Mapping of a Cellulose-Deficient Mutant Named dwarf1-1 in Sorghum bicolor to the Green Revolution Gene gibberellin20-oxidase Reveals a Positive Regulatory Association between Gibberellin and Cellulose Biosynthesis.

Here, we show a mechanism for expansion regulation through mutations in the green revolution gene gibberellin20 (GA20)-oxidase and show that GAs control biosynthesis of the plants main structural polymer cellulose. Within a 12,000 mutagenized Sorghum bicolor plant population, we identified a single cellulose-deficient and male gametophyte-dysfunctional mutant named dwarf1-1 (dwf1-1). Through the Sorghum propinquum male/dwf1-1 female F2 population, we mapped dwf1-1 to a frameshift in GA20-oxidase. Assessment of GAs in dwf1-1 revealed ablation of GA. GA ablation was antagonistic to the expression of three specific cellulose synthase genes resulting in cellulose deficiency and growth dwarfism, which were complemented by exogenous bioactive gibberellic acid application. Using quantitative polymerase chain reaction, we found that GA was positively regulating the expression of a subset of specific cellulose synthase genes. To cross reference data from our mapped Sorghum sp. allele with another monocotyledonous plant, a series of rice (Oryza sativa) mutants involved in GA biosynthesis and signaling were isolated, and these too displayed cellulose deficit. Taken together, data support a model whereby suppressed expansion in green revolution GA genes involves regulation of cellulose biosynthesis.

Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1A903V and CESA3T942I of cellulose synthase.

The mechanisms underlying the biosynthesis of cellulose in plants are complex and still poorly understood. A central question concerns the mechanism of microfibril structure and how this is linked to the catalytic polymerization action of cellulose synthase (CESA). Furthermore, it remains unclear whether modification of cellulose microfibril structure can be achieved genetically, which could be transformative in a bio-based economy. To explore these processes in planta, we developed a chemical genetic toolbox of pharmacological inhibitors and corresponding resistance-conferring point mutations in the C-terminal transmembrane domain region of CESA1(A903V) and CESA3(T942I) in Arabidopsis thaliana. Using (13)C solid-state nuclear magnetic resonance spectroscopy and X-ray diffraction, we show that the cellulose microfibrils displayed reduced width and an additional cellulose C4 peak indicative of a degree of crystallinity that is intermediate between the surface and interior glucans of wild type, suggesting a difference in glucan chain association during microfibril formation. Consistent with measurements of lower microfibril crystallinity, cellulose extracts from mutated CESA1(A903V) and CESA3(T942I) displayed greater saccharification efficiency than wild type. Using live-cell imaging to track fluorescently labeled CESA, we found that these mutants show increased CESA velocities in the plasma membrane, an indication of increased polymerization rate. Collectively, these data suggest that CESA1(A903V) and CESA3(T942I) have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization.

The opinions of farm animal veterinarians in Ireland on antibiotic use and their role in antimicrobial stewardship.

BACKGROUND: Antibiotic use and resistance in animal production are a concern to public health, and there is an urgent need to reduce antibiotic use in farm animals. To prevent blame shifting, professionals from human medicine, animal medicine and environmental backgrounds must collaborate to tackle this issue. Veterinarians are typically responsible for overseeing and prescribing antibiotic use in animals. There are currently no available studies on the opinions of Irish farm animal veterinarians on antibiotic use, reduction opportunities and their relationships with farmers. A digital survey was developed and sent out to Irish farm animal veterinarians. This paper presents the results of a cross-sectional study of Irish farm animal veterinarians' attitudes towards antimicrobial stewardship, their prescribing behaviours, antibiotic reduction opportunities and their attitudes for the future of antibiotic use. The veterinarian-farmer relationship is examined and potential interventions to reduce antibiotic use on farms are identified. RESULTS: In total, 114 complete questionnaires were received, representing approximately 11 per cent of all farm animal veterinarians in Ireland. Respondents were aware of the problem of antibiotic resistance and recognise their role in the fight against it. They realise what actions they must take to reduce antibiotic use and identify barriers that prevent their farmer clients from implementing their advice. Many of them say that they can reduce antibiotic use on farms in the future, but some remain doubtful. There was no statistical difference between veterinarians that had less experience working than those that had more experience in their attitudes towards future reduction in antibiotic use. CONCLUSION: Most of the respondents seek to use antibiotics as judiciously as they can. The majority agree that antibiotic overuse is the main contributor to antibiotic resistance. Possible solutions to reduce antibiotic use include the development of antibiotic treatment guidelines, assigning one unique practice to each farm and compulsory CPD (Continuous Professional Development) courses.

Auxin as a player in the biocontrol of Fusarium head blight disease of barley and its potential as a disease control agent.

BACKGROUND: Mechanisms involved in the biological control of plant diseases are varied and complex. Hormones, including the auxin indole acetic acid (IAA) and abscisic acid (ABA), are essential regulators of a multitude of biological functions, including plant responses to biotic and abiotic stressors. This study set out to determine what hormones might play a role in Pseudomonas fluorescens -mediated control of Fusarium head blight (FHB) disease of barley and to determine if biocontrol-associated hormones directly affect disease development. RESULTS: A previous study distinguished bacterium-responsive genes from bacterium-primed genes, distinguished by the fact that the latter are only up-regulated when both P. fluorescens and the pathogen Fusarium culmorum are present. In silico analysis of the promoter sequences available for a subset of the bacterium-primed genes identified several hormones, including IAA and ABA as potential regulators of transcription. Treatment with the bacterium or pathogen resulted in increased IAA and ABA levels in head tissue; both microbes had additive effects on the accumulation of IAA but not of ABA. The microbe-induced accumulation of ABA preceded that of IAA. Gene expression analysis showed that both hormones up-regulated the accumulation of bacterium-primed genes. But IAA, more than ABA up-regulated the transcription of the ABA biosynthesis gene NCED or the signalling gene Pi2, both of which were previously shown to be bacterium-responsive rather than primed. Application of IAA, but not of ABA reduced both disease severity and yield loss caused by F. culmorum, but neither hormone affect in vitro fungal growth. CONCLUSIONS: Both IAA and ABA are involved in the P. fluorescens-mediated control of FHB disease of barley. Gene expression studies also support the hypothesis that IAA plays a role in the primed response to F. culmorum. This hypothesis was validated by the fact that pre-application of IAA reduced both symptoms and yield loss asssociated with the disease. This is the first evidence that IAA plays a role in the control of FHB disease and in the bacterial priming of host defences.

Impacts of the Invasive Impatiens glandulifera: Lessons Learned from One of Europe's Top Invasive Species.

Biological invasions are renowned for their negative ecological and economic implications, however from studying invasions invaluable insights can be gained in the fields of ecology and evolution- potentially contributing towards conservation plans to deal, not only with biological invasion, but with other concerning issues, such as climate change. Impatiens glandulifera, or Himalayan balsam, is widely considered to be a highly problematic invasive, having spread across more than thirty countries during the past century. This paper will examine the findings which have arose from studying I. glandulifera and its impacts on the invaded ecosystem.

Enzymatic response to cadmium by Impatiens glandulifera: A preliminary investigation.

This paper aims to develop our understanding of the effect of cadmium (Cd) on Impatiens glandulifera, a recently identified potential Cd hyperaccumulator. Impatiens glandulifera plants were exposed to three concentrations of Cd (20, 60 and 90 mg/kg) and were sampled at two timepoints (one and seven days) to investigate the stress response of I. glandulifera to Cd. Cd can induce oxidative stress in plants, triggering overproduction of reactive oxygen species (ROS). The level of activity of catalase (CAT) and ascorbate peroxidase (APX), two crucial antioxidant enzymes responsible for detoxifying ROS, were found to increase in a concentration dependent manner. Though there was no change observed in the level of superoxide dismutase (SOD) activity, the activity of glutathione S-transferase (GST), involved in detoxifying and sequestering Cd, increased after exposure to Cd. Cd did not appear to impact the levels of proline and photosynthetic pigments, indicating the plants weren't stressed by the presence of Cd. These results suggest that the rapid response observed in enzyme activity aid the efficacious mitigation of the toxic effects of Cd, preventing significant physiological stress in I. glandulifera.

Lipid transfer proteins and protease inhibitors as key factors in the priming of barley responses to Fusarium head blight disease by a biocontrol strain of Pseudomonas fluorescens.

Strains of non-pathogenic pseudomonad bacteria, can elicit host defence responses against pathogenic microorganisms. Pseudomonas fluorescens strain MKB158 can protect cereals from pathogenesis by Fusarium fungi, including Fusarium head blight which is an economically important disease due to its association with both yield loss and mycotoxin contamination of grain. Using the 22 K barley Affymetrix chip, trancriptome studies were undertaken to determine the local effect of P. fluorescens strain MKB158 on the transcriptome of barley head tissue, and to discriminate transcripts primed by the bacterium to respond to challenge by Fusarium culmorum, a causal agent of the economically important Fusarium head blight disease of cereals. The bacterium significantly affected the accumulation of 1203 transcripts and primed 74 to positively, and 14 to negatively, respond to the pathogen (P = 0.05). This is the first study to give insights into bacterium priming in the Triticeae tribe of grasses and associated transcripts were classified into 13 functional classes, associated with diverse functions, including detoxification, cell wall biosynthesis and the amplification of host defence responses. In silico analysis of Arabidopsis homologs of bacterium-primed barley genes indicated that, as is the case in dicots, jasmonic acid plays a role in pseudomonad priming of host responses. Additionally, the transcriptome studies described herein also reveal new insights into bacterium-mediated priming of host defences against necrotrophs, including the positive effects on grain filling, lignin deposition, oxidative stress responses, and the inhibition of protease inhibitors and proteins that play a key role in programmed cell death.

Phloroglucinol Mediated Plant Regeneration of Ornithogalum dubium as the Sole "Hormone-Like Supplement" in Plant Tissue Culture Long-Term Experiments.

Tissue culture is an essential requirement in plant science to preserve genetic resources and to expand naturally occurring germplasm. A variety of naturally occurring and synthetic hormones are available to induce the processes of dedifferentiation and redifferentiation. Not all plant material is susceptible to tissue culture, and often complex media and hormone requirements are needed to achieve successful plant propagations. The availability of new hormones or chemicals acting as hormones are critical to the expansion of tissue culture potentials. Phloroglucinol has been shown to have certain hormone-like properties in a variety of studies. Ornithogalum dubium, an important geophyte species, was used to characterise the potential of phloroglucinol as the sole plant-like hormone in a tissue culture experiment. Tissue culture, plant regeneration, total phenolic and genetic variability were established by applying a variety of methods throughout long-term experiments. Phloroglucinol did induce callus formation and plant regeneration when used as the sole supplement in the media at a rate of 37%, thus demonstrating auxin/cytokines-like properties. Callus formation was of 3 types, friable and cellular, hard and compact, and a mixture of the two. The important finding was that direct somatogenesis did occur albeit more frequently on younger tissue, whereby rates of induction were up to 52%. It is concluded that phloroglucinol acts as a "hormone-like" molecule and can trigger direct embryogenesis without callus formation.

The involvement of J-protein AtDjC17 in root development in Arabidopsis.

In a screen for root hair morphogenesis mutants in Arabidopsis thaliana L. we identified a T-DNA insertion within a type III J-protein AtDjC17 caused altered root hair development and reduced hair length. Root hairs were observed to develop from trichoblast and atrichoblast cell files in both Atdjc17 and 35S::AtDJC17. Localization of gene expression in the root using transgenic plants expressing proAtDjC17::GUS revealed constitutive expression in stele cells. No AtDJC17 expression was observed in epidermal, endodermal, or cortical layers. To explore the contrast between gene expression in the stele and epidermal phenotype, hand cut transverse sections of Atdjc17 roots were examined showing that the endodermal and cortical cell layers displayed increased anticlinal cell divisions. Aberrant cortical cell division in Atdjc17 is proposed as causal in ectopic root hair formation via the positional cue requirement that exists between cortical and epidermal cell in hair cell fate determination. Results indicate a requirement for AtDJC17 in position-dependent cell fate determination and illustrate an intriguing requirement for molecular co-chaperone activity during root development.

Experimental approaches to study plant cell walls during plant-microbe interactions.

Plant cell walls provide physical strength, regulate the passage of bio-molecules, and act as the first barrier of defense against biotic and abiotic stress. In addition to providing structural integrity, plant cell walls serve an important function in connecting cells to their extracellular environment by sensing and transducing signals to activate cellular responses, such as those that occur during pathogen infection. This mini review will summarize current experimental approaches used to study cell wall functions during plant-pathogen interactions. Focus will be paid to cell imaging, spectroscopic analyses, and metabolic profiling techniques.

Mutagenesis breeding for increased 3-deoxyanthocyanidin accumulation in leaves of Sorghum bicolor (L.) Moench: a source of natural food pigment.

Natural food colorants with functional properties are of increasing interest. Prior papers indicate the chemical suitability of sorghum leaf 3-deoxyanthocyanidins as natural food colorants. Via mutagenesis-assisted breeding, a sorghum variety that greatly overaccumulates 3-deoxyanthocyanidins of leaf tissue, named REDforGREEN (RG), has been isolated and characterized. Interestingly, RG not only caused increased 3-deoxyanthocyanidins but also caused increased tannins, chlorogenic acid, and total phenolics in the leaf tissue. Chemical composition of pigments was established through high-performance liquid chromatography (HPLC) that identified luteolinidin (LUT) and apigeninidin (APG) as the main 3-deoxyanthocianidin species. Specifically, 3-deoxyanthocianidin levels were 1768 µg g⁻¹ LUT and 421 µg g⁻¹ APG in RG leaves compared with trace amounts in wild type, representing 1000-fold greater levels in the mutant leaves. Thus, RG represents a useful sorghum mutagenesis variant to develop as a functionalized food colorant.

Comparative feedstock analysis in Setaria viridis L. as a model for C4 bioenergy grasses and Panicoid crop species.

Second generation feedstocks for bioethanol will likely include a sizable proportion of perennial C4 grasses, principally in the Panicoideae clade. The Panicoideae contain agronomically important annual grasses including Zea mays L. (maize), Sorghum bicolor (L.) Moench (sorghum), and Saccharum officinarum L. (sugar cane) as well as promising second generation perennial feedstocks including Miscanthus×giganteus and Panicum virgatum L. (switchgrass). The underlying complexity of these polyploid grass genomes is a major limitation for their direct manipulation and thus driving a need for rapidly cycling comparative model. Setaria viridis (green millet) is a rapid cycling C4 panicoid grass with a relatively small and sequenced diploid genome and abundant seed production. Stable, transient, and protoplast transformation technologies have also been developed for Setaria viridis making it a potentially excellent model for other C4 bioenergy grasses. Here, the lignocellulosic feedstock composition, cellulose biosynthesis inhibitor response and saccharification dynamics of Setaria viridis are compared with the annual sorghum and maize and the perennial switchgrass bioenergy crops as a baseline study into the applicability for translational research. A genome-wide systematic investigation of the cellulose synthase-A genes was performed identifying eight candidate sequences. Two developmental stages; (a) metabolically active young tissue and (b) metabolically plateaued (mature) material are examined to compare biomass performance metrics.

Sorghum mutant RG displays antithetic leaf shoot lignin accumulation resulting in improved stem saccharification properties.

BACKGROUND: Improving saccharification efficiency in bioenergy crop species remains an important challenge. Here, we report the characterization of a Sorghum (Sorghum bicolor L.) mutant, named REDforGREEN (RG), as a bioenergy feedstock. RESULTS: It was found that RG displayed increased accumulation of lignin in leaves and depletion in the stems, antithetic to the trend observed in wild type. Consistent with these measurements, the RG leaf tissue displayed reduced saccharification efficiency whereas the stem saccharification efficiency increased relative to wild type. Reduced lignin was linked to improved saccharification in RG stems, but a chemical shift to greater S:G ratios in RG stem lignin was also observed. Similarities in cellulose content and structure by XRD-analysis support the correlation between increased saccharification properties and reduced lignin instead of changes in the cellulose composition and/or structure. CONCLUSION: Antithetic lignin accumulation was observed in the RG mutant leaf-and stem-tissue, which resulted in greater saccharification efficiency in the RG stem and differential thermochemical product yield in high lignin leaves. Thus, the red leaf coloration of the RG mutant represents a potential marker for improved conversion of stem cellulose to fermentable sugars in the C4 grass Sorghum.

Evidence of genotype dependency within Agrobacterium tumefaciens in relation to the integration of vector backbone sequence in transgenic Phytophthora infestans-tolerant potato.

In this study the effect of Agrobacterium tumefaciens genotype of two strains AGL1 and LBA4404 was investigated in regard to the propensity for backbone integration during the transformation of potato for blight tolerance conferred by the resistant to blight (RB) gene carried by the vector pCLDO4541. A PCR based walking approach was employed to identify left and right backbone sequences as well as for selected genes carried on the plasmid backbone. It was found that adjacent to the left border insertion site, the integration of backbone sequence was greater for AGL1 than for LBA4404; however, the opposite was observed with regards to the right border T-DNA junction. Considering both T-DNA borders LBA4404 was found to have a two fold greater integration potential for backbone than the AGL1. The possibility of only backbone integration in T-DNA negative plants was also investigated with the average rate of integration between the two strains calculated at 4.2% with LBA4404 recording a three fold greater occurrence of backbone integration than AGL1. In summary, evidence of Agrobacterium genotype dependency showed that LBA4404 has greater potential to integrate non-T-DNA vector sequence than AGL1 and this should be taken into account when utilising the listed A. tumefaciens genotypes in generating transgenic potato. Additionally, the application of a PCR and primer walking system proved to be reliable and allows for fine detailed studies of backbone sequence integration of transgenic plant.

Facilitating co-existence by tracking gene dispersal in conventional potato systems with microsatellite markers.

Based on international findings, Irish co-existence guidelines for the cultivation of GM potato stipulate that an isolation distance of 20 m is required to minimize the spread of transgenic pollen in accordance with required labeling thresholds. As potato tolerant to Phytophthora infestans is the most applicable GM crop from an Irish context, we tested the efficacy of this isolation distance under Irish environmental conditions using the conventional variety Désirée as a pollen donor and the male-sterile variety British Queen as a pollen receptor. Gene flow was determined by scoring for berry presence on receptor plants and confirmed using a microsatellite marker system designed to assess paternity in F(1) seedlings. 99.1% of seedlings recovered were identified as having Désirée paternity. Whereas 19.9% (140/708) of total berries formed on receptor plants occurred at a distance of 21 m from the pollen source, only 4 of these berries bore viable true potato seed (TPS), from which 23 TPS germinated. TPS-bearing berry formation was negatively correlated with distance from the pollen source, and although overall distribution of berries and seeds was non-random across the plot, no significant correlation was evident with respect to wind direction. Microsatellite markers were also used to confirm that the foraging beetle Meligethes aeneus is a vector for the transmission of potato pollen, but a more detailed statistical analysis of this dataset was limited by inclement weather during the trial. To conclude, we recommend that a two-tiered system be established in regard to establishing isolation distances for the experimental trial and commercial cultivation of GM potato in Ireland, and that responsible crop management be adopted to minimize the establishment of TPS-derived volunteers, which we have noted will emerge through a rotation as a result of pollen-mediated gene flow.