Eckhard Tacke

Influence of transposable elements on the structure and function of the A1 gene of Zea mays

The structure of the A1 gene of Zea mays was determined by sequencing cDNA and genomic clones. The gene is composed of four exons and three short introns. The 40.1‐kd A1 protein is an NADPH‐dependent reductase. Germinal derivatives of the mutable a1‐ml allele with either recessive or wild‐type phenotype have been isolated. Sequence analysis of these revertant alleles indicates that frame‐shift mutations abolish A1 gene function, whereas one additional amino acid within the protein sequence still allows wild‐type gene expression. The presence of a second, promoter‐like structure, upstream of the functional A1 gene promoter is discussed with respect to its possible involvement in differential expression of the A1 gene. The structure of the a1‐m2 8004, 3456 and 4412 alleles, featuring distinguishable phenotypes in the presence of Spm(En), was also determined. In all alleles the 1080‐bp‐long inhibitor (I) element is located 15 bp upstream of the CAAT box of the A1 gene promoter. The unusual response of al‐m2 alleles to trans‐active signals of the Spm(En) element is discussed with respect to the position of the I inserts. Also presented are data on the structure and insertion sites of transposable elements determined by cloning and sequencing of the mutable a1 alleles a1‐mpapu, a1‐mr 102 and al‐ml.

Infections with various types of organisms stimulate transcription from a short promoter fragment of the potato gst1 gene.

By histochemical GUS staining, we demonstrate that transcription from a short promoter fragment of the potato gst1 gene is locally induced after infection of a host plant with various types of pathogenic or symbiotic organisms. This regulatory unit is not active in noninfected tissues, except root apices and senescing leaves. Measuring the expression of a fusion between the promoter fragment and the gus gene in transgenic plants, therefore, allows comparison of the induction of defense reactions in different types of plant-microbe interactions, in one and the same plant.

The potato leafroll virus 17K movement protein is phosphorylated by a membrane-associated protein kinase from potato with biochemical features of protein kinase C

The 17 kDa protein (pr17), the phloem‐limited movement protein (MP) of potato leafroll luteovirus (PLRV), is associated with membranous structures and localized to plasmodesmata [Tacke et al. (1993) Virology 197, 274–282; Schmitz, J. (1995) Ph.D. Thesis, University of Cologne]. In planta the protein is predominantly present in its phosphorylated form, but it is rapidly dephosphorylated during isolation under native conditions. In an effort to examine the nature of the protein kinase(s) involved in the phosphorylation reaction, pr17 deletion mutants were expressed as fusion proteins in a bacterial expression vector system and tested for their ability to be phosphorylated by potato membrane preparations as well as by commercially available kinases. A fusion protein containing the nucleic acid‐binding, basic, C‐proximal domain (pr17C1) was identified to be phosphorylated by a Ca2+‐ and phospholipid‐dependent, membrane‐associated protein kinase. This protein kinase activity was inhibited by the addition of (19–36) protein kinase C (PKC) inhibitory peptide, known to be a highly specific inhibitor of mammalian PKC. Moreover, also the mammalian PKC from rat was able to phosphorylate pr17 in vitro. The results suggest that phosphorylation of pr17 takes place at membranous structures, possibly at the deltoid plasmodesmata connecting the sieve cell‐companion cell complex of the phloem, by the activity of PKC‐related, membrane‐associated protein kinase activity.

Ribosomal frameshifting in plants : a novel signal directs the -1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus

The 5.8 kb RNA genome of potato leafroll luteovirus (PLRV) contains two overlapping open reading frames, ORF2a and ORF2b, which are characterized by helicase and RNA polymerase motifs, respectively, and possibly represent the viral replicase. Within the overlap, ORF2b lacks an AUG translational start codon and is therefore presumably translated by −1 ribosomal frameshifting as a transframe protein with ORF2a. This hypothesis was studied by introducing the putative frameshift region into an internal position of the beta‐glucuronidase (GUS) gene and testing for the occurrence of frameshifting in vivo by transient expression of GUS activity in potato protoplasts as well as in vitro by translation in the reticulocyte system. Both experimental approaches demonstrate that a −1 frameshift occurs at a frequency of approximately 1%. Site‐directed mutagenesis identified the frameshift region and the involvement of the novel heptanucleotide motif UUUAAAU in conjunction with an adjacent stem‐loop structure. Part of this stem‐loop encodes a basic region in the ORF2b moiety of the transframe protein which was shown by binding experiments with PLRV RNA to represent a nucleic acid‐binding domain. These data support a possible biological significance of the frameshift to occur at this position of the large overlap by including the putative RNA template‐binding site of the PLRV replicase in the ORF2a/ORF2b transframe protein.

Single Nucleotide Polymorphisms in the Allene Oxide Synthase 2 Gene Are Associated With Field Resistance to Late Blight in Populations of Tetraploid Potato Cultivars

The oomycete Phytophthora infestans causes late blight, the most relevant disease of potato (Solanum tuberosum) worldwide. Field resistance to late blight is a complex trait. When potatoes are cultivated under long day conditions in temperate climates, this resistance is correlated with late plant maturity, an undesirable characteristic. Identification of natural gene variation underlying late blight resistance not compromised by late maturity will facilitate the selection of resistant cultivars and give new insight in the mechanisms controlling quantitative pathogen resistance. We tested 24 candidate loci for association with field resistance to late blight and plant maturity in a population of 184 tetraploid potato individuals. The individuals were genotyped for 230 single nucleotide polymorphisms (SNPs) and 166 microsatellite alleles. For association analysis we used a mixed model, taking into account population structure, kinship, allele substitution and interaction effects of the marker alleles at a locus with four allele doses. Nine SNPs were associated with maturity corrected resistance (P < 0.001), which collectively explained 50% of the genetic variance of this trait. A major association was found at the StAOS2 locus encoding allene oxide synthase 2, a key enzyme in the biosynthesis of jasmonates, plant hormones that function in defense signaling. This finding supports StAOS2 as being one of the factors controlling natural variation of pathogen resistance.

Single nucleotide polymorphism (SNP) genotyping as basis for developing a PCR-based marker highly diagnostic for potato varieties with high resistance to Globodera pallida pathotype Pa2/3

Globodera pallida is a parasitic root cyst nematode of potato, which causes reduction of crop yield and quality in infested fields. Field populations of G. pallida containing mixtures of pathotypes Pa2 and Pa3 (Pa2/3) are currently most relevant for potato cultivation in middle Europe. Genes for resistance to G. pallida have been introgressed into the cultivated potato gene pool from the wild, tuber bearing Solanum species S. spegazzinii and S. vernei. Selection of resistant genotypes in breeding programs is hampered by the fact that the phenotypic evaluation of resistance to G. pallida is time consuming, costly and often ambiguous. DNA-based markers diagnostic for resistance to G. pallida would facilitate the development of resistant varieties. A tetraploid F1 hybrid family SR-Gpa segregating for quantitative resistance to G.␣pallida was developed and evaluated for resistance to G. pallida population ‘Chavornay’. Two subpopulations of 30 highly resistant and 30 susceptible individuals were selected and genotyped for 96 single nucleotide polymorphism (SNP) markers tagging 12 genomic regions on 10 potato chromosomes. Seven SNPs were found significantly linked to the nematode resistance, which were all located within a resistance ‘hotspot’ on potato chromosome V. A haplotype model for these seven SNPs was deduced from the SNP patterns observed in the SR-Gpa family. A PCR assay ‘HC’ was developed, which specifically detected the SNP haplotype c that was linked with high levels of nematode resistance. The HC marker was only found in accessions of S.␣vernei. Screening with the HC marker 34 potato varieties resistant to G. pallida pathotypes Pa2 and/or Pa3 and 22 susceptible varieties demonstrated that the HC marker was highly diagnostic for presence of high levels of resistance to G. pallida pathotype Pa2/Pa3.

Plant viruses as model systems for the study of non-canonical translation mechanisms in higher plants.

During protein synthesis the translational apparatus of the cell performs three basic steps, (i) initiation by the recognition of translational start codons, (ii) elongation of the nascent polypeptide chain by the sequential decoding of triplets within an open reading frame (ORF) and (iii) termination at appropriate stop codons. This canonical sequence of events is controlled firstly by the primary structure of mRNAs and the interactions of coding triplets with the anticodons of tRNAs. In addition, for prokaryotes ample evidence has accumulated to demonstrate that ribosomes, both by themselves and through interaction with the elongation factor EF-Tu, play an important role in the initial selection and proofreading of the appropriate aminoacyl-tRNA (Maden, 1993; Powers & Noller, 1994). Lastly, translational efficiency and specificity is modulated by 5′ or 3′ untranslated mRNA regions, in that primary (enhancer sequences), secondary (stable stem-loops) and tertiary (pseudoknots) structures or small ORFs in the untranslated region influence the expression of the major ORF(s) (for a recent review on post-transcriptional regulation of plant gene expression see Gallie, 1993).

Association genetics in Solanum tuberosum provides new insights into potato tuber bruising and enzymatic tissue discoloration

BackgroundMost agronomic plant traits result from complex molecular networks involving multiple genes and from environmental factors. One such trait is the enzymatic discoloration of fruit and tuber tissues initiated by mechanical impact (bruising). Tuber susceptibility to bruising is a complex trait of the cultivated potato (Solanum tuberosum) that is crucial for crop quality. As phenotypic evaluation of bruising is cumbersome, the application of diagnostic molecular markers would empower the selection of low bruising potato varieties. The genetic factors and molecular networks underlying enzymatic tissue discoloration are sparsely known. Hitherto there is no association study dealing with tuber bruising and diagnostic markers for enzymatic discoloration are rare.ResultsThe natural genetic diversity for bruising susceptibility was evaluated in elite middle European potato germplasm in order to elucidate its molecular basis. Association genetics using a candidate gene approach identified allelic variants in genes that function in tuber bruising and enzymatic browning. Two hundred and five tetraploid potato varieties and breeding clones related by descent were evaluated for two years in six environments for tuber bruising susceptibility, specific gravity, yield, shape and plant maturity. Correlations were found between different traits. In total 362 polymorphic DNA fragments, derived from 33 candidate genes and 29 SSR loci, were scored in the population and tested for association with the traits using a mixed model approach, which takes into account population structure and kinship. Twenty one highly significant (p < 0.001) and robust marker-trait associations were identified.ConclusionsThe observed trait correlations and associated marker fragments provide new insight in the molecular basis of bruising susceptibility and its natural variation. The markers diagnostic for increased or decreased bruising susceptibility will facilitate the combination of superior alleles in breeding programs. In addition, this study presents novel candidates that might control enzymatic tissue discoloration and tuber bruising. Their validation and characterization will increase the knowledge about the underlying biological processes.

Validation of candidate gene markers for marker-assisted selection of potato cultivars with improved tuber quality

Tuber yield, starch content, starch yield and chip color are complex traits that are important for industrial uses and food processing of potato. Chip color depends on the quantity of reducing sugars glucose and fructose in the tubers, which are generated by starch degradation. Reducing sugars accumulate when tubers are stored at low temperatures. Early and efficient selection of cultivars with superior yield, starch yield and chip color is hampered by the fact that reliable phenotypic selection requires multiple year and location trials. Application of DNA-based markers early in the breeding cycle, which are diagnostic for superior alleles of genes that control natural variation of tuber quality, will reduce the number of clones to be evaluated in field trials. Association mapping using genes functional in carbohydrate metabolism as markers has discovered alleles of invertases and starch phosphorylases that are associated with tuber quality traits. Here, we report on new DNA variants at loci encoding ADP-glucose pyrophosphorylase and the invertase Pain-1, which are associated with positive or negative effect with chip color, tuber starch content and starch yield. Marker-assisted selection (MAS) and marker validation were performed in tetraploid breeding populations, using various combinations of 11 allele-specific markers associated with tuber quality traits. To facilitate MAS, user-friendly PCR assays were developed for specific candidate gene alleles. In a multi-parental population of advanced breeding clones, genotypes were selected for having different combinations of five positive and the corresponding negative marker alleles. Genotypes combining five positive marker alleles performed on average better than genotypes with four negative alleles and one positive allele. When tested individually, seven of eight markers showed an effect on at least one quality trait. The direction of effect was as expected. Combinations of two to three marker alleles were identified that significantly improved average chip quality after cold storage and tuber starch content. In F1 progeny of a single-cross combination, MAS with six markers did not give the expected result. Reasons and implications for MAS in potato are discussed.

Novel candidate genes influencing natural variation in potato tuber cold sweetening identified by comparative proteomics and association mapping

BackgroundHigher plants evolved various strategies to adapt to chilling conditions. Among other transcriptional and metabolic responses to cold temperatures plants accumulate a range of solutes including sugars. The accumulation of the reducing sugars glucose and fructose in mature potato tubers during exposure to cold temperatures is referred to as cold induced sweetening (CIS). The molecular basis of CIS in potato tubers is of interest not only in basic research on plant adaptation to environmental stress but also in applied research, since high amounts of reducing sugars affect negatively the quality of processed food products such as potato chips. CIS-tolerance varies considerably among potato cultivars. Our objective was to identify by an unbiased approach genes and cellular processes influencing natural variation of tuber sugar content before and during cold storage in potato cultivars used in breeding programs. We compared by two-dimensional polyacrylamide gel electrophoresis the tuber proteomes of cultivars highly diverse for CIS. DNA polymorphisms in genomic sequences encoding differentially expressed proteins were tested for association with tuber starch content, starch yield and processing quality.ResultsPronounced natural variation of CIS was detected in tubers of a population of 40 tetraploid potato cultivars. Significant differences in protein expression were detected between CIS-tolerant and CIS-sensitive cultivars before the onset as well as during cold storage. Identifiable differential proteins corresponded to protease inhibitors, patatins, heat shock proteins, lipoxygenase, phospholipase A1 and leucine aminopeptidase (Lap). Association mapping based on single nucleotide polymorphisms supported a role of Lap in the natural variation of the quantitative traits tuber starch and sugar content.ConclusionsThe combination of comparative proteomics and association genetics led to the discovery of novel candidate genes for influencing the natural variation of quantitative traits in potato tubers. One such gene was a leucine aminopeptidase not considered so far to play a role in starch sugar interconversion. Novel SNP’s diagnostic for increased tuber starch content, starch yield and chip quality were identified, which are useful for selecting improved potato processing cultivars.