3'-(Phenyl alkynyl) analogs of abscisic acid: synthesis and biological activity of potent ABA antagonists.

We report here the synthesis and biological testing of 3'-(phenyl alkynyl) abscisic ABA analogs, a new class of potent ABA antagonists. These ABA analogs incorporate a rigid framework of eight carbon atoms attached at the 3'-carbon atom of ABA that prevents folding of the ABA analog-bound receptor required for ABA signalling. The two-step synthesis is based upon the optimized conversion of natural (S)-ABA to 3'-iodo ABA which can be coupled to phenyl acetylenes using Sonogashira conditions, or to styryl compounds through Suzuki chemistry. The parent 3'-(phenyl alkynyl) ABA analog 7 was obtained in 29% yield, 74% yield based on recovered starting material. In a lentil seed germination assay, compound 7 was found to have more potent activity than other known 3'-substituted ABA antagonists to date. In a structure activity study parasubstituted phenyl alkynyl analogs had comparable activity to the analog 7 while the 3'-styryl ABA 18 was only slightly less active. Analog 7 overcame ABA inhibition of germination and seedling growth in a wide range of mono and dicot plant species, including canola, lentil, soybean, rice, wheat, barley, cannabis and canary seed. 3'-(Phenyl alkynyl) ABA analogs have numerous potential practical agricultural applications including promoting ripening of crops, dormancy breaking of seeds and woody perennials, as well as promoting seed germination, and growth under stress conditions as demonstrated in this report.

Comparative RNA-Seq analysis reveals genes associated with masculinization in female Cannabis sativa.

MAIN CONCLUSION: Using RNA profiling, we identified several silver thiosulfate-induced genes that potentially control the masculinization of female Cannabis sativa plants. Genetically female Cannabis sativa plants normally bear female flowers, but can develop male flowers in response to environmental and developmental cues. In an attempt to elucidate the molecular elements responsible for sex expression in C. sativa plants, we developed genetically female lines producing both female and chemically-induced male flowers. Furthermore, we carried out RNA-Seq assays aimed at identifying differentially expressed genes responsible for male flower development in female plants. The results revealed over 10,500 differentially expressed genes, of which around 200 potentially control masculinization of female cannabis plants. These genes include transcription factors and other genes involved in male organ (i.e., anther and pollen) development, as well as genes involved in phytohormone signalling and male-biased phenotypes. The expressions of 15 of these genes were further validated by qPCR assay confirming similar expression patterns to that of RNA-Seq data. These genes would be useful for understanding predisposed plants producing flowers of both sex types in the same plant, and help breeders to regulate the masculinization of female plants through targeted breeding and plant biotechnology.

Seed specific expression and analysis of recombinant human adenosine deaminase (hADA) in three host plant species.

The plant seed is a leading platform amongst plant-based storage systems for the production of recombinant proteins. In this study, we compared the activity of human adenosine deaminase (hADA) expressed in transgenic seeds of three different plant species: pea (Pisum sativum L.), Nicotiana benthamiana L. and tarwi (Lupinus mutabilis Sweet). All three species were transformed with the same expression vector containing the hADA gene driven by the seed-specific promoter LegA2 with an apoplast targeting pinII signal peptide. During the study, several independent transgenic lines were generated and screened from each plant species and only lines with a single copy of the gene of interest were used for hADA expression analysis. A stable transgenic canola line expressing the ADA protein, under the control of 35S constitutive promoter was used as both as a positive control and for comparative study with the seed specific promoter. Significant differences were detected in the expression of hADA. The highest activity of the hADA enzyme (Units/g seed) was reported in tarwi (4.26 U/g) followed by pea (3.23 U/g) and Nicotiana benthamiana (1.69 U/g). The expression of mouse ADA in canola was very low in both seed and leaf tissue compared to other host plants, confirming higher activity of seed specific promoter. Altogether, these results suggest that tarwi could be an excellent candidate for the production of valuable recombinant proteins.

The case for plant-made veterinary immunotherapeutics.

The excessive use of antibiotics in food animal production has contributed to resistance in pathogenic bacteria, thereby triggering regulations and consumer demands to limit their use. Alternatives for disease control are therefore required that are cost-effective and compatible with intensive production. While vaccines are widely used and effective, they are available against a minority of animal diseases, and development of novel vaccines and other immunotherapeutics is therefore needed. Production of such proteins recombinantly in plants can provide products that are effective and safe, can be orally administered with minimal processing, and are easily scalable with a relatively low capital investment. The present report thus advocates the use of plants for producing vaccines and antibodies to protect farm animals from diseases that have thus far been managed with antibiotics; and highlights recent advances in product efficacy, competitiveness, and regulatory approval.

Bringing plant-based veterinary vaccines to market: Managing regulatory and commercial hurdles.

The production of recombinant vaccines in plants may help to reduce the burden of veterinary diseases, which cause major economic losses and in some cases can affect human health. While there is abundant research in this area, a knowledge gap exists between the ability to create and evaluate plant-based products in the laboratory, and the ability to take these products on a path to commercialization. The current report, arising from a workshop sponsored by an Organisation for Economic Co-operation and Development (OECD) Co-operative Research Programme, addresses this gap by providing guidance in planning for the commercialization of plant-made vaccines for animal use. It includes relevant information on developing business plans, assessing market opportunities, manufacturing scale-up, financing, protecting and using intellectual property, and regulatory approval with a focus on Canadian regulations.

LINE-1 hypomethylation in cancer is highly variable and inversely correlated with microsatellite instability.

BACKGROUND: Alterations in DNA methylation in cancer include global hypomethylation and gene-specific hypermethylation. It is not clear whether these two epigenetic errors are mechanistically linked or occur independently. This study was performed to determine the relationship between DNA hypomethylation, hypermethylation and microsatellite instability in cancer. METHODOLOGY/PRINCIPAL FINDINGS: We examined 61 cancer cell lines and 60 colorectal carcinomas and their adjacent tissues using LINE-1 bisulfite-PCR as a surrogate for global demethylation. Colorectal carcinomas with sporadic microsatellite instability (MSI), most of which are due to a CpG island methylation phenotype (CIMP) and associated MLH1 promoter methylation, showed in average no difference in LINE-1 methylation between normal adjacent and cancer tissues. Interestingly, some tumor samples in this group showed increase in LINE-1 methylation. In contrast, MSI-showed a significant decrease in LINE-1 methylation between normal adjacent and cancer tissues (P<0.001). Microarray analysis of repetitive element methylation confirmed this observation and showed a high degree of variability in hypomethylation between samples. Additionally, unsupervised hierarchical clustering identified a group of highly hypomethylated tumors, composed mostly of tumors without microsatellite instability. We extended LINE-1 analysis to cancer cell lines from different tissues and found that 50/61 were hypomethylated compared to peripheral blood lymphocytes and normal colon mucosa. Interestingly, these cancer cell lines also exhibited a large variation in demethylation, which was tissue-specific and thus unlikely to be resultant from a stochastic process. CONCLUSION/SIGNIFICANCE: Global hypomethylation is partially reversed in cancers with microsatellite instability and also shows high variability in cancer, which may reflect alternative progression pathways in cancer.

DNA methylation changes after 5-aza-2'-deoxycytidine therapy in patients with leukemia.

5-Aza-2'-deoxycytidine (decitabine) is postulated to have clinical activity in myeloid leukemias via its ability to inhibit DNA methylation. To study this, we examined DNA methylation in patients with leukemia treated with decitabine. Five days after the treatment, total genomic 5-methylcytosine/cytosine decreased on average by 14% (from 4.3% to 3.7%), whereas methylation of repetitive DNA elements showed a mean decrease of 9% and 16% for Alu and long interspersed nucleotide elements, respectively. Methylation decreased linearly with increasing doses between 5 and 20 mg/m(2)/d (r = 0.88; P = 0.05) but showed a plateau above that. Hypomethylation correlated with response in patients with acute myelogenous leukemia treated with low doses (5-20 mg/m(2)/d), but patients with chronic myelogenous leukemia treated with high doses (100-180 mg/m(2)/d) showed no such correlation. Aberrant methylation of p15 (>10%) was found in 27% of patients, and 80% of these showed a decrease by at least one third, but this did not correlate with response. The imprinted gene H19 showed little change in methylation after decitabine. In conclusion, we show dose-dependent hypomethylation after decitabine at low doses. Increasing the dose, which has been shown previously to result in a reduced response rate, was not accompanied by further hypomethylation.

A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements.

We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15 000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2'deoxycytidine (DAC), where we found a 1-16% decrease in Alu element and 18-60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation.