Assessing data analysis techniques in a high-throughput meiosis-like induction detection system.

BACKGROUND: Strategies to understand meiotic processes have relied on cytogenetic and mutant analysis. However, thus far in vitro meiosis induction is a bottleneck to laboratory-based plant breeding as factor(s) that switch cells in crops species from mitotic to meiotic divisions are unknown. A high-throughput system that allows researchers to screen multiple candidates for their meiotic induction role using low-cost microfluidic devices has the potential to facilitate the identification of factors with the ability to induce haploid cells that have undergone recombination (artificial gametes) in cell cultures. RESULTS: A data analysis pipeline and a detailed protocol are presented to screen for plant meiosis induction factors in a quantifiable and efficient manner. We assessed three data analysis techniques using spiked-in protoplast samples (simulated gametes mixed into somatic protoplast populations) of flow cytometry data. Polygonal gating, which was considered the "gold standard", was compared to two thresholding methods using open-source analysis software. Both thresholding techniques were able to identify significant differences with low spike-in concentrations while also being comparable to polygonal gating. CONCLUSION: Our study provides details to test and analyze candidate meiosis induction factors using available biological resources and open-source programs for thresholding. RFP (PE.CF594.A) and GFP (FITC.A) were the only channels required to make informed decisions on meiosis-like induction and resulted in detection of cell population changes as low as 0.3%, thus enabling this system to be scaled using microfluidic devices at low costs.

The impact of 'framing' in the adoption of GM crops.

Genetically modified (GM) crops offer significant advantages in our crop improvement programs because they are created using a more targeted approach which is not possible in traditional breeding methods. Human benefit is one of the main objectives of crop improvement but the legal framework for the introduction of GM crops and the depiction and portrayal of GM crops in the media create barriers to these benefits. This article attempts to highlight the barriers to GM crop adoption particularly focusing on the idea of "framing" and the way GM technology is framed in the media. The resulting public health, economic, and ecological concerns about genetically modified plants, along with the reality of these misconceptions, are discussed with specific examples. Finally, we propose potential routes toward increased acceptance of GM crops.

Overcoming roadblocks for in vitro nurseries in plants: induction of meiosis.

Efforts to increase genetic gains in breeding programs of flowering plants depend on making genetic crosses. Time to flowering, which can take months to decades depending on the species, can be a limiting factor in such breeding programs. It has been proposed that the rate of genetic gain can be increased by reducing the time between generations by circumventing flowering through the in vitro induction of meiosis. In this review, we assess technologies and approaches that may offer a path towards meiosis induction, the largest current bottleneck for in vitro plant breeding. Studies in non-plant, eukaryotic organisms indicate that the in vitro switch from mitotic cell division to meiosis is inefficient and occurs at very low rates. Yet, this has been achieved with mammalian cells by the manipulation of a limited number of genes. Therefore, to experimentally identify factors that switch mitosis to meiosis in plants, it is necessary to develop a high-throughput system to evaluate a large number of candidate genes and treatments, each using large numbers of cells, few of which may gain the ability to induce meiosis.

Haploid male fertility is restored by parallel spindle genes in Arabidopsis thaliana.

Doubled haploid technology can accelerate plant breeding and its two main steps are haploid induction and subsequent doubled haploid production from fertile haploid plants. Although haploid female fertility is present to some extent in plants, the lack of haploid male fertility is a bottleneck. Herein, we demonstrate that mutations in the parallel spindle genes are sufficient to restore haploid male fertility in Arabidopsis with no impact on haploid female fertility.

Protocols for In Vivo Doubled Haploid (DH) Technology in Maize Breeding: From Haploid Inducer Development to Haploid Genome Doubling.

Doubled haploid (DH) technology reduces the time required to obtain homozygous genotypes and accelerates plant breeding among other advantages. It is established in major crop species such as wheat, barley, maize, and canola. DH lines can be produced by both in vitro and in vivo methods and the latter is focused here. The major steps involved in in vivo DH technology are haploid induction, haploid selection/identification, and haploid genome doubling. Herein, we elaborate on the various steps of DH technology in maize breeding from haploid induction to haploid genome doubling to produce DH lines. Detailed protocols on the following topics are discussed: in vivo haploid inducer line development, haploid selection using seed and root color markers and automated seed sorting based on embryo oil content using QSorter, artificial genome doubling, and the identification of genotypes with spontaneous haploid genome doubling (SHGD) ability.

Maize Zmcyp710a8 Mutant as a Tool to Decipher the Function of Stigmasterol in Plant Metabolism.

Sterols are integral components of membrane lipid bilayers in eukaryotic organisms and serve as precursors to steroid hormones in vertebrates and brassinosteroids (BR) in plants. In vertebrates, cholesterol is the terminal sterol serving both indirect and direct roles in cell signaling. Plants synthesize a mixture of sterols including cholesterol, sitosterol, campesterol, and stigmasterol but the signaling role for the free forms of individual plant sterols is unclear. Since stigmasterol is the terminal sterol in the sitosterol branch and produced from a single enzymatic step, modifying stigmasterol concentration may shed light on its role in plant metabolism. Although Arabidopsis has been the model of choice to study sterol function, the functional redundancy of AtCYP710A genes and the presence of brassicasterol may hinder our ability to test the biological function of stigmasterol. We report here the identification and characterization of ZmCYP710A8, the sole maize C-22 sterol desaturase involved in stigmasterol biosynthesis and the identification of a stigmasterol-free Zmcyp710a8 mutant. ZmCYP710A8 mRNA expression pattern correlated with transcripts for several sterol biosynthesis genes and loss of stigmasterol impacted sterol composition. Exogenous stigmasterol also had a stimulatory effect on mRNA for ZmHMGR and ZmSMT2. This demonstrates the potential of Zmcyp710a8 in understanding the role of stigmasterol in modulating sterol biosynthesis and global cellular metabolism. Several amino acids accumulate in the Zmcyp710a8 mutant, offering opportunity for genetic enhancement of nutritional quality of maize. Other cellular metabolites in roots and shoots of maize and Arabidopsis were also impacted by genetic modification of stigmasterol content. Yet lack of obvious developmental defects in Zmcyp710a8 suggest that stigmasterol might not be essential for plant growth under normal conditions. Nonetheless, the Zmcyp710a8 mutant reported here is of great utility to advance our understanding of the additional roles of stigmasterol in plant metabolism. A number of biological and agronomic questions can be interrogated using this tool such as gene expression studies, spatio-temporal localization of sterols, cellular metabolism, pathway regulation, physiological studies, and crop improvement.

Why Do Plants Convert Sitosterol to Stigmasterol?

A direct role for cholesterol signaling in mammals is clearly established; yet, the direct role in signaling for a plant sterol or sterol precursor is unclear. Fluctuations in sitosterol and stigmasterol levels during development and stress conditions suggest their involvement in signaling activities essential for plant development and stress compensation. Stigmasterol may be involved in gravitropism and tolerance to abiotic stress. The isolation of stigmasterol biosynthesis mutants offers a promising tool to test the function of sterol end products in signaling responses to developmental and environmental cues.

Characterization of Two Arabidopsis L-Gulono-1,4-lactone Oxidases, AtGulLO3 and AtGulLO5, Involved in Ascorbate Biosynthesis.

L-Ascorbic acid (AsA, vitamin C) is an essential antioxidant for plants and animals. There are four known ascorbate biosynthetic pathways in plants: the L-galactose, L-gulose, D-galacturonate, and myo-inositol routes. These pathways converge into two AsA precursors: L-galactono-1,4-lactone and L-gulono-1,4-lactone (L-GulL). This work focuses on the study of L-gulono-1,4-lactone oxidase (GulLO), the enzyme that works at the intersect of the gulose and inositol pathways. Previous studies have shown that feeding L-gulono-1,4-lactone to multiple plants leads to increased AsA. There are also reports showing GulLO activity in plants. We describe the first detailed characterization of a plant enzyme specific to oxidize L-GulL to AsA. We successfully purified a recombinant Arabidopsis GulLO enzyme (called AtGulLO5) in a transient expression system. The biochemical properties of this enzyme are similar to the ones of bacterial isozymes in terms of substrate specificity, subcellular localization, use of flavin adenine dinucleotide (FAD) as electron acceptor, and specific activity. AtGulLO5 is an exclusive dehydrogenase with an absolute specificity for L-GulL as substrate thus differing from the existing plant L-galactono-1,4-lactone dehydrogenases and mammalian GulLOs. Feeding L-GulL to N. benthamiana leaves expressing AtGulLO5 constructs led to increased foliar AsA content, but it was not different from that of controls, most likely due to the observed low catalytic efficiency of AtGulLO5. Similar results were also obtained with another member of the AtGulLO family (AtGulLO3) that appears to have a rapid protein turnover. We propose that AsA synthesis through L-GulL in plants is regulated at the post-transcriptional level by limiting GulLO enzyme availability.

Recent progress on the characterization of aldonolactone oxidoreductases.

L-Ascorbic acid (ascorbate, AsA, vitamin C) is essential for animal and plant health. Despite our dependence on fruits and vegetables to fulfill our requirement for this vitamin, the metabolic network leading to its formation in plants is just being fully elucidated. There is evidence supporting the operation of at least four biosynthetic pathways leading to AsA formation in plants. These routes use D-mannose/L-galactose, L-gulose, D-galacturonate, and myo-inositol as the main precursors. This review focuses on aldonolactone oxidoreductases, a subgroup of the vanillyl alcohol oxidase (VAO; EC 1.1.3.38) superfamily, enzymes that catalyze the terminal step in AsA biosynthesis in bacteria, protozoa, animals, and plants. In this report, we review the properties of well characterized aldonolactone oxidoreductases to date. A shared feature in these proteins is the presence of a flavin cofactor as well as a thiol group. The flavin cofactor in many cases is bound to the N terminus of the enzymes or to a recently discovered HWXK motif in the C terminus. The binding between the flavin moiety and the protein can be either covalent or non-covalent. Substrate specificity and subcellular localization differ among the isozymes of each kingdom. All oxidases among these enzymes possess dehydrogenase activity, however, exclusive dehydrogenases are also found. We also discuss recent evidence indicating that plants have both L-gulono-1,4-lactone oxidases and L-galactono-1,4-lactone dehydrogenases involved in AsA biosynthesis.