated by salicylic acid appear to be suppressed. Moreover, the activities of the antioxidant enzymes catalase, peroxidase, superoxide dismutase and oxalic acid oxidase, but not NADPH oxidase, were suppressed by OA stress. Our findings are discussed within the context of the proposed role(s) of OA during infection by Sclerotinia sclerotiorum and subsequent disease progression.

Sclerotinia sclerotiorum (Lib.) de Bary is a phytopathogenic fungus capable of causing significant yield losses in numerous crops, including canola, in which the fungus causes sclerotinia stem rot. Immunological detection methods to rapidly determine the presence of S. sclerotiorum on plants may provide growers with a viable diagnostic tool to aid with fungicide use decisions. This paper discusses the generation of a monomeric and dimeric single-chain, variable fragment (scFv) antibody with affinity for S. sclerotiorum using phage display technology. The bacterially expressed and purified scFv is shown to bind S. sclerotiorum with some cross-reactivity with the closely related phytopathogen Botrytis cinerea (Pers.:Fr.). The dimeric scFv displayed improved binding to the fungus as compared to the monomer and could detect the presence of mycelia in inoculated canola petals. To the authors' knowledge, this is the first report of a scFv dimer with affinity for S. sclerotiorum that has the potential for use in the development of a new diagnostic test.

To better understand the pathogen-stress response of Brassica species against the ubiquitous hemi-biotroph fungus Leptosphaeria maculans, we conducted a comparative proteomic analysis between blackleg-susceptible Brassica napus and blackleg-resistant Brassica carinata following pathogen inoculation. We examined temporal changes (6, 12, 24, 48 and 72 h) in protein profiles of both species subjected to pathogen-challenge using two-dimensional gel electrophoresis. A total of 64 proteins were found to be significantly affected by the pathogen in the two species, out of which 51 protein spots were identified using tandem mass spectrometry. The proteins identified included antioxidant enzymes, photosynthetic and metabolic enzymes, and those involved in protein processing and signaling. Specifically, we observed that in the tolerant B. carinata, enzymes involved in the detoxification of free radicals increased in response to the pathogen whereas no such increase was observed in the susceptible B. napus. The expression of genes encoding four selected proteins was validated using quantitative real-time PCR and an additional one by Western blotting. Our findings are discussed with respect to tolerance or susceptibility of these species to the pathogen.

Triticum aestivum ‘Opata’ is an elite hard red spring wheat that has been used as a parent of the ITMI (International Triticeae Mapping Inititative) mapping population and also in the production of synthetically derived hexaploid wheats, some of which (following selection) show increased drought tolerance relative to Opata. Here, we describe the response of Opata roots to water withholding, using physiological variables and oligonucleotide microarrays. We identified 394 distinct transcripts whose abundance differed (p ≤ 0.05) at least 1.5-fold between water-limited and control roots of Opata, of which 190 transcripts increased and 204 decreased following water limitation. In addition to previously characterized markers of abiotic stress and many genes of unknown function, we identified multiple putative glucanases and class III peroxidases as being particularly responsive to stress. We also compared these data to previously described microarray analyses of Opata’s more drought-tolerant, synthetic-derived progeny, and found a relatively high correlation (r = 0.7) between responsive transcripts in the two genotypes, despite differing physiological responses. Some of the transcripts that we confirmed by qRT-PCR as being differentially expressed between Opata and the more tolerant synthetic-derived genotype under stress include a class III peroxidase, an AP2-family transcription factor, and several transcripts of unknown function.

Sclerotinia stem rot, caused by the necrotrophic fungal pathogen Sclerotinia sclerotiorum, is a serious disease of canola (Brassica napus L.). To increase the understanding of the B. napus- S. sclerotiorum interaction, proteins potentially involved in mediating this interaction were identified and characterized. Upon infection of canola leaves by S. sclerotiorum, necrosis of host leaves was observed by 12 h and rapidly progressed during the later time points. These morphological observations were supported by microscopic study performed at different time points after pathogen challenge. Leaf proteins were extracted and analyzed by 2-DE, which revealed the modulation of 32 proteins (12 down- and 20 up-regulated). The identities of these proteins were established by ESI-q-TOF MS/MS and included proteins involved in photosynthesis and metabolic pathways, protein folding and modifications, hormone signaling, and antioxidant defense. Gene expression analysis of selected genes was performed by qRT-PCR, whereas the elevated levels of the antioxidant enzymes peroxidase and superoxide dismutase were validated by enzyme assays. To the authors' best knowledge, this is the first proteomics-based investigation of B. napus-S. sclerotiorum interaction, and the roles of many of the proteins identified are discussed within the context of this pathosystem.

Clubroot, caused by the obligate parasite Plasmodiophora brassicae , has emerged as an important new disease of canola ( Brassica napus ) in Alberta, Canada. Changes in the root protein profile were examined by two-dimensional gel electrophoresis at 12, 24, 48 and 72 h after inoculation of a susceptible canola genotype with the pathogen. A total of 20 protein spots were identified as either increasing (13 spots) or decreasing (7 spots) in intensity relative to water-treated controls. The identities of these proteins were established by MS/MS, and included proteins involved in lignin biosynthesis, cytokinin metabolism, glycolysis, intracellular calcium homeostasis, and the detoxification of ROS. Decreased abundance of adenosine kinase, which is involved in cytokinin homeostasis, supported previous reports indicating a key role for cytokinin in the early phases of clubroot infection. An approximately sixfold reduction in caffeoyl-CoA O -methyltransferase abundance suggested a reduction in host lignin biosynthesis after pathogen challenge, and is consistent with the compatible nature of the interaction examined. Interestingly, levels of enzymes involved in ROS metabolism declined sharply at 12 h after inoculation, but increased at 24–72 h. Collectively, these data suggest major changes in canola metabolism shortly after challenge by the pathogen, which may result in the susceptibility of the host.

Pathogenesis-related 10 (PR 10) proteins are induced in a number of angiosperms and gymnosperms following exposure to abiotic or biotic stresses, but their biological function remains unknown. However, the constitutive expression of a PR 10 cDNA has been reported to enhance germination and abiotic stress tolerance in Arabidopsis thaliana. In the present study, we report the constitutive expression of Pisum sativum ABA-responsive 17 (ABR17/PR 10.4) cDNA in an important agricultural species, Brassica napus (canola,) to determine its effects on germination as well as other characteristics. We observed significantly greater germination in the transgenic line at 275 mM NaCl, 5 °C, and 10 °C + 75 mM NaCl when compared to the wild type. In addition, we observed a significantly greater rate of flowering, significantly earlier flowering and greater height in the transgenic line when compared to the wild type at 42 days after planting. Proteome analysis of our transgenic line revealed surprisingly few transgene-induced changes but identified carbonic anhydrase, which may be potentially responsible for some of the observed characteristics, as being elevated. Our study demonstrates that ABR17 promotes germination and early flowering in canola and possible applications are discussed.

To investigate molecular changes in canola (Brassica napus L.) accompanying its interaction with the fungal pathogen Sclerotinia sclerotiorum, we investigated pathogen-induced changes in gene expression using microarrays. This study led to the identification of a number of interesting canola genes that were responsive to the pathogen-challenge at three different time points. More than 300 transcripts were detected by our studies as being increased or decreased in abundance at least two-fold as compared to uninoculated controls. Pathogen-responsive transcripts included those putatively associated with JA biosynthesis and signaling, reactive oxygen species metabolism, and cell wall structure and function. These genes may play important roles in mediating plant responses to the pathogen. We selected 11 genes that exhibited an increase, and 4 genes that exhibited a decrease in transcript abundance, for validation by quantitative real-time PCR. Results from qRT-PCR analysis demonstrated a similar trend as observed by microarray analysis and highlighted the potential involvement of these genes in mediating plant responses to the pathogen.

The role of cytokinins (CKs) in mediating the previously observed ABR17-mediated enhancement of germination of Arabidopsis thaliana under salinity and low-temperature stresses has been evaluated. We determined the endogenous concentrations of CK in the three transgenic and wild-type seedlings, which indicated that the transgenic seedlings had higher endogenous concentrations of CK. Furthermore, the relative levels of expression of ABR17 cDNA and the primary CK response gene, ARR5, were evaluated in the transgenic and wild-type seedlings by quantitative real-time polymerase chain reaction (RT-PCR). Our results indicated that two of the three independently derived transgenic plants possessed higher levels of ABR17 transcripts, which correlated well with increased ARR5 expression, further supporting a possible role for CK in mediating the observed phenomenon. In addition, the exogenous application of various CKs on the germination of wild-type A. thaliana under these abiotic stress conditions enhanced its germination. Finally, the ribonuclease (RNase) activity of the pea ABR17 protein was also demonstrated after expression of its cDNA in Escherichia coli, purification of the recombinant protein, and in vitro RNase assays. Our findings are discussed within the context of ABR17-mediated enhancement of endogenous CK concentrations, the involvement of CKs in germination under abiotic stress, as well as the role of the RNase activity of ABR17 protein in mediating the observed effects.

Two Brassica lines derived from an interspecific cross between Brassica napus and B. carinata were evaluated for tolerance to the fungal pathogen Alternaria brassicae. Pathogen-induced chlorosis and necrosis spread significantly in one linewhereas it remained localized in the other. Proteome-level changes in response to the fungal pathogen were investigated using two-dimensional electrophoresis. Levels of 48 proteins were significantly affected at various time points in the tolerant line (41 up-regulated and 7 down-regulated). In contrast, in the susceptible line, we observed the levels of 23 proteins to be significantly affected with 4 increasing and 19 decreasing. We were successful in establishing the identities of 38 proteins and those identified from the tolerant line included enzymes involved in the generation of reactive oxygen species (ROS), ROS mediated signaling, auxin signal transduction and metabolic pathways. Proteome-level results suggesting a role for ROS mediated auxin signaling in this pathosystem was further investigated and confirmed using quantitative real-time PCR. Our findings are discussed within the context of A. brassicae‐B. napus interaction and, tolerance to this pathogen. # 2006 Elsevier Ireland Ltd. All rights reserved.

Non-coding RNAs (ncRNAs) are a family of regulatory RNAs, which do not encode mRNA, rRNA or tRNA, found in a variety of organisms including plants. Different classes of ncRNAs have been identified based on their length and their position in the genome, including small ncRNAs (microRNAs and small-interfering RNAs), natural antisense transcripts (NATs), and long intronic/intergenic ncRNAs (lncRNAs, 200nt or longer). Recent advances in next-generation sequencing technologies and computational analysis for transcriptome profiling have led to the genome-wide identification of ncRNAs. Functional characterization of these ncRNAs has implicated them to play a role in a wide range of cellular functions, such as epigenetic silencing, transcriptional regulation, and RNA metabolism. Emerging evidence suggest that several lncRNAs play important roles in many fundamental biological processes including growth and development as well as abiotic stress responses. Recent findings on the roles of lncRNAs in the aforementioned plant processes are summarized in this chapter.

The constitutive expression of a cDNA encoding a pea (Pisum sativum L.) PR 10 protein in Brassica napus leading to an enhancement of germination under saline conditions has been previously reported. In order to understand the biochemical function of this pea PR 10 protein, its cDNA has been expressed in Escherichia coli and the recombinant protein purified to homogeneity. Ribonuclease activity of the recombinant pea PR 10 protein has been demonstrated for the first time using an in-solution as well as an in-gel RNA degradation assay. Furthermore, in order to characterize the changes brought about as a result of the constitutive expression of the pea PR 10 cDNA in B. napus, we have measured the endogenous concentrations of several phytohormones. Increased cytokinin and, decreased abscisic acid (ABA) were observed in 7-day-old transgenic seedlings whereas no significant changes in the concentrations of gibberellin (GA) or indoleacetic acid (IAA) were observed at this stage of growth and development. The potential role(s) of PR 10 proteins with RNase activity and elevated cytokinins during plant stress responses as well as the possible relationship between PR 10 protein and changes in cytokinin concentrations are discussed.

Canola (Brassica napus L.) is planted in early spring and must survive both low soil temperatures and periods of wet weather. Shoot effects result from both low root temperature and low aeration, but little is known about the interaction between these two environmental factors, particularly with respect to changes in gene products. In this study, canola plants ('46A65') were treated in solution culture. Shoot temperatures were maintained at day/night temperatures of 24/18 degrees C, while roots were maintained at an ambient temperature of 24/18 degrees C, or cooled to 10 degrees C. Roots were either aerated, or not aerated to create hypoxic treatments. Plants with roots in cold and hypoxic solution accumulated starch in the root, and had greater reductions in fresh weight and leaf area than those in either cold or hypoxic treatments alone. Twenty-one changes in protein expression were also found in the cold hypoxic treatment, 17 of which were not found in either cold or hypoxic treatments alone. Gene products up-regulated in leaves included cytochrome oxidase Subunit I (COX1) in response to hypoxia, elongation factor eEF1 gamma chain in plants with cooled roots, and chaperonin 10 when roots were cooled without aeration. Results demonstrate the interaction between multiple stresses on a molecular level, and suggest that flooding under cool soil temperatures will be more detrimental to canola than that which occurs at warmer temperatures.