Henrik Brinch-Pedersen

Engineering crop plants: getting a handle on phosphate

In plant seeds, most of the phosphate is in the form of phytic acid. Phytic acid is largely indigestible by monogastric animals and is the single most important factor hindering the uptake of a range of minerals. Engineering crop plants to produce a heterologous phytase improves phosphate bioavailability and reduces phytic acid excretion. This reduces the phosphate load on agricultural ecosystems and thereby alleviates eutrophication of the aquatic environment. Improved phosphate availability also reduces the need to add inorganic phosphate, a non-renewable resource. Iron and zinc uptake might be improved, which is significant for human nutrition in developing countries.

TAL effector nucleases induce mutations at a pre-selected location in the genome of primary barley transformants

Transcription activator-like effector nucleases (TALENs) enable targeted mutagenesis in a variety of organisms. The primary advantage of TALENs over other sequence-specific nucleases, namely zinc finger nucleases and meganucleases, lies in their ease of assembly, reliability of function, and their broad targeting range. Here we report the assembly of several TALENs for a specific genomic locus in barley. The cleavage activity of individual TALENs was first tested in vivo using a yeast-based, single-strand annealing assay. The most efficient TALEN was then selected for barley transformation. Analysis of the resulting transformants showed that TALEN-induced double strand breaks led to the introduction of short deletions at the target site. Additional analysis revealed that each barley transformant contained a range of different mutations, indicating that mutations occurred independently in different cells.

A microarray-based comparative analysis of gene expression profiles during grain development in transgenic and wild type wheat.

Global, comparative gene expression analysis is potentially a very powerful tool in the safety assessment of transgenic plants since it allows for the detection of differences in gene expression patterns between a transgenic line and the mother variety. In the present study, we compared the gene expression profile in developing seeds of wild type wheat and wheat transformed for endosperm-specific expression of an Aspergillus fumigatus phytase. High-level expression of the phytase gene was ensured by codon modification towards the prevalent codon usage of wheat genes and by using the wheat 1DX5HMW glutenin promoter for driving transgene expression. A 9K wheat unigene cDNA microarray was produced from cDNA libraries prepared mainly from developing wheat seed. The arrays were hybridised to flourescently labelled cDNA prepared from developing seeds of the transgenic wheat line and the mother variety, Bobwhite, at three developmental stages. Comparisons and statistical analyses of the gene expression profiles of the transgenic line vs. that of the mother line revealed only slight differences at the three developmental stages. In the few cases where differential expression was indicated by the statistical analysis it was primarily genes that were strongly expressed over a shorter interval of seed development such as genes encoding storage proteins. Accordingly, we interpret these differences in gene expression levels to result from minor asynchrony in seed development between the transgenic line and the mother line. In support of this, real time PCR validation of results from selected genes at the late developmental stage could not confirm differential expression of these genes. We conclude that the expression of the codon-modified A.␣fumigatus phytase gene in the wheat seed had no significant effects on the overall gene expression patterns in the developing seed.

Engineering of the aspartate family biosynthetic pathway in barley (Hordeum vulgare L.) by transformation with heterologous genes encoding feed-back-insensitive aspartate kinase and dihydrodipicolinate synthase

In prokaryotes and plants the synthesis of the essential amino acids lysine and threonine is predominantly regulated by feed-back inhibition of aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). In order to modify the flux through the aspartate family pathway in barley and enhance the accumulation of the corresponding amino acids, we have generated transgenic barley plants that constitutively express mutant Escherichia coli genes encoding lysine feed-back insensitive forms of AK and DHPS. As a result, leaves of primary transformants (T0) exhibited a 14-fold increase of free lysine and an 8-fold increase in free methionine. In mature seeds of the DHPS transgenics, there was a 2-fold increase in free lysine, arginine and asparagine and a 50% reduction in free proline, while no changes were observed in the seeds of the two AK transgenic lines analysed. When compared to that of control seeds, no differences were observed in the composition of total amino acids. The introduced genes were inherited in the T1 generation where enzymic activities revealed a 2.3-fold increase of AK activity and a 4.0–9.5-fold increase for DHPS. T1 seeds of DHPS transformants showed the same changes in free amino acids as observed in T0 seeds. It is concluded that the aspartate family pathway may be genetically engineered by the introduction of genes coding for feed-back-insensitive enzymes, preferentially giving elevated levels of lysine and methionine.

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. Preformed wheat PAPhy was localized to the protein crystalloid of the aleurone vacuole. Phylogenetic analyses indicated that PAPhys possess four conserved domains unique to the PAPhys. In barley and wheat, the PAPhy genes can be grouped as PAPhy_a or PAPhy_b isogenes (barley, HvPAPhy_a, HvPAPhy_b1, and HvPAPhy_b2; wheat, TaPAPhy_a1, TaPAPhy_a2, TaPAPhy_b1, and TaPAPhy_b2). In rice and maize, only the b type (OsPAPhy_b and ZmPAPhy_b, respectively) were identified. HvPAPhy_a and HvPAPhy_b1/b2 share 86% and TaPAPhya1/a2 and TaPAPhyb1/b2 share up to 90% (TaPAPhy_a2 and TaPAPhy_b2) identical amino acid sequences. despite of this, PAPhy_a and PAPhy_b isogenes are differentially expressed during grain development and germination. In wheat, it was demonstrated that a and b isogene expression is driven by different promoters (approximately 31% identity). TaPAPhy_a/b promoter reporter gene expression in transgenic grains and peptide mapping of TaPAPhy purified from wheat bran and germinating grains confirmed that the PAPhy_a isogene set present in wheat/barley but not in rice/maize is the origin of high phytase activity in mature grains.

Iron transport, deposition and bioavailability in the wheat and barley grain

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 2000; Brinch-Pedersen et al. J Cereal Sci 46, 308-326 2007). 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. 2009). In the present paper we will provide a tentative and preliminary roadmap for iron trafficking in the barley grain.

Concerted Action of Endogenous and Heterologous Phytase on Phytic Acid Degradation in Seed of Transgenic Wheat (Triticum aestivum L.)

Expression of heterologous phytases in crops offers a great potential for improving phosphate and mineral bio-availability in food and feed. In this context it is of relevance to describe the concerted action of endogenous and hetrologous phytases on the transgenic seed inositol phosphate profile. Here we report metal-dye detection HPLC analysis of inositol phosphate degradation in flour from transgenic wheat materials possessing wheat endogenous 6-phytase [EC 3.1.3.26] and Aspergillus 3-phytase [EC 3.1.3.8] activities under the control of the maize ubiquitin-1 promoter and the wheat high molecular weight glutenin subunit 1DX5 promoter respectively. During 50 min incubation there is an accumulation of InsP5 to InsP2 breakdown products in non-transgenic material. Aspergillus niger phytase specific breakdown products are transiently detected in transgenic material but after 50 min incubation virtually all InsP5, InsP4 and InsP3 isomers are hydrolysed.

Cloning and Characterization of Purple Acid Phosphatase Phytases from Wheat ( Triticum aestivum L.), Barley ( Hordeum vulgare L.), Maize ( Zea maize L.) and Rice ( Oryza sativa L.)

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. Preformed wheat PAPhy was localized to the protein crystalloid of the aleurone vacuole. Phylogenetic analyses indicated that PAPhys possess four conserved domains unique to the PAPhys. In barley and wheat, the PAPhy genes can be grouped as PAPhy_a or PAPhy_b isogenes (barley, HvPAPhy_a, HvPAPhy_b1, and HvPAPhy_b2; wheat, TaPAPhy_a1, TaPAPhy_a2, TaPAPhy_b1, and TaPAPhy_b2). In rice and maize, only the b type (OsPAPhy_b and ZmPAPhy_b, respectively) were identified. HvPAPhy_a and HvPAPhy_b1/b2 share 86% and TaPAPhya1/a2 and TaPAPhyb1/b2 share up to 90% (TaPAPhy_a2 and TaPAPhy_b2) identical amino acid sequences. despite of this, PAPhy_a and PAPhy_b isogenes are differentially expressed during grain development and germination. In wheat, it was demonstrated that a and b isogene expression is driven by different promoters (approximately 31% identity). TaPAPhy_a/b promoter reporter gene expression in transgenic grains and peptide mapping of TaPAPhy purified from wheat bran and germinating grains confirmed that the PAPhy_a isogene set present in wheat/barley but not in rice/maize is the origin of high phytase activity in mature grains.

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. The marker gene elimination method was used to obtain marker-free plant lines. Here, the gene of interest and the selection gene are flanked by their own T-DNA borders to allow unlinked integration of the two genes. We analysed the transformants for co-transformation efficiency, increased phytase activities in the grain, integration of the kanamycin resistance gene of the vector-backbone and segregation between the HvPAPhy_a insert and the hygromycin resistance gene. The frequencies of the four parameters imply that it should be possible to select 11 potentially cisgenic T(1) -lines out of the 72 T(0) -lines obtained, indicating that the generation of cisgenic barley is possible at reasonable frequencies with present methods. We selected two potential cisgenic lines with a single extra copy of the HvPAPhy_a insert for further analysis. Seeds from plants homozygous for the insert showed 2.6- and 2.8-fold increases in phytase activities and the activity levels were stable over the three generations analysed. In one of the selected lines, the flanking sequences from both the left and right T-DNA borders were analysed. These sequences confirmed the absence of truncated vector-backbone sequences linked to the borders. The described line should therefore be classified as cisgenic.

Transformation of barley by microinjection into isolated zygote protoplasts

Barley zygote protoplasts were mechanically isolated, embedded in agarose droplets, and microinjected with a rice actin promoter Act1–gusA-nos gene construct. On average 62% of the cells survived the injection and of these 55% continued development into embryo-like structures and eventually to plants. PCR screening for the presence of a 307-bp fragment in the middle of the gusA gene showed that on average 21% of the derived structures contained this fragment. However, among the hundreds of injected zygotes, derived structures and regenerants we only found significant GUS expression in two cases (embryo-like structures nine days after injection). Two lines of green plants, derived from zygotes microinjected with linearized plasmid (line A147-1) or an isolated Act1–gusA-nos gene cassette (line A166-h) proved to be transgenic. Line A147-1 appeared to contain a single and intact copy of the expression cassette but a PCR based progeny analysis indicated the presence of additional shorter fragments of the cassette. Line A166-h appeared to contain a single fragment of the gusA gene that was transferred to the progeny as a single Mendelian trait. One additional fragment of the gusA gene was identified in this line. The present data show that transformation of barley by microinjection of DNA into isolated zygotes is feasible but also that gene expression rarely is achieved, possibly due to degradation of the introduced DNA.