Toward eradication of B-vitamin deficiencies : considerations for crop biofortification

'Hidden hunger' involves insufficient intake of micronutrients and is estimated to affect over two billion people on a global scale. Malnutrition of vitamins and minerals is known to cause an alarming number of casualties, even in the developed world. Many staple crops, although serving as the main dietary component for large population groups, deliver inadequate amounts of micronutrients. Biofortification, the augmentation of natural micronutrient levels in crop products through breeding or genetic engineering, is a pivotal tool in the fight against micronutrient malnutrition (MNM). Although these approaches have shown to be successful in several species, a more extensive knowledge of plant metabolism and function of these micronutrients is required to refine and improve biofortification strategies. This review focuses on the relevant B-vitamins (B1, B6, and B9). First, the role of these vitamins in plant physiology is elaborated, as well their biosynthesis. Second, the rationale behind vitamin biofortification is illustrated in view of pathophysiology and epidemiology of the deficiency. Furthermore, advances in biofortification, via metabolic engineering or breeding, are presented. Finally, considerations on B-vitamin multi-biofortified crops are raised, comprising the possible interplay of these vitamins in planta

Ethylene

Ethylene is a volatile 2-carbon atom molecule with hormonal properties in plants. In cross-talk with other hormones, it plays a pivotal role in controlling plant growth, through regulation of cell expansion. In addition to stimulating germination and flowering in certain species, it is also well known as a regulator of flower and leaf abscission as well as of fruit ripening in climacteric species. Consequently, control of ethylene production is of paramount importance in agri- and horticulture. Molecular genetics has allowed profound understanding of the mechanism of the biosynthetic and response pathways of ethylene, creating ample possibilities to control ethylene status through genetic modification or marker-assisted breeding or through the use of small molecules that target key players in the pathway

The first comprehensive LC–MS/MS method allowing dissection of the thiamine pathway in plants

Arabidopsis thaliana serves as a model plant for genetic research, including vitamin research. When aiming at engineering the thiamine (vitamin B1) pathway in plants, the availability of tools that allow the quantitative determination of different intermediates in the biosynthesis pathway is of pivotal importance. This is a challenge, given the nature of the compounds and the minute quantities of genetically engineered material that may be available for analysis. Here, we report on the first LC–MS/MS method for the simultaneous quantification of thiamine, its mono- and diphosphate derivatives and its precursors 4-methyl-5-(2-hydroxyethyl) thiazole (HET) and 4-amino-2-methyl-5-hydroxymethylpyrimidine (HMP). This method was optimized and validated for the quantitative determination of these analytes in Arabidopsis thaliana. All analytes were chromatographically separated within less than 2.5 min during an 8 min run. No unacceptable interferences were found. The method was fully validated based on international guidelines. Accuracy (%bias) and total imprecision (%CV) were within preset acceptance criteria for all analytes in both QC and real samples. All analytes were stable in extracted samples when stored for 48 h at 4 °C (autosampler stability) and when reanalyzed after storage at −80 °C and −20 °C for 2 weeks (freeze/thaw stability). We demonstrated the start material should be stored at −80 °C to ensure stability of all analytes during short- and long-term storage (up to 3 months). The validity and applicability of the developed procedure was demonstrated via its successful application on Arabidopsis lines, genetically engineered to enhance thiamine content

1-aminocyclopropane-1-carboxylic acid (ACC) in plants: more than just the precursor of ethylene!

Ethylene is a simple two carbon atom molecule with profound effects on plants. There are quite a few review papers covering all aspects of ethylene biology in plants, including its biosynthesis, signaling and physiology. This is merely a logical consequence of the fascinating and pleiotropic nature of this gaseous plant hormone. Its biochemical precursor, 1-aminocyclopropane-1-carboxylic acid (ACC) is also a fairly simple molecule, but perhaps its role in plant biology is seriously underestimated. This triangularly shaped amino acid has many more features than just being the precursor of the lead-role player ethylene. For example, ACC can be conjugated to three different derivatives, but their biological role remains vague. ACC can also be metabolized by bacteria using ACC-deaminase, favoring plant growth and lowering stress susceptibility. ACC is also subjected to a sophisticated transport mechanism to ensure local and long-distance ethylene responses. Last but not least, there are now a few exciting studies where ACC has been reported to function as a signal itself, independently from ethylene. This review puts ACC in the spotlight, not to give it the lead-role, but to create a picture of the stunning co-production of the hormone and its precursor

Optimization of non-denaturing protein extraction conditions for plant PPR proteins

[EN] Pentatricopeptide repeat proteins are one of the major protein families in flowering plants, containing around 450 members. They participate in RNA editing and are related to plant growth, development and reproduction, as well as to responses to ABA and abiotic stresses. Their characteristics have been described in silico; however, relatively little is known about their biochemical properties. Different types of PPR proteins, with different tasks in RNA editing, have been suggested to interact in an editosome to complete RNA editing. Other non-PPR editing factors, such as the multiple organellar RNA editing factors and the organelle RNA recognition motif-containing protein family, for example, have also been described in plants. However, while evidence on protein interactions between non-PPR RNA editing proteins is accumulating, very few PPR protein interactions have been reported; possibly due to their high instability. In this manuscript, we aimed to optimize the conditions for non denaturing protein extraction of PPR proteins allowing in vivo protein analyses, such as interaction assays by co-immunoprecipitation. The unusually high protein degradation rate, the aggregation properties and the high pl, as well as the ATP-dependence of some PPR proteins, are key aspects to be considered when extracting PPR proteins in a non-denatured state. During extraction of PPR proteins, the use of proteasome and phosphatase inhibitors is critical. The use of the ATP-cofactor reduces considerably the degradation of PPR proteins. A short centrifugation step to discard cell debris is essential to avoid PPR precipitation; while in some cases, addition of a reductant is needed, probably caused by the pl/pH context. This work provides an easy and rapid optimized non-denaturing total protein extraction protocol from plant tissue, suitable for polypeptides of the PPR family.This work was supported by Research Foundation Flanders (FWO) (project G.0C84.14N). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.r D.V.D.S. gratefully acknowledges Ghent University and the Research Foundation Flanders (FWO) for financial support (project G.0084.14N). N.A.-C. and D.V.D.S. acknowledge Maria Helena S. Goldman for the N. benthamiana seeds, helpful suggestions regarding experimental design and data interpretation, and critical reading of the manuscript.Andrés-Colás, N.; Van Der Straeten, D. (2017). Optimization of non-denaturing protein extraction conditions for plant PPR proteins. PLoS ONE. 12(11):1-15. https://doi.org/10.1371/journal.pone.0187753S1151211Barkan, A., & Small, I. (2014). Pentatricopeptide Repeat Proteins in Plants. 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(2013). MEF10 is required for RNA editing at nad2-842 in mitochondria of Arabidopsis thaliana and interacts with MORF8. Plant Molecular Biology, 81(4-5), 337-346. doi:10.1007/s11103-012-0003-2Hu, J., Wang, K., Huang, W., Liu, G., Gao, Y., Wang, J., … Zhu, Y. (2012). The Rice Pentatricopeptide Repeat Protein RF5 Restores Fertility in Hong-Lian Cytoplasmic Male-Sterile Lines via a Complex with the Glycine-Rich Protein GRP162. The Plant Cell, 24(1), 109-122. doi:10.1105/tpc.111.093211Sun, T., Germain, A., Giloteaux, L., Hammani, K., Barkan, A., Hanson, M. R., & Bentolila, S. (2013). An RNA recognition motif-containing protein is required for plastid RNA editing in Arabidopsis and maize. Proceedings of the National Academy of Sciences, 110(12), E1169-E1178. doi:10.1073/pnas.1220162110Sun, T., Shi, X., Friso, G., Van Wijk, K., Bentolila, S., & Hanson, M. R. (2015). A Zinc Finger Motif-Containing Protein Is Essential for Chloroplast RNA Editing. 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Metabolic engineering of micronutrients in crop plants

Micronutrient deficiency is a widespread phenomenon, most prevalent in developing countries. Being causally linked to the occurrence of a range of diseases, it affects billions of people worldwide. Enhancing the content of micronutrients in crop products through biotechnology is a promising technique to fight micronutrient malnutrition worldwide. Micronutrient fortification of food products has been implemented in a number of Western countries, but remains inaccessible for poor rural populations in a major part of the developing world. Moreover, evidence of the negative impacts of this practice on human health, at least for some vitamins, is accumulating. Biofortification of crop plants-the enhancement of vitamins and minerals through plant biotechnology-is a promising alternative or complement in the battle against micronutrient deficiencies. Owing to a growing knowledge about vitamin metabolism, as well as mineral uptake and reallocation in plants, it is today possible to enhance micronutrient levels in crop plants, offering a sustainable solution to populations with a suboptimal micronutrient intake

Differential coupling of gibberellin responses by Rht-B1c suppressor alleles and Rht-B1b in wheat highlights a unique role for the DELLA N-terminus in dormancy

During the Green Revolution, substantial increases in wheat (Triticum aestivum) yields were realized, at least in part, through the introduction of the Reduced height (Rht)-B1b and Rht-D1b semi-dwarfing alleles. In contrast to Rht-B1b and Rht-D1b, the Rht-B1c allele is characterized by extreme dwarfism and exceptionally strong dormancy. Recently, 35 intragenic Rht-B1c suppressor alleles were created in the spring wheat cultivar Maringa, and termed overgrowth (ovg) alleles. Here, 14 ovg alleles with agronomically relevant plant heights were reproducibly classified into nine tall and five semi-dwarf alleles. These alleles differentially affected grain dormancy, internode elongation rate, and coleoptile and leaf lengths. The stability of these ovg effects was demonstrated for three ovg alleles in different genetic backgrounds and environments. Importantly, two semi-dwarf ovg alleles increased dormancy, which correlated with improved pre-harvest sprouting (PHS) resistance. Since no negative effects on grain yield or quality were observed, these semi-dwarf ovg alleles are valuable for breeding to achieve adequate height reduction and protection of grain quality in regions prone to PHS. Furthermore, this research highlights a unique role for the first 70 amino acids of the DELLA protein, encoded by the Rht-1 genes, in grain dormancy

Ethylene and hormonal cross talk in vegetative growth and development

Ethylene is a gaseous plant hormone that most likely became a functional hormone during the evolution of charophyte green algae, prior to land colonization. From this ancient origin, ethylene evolved into an important growth regulator that is essential for myriad plant developmental processes. In vegetative growth, ethylene appears to have a dual role, stimulating and inhibiting growth, depending on the species, tissue, and cell type, developmental stage, hormonal status, and environmental conditions. Moreover, ethylene signaling and response are part of an intricate network in cross talk with internal and external cues. Besides being a crucial factor in the growth control of roots and shoots, ethylene can promote flowering, fruit ripening and abscission, as well as leaf and petal senescence and abscission and, hence, plays a role in virtually every phase of plant life. Last but not least, together with jasmonates, salicylate, and abscisic acid, ethylene is important in steering stress responses

Determination of five folate monoglutamates in rodent diets

A method for the quantitative determination of folates in rodent diets is very important for correct interpretation of folate intake during feeding trials, given the possible discrepancy between the actual folate concentration in the diet and that mentioned on the product sheet. Liquid chromatography tandem-mass spectrometry is the method of choice to differentiate and quantify the individual folate species present. This discrepancy may be accounted for by, e.g., inaccurate folic acid supplementation and/or the presence of endogenous reduced and substituted folates. We developed a method, validated based on FDA guidelines, that allows the measurement of added and endogenous folates by quantitative determination of 5 folate monoglutamates with linear ranges from 8 mu g to 2 mg/kg feed. This information, combined with feed intake data, allows insight into the actual folate intake in animal feeding studies. The relevance of this method was illustrated by the analysis of several feed samples of varying composition, by the investigation of the effect of casein incorporation, and by evaluating the variability of the folate content between pellets and production batches

Tracking multiple objects using intensity-GVF snakes

Active contours or snakes are widely used for segmentation and tracking. Multiple object tracking remains a difficult task, characterised by a trade off between increasing the capturing range of edges of the object of interest, and decreasing the capturing range of other edges. We propose a new external force field which is calculated for every object independently. This new force field uses prior knowledge about the intensity of the object of interest. Using this extra information, this new force field helps in discriminating between edges of interest and other objects. For this new force field, the expected intensity of an object must be estimated. We propose a technique which calculates this estimation out of the image