Lab-scale preparation and QC of phytase assay substrate from rice bran

Sodium phytate is the most commonly used substrate for phytase assays. The current data documents the composition of sodium phytate prepared from rice bran on the lab scale. Two batches (A and B) of rice bran sodium phytate were analyzed for their elemental composition by ICP-SFMS. The preparation of the two batches differed by the introduction of an extra step to reduce contaminating magnesium for batch B. Both batches and additional samples of commercial and recrystallized commercial sodium phytate were also analyzed for their inositol phosphate profile by anion exchange HPLC

Challenges and potentials of new breeding techniques in Cannabis sativa

Cannabis sativa L. is an ancient crop used for fiber and seed production and not least for its content of cannabinoids used for medicine and as an intoxicant drug. Due to the psychedelic effect of one of the compounds, tetrahydrocannabinol (THC), many countries had regulations or bands on Cannabis growing, also as fiber or seed crop. Recently, as many of these regulations are getting less tight, the interest for the many uses of this crop is increasing. Cannabis is dioecious and highly heterogenic, making traditional breeding costly and time consuming. Further, it might be difficult to introduce new traits without changing the cannabinoid profile. Genome editing using new breeding techniques might solve these problems. The successful use of genome editing requires sequence information on suitable target genes, a genome editing tool to be introduced into plant tissue and the ability to regenerate plants from transformed cells. This review summarizes the current status of Cannabis breeding, uncovers potentials and challenges of Cannabis in an era of new breeding techniques and finally suggests future focus areas that may help to improve our overall understanding of Cannabis and realize the potentials of the plant

UCE: A uracil excision (USER™)-based toolbox for transformation of cereals

<p>Abstract</p> <p>Background</p> <p>Cloning of gene casettes and other DNA sequences into the conventional vectors for biolistic or <it>Agrobacterium</it>-mediated transformation is hampered by a limited amount of unique restriction sites and by the difficulties often encountered when ligating small single strand DNA overhangs. These problems are obviated by "The Uracil Specific Excision Reagent (USER™)" technology (New England Biolabs) which thus offers a new and very time-efficient method for engineering of big and complex plasmids.</p> <p>Results</p> <p>By application of the USER™ system, we engineered a collection of binary vectors, termed UCE (USER cereal), ready for use in cloning of complex constructs into the T-DNA. A series of the vectors were tested and shown to perform successfully in <it>Agrobacterium</it>-mediated transformation of barley (<it>Hordeum vulgare </it>L.) as well as in biolistic transformation of endosperm cells conferring transient expression.</p> <p>Conclusions</p> <p>The USER™ technology is very well suited for generating a toolbox of vectors for transformation and it opens an opportunity to engineer complex vectors, where several genetic elements of different origin are combined in a single cloning reaction.</p

Optimized barley phytase gene expression by focused FIND-IT screening for mutations in cis-acting regulatory elements

Introduction: Induced modification of plant gene expression is of both fundamental and applied importance. Cis-acting regulatory elements (CREs) are major determinants of the spatiotemporal strength of gene expression. Yet, there are few examples where induced genetic variation in predetermined CREs has been exploited to improve or investigate crop plants. Methods: The digital PCR based FIND-IT technology was applied to discover barley mutants with CRE variants in the promoter of the nutritional important barley grain phytase (PAPhy_a) gene. Results and discussion: Mutants with higher or lower gene expression and ultimately higher or lower mature grain phytase activity (MGPA), respectively, were discovered. Field trials and inositol phosphate profiling during germination showed that PAPhy_a does not influence agronomic performance under the trial conditions but it does shorten the lag time of phosphate mobilization during germination. Higher endogenous MGPA is an improvement of grain quality for feed use as it improves the phosphate bioavailability for monogastric animals. Moreover, as the targeted CRE motifs of the PAPhy_a promoter are shared with a range of seed expressed genes like key cereal and legume storage genes, the current results demonstrates a concept for modulating individual gene expression levels of a range of seed genes

Scandinavian perspectives on plant gene technology: applications, policies and progress

Plant research and breeding has a long and successful history in the Scandinavian countries, Denmark, Finland, Norway and Sweden. Researchers in the region have been early in adopting plant gene technologies as they developed. This review gives a background, as well as discuss the current and future progress of plant gene technology in these four countries. Country-specific details of the regulation of genetically modified plants are described, as well as similarities and differences in the approach to regulation of novel genome-editing techniques. Also, the development of a sustainable bioeconomy may encompass the application of plant gene technology and we discuss whether or not this is reflected in current associated national strategies. In addition, country-specific information about the opinion of the public and other stakeholders on plant gene technology is presented, together with a country-wise political comparison and a discussion of the potential reciprocal influence between public opinion and the political process of policy development. The Scandinavian region is unique in several aspects, such as climate and certain agriculturally related regulations, and at the same time the region is vulnerable to changes in plant breeding investments due to the relatively small market sizes. It is therefore important to discuss the role and regulation of innovative solutions in Scandinavian plant research and breeding.Peer reviewe

Structure of a cereal purple acid phytase provides new insights to phytate degradation in plants

Grain phytate, a mixed metal ion salt of inositol hexakisphosphate, accounts for 60%–80% of stored phosphorus in plants and is a potent antinutrient of non-ruminant animals including humans. Through neofunctionalization of purple acid phytases (PAPhy), some cereals such as wheat and rye have acquired particularly high mature grain phytase activity. As PAPhy activity supplies phosphate, liberates metal ions necessary for seedling emergence, and obviates antinutrient effects of phytate, its manipulation and control are targeted crop traits. Here we show the X-ray crystal structure of the b2 isoform of wheat PAPhy induced during germination. This high-resolution crystal structure suggests a model for phytate recognition that, validated by molecular dynamics simulations, implicates elements of two sequence inserts (termed PAPhy motifs) relative to a canonical metallophosphoesterase (MPE) domain in forming phytate-specific substrate specificity pockets. These motifs are well conserved in PAPhys from monocot cereals, enzymes which are characterized by high specificity for phytate. Tested by mutagenesis, residues His229 in PAPhy motif 4 and Lys410 in the MPE domain, both conserved in PAPhys, are found to strongly influence phytase activity. These results explain the observed phytase activity of cereal PAPhys and open the way to the rational engineering of phytase activity in planta

Editorial: Wheat biofortification to alleviate global malnutrition

According to the latest FAO report on the state of food security and nutrition in the world (1), more than 720 million people faced hunger, and around 3 billion people did not have access to a healthy diet. All these problematics, exacerbated by the current COVID-19 crisis, led to an increase in the number of people affected by the so-called hidden hunger, caused by an inadequate intake of essential micronutrients (MNs) such as iron (Fe), zinc (Zn), selenium (Se) and provitamin A. Biofortification, intended as the improvement of the nutritional quality of food crops through either conventional breeding, agronomic practices ormodern biotechnologies, represents a sustainable, costeffective and long-term approach to alleviate micronutrient-deficiency. Staple crops are typically the major target of most biofortification studies, given their central role in human diet. Wheat, specifically, contributes to around 20% of the total energy and protein intake and to around 30% of the Fe and Zn intake worldwide. However, the current level of MNs present in most wheat-derived food products is not enough to meet the minimum daily intake, especially in the poorest regions of the world. For these reasons, continuing to work on wheat biofortification is fundamental to ensure the production of nutritious and sustainable food and to contribute to the reduction of MNs deficiency

Molecular Advances on Phytases in Barley and Wheat

Phytases are pro-nutritional enzymes that hydrolyze phytate and make associated nutrients, such as phosphorous, iron, and zinc, bioavailable. Single-stomached animals and humans depend on phytase supplied through the diet or the action of phytase on the food before ingestion. As a result, phytases&#8212;or lack thereof&#8212;have a profound impact on agricultural ecosystems, resource management, animal health, and public health. Wheat, barley and their Triticeae relatives make exceptionally good natural sources of phytase. This review highlights advances in the understanding of the molecular basis of the phytase activity in wheat and barley, which has taken place over the past decade. It is shown how the phytase activity in the mature grains of wheat and barley can be ascribed to the PAPhy_a gene, which exists as a single gene in barley and in two or three homeologous copies in tetra- and hexaploid wheat, respectively. It is discussed how understanding the function and regulation of PAPhy_a may support the development of improved wheat and barley with even higher phytase activity

Globoids and Phytase: The Mineral Storage and Release System in Seeds

Phytate and phytases in seeds are the subjects of numerous studies, dating back as far as the early 20th century. Most of these studies concern the anti-nutritional properties of phytate, and the prospect of alleviating the effects of phytate with phytase. As reasonable as this may be, it has led to a fragmentation of knowledge, which hampers the appreciation of the physiological system at hand. In this review, we integrate the existing knowledge on the chemistry and biosynthesis of phytate, the globoid cellular structure, and recent advances on plant phytases. We highlight that these components make up a system that serves to store and&mdash;in due time&mdash;release the seed&rsquo;s reserves of the mineral nutrients phosphorous, potassium, magnesium, and others, as well as inositol and protein. The central component of the system, the phytate anion, is inherently rich in phosphorous and inositol. The chemical properties of phytate enable it to sequester additional cationic nutrients. Compartmentalization and membrane transport processes regulate the buildup of phytate and its associated nutrients, resulting in globoid storage structures. We suggest, based on the current evidence, that the degradation of the globoid and the mobilization of the nutrients also depend on membrane transport processes, as well as the enzymatic action of phytase