A Brassica exon array for whole-transcript gene expression profiling

Affymetrix GeneChip (R) arrays are used widely to study transcriptional changes in response to developmental and environmental stimuli. GeneChip (R) arrays comprise multiple 25-mer oligonucleotide probes per gene and retain certain advantages over direct sequencing. For plants, there are several public GeneChip (R) arrays whose probes are localised primarily in 39 exons. Plant whole-transcript (WT) GeneChip (R) arrays are not yet publicly available, although WT resolution is needed to study complex crop genomes such as Brassica, which are typified by segmental duplications containing paralogous genes and/or allopolyploidy. Available sequence data were sampled from the Brassica A and C genomes, and 142,997 gene models identified. The assembled gene models were then used to establish a comprehensive public WT exon array for transcriptomics studies. The Affymetrix GeneChip (R) Brassica Exon 1.0 ST Array is a 5 mu M feature size array, containing 2.4 million 25-base oligonucleotide probes representing 135,201 gene models, with 15 probes per gene distributed among exons. Discrimination of the gene models was based on an E-value cut-off of 1E(-5), with <= 98 sequence identity. The 135 k Brassica Exon Array was validated by quantifying transcriptome differences between leaf and root tissue from a reference Brassica rapa line (R-o-18), and categorisation by Gene Ontologies (GO) based on gene orthology with Arabidopsis thaliana. Technical validation involved comparison of the exon array with a 60-mer array platform using the same starting RNA samples. The 135 k Brassica Exon Array is a robust platform. All data relating to the array design and probe identities are available in the public domain and are curated within the BrassEnsembl genome viewer at http://www.brassica.info/BrassEnsembl/index.html.

Overexpression of coconut AINTEGUMENTA-like gene, CnANT, promotes in vitro regeneration in transgenic Arabidopsis

Knowledge of the molecular biological changes underlying the process of embryogenesis is important for the improvement of somatic embryogenesis of coconut. Among the transcription factors that control the transition from vegetative to embryogenic growth, members of APETALA2/Ethylene-responsive element binding protein domain family play an important role in promoting embryo development. Significant insights into the role of AP2 genes have been obtained by the ectopic expression of AP2 sub family genes in transgenic Arabidopsis. A homolog of the AINTEGUMENTA-like gene that encodes the two AP2 domains and the linker region was identified in the coconut genome. Phylogenetic analysis showed that this gene, CnANT, encodes a protein that branched with BABY BOOM/PLETHORA clade in the AINTEGUMENTA-like major clade and was similar to the oil palm EgAP2-1 protein. According to real time RT-PCR results, higher expression of CnANT was observed in more mature zygotic embryos. Also, high CnANT expression was recorded in embryogenic callus compared to other stages of somatic embryogenesis. We examined the effect of ectopic CnANT expression on the development and regenerative capacity of transgenic Arabidopsis. Overexpression of CnANT in Arabidopsis induced hormone free regeneration of explants. Furthermore, ectopic expression of CnANT enhanced regeneration in vitro and suggested a role for this gene in cell proliferation during in vitro culture.

Cannabidivarin (CBDV) suppresses pentylenetetrazole (PTZ)-induced increases in epilepsy-related gene expression

To date, anticonvulsant effects of the plant cannabinoid, cannabidivarin (CBDV), have been reported in several animal models of seizure. However, these behaviourally observed anticonvulsant effects have not been confirmed at the molecular level. To examine changes to epilepsy-related gene expression following chemical convulsant treatment and their subsequent control by phytocannabinoid administration, we behaviourally evaluated effects of CBDV (400 mg/kg, p.o.) on acute, pentylenetetra- zole (PTZ: 95 mg/kg, i.p.)-induced seizures, quantified expression levels of several epilepsy-related genes (Fos, Casp 3, Ccl3, Ccl4, Npy, Arc, Penk, Camk2a, Bdnf and Egr1) by qPCR using hippocampal, neocortical and prefrontal cortical tissue samples before examining correlations between expression changes and seizure severity. PTZ treatment alone produced generalised seizures (median: 5.00) and significantly increased expression of Fos, Egr1, Arc, Ccl4 and Bdnf. Consistent with previous findings, CBDV significantly decreased PTZ-induced seizure severity (median: 3.25) and increased latency to the first sign of seizure. Furthermore, there were correlations between reductions of seizure severity and mRNA expression of Fos, Egr1, Arc, Ccl4 and Bdnf in the majority of brain regions in the CBDV+PTZ treated group. When CBDV treated animals were grouped into CBDV responders (criterion: seizure severity ≤ 3.25) and non-responders (criterion: seizure severity >3.25), PTZ-induced increases of Fos, Egr1, Arc, Ccl4 and Bdnf expression were suppressed in CBDV re- sponders. These results provide the first molecular confirmation of behaviourally observed effects of the non-psychoactive, anticonvulsant cannabinoid, CBDV, upon chemically-induced seizures and serve to underscore its suitability for clinical development.

Alterations in predicted regulatory and coding regions of the sterol 14α-demethylase gene (CYP51) confer decreased azole sensitivity in the oilseed rape pathogen Pyrenopeziza brassicae

The incidence and severity of light leaf spot epidemics caused by the ascomycete fungus Pyrenopeziza brassicae on UK oilseed rape crops is increasing. The disease is currently controlled by a combination of host resistance, cultural practices and fungicide applications. We report decreases in sensitivities of modern UK P. brassicae isolates to the azole (imidazole and triazole) class of fungicides. By cloning and sequencing the P. brassicae CYP51 (PbCYP51) gene, encoding the azole target sterol 14α-demethylase, we identified two non-synonymous mutations encoding substitutions G460S and S508T associated with reduced azole sensitivity. We confirmed the impact of the encoded PbCYP51 changes on azole sensitivity and protein activity by heterologous expression in a Saccharomyces cerevisiae mutant YUG37::erg11 carrying a controllable promoter of native CYP51 expression. In addition, we identified insertions in the predicted regulatory regions of PbCYP51 in isolates with reduced azole sensitivity. The presence of these insertions was associated with enhanced transcription of PbCYP51 in response to sub-inhibitory concentrations of the azole fungicide tebuconazole. Genetic analysis of in vitro crosses of sensitive and resistant isolates confirmed the impact of PbCYP51 alterations in coding and regulatory sequences on a reduced sensitivity phenotype, as well as identifying a second major gene at another locus contributing to resistance in some isolates. The least sensitive field isolates carry combinations of upstream insertions and non-synonymous mutations, suggesting PbCYP51 evolution is on-going and the progressive decline in azole sensitivity of UK P. brassicae populations will continue. The implications for the future control of light leaf spot are discussed.

An optimized method for the extraction of bacterial mRNA from plant roots infected with Escherichia coli O157:H7

Analysis of microbial gene expression during host colonization provides valuable information on the nature of interaction, beneficial or pathogenic, and the adaptive processes involved. Isolation of bacterial mRNA for in planta analysis can be challenging where host nucleic acid may dominate the preparation, or inhibitory compounds affect downstream analysis, e.g., quantitative reverse transcriptase PCR (qPCR), microarray, or RNA-seq. The goal of this work was to optimize the isolation of bacterial mRNA of food-borne pathogens from living plants. Reported methods for recovery of phytopathogen-infected plant material, using hot phenol extraction and high concentration of bacterial inoculation or large amounts of infected tissues, were found to be inappropriate for plant roots inoculated with Escherichia coli O157:H7. The bacterial RNA yields were too low and increased plant material resulted in a dominance of plant RNA in the sample. To improve the yield of bacterial RNA and reduce the number of plants required, an optimized method was developed which combines bead beating with directed bacterial lysis using SDS and lysozyme. Inhibitory plant compounds, such as phenolics and polysaccharides, were counteracted with the addition of high-molecular-weight polyethylene glycol and hexadecyltrimethyl ammonium bromide. The new method increased the total yield of bacterial mRNA substantially and allowed assessment of gene expression by qPCR. This method can be applied to other bacterial species associated with plant roots, and also in the wider context of food safety.

Investigations into plant biochemical wound-response pathways involved in the production of aphid-induced plant volatiles

Feeding damage to plants by insect herbivores induces the production of plant volatiles, which are attractive to the herbivores natural enemies. Little is understood about the plant biochemical pathways involved in aphid-induced plant volatile production. The aphid parasitoid Diaeretiella rapae can detect and respond to aphid-induced volatiles produced by Arabidopsis thaliana. When given experience of those volatiles, it can learn those cues and can therefore be used as a novel biosensor to detect them. The pathways involved in aphid-induced volatile production were investigated by comparing the responses of D. rapae to volatiles from a number of different transgenic mutants of A. thaliana, mutated in their octadecanoid, ethylene or salicylic acid wound-response pathways and also from wild-type plants. Plants were either undamaged or infested by the peach-potato aphid, Myzus persicae. It is demonstrated that the octadecanoid pathway and specifically the COI1 gene are required for aphid-induced volatile production. The presence of salicylic acid is also involved in volatile production. Using this model system, in combination with A. thaliana plants with single point gene mutations, has potential for the precise dissection of biochemical pathways involved in the production of aphid-induced volatiles

The identification of genes important in pseudomonas syringae pv. phaseolicola plant colonisation using in vitro screening of transposon libraries

The bacterial plant pathogen Pseudomonas syringae pv. phaseolicola (Pph) colonises the surface of common bean plants before moving into the interior of plant tissue, via wounds and stomata. In the intercellular spaces the pathogen proliferates in the apoplastic fluid and forms microcolonies (biofilms) around plant cells. If the pathogen can suppress the plant’s natural resistance response, it will cause halo blight disease. The process of resistance suppression is fairly well understood, but the mechanisms used by the pathogen in colonisation are less clear. We hypothesised that we could apply in vitro genetic screens to look for changes in motility, colony formation, and adhesion, which are proxies for infection, microcolony formation and cell adhesion. We made transposon (Tn) mutant libraries of Pph strains 1448A and 1302A and found 106/1920 mutants exhibited alterations in colony morphology, motility and biofilm formation. Identification of the insertion point of the Tn identified within the genome highlighted, as expected, a number of altered motility mutants bearing mutations in genes encoding various parts of the flagellum. Genes involved in nutrient biosynthesis, membrane associated proteins, and a number of conserved hypothetical protein (CHP) genes were also identified. A mutation of one CHP gene caused a positive increase in in planta bacterial growth. This rapid and inexpensive screening method allows the discovery of genes important for in vitro traits that can be correlated to roles in the plant interaction

Transcriptomic analysis of Enterohaemorrhagic Escherichia coli O157:H7 in response to plant extracts

Enterohaemorrhagic Escherichia coli (EHEC) are a group of food and contact-borne pathogens responsible for haemorrhagic colitis. The bacteria can be transmitted by contaminated meat, but importantly, also by plants. The bacteria can use plants as an alternative host, where they associate with both the leaves and the roots. Colonisation in the rhizosphere of plants is thought to be the main habitat for colonisation. Four different plant species, commonly associated with EHEC outbreaks, were infected with EHEC O157:H7 isolates Sakai and TUV 93-0 over ten days to assess the colonisation potential of the bacteria in both the phyllosphere and rhizosphere of plants. The rhizosphere was found to sustain a higher population level of bacteria over time in comparison to the phyllosphere, yet both strains were unable to utilize root exudates for growth. Global gene expression changes of EHEC O157:H7 strain Sakai were measured in response to plant extracts such as leaf lysates, root exudates and leaf cell wall polysaccharides from spinach cultivar Amazon and lettuce cultivar Salinas. Microarrays analysis showed a significant change in expression of 17 % of genes on exposure to leaf lysates of spinach. A more specific response was seen to spinach leaf cell wall polysaccharides with only a 1.5 % change. In contrast, when exposed to lettuce leaf cell wall polysaccharides a higher change of 4.8 % was seen. Genes that were differentially expressed belonged to multiple functional groups, including metabolism, indicating the utilization of plant-specific polysaccharides. Several areas of further investigation have been determined from this project, including the importance of culturing bacterial strains at a relevant temperature, the proposed lack of the type III secretion system in plant colonization by EHEC O157:H7 and the utilization of plant components for growth and persistence in the plant environment.

Inter-plant communication through mycorrhizal networks mediates complex adaptive behaviour in plant communities

Adaptive behaviour of plants, including rapid changes in physiology, gene regulation and defence response, can be altered when linked to neighbouring plants by a mycorrhizal network (MN). Mechanisms underlying the behavioural changes include mycorrhizal fungal colonization by the MN or interplant communication via transfer of nutrients, defence signals or allelochemicals. We focus this review on our new findings in ectomycorrhizal ecosystems, and also review recent advances in arbuscular mycorrhizal systems. We have found that the behavioural changes in ectomycorrhizal plants depend on environmental cues, the identity of the plant neighbour and the characteristics of the MN. The hierarchical integration of this phenomenon with other biological networks at broader scales in forest ecosystems, and the consequences we have observed when it is interrupted, indicate that underground ‘tree talk’ is a foundational process in the complex adaptive nature of forest ecosystems.

A tillering inhibition gene influences root-shoot carbon partitioning and pattern of water use to improve wheat productivity in rainfed environments

Genetic modification of shoot and root morphology has potential to improve water and nutrient 19 uptake of wheat crops in rainfed environments. Near-isogenic lines (NILs) varying for a tillering 20 inhibition (tin) gene and representing multiple genetic backgrounds were investigated in contrasting 21 controlled environments for shoot and root growth. Leaf area, shoot and root biomass were similar 22 until tillering whereupon reduced tillering in tin-containing NILs produced reductions of up to 60% in 23 total leaf area and biomass, and increases in total root length of up to 120% and root biomass to 24 145%. Together, root-to-shoot ratio increased two-fold with the tin gene. The influence of tin on shoot 25 and root growth was greatest in the cv. Banks genetic background, particularly in the biculm-selected 26 NIL, and was typically strongest in cooler environments. A separate de-tillering study confirmed 27 greater root-to-shoot ratios with regular tiller removal in non-tin containing genotypes. In validating 28 these observations in a rainfed field study, the tin allele had a negligible effect on seedling growth but 29 was associated with significantly (P<0.05) reduced tiller number (-37%), leaf area index (-26%) and 30 spike number (-35%) to reduce plant biomass (-19%) at anthesis. Root biomass, root-to-shoot ratio at 31 early stem elongation and root depth at maturity were increased in tin-containing NILs. Soil water use 32 was slowed in tin-containing NILs resulting in greater water availability, greater stomatal 33 conductance, cooler canopy temperatures and maintenance of green leaf area during grain-filling. 34 Together these effects contributed to increases in harvest index and grain yield. In both the controlled 35 and field environments, the tin gene was commonly associated with increased root length and biomass 36 but the significant influence of genetic background and environment suggests careful assessment of 37 tin-containing progeny in selection for genotypic increases in root growth.

Production of bioactive diterpenoids in the Euphorbiaceae depends on evolutionarily conserved gene clusters

The Euphorbiaceae produce a diverse range of diterpenoids, many of which have pharmacological activities. These diterpenoids include ingenol mebutate, which is licensed for the treatment of a precancerous skin condition (actinic keratosis), and phorbol derivatives such as resiniferatoxin and prostratin, which are undergoing investigation for the treatment of severe pain and HIV, respectively. Despite the interest in these diterpenoids, their biosynthesis is poorly understood at present, with the only characterized step being the conversion of geranylgeranyl pyrophosphate into casbene. Here, we report a physical cluster of diterpenoid biosynthetic genes from castor (Ricinus communis), including casbene synthases and cytochrome P450s from the CYP726A subfamily. CYP726A14, CYP726A17, and CYP726A18 were able to catalyze 5-oxidation of casbene, a conserved oxidation step in the biosynthesis of this family of medicinally important diterpenoids. CYP726A16 catalyzed 7,8-epoxidation of 5-keto-casbene and CYP726A15 catalyzed 5-oxidation of neocembrene. Evidence of similar gene clustering was also found in two other Euphorbiaceae, including Euphorbia peplus, the source organism of ingenol mebutate. These results demonstrate conservation of gene clusters at the higher taxonomic level of the plant family and that this phenomenon could prove useful in further elucidating diterpenoid biosynthetic pathways.

Linking alkaline phosphatase activity with bacterial phoD gene abundance in soil from a long-term management trial

Changes in land management practices may have significant implications for soil microbial communities important in organic P turnover. Soil bacteria can increase plant P availability by excreting phosphatase enzymes which catalyze the hydrolysis of ester-phosphate bonds. Examining the diversity and abundance of alkaline phosphatase gene harboring bacteria may provide valuable insight into alkaline phosphatase production in soils. This study examined the effect of 20 years of no input organic (ORG), organic with composted manure (ORG + M), conventional (CONV) and restored prairie (PRA) management on soil P bioavailability, alkaline phosphatase activity (ALP), and abundance and diversity of ALP gene (phoD) harboring bacteria in soils from the northern Great Plains of Canada. Management system influenced bioavailable P (P < 0.001), but not total P, with the lowest concentrations in the ORG systems and the highest in PRA. Higher rates of ALP were observed in the ORG and ORG + M treatments with a significant negative correlation between bioavailable P and ALP in 2011 (r2 = 0.71; P = 0.03) and 2012 (r2 = 0.51; P = 0.02), suggesting that ALP activity increased under P limiting conditions. The phoD gene abundance was also highest in ORG and ORG + M resulting in a significant positive relationship between bacterial phoD abundance and ALP activity (r2 = 0.71; P = 0.009). Analysis of phoD bacterial community fingerprints showed a higher number of species in CONV compared to ORG and ORG + M, contrary to what was expected considering greater ALP activity under ORG management. In 2012, banding profiles of ORG + M showed fewer phoD bacterial species following the second manure application, although ALP activity is higher than in 2011. This indicates that a few species may be producing more ALP and that quantitative gene analysis was a better indicator of activity than the number of species present.

Interaction of plant growth regulators and reactive oxygen species to regulate petal senescence in wallflowers (Erysimum linifolium)

Background In many species floral senescence is coordinated by ethylene. Endogenous levels rise, and exogenous application accelerates senescence. Furthermore, floral senescence is often associated with increased reactive oxygen species, and is delayed by exogenously applied cytokinin. However, how these processes are linked remains largely unresolved. Erysimum linifolium (wallflower) provides an excellent model for understanding these interactions due to its easily staged flowers and close taxonomic relationship to Arabidopsis. This has facilitated microarray analysis of gene expression during petal senescence and provided gene markers for following the effects of treatments on different regulatory pathways. Results In detached Erysimum linifolium (wallflower) flowers ethylene production peaks in open flowers. Furthermore senescence is delayed by treatments with the ethylene signalling inhibitor silver thiosulphate, and accelerated with ethylene released by 2-chloroethylphosphonic acid. Both treatments with exogenous cytokinin, or 6-methyl purine (which is an inhibitor of cytokinin oxidase), delay petal senescence. However, treatment with cytokinin also increases ethylene biosynthesis. Despite the similar effects on senescence, transcript abundance of gene markers is affected differentially by the treatments. A significant rise in transcript abundance of WLS73 (a putative aminocyclopropanecarboxylate oxidase) was abolished by cytokinin or 6-methyl purine treatments. In contrast, WFSAG12 transcript (a senescence marker) continued to accumulate significantly, albeit at a reduced rate. Silver thiosulphate suppressed the increase in transcript abundance both of WFSAG12 and WLS73. Activity of reactive oxygen species scavenging enzymes changed during senescence. Treatments that increased cytokinin levels, or inhibited ethylene action, reduced accumulation of hydrogen peroxide. Furthermore, although auxin levels rose with senescence, treatments that delayed early senescence did not affect transcript abundance of WPS46, an auxin-induced gene. Conclusions A model for the interaction between cytokinins, ethylene, reactive oxygen species and auxin in the regulation of floral senescence in wallflowers is proposed. The combined increase in ethylene and reduction in cytokinin triggers the initiation of senescence and these two plant growth regulators directly or indirectly result in increased reactive oxygen species levels. A fall in conjugated auxin and/or the total auxin pool eventually triggers abscission.

Analysis of the genetic diversity and structure across a wide range of germplasm reveals prominent gene flow in apple at the European level

Abstract Background: The amount and structure of genetic diversity in dessert apple germplasm conserved at a European level is mostly unknown, since all diversity studies conducted in Europe until now have been performed on regional or national collections. Here, we applied a common set of 16 SSR markers to genotype more than 2,400 accessions across 14 collections representing three broad European geographic regions (North+East, West and South) with the aim to analyze the extent, distribution and structure of variation in the apple genetic resources in Europe. Results: A Bayesian model-based clustering approach showed that diversity was organized in three groups, although these were only moderately differentiated (FST=0.031). A nested Bayesian clustering approach allowed identification of subgroups which revealed internal patterns of substructure within the groups, allowing a finer delineation of the variation into eight subgroups (FST=0.044). The first level of stratification revealed an asymmetric division of the germplasm among the three groups, and a clear association was found with the geographical regions of origin of the cultivars. The substructure revealed clear partitioning of genetic groups among countries, but also interesting associations between subgroups and breeding purposes of recent cultivars or particular usage such as cider production. Additional parentage analyses allowed us to identify both putative parents of more than 40 old and/or local cultivars giving interesting insights in the pedigree of some emblematic cultivars. Conclusions: The variation found at group and sub-group levels may reflect a combination of historical processes of migration/selection and adaptive factors to diverse agricultural environments that, together with genetic drift, have resulted in extensive genetic variation but limited population structure. The European dessert apple germplasm represents an important source of genetic diversity with a strong historical and patrimonial value. The present work thus constitutes a decisive step in the field of conservation genetics. Moreover, the obtained data can be used for defining a European apple core collection useful for further identification of genomic regions associated with commercially important horticultural traits in apple through genome-wide association studies.