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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. [ABSTRACT FROM AUTHOR]

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 (PAPhy) in a common ancestor. Increased gene copy number of the PAPhy_a 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. However, barley only contains one HvPAPhy_a gene and the MGPA levels needs to be increased to substitute for the addition of microbial phytases to the feed. A substantial increase in MGPA for cisgenic barley was achieved with one extra homozygous HvPAPhy_a insert when the plants were grown in the greenhouse. In the current study, the stability of increased MGPA was confirmed in open field grown cisgenic barley. Furthermore, the gene dose response of phytase cisgenes from three different cisgenic barley plants were horizontally stacked. Cisgenic barley with 0, 1, 2, 3, 4, and 6 extra HvPAPhy_a inserts demonstrated a perfect positive linear correlation with the level of MGPA. The current study provides new insight into the potential of stacking of cisgenes in crops and suggests cisgene stacking as a versatile strategy for crop improvement. [ABSTRACT FROM AUTHOR]

The phytase purple acid phosphatase (Hv PAPhy_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 T2-generation of Hv PAPhy_a transformed barley showed phytase activity increases up to 19-fold (29 000 phytase units ( FTU) per kg in mature grains). Moreover, also in green leaves and mature dry straw, phytase activities were increased significantly by 110-fold (52 000 FTU/kg) and 57-fold (51 000 FTU/kg), respectively. The Hv PAPhy_a-transformed barley plants with high phytase activities possess triple potential utilities for the improvement of phosphate bioavailability. First of all, the utilization of the mature grains as feed to increase the release of bio-available phosphate and minerals bound to the phytate of the grains; secondly, the utilization of the powdered straw either directly or phytase extracted hereof as a supplement to high phytate feed or food; and finally, the use of the stubble to be ploughed into the soil for mobilizing phytate-bound phosphate for plant growth. [ABSTRACT FROM AUTHOR]

Abstract: The characterization of the full complement of wheat ferritins show that the modern hexaploid wheat genome contains two ferritin genes, TaFer1 and TaFer2, each represented by three homeoalleles and placed on chromosome 5 and 4, respectively. The two genes are differentially regulated and expressed. The TaFer1 genes are, except in the endosperm, the most abundantly expressed and regulated by iron and abscisic acid status. The promoter of TaFer1, in contrast to TaFer2, has iron- and ABA-responsive elements, supporting the expression data. The TaFer1 and TaFer2 genes encode two isoforms, probably functional different and acting in heteropolymer structures of ferritin in cereals. Iron biofortification of the wheat grain is possible. Endosperm targeted intragenic overexpressing of the TaFer1-A gene results in a 50–85% higher iron content in the grain. [Copyright &y& Elsevier]

Background: Cloning of gene casettes and other DNA sequences into the conventional vectors for biolistic or Agrobacterium-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 timeefficient method for engineering of big and complex plasmids. Results: 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 Agrobacterium-mediated transformation of barley (Hordeum vulgare L.) as well as in biolistic transformation of endosperm cells conferring transient expression. Conclusions: 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. [ABSTRACT FROM AUTHOR]

In recent years, the increasing knowledge on the molecular mechanisms underlying mineral uptake, transport, homeostasis and deposition within plants, has paved the way for a more targeted approach to improving the nutrient status of crop plants based on biotechnology. In the present paper we will briefly review existing knowledge on the distribution and transport pathways of iron in the two small grained cereals, barley and wheat, and focus on the efforts made to increase the iron content in cereals in general. However, mineral content is not the only factor of relevance for improving the nutritional status of poor populations. It is thus well documented that a number of plant components can act either as promoters or inhibitors of mineral uptake in the human digestive system (Frossard et al. J Sci Food Agric 80, 817-879 ; Brinch-Pedersen et al. J Cereal Sci 46, 308-326 ). The nutritional impact of increasing mineral content accordingly has to be seen in the context of mineral bioavailability. Finally, we will briefly report on recent data from barley, where laser capture microdissection of the different grain tissues combined with gene expression profiling has provided some insight into metal transport and deposition (Tauris et al. ). In the present paper we will provide a tentative and preliminary roadmap for iron trafficking in the barley grain. [ABSTRACT FROM AUTHOR]

Abstract: The present paper summarizes the current state of knowledge on molecular genetic approaches to increasing iron and zinc availability and vitamin content in cereals. We have also attempted to integrate the scientific issues into the wider context of human nutrition. In the cereal grain, iron and zinc are preferentially stored together with phytate in membrane-enclosed globoids in the protein storage vacuole (PSV) found in the aleurone and the embryo scutellum. The PSV is accordingly central for understanding mineral deposition during grain filling and mobilization of minerals during germination. Recent studies in Arabidopsis have led to the first identification of iron and zinc transporters of the PSV and further illustrate some of the dynamics associated with mineral and phytate transport and deposition into the vacuole. This provides new opportunities for modulating iron and zinc deposition in the cereal grain. Current strategies towards increasing the iron content of the endosperm are largely based on the expression of legume ferritin genes in an endosperm-specific manner. However, it is apparent that this approach, at least in rice, only allows a two- to three-fold increase in the iron content of the grain due to exhaustion of the iron stores in leaves. Further increases thus have to rely on additional uptake and transport of iron from the root. Phytate is generally considered to be the single most important anti-nutritional factor for iron and zinc availability. In the current paper we summarize attempts to increase phytase activity in the grain by transformation and evaluate the potential of this approach as well as the reduction of phytate biosynthesis for improving the bioavailability of iron and zinc. Vitamins constitute the second important group of micronutrients in grain and we discuss current efforts to increase the amounts of provitamin A, vitamin C and vitamin E. [Copyright &y& Elsevier]

Fusarium head blight (FHB) causes substantial losses of yield and quality in grains, both in the field and in post-harvest storage. To date, adequate natural genetic resistance is not available for the control of FHB. This study reports the cloning and overexpression of a barley (Hordeum vulgare L.) antifungal gene, nepenthesin 1 (HvNEP-1), in the endosperm of barley grains. Transgenic barley lines overexpressing HvNEP-1 substantially reduced FHB severity and disease progression after inoculation with Fusarium graminearum or Fusarium culmorum. The transgenic barley also showed reduced accumulation of the mycotoxin deoxynivalenol (DON) in grain, far below the minimum value allowable for food. Semi-field evaluation of four HvNEP-1 transgenic lines revealed substantial reduction of FHB severity and progression as compared with the control H. vulgare cultivar Golden promise (GP) plants. Our study demonstrated the utility of HvNEP-1 for the control of FHB in barley, and possibly other grains such as wheat and maize. [ABSTRACT FROM AUTHOR]

Abstract: Cereal purple acid phosphatase-type phytases, PAPhy, play an essential role in making phosphate accessible to mammalian digestion and reducing the environmental impact of manure. Studying the potential of PAPhy requires easy access to the enzymes. For that purpose wheat and barley isophytases have been expressed in Pichia pastoris from constructs encoding the alpha-mating factor at the N-termini and a His6 tag before the stop codon in all constructs. A protein chemical study of a C-terminally truncated recombinant wheat phytase, r-TaPAPhy_b2, was carried out to clarifying the posttranslational processing of proteins secreted from P. pastoris. Extensive mass spectrometric sequencing of tryptic, chymotryptic and AspN derived peptides of both the native and endoH deglycosylated forms showed: (i) All mating factor derived sequence had been removed and further unspecific proteolysis left highly heterogeneous N-terminal variant forms of r-TaPAPhy; (ii) The His6 tag had been retained or slightly truncated; (iii) All seven potential N-glycan sites were glycosylated except for two sites which were partially glycosylated by ca. 90% and 30%; (iv) Among the nine cysteine residues of this phytase, the most N-terminal residue is free, whereas the remaining eight appear to be disulfide bonded. It is noteworthy that already the first step in ESI-MS/MS sequencing had fragmented the hyper glycosylated peptides into free Z, Y and X mass spectrometric glycan fragments attached to the peptide. [Copyright &y& Elsevier]