Optimized barley phytase gene expression by focused FIND-IT screening for mutations in -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.

New Hope for Genome Editing in Cultivated Grasses: CRISPR Variants and Application.

With the advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) mediated genome editing, crop improvement has progressed significantly in recent years. In this genome editing tool, CRISPR-associated Cas nucleases are restricted to their target of DNA by their preferred protospacer adjacent motifs (PAMs). A number of CRISPR-Cas variants have been developed e.

Genome Editing-accelerated Re-Domestication (GEaReD) - A new major direction in plant breeding.

Background: The effects of climate change, soil depletion, a growing world population putting pressure on food safety and security are major challenges for agriculture in the 21st century. The breeding success of the green revolution has decelerated and current programs can only offset the yield affecting factors.

Purpose And Scope: New approaches are urgently needed and "Genome Editing-accelerated Re-Domestication" (GEaReD) is proposed as a major new direction in plant breeding.

Barley Nepenthesin-Like Aspartic Protease Degrades Phytase, Impairs Toxin Production, and Suppresses the Fungal Growth.

Nepenthesins are categorized under the subfamily of the nepenthesin-like plant aspartic proteases (PAPs) that form a distinct group of atypical PAPs. This study describes the effect of nepenthesin 1 () protease from barley ( L.) on fungal histidine acid phosphatase (HAP) phytase activity.

Horizontal Stacking of _a Cisgenes in Barley Is a Potent Strategy for Increasing Mature Grain Phytase Activity.

Mature grain phytase activity (MGPA) in the Triticea tribe cereals has evolved through gene duplications and neo-functionalization of the purple acid phosphatase phytase gene () in a common ancestor. Increased gene copy number of the gene expressed during seed development has augmented the MGPA in cereals like rye and wheat. PAPhy_a phytase is highly stable and a potent enzyme in feed.

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.

A novel wheat q' allele identified by forward genetic in silico TILLING.

The major wheat domestication allele Q (encoding an APETALA2 like transcription factor) is responsible for the free threshing and square-headed spikes of modern wheat. Wild type q and null q' alleles cause a reversal to the speltoid phenotype. Q pleiotropically affects additional yield and quality traits so genetic variation in Q and its interaction partners remain important for crop improvement.

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-or lack thereof-have a profound impact on agricultural ecosystems, resource management, animal health, and public health.

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

Phytases are involved in the phosphate acquisition and remobilization in plants, microbes and animals. They have become important technical enzymes in the feed industry and are used to make phosphate, present in animal feed as phytate, available for monogastric animal nutrition. Phytases may also be beneficial to human nutrition because phytate is known to interfere with the uptake of important micronutrients.

Immobilisation of barley aleurone layers enables parallelisation of assays and analysis of transient gene expression in single cells.

The barley aleurone layer is an established model system for studying phytohormone signalling, enzyme secretion and programmed cell death during seed germination. Most analyses performed on the aleurone layer are end-point assays based on cell extracts, meaning each sample is only analysed at a single time point. By immobilising barley aleurone layer tissue on polydimethylsiloxane pillars in the lid of a multiwell plate, continuous monitoring of living tissue is enabled using multiple non-destructive assays in parallel.

Barley HvPAPhy_a as transgene provides high and stable phytase activities in mature barley straw and in grains.

The phytase purple acid phosphatase (HvPAPhy_a) expressed during barley seed development was evaluated as transgene for overexpression in barley. The phytase was expressed constitutively driven by the cauliflower mosaic virus 35S-promoter, and the phytase activity was measured in the mature grains, the green leaves and in the dry mature vegetative plant parts left after harvest of the grains. The T -generation of HvPAPhy_a transformed barley showed phytase activity increases up to 19-fold (29 000 phytase units (FTU) per kg in mature grains).

The PARS sequence increase the efficiency of stable Pichia pastoris transformation.

The methylotrophic yeast Pichia pastoris is a popular host for recombinant expression of proteins. Plasmids containing the Pichia autonomously replicating sequence (PARS) transform P. pastoris with higher efficiency than linear DNA equipped with termini designed for homologous recombination.

A novel approach to the generation of seamless constructs for plant transformation.

Background: When creating plant transformation vectors, full control of nucleotides flanking the insert in the final construct may be desirable. Modern ligase-independent methods for DNA-recombination are based on linearization by classical type II restriction endonucleases (REs) alone or in combination with nicking enzymes leaving residual nucleotides behind in the final construct. We here explore the use of type IIS and type IIB REs for vector linearization that combined with sequence and ligase-independent cloning (SLIC) overcomes this problem and promotes seamless gene-insertion in vectors.

High mature grain phytase activity in the Triticeae has evolved by duplication followed by neofunctionalization of the purple acid phosphatase phytase (PAPhy) gene.

The phytase activity in food and feedstuffs is an important nutritional parameter. Members of the Triticeae tribe accumulate purple acid phosphatase phytases (PAPhy) during grain filling. This accumulation elevates mature grain phytase activities (MGPA) up to levels between ~650 FTU/kg for barley and 6000 FTU/kg for rye.

Cisgenic barley with improved phytase activity.

The cisgenesis concept implies that plants are transformed only with their own genetic materials or genetic materials from closely related species capable of sexual hybridization. Furthermore, foreign sequences such as selection genes and vector-backbone sequences should be absent. We used a barley phytase gene (HvPAPhy_a) expressed during grain filling to evaluate the cisgenesis concept in barley.

Cloning and characterization of purple acid phosphatase phytases from wheat, barley, maize, and rice.

Barley (Hordeum vulgare) and wheat (Triticum aestivum) possess significant phytase activity in the mature grains. Maize (Zea mays) and rice (Oryza sativa) possess little or virtually no preformed phytase activity in the mature grain and depend fully on de novo synthesis during germination. Here, it is demonstrated that wheat, barley, maize, and rice all possess purple acid phosphatase (PAP) genes that, expressed in Pichia pastoris, give fully functional phytases (PAPhys) with very similar enzyme kinetics.

A novel family of plant DNA-binding proteins containing both HMG-box and AT-rich interaction domains.

The A/T-rich interaction domain (ARID) and the HMG-box domain represent DNA-interaction modules that are found in sequence-specific as well as nonsequence-specific DNA-binding proteins. Both domains are found in a variety of DNA-interacting proteins in a wide range of eukaryotic organisms. Proteins that contain both an ARID and an HMG-box domain, here termed ARID-HMG proteins, appear to be specific for plants.