Fritz Kreuzaler

Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana

We introduced the Escherichia coli glycolate catabolic pathway into Arabidopsis thaliana chloroplasts to reduce the loss of fixed carbon and nitrogen that occurs in C3 plants when phosphoglycolate, an inevitable by-product of photosynthesis, is recycled by photorespiration. Using step-wise nuclear transformation with five chloroplast-targeted bacterial genes encoding glycolate dehydrogenase, glyoxylate carboligase and tartronic semialdehyde reductase, we generated plants in which chloroplastic glycolate is converted directly to glycerate. This reduces, but does not eliminate, flux of photorespiratory metabolites through peroxisomes and mitochondria. Transgenic plants grew faster, produced more shoot and root biomass, and contained more soluble sugars, reflecting reduced photorespiration and enhanced photosynthesis that correlated with an increased chloroplastic CO2 concentration in the vicinity of ribulose-1,5-bisphosphate carboxylase/oxygenase. These effects are evident after overexpression of the three subunits of glycolate dehydrogenase, but enhanced by introducing the complete bacterial glycolate catabolic pathway. Diverting chloroplastic glycolate from photorespiration may improve the productivity of crops with C3 photosynthesis.

Identification of Genes Required for the Function of Non-Race-Specific mlo Resistance to Powdery Mildew in Barley.

Recessive alleles (mlo) of the Mlo locus in barley mediate a broad, non-race-specific resistance reaction to the powdery mildew fungus Erysiphe graminis f sp hordei. A mutational approach was used to identify genes that are required for the function of mlo. Six susceptible M2 individuals were isolated after inoculation with the fungal isolate K1 from chemically mutagenized seed carrying the mlo-5 allele. Susceptibility in each of these individuals is due to monogenic, recessively inherited mutations in loci unlinked to mlo. The mutants identify two unlinked complementation groups, designated Ror1 and Ror2 (required for mlo-specified resistance). Both Ror genes are required for the function of different tested mlo alleles and for mlo function after challenge with different isolates of E. g. f sp hordei. A quantitative cytological time course analysis revealed that the host cell penetration efficiency in the mutants is intermediate compared with mlo-resistant and Mlo-susceptible genotypes. Ror1 and Ror2 mutants could be differentiated from each other by the same criterion. The spontaneous formation of cell wall appositions in mlo plants, a subcellular structure believed to represent part of the mlo defense, is suppressed in mlo/ror genotypes. In contrast, accumulation of major structural components in the appositions is seemingly unaltered. We conclude that there is a regulatory function for the Ror genes in mlo-specified resistance and propose a model in which the Mlo wild-type allele functions as a negative regulator and the Ror genes act as positive regulators of a non-race-specific resistance response.

Reduced virus infectivity inN. tabacum secreting a TMV-specific full-size antibody

We developed a new concept of controlling plant virus infection based on the expression and secretion of full-size antibodies in plants. The neotope-specific anti-Tobacco Mosaic Virus (anti-TMV) antibody mAb24 has a high affinity towards epitopes present only on the surface of intact tobacco mosaic virions. The infectivity of the virus is inhibited almost completely if TMV is adsorbedin vitro at ratios as low as 300 antibody molecules per virion prior to inoculation. Cloned full-size cDNAs of mAb24 heavy and light chains were integrated into the plant expression vector pSS in tandem array and used for transformation ofNicotiana tabacum. The resulting transgenic tobacco plants expressed heavy- and light-chains of mAb24 which were assembled into functional antibodies and exported to the intercellular space. TMV specificity and affinity of the plant-produced antibody (mAb24-P) was not altered when compared to the original murine mAb24. F1 progenies segregated 3:1 with respect to antibody secretion and showed up to two-fold higher expression levels compared to the F0 plants. Upon infection with TMV F1 plants producing mAb24-P showed a reduction of necrotic lesion numbers which is correlated with the amount of antibody produced in transgenic plants.

Fusion proteins comprising a Fusarium -specific antibody linked to antifungal peptides protect plants against a fungal pathogen

In planta expression of recombinant antibodies recognizing pathogen-specific antigens has been proposed as a strategy for crop protection. We report the expression of fusion proteins comprising a Fusarium-specific recombinant antibody linked to one of three antifungal peptides (AFPs) as a method for protecting plants against fungal diseases. A chicken-derived single-chain antibody specific to antigens displayed on the Fusarium cell surface was isolated from a pooled immunocompetent phage display library. This recombinant antibody inhibited fungal growth in vitro when fused to any of the three AFPs. Expression of the fusion proteins in transgenic Arabidopsis thaliana plants conferred high levels of protection against Fusarium oxysporum f.sp. matthiolae, whereas plants expressing either the fungus-specific antibody or AFPs alone exhibited only moderate resistance. Our results demonstrate that antibody fusion proteins may be used as effective and versatile tools for the protection of crop plants against fungal infection.

Cloning and nucleotide sequence of the Escherichia coli K-12 ppsA gene, encoding PEP synthase.

SummaryWe have cloned and sequenced the Escherichia coli K-12 ppsA gene. The ppsA gene codes for PEP synthase, which converts pyruvate into phosphoenolpyruvate (PEP), an essential step in gluconeogenesis when pyruvate or lactate are used as a carbon source. The open reading frame consists of 792 amino acids and shows homology with other phosphohistidine-containing enzymes that catalyze the conversion between pyruvate and PEP. These enzymes include pyruvate, orthophosphate dikinases from plants and Bacteroides symbiosus and Enzyme I of the bacterial PEP:carbohydrate phosphotransferase system.

Solanum tuberosum double transgenic expressing phosphoenolpyruvate carboxylase and NADP-malic enzyme display reduced electron requirement for CO2 fixation

Abstract The cDNA of the NADP-dependent malic enzyme gene Me 2 from the C 3 plant Flaveria pringlei was used for expression in Escherichia coli and Solanum tuberosum . A chimeric GST-Me 2 gene complemented a malic enzyme deficient E. coli mutant. Two potato lines were transformed with Me 2-cDNA constructs, one line already overexpressing the phospho enol pyruvate carboxylase gene ( ppc ) from Corynebacterium glutamicum . Both genes were under the control of the constitutive 35S CaMV promoter. Increased levels of malic enzyme (ME) were found in chloroplasts of transformants. Western blot analysis indicated that the ME transit sequence was cleaved. Expression of both genes led to a significantly reduced electron requirement for apparent CO 2 assimilation (e/A) at higher temperature. At low temperatures (15°C) 11 electrons per CO 2 assimilated (e/A) were measured in controls, single transformants ( ppc or Me 2) and double transformants ( ppc and Me 2). However, when leaf temperature was raised to 36°C electron requirement of the double transformants (15 e/A) was 65% of controls or single transformants (23 e/A). Thus, the temperature dependent increase in electron requirement was reduced in the double transformants suggesting a suppression in the oxygenation reaction of Rubisco and with it presumably in the rate of photorespiratory CO 2 release which is more marked at high light and high temperatures.

Characterization of a wheat class Ib chitinase gene differentially induced in isogenic lines by infection with Puccinia graminis

Abstract Plant chitinases have been recognized to be involved in defense against pathogens. A class Ib genomic chitinase gene was isolated from a wheat (Triticum aestivum L.) genomic library using a wheat chitinase sequence amplified from DNA by PCR. The nucleotide sequence analysis showed that the wheat chitinase gene (Wchl) contains a 960-bp long coding region with no intron. The derived polypeptide includes a signal peptide, a cysteine-rich domain and a catalytic domain without a carboxy terminal extension. Primer extension experiments identified a single transcription start site 33 bp upstream of the translation initiation site. Southern blot analysis indicated that Wchl is one member of a small gene family in wheat. Northern blot hybridization and histological investigation of wheat leaves infected by Puccinia graminis f. sp. tritici revealed a close correlation between the accumulation of the chitinase transcripts and the inhibition of fungal growth when two isogenic wheat lines (Prelude Sr6 and Prelude sr6) differing at the Sr6 locus for race-specific incompatibility with the fungus were studied. The massive accumulation of the chitinase mRNA occurred only in the incompatible Sr6/P6 interaction, in parallel with a severe inhibition of mycelial growth in the leaves. In contrast, in the compatible sr6/P6 interaction the fungus grew rapidly throughout the leaf tissue and the chitinase transcripts were not detected. The chitinase mRNA was detectable in non-infected stems but not in non-infected leaves or roots.

Quantification of photosynthetic gene expression in maize C3 and C4 tissues by real-time PCR

Carbon assimilation in maize follows the C4 mechanism. This requires the tissue-specific and light-induced expression of a set of different genes involved in CO2 fixation as well as adaptations in the leaf anatomy including a reduced distance between vascular bundles compared to C3 plants. However, several maize tissues exist with larger bundle distances and there is significant evidence that CO2 fixation follows the C3 mechanism in these tissues. We isolated maize C3 and C4 tissues and quantified the accumulation of mRNAs encoding PEPC, ME, the small subunit of Rubisco, and PPDK. For this, primer systems for the specific and sensitive detection by real-time PCR were established. The observed patterns show the expected distribution for foliar leaf tissues. Also in total husk leaves, all transcripts under investigation were detected, albeit at a lower level. When mesophyll cells which are located distant from bundles were isolated from husk leaves, only accumulation of RbcS was observed. Comparing the expression of two genes encoding for isoenzymes of the small subunit of RbcS in the different tissues differential patterns of relative transcript abundance were observed. Transcripts for the DOF1 transcription factor involved in the activation of photosynthetic genes in maize were found in leaf tissues performing both C4 and C3 photosynthesis with highest accumulation levels in C4 mesophyll cells, whereas the homologous DOF2 gene was not expressed in any of the investigated samples. The results provide novel insights into the regulation of C3 and C4 carbon fixation pathways in maize.

Cloning, sequence analysis and expression of a cDNA encoding active phosphoenolpyruvate carboxylase of the C3 plant Solanum tuberosum

A cDNA coding for phosphoenolpyruvate carboxylase (PEPC) was isolated from a cDNA library from Solanum tuberosum and the sequence of the cDNA was determined. It was inserted into a bacterial expression vector and a PEPC-Escherichia coli mutant could be complemented by the cDNA construct. A functional fusion protein could be synthesized in E. coli. The properties of this PEPC protein clearly resembled those of typical C3 plant enzymes.

Metabolic engineering for p-coumaryl alcohol production in Escherichia coli by introducing an artificial phenylpropanoid pathway.

The plant polymer lignin is the greatest source of aromatic chemical structures on earth. Hence, the chemically diverse lignin monomers are valuable raw materials for fine chemicals, materials synthesis, and food and flavor industries. However, extensive use of this natural resource is hampered by the large number of different lignin monomers and the complex and irregular structure of lignin, which renders current processes for its chemical or enzymatic degradation inefficient. The microbial production of lignin monomers from renewable resources represents a promising alternative to lignin degradation, which could meet the demand for aromatic chemical structures. In this study, we describe the functional introduction of an artificial phenylpropanoid pathway into Escherichia coli, achieved by transferring several genes from plants and microbes. The established chimeric pathway efficiently converts l‐tyrosine into the lignin precursor molecule p‐coumaryl alcohol.