Reinhard Töpfer

Modification of Plant Lipid Synthesis

Genetic engineering of new storage oils and fats has produced oil crop plants with fatty acid compositions unattainable by plant breeding alone. The combination of classical breeding methods with molecular techniques provides new ways for designing oils for food and nonfood uses. Alterations in the position and number of double bonds, variation in fatty acid chain length, and the introduction of desired functional groups have already been achieved in model systems. Short-term prospects include crops such as rapeseed or soybean engineered to have greater than 70 to 80 percent medium-chain fatty acids by content, greater than 90 percent oleic acid, and high erucic acid content, and engineered to form ricinoleic acid in seed storage tissues.

Uptake and transient expression of chimeric genes in seed-derived embryos.

Uptake of DNA in dry and viable embryos of wheat by imbibition in DNA solution was detected by monitoring the transient expression of chimeric genes. Gene expression vectors used in this study contained a neomycin phosphotransferase (NPT) II reporter gene fused to various promoters. Some of the chimeric neo genes were shown to yield reproducibly NPT II activity in germinating embryos. This NPT II activity was increased markedly when the neo genes were carried by a vector capable of autonomous replication. Dimers of wheat dwarf virus, a monopartite gemini virus, were thus shown to be effective in amplifying the transient expressed NPT II activity in embryos of several cereals. These and other observations indicate that the observed transient expression really results from DNA uptake and expression in plant embryo cells and is not due to contaminating microorganisms.

Transient gene expression in tobacco protoplasts: II. Comparison of the reporter gene systems for CAT, NPT II, and GUS

The reporter genes for Chloramphenicolacetyltransferase (CAT), Neomycinphosphotransferase-(NPT)-II and β-Glucuronidase (GUS) were compared in transient gene expression experiments in tobacco mesophyll protoplasts. For this purpose, nearly identical chimeric genes controlled by the CaMV 35 S promoter were constructed. The detection level of each system was determined yielding the following order of relative sensitivity: CAT

Transient gene expression in tobacco protoplasts. I: Time course of CAT appearance

The early events of transient gene expression have been investigated monitoring CAT activity in tobacco protoplasts encoded by the recombinant plasmid pRT101cat. The first appearance of CAT activity was observed within 30 minutes after the outset of cultivation, and maximal values were obtained between four and 24 hours. CAT expression, at the level of RNA synthesis, could not be inhibited by cordycepin (3′deoxyadenine) added one hour after protoplast plating, whereas cycloheximide, an inhibitor of protein synthesis, showed an influence during the first four hours. This indicates a rapid decay of biologically active forms of both the DNA transferred and the CAT-mRNA synthesized within the first hours. These results suggest that in the tobacco protoplast system CAT protein stability lasts up to two weeks rather than a continuous synthesis of new enzyme.

[6] Expression vectors for high-level gene expression in dicotyledonous and monocotyledonous plants

Publisher Summary This chapter describes the sets of vectors that are derivatives of the expression vector cassette pRT100, which uses the CaMV 35S RNA promoter in combination with various reporter and selectable marker genes. It introduces a set of expression vectors for enhanced gene expression in monocotyledonous plants, especially agronomically important cereals. These vectors have been constructed as basic tools applicable for transient gene expression, as well as for stable integration of foreign genes into plant genomes. Improvement of the cassette pRT101 cat to produce pRT-ex/s-int/s-cat gives a construct, leading to a high level of gene expression in monocotyledonous plants and in particular in agronomically important cereals. The construct pRT-ex/s-int/s-cat has been constructed to facilitate the replacement of the CAT-coding region, such as a Bam HI or Xba I fragment, or an exchange of the promoter region, such as a Hinc II /Xho I or Hinc II /Kpn I fragment. The entire chimeric gene can be transferred to other vectors using Pst I or Sph I. Moreover, the construct pRT-ex/s-int/scat might be helpful for the analysis of plant promoters or for the achievement of high levels of gene product in transgenic plants.

Efficient regeneration of Brassica oleracea hypocotyl protoplasts and high frequency genetic transformation by direct DNA uptake.

SummaryEfficient regeneration (80%) and high frequency genetic transformation (10–33%) were achieved by culturing protoplasts isolated from hypocotyl tissues of six day old Brassica oleracea seedlings and by subjecting these protoplasts to PEG mediated direct plasmid uptake. Three different plasmid vectors carrying marker genes for resistance to methotrexate (dhfr), hygromycin (hpt) and phosphinotricin (bar) were constructed and used for transformation. Large number of normal, fertile transformants were obtained with vectors carrying hpt and bar genes. No transformants could be regenerated for resistance to methotrexate as it severely suppressed shoot differentiation.

Three different cDNAs encoding acyl carrier proteins from Cuphea lanceolata.

Mature seeds of Cuphea lanceolata contain 83% capric acid in storage triacylglycerols (Graham, 1989). However, the regulation of the biosynthesis of medium-chain fatty acids in the genus Cuphea is not known. ACP plays an essential role as a cofactor in the plastid-located machinery of plant fatty acid biosynthesis. The growing acyl chain is covalently bound via a thioester to the ACP‘s 4’-phosphopantetheine group. In recent years a number of ACP genes and cDNAs from different plants have been isolated (for review, see Topfer and Martini, 1994). ACP isoforms with different pattems of tissue-specific expression have been characterized from Spinacia oleracea (Ohlrogge and Kuo, 1985), Hordeum vulgare (Hansen, 1987), and Arubidopsis thaliana (HlouBek-Radojsk et al., 1992). Recently, protein sequences for two ACP isoforms of C. lanceolata have been reported (Kopka et al., 1993). We performed reverse transcriptase-PCR on C. lanceolata poly(A)+ RNA using degenerate oligonucleotides as primer pairs designed from these sequences. The resulting amplification product was used to screen a C. lanceolata embryo-specific XZAP-cDNA library. Here we present three complete cDNA clones encoding ACP (Table I). The probable full-size cDNAs cover, respectively, 810 bp (Ac1l;Cl-lu), 733 bp (Ac1l;Cl-lb), and 688 bp (Acl2;Cl-lc). The clones are very similar to each other; a comparison of the deduced amino acid sequences of the mature proteins revealed a homology of greater than 96%. The mature proteins corresponding to Acl2;CI-Za and AcZl;Cl-Zb are composed of 84 amino acids, whereas Acl2;Cl-lc is 83 amino acids long. In addition, each protein contains a Ser-rich transit peptide of 56 (Ac1l;Cl-lu), 53 (Acl2;CZ-lb), or 60 (AcZl;Cl-lc) amino acids, respectively. We predict that a11 clones contain between 30 and 49 nucleotides of 5’ untranslated DNA, with the first ATG most likely representing the translational start site in a11 clones described. The predicted amino acid sequences based on the cDNAs a11 show strong homology to the protein sequence ACPl of Kopka et al. (1993); however, none is identical. The predicted stop codon is followed by 3’ untranslated sequences of 319, 249, and 212 nucleotides,

A gene encoding acetyl-coenzyme A carboxylase from Brassica napus.

ACCase (EC 6.4.1.2) is one of the key regulatory enzymes in fatty acid biosynthesis catalyzing the formation of malonyl-COA from acetyl-COA and bicarbonate in an ATPdependent reaction providing the substrate for fatty acid synthesis. ACCase from plants is proposed to be a dimer consisting of identical subunits of larger than 200 kD (Egli et al., 1993; Gomicki and Haselkom, 1993). To date the sequence of a plant ACCase gene has not been reported. Here we describe the sequence of an ACCase gene from rapeseed (Brassica napus) (Table I). Based on regions conserved between ACCase from chicken and Escherichia coli BC (Kondo et al., 1991), a specific hybridization probe was generated by PCR from cDNAs synthesized from poly(A+) RNA of immature seeds of B. napus. Degenerate oligonucleotide primers for PCR were deduced from amino acids 305 to 312 and 384 to 391 of chicken ACCase and resulted in amplification of a 260-bp fragment covering the exon sequences in the region of nucleotides 4300 to 4813 of the rapeseed sequence reported here. This fragment encodes 84 amino acids showing 88.4 and 65.1% similarity to ACCase of chicken and BC from E. coli, respectively, and was used as a probe for the isolation of clones from a rapeseed genomic library constructed in A-FIX I1 (Stratagene, La Jolla, CA). Fragments of two overlapping genomic clones were subcloned and sequenced. A comparison with protein sequences of the ACCase from chicken, yeast, Cyclotella cypt ica , and E. coli allowed us to postulate the presence and location of 31 introns. Potential promoter elements were localized at position 2283 to 2286 (CAAT box) and 2416 to 2419 (TATA box). A putative ATG start codon is located at position 2506 to 2508. The Lys residue in the common motif for biotinyla-

Isolation and characterization of a cDNA fromCuphea lanceolata encoding a β-ketoacyl-ACP reductase

SummaryA cDNA encoding β-ketoacyl-ACP reductase (EC 1.1.1.100), an integral part of the fatty acid synthase type II, was cloned fromCuphea lanceolata. This cDNA of 1276 by codes for a polypeptide of 320 amino acids with 63 N-terminal residues presumably representing a transit peptide and 257 residues corresponding to the mature protein of 27 kDa. The encoded protein shows strong homology with the amino-terminal sequence and two tryptic peptides from avocado mesocarp β-ketoacyl-ACP reductase, and its total amino acid composition is highly similar to those of the β-ketoacyl-ACP reductases of avocado and spinach. Amino acid sequence homologies to polyketide synthase, β-ketoreductases and short-chain alcohol dehydrogenases are discussed. An engineered fusion protein lacking most of the transit peptide, which was produced inEscherichia coli, was isolated and proved to possess β-ketoacyl-ACP reductase activity. Hybridization studies revealed that inC. lanceolata β-ketoacyl-ACP reductase is encoded by a small family of at least two genes and that members of this family are expressed in roots, leaves, flowers and seeds.

Plant regeneration from cultured fertilized barley ovules

Abstract A method for the culture of barley ( Hordeum vulgare ) ovules isolated directly after fertilization is described. Up to 28% of these ovules contained a poorly developed embryo after 10–12 days of culture. After dissection these embryos were cultured and about 20% of them yielded mature plants. Our observations indicate that plant regeneration from cultured ovules, isolated 1–2 h after pollination, is possible. Similar methods also allow culture and regeneration of wheat plants from ovules isolated after 2 days post anthesis.