Søren Knudsen

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.

Transformation of the developing barley endosperm by particle bombardment.

Delivery of DNA into intact cells of the developing barley (Hordeum vulgare L.) endosperm was performed with the BIOLISTIC particle gun. It is shown that the proximal 532 base pairs (bp) of the upstream region of a B1-hordein gene drive the expression of the β-glucuronidase (GUS) gene (uidA) in sub-aleurone and starchy-endosperm cells but not in cells devoid of starch, i.e. developing aleurone cells. The 35S promoter from cauliflower mosaic virus was active in all three cell types. This cell-specific activity of the hordein promoter was verified by a detailed histological study of the regions of the extruded endosperms expressing the uidA gene. The analysis included a histological study of the developing endosperm as a base for classifying the different cell types in the developing endosperm.

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.

Amber codon suppression: the in vivo and in vitro analysis of two C-hordein genes from barley

A 1420 bp genomic fragment (λ-horl-17) encompassing a Hor-1 gene encoding a C-hordein polypeptide is presented. The deduced amino acid sequence is 261 residues long. It comprises a 20 amino acid signal peptide, unique NH2- and COOH-terminal regions and a coding region comprised of pentapeptide (PQQPY) and octapeptide (PQQPFPQQ) repeat motifs. The 431 bp of 5′ non-coding region contains a ‘TATA box’ at −105, a ‘CACA box’ (−181 to −201) and a −300 prolamin element. In the 3′ noncoding region there are two putative polyadenylation signals located 88 and 142 bp downstream of the stop codon.The structure of λ-hor1-17 is compared with that of another gene (λ-hor1-14) encoding a C-hordein polypeptide, which contains an amber codon interrupting the ORF. A functional assay in which the 5′ non-coding regions of the two genes were fused to the β-glucuronidase (GUS) gene demonstrated that both genes were transcriptionally active and that circa 430 bp of the C-hordein promoters were sufficient to drive the expression of the GUS gene in developing barley endosperms. It also demonstrated that both promoters had transcriptional efficiencies comparable with that of the 35S CaMV promoter. The in vitro translation of the coding region of λ-hor1-14 in the wheat germ system showed that the premature stop codon could be partially suppressed. The suppression was also demonstrated in a transient expression assay in vivo using isolated barley endosperms.

Regulation of Storage Protein Synthesis in Cereal Seeds: Developmental and Nutritional Aspects

Summary The major seed storage proteins of cereals are deposited in the maturation phase of endosperm development, starting at 12 days after pollination (dap) in maize and continuing up to around 30dap. Cell types-pecific expression in the endosperm is conferred by a multicomponent promoter motif, the endosperm box sequence. Proteins interacting at this motif include the gene product of the regulatory gene Opaque-2 (O2). O2 encodes a b2IP transcription factor, which is capable of activating its target promoters, 22kD α-zein and b-32, in a number of experimental systems. Storage protein accumulation, and storage protein promoter activity, are modulated according to available levels of nitrogen (N). In barley, nitrogen regulation is mediated by the endosperm box via interaction of the endosperm and GCN 4 motifs. The nitrogen- responsive components of the 22kd zein promoter have been shown to include the O2-binding sites Z1–Z3 but to display N-responsiveness even in the absence of O2 protein.