Identification and reproducibility of diagnostic DNA markers for tuber starch and yield optimization in a novel association mapping population of potato (Solanum tuberosum L.).

KEY MESSAGE: SNPs in candidate genes Pain - 1, InvCD141 (invertases), SSIV (starch synthase), StCDF1 (transcription factor), LapN (leucine aminopeptidase), and cytoplasm type are associated with potato tuber yield, starch content and/or starch yield. Tuber yield (TY), starch content (TSC), and starch yield (TSY) are complex characters of high importance for the potato crop in general and for industrial starch production in particular. DNA markers associated with superior alleles of genes that control the natural variation of TY, TSC, and TSY could increase precision and speed of breeding new cultivars optimized for potato starch production. Diagnostic DNA markers are identified by association mapping in populations of tetraploid potato varieties and advanced breeding clones. A novel association mapping population of 282 genotypes including varieties, breeding clones and Andean landraces was assembled and field evaluated in Northern Spain for TY, TSC, TSY, tuber number (TN) and tuber weight (TW). The landraces had lower mean values of TY, TW, TN, and TSY. The population was genotyped for 183 microsatellite alleles, 221 single nucleotide polymorphisms (SNPs) in fourteen candidate genes and eight known diagnostic markers for TSC and TSY. Association test statistics including kinship and population structure reproduced five known marker-trait associations of candidate genes and discovered new ones, particularly for tuber yield and starch yield. The inclusion of landraces increased the number of detected marker-trait associations. Integration of the present association mapping results with previous QTL linkage mapping studies for TY, TSC, TSY, TW, TN, and tuberization revealed some hot spots of QTL for these traits in the potato genome. The genomic positions of markers linked or associated with QTL for complex tuber traits suggest high multiplicity and genome wide distribution of the underlying genes.

Evidence for expression level-dependent modulation of carbohydrate status and viral resistance by the potato leafroll virus movement protein in transgenic tobacco plants.

High-level constitutive expression of the cell-to-cell movement protein from the phloem-restricted potato leafroll virus (PLRV-MP17) in transgenic tobacco plants leads to growth retardation and severe phenotypic changes of source leaves paralleled by a drastic accumulation of soluble sugars and starch (Herbers et al., 1997). To investigate whether the MP17-induced alteration in carbon metabolism is related to the targeting and modification of specific plasmodesmata (Pd) or is rather due to pleiotropic effects caused by high MP17 protein amounts, non-phenotypic tobacco plants expressing a MP17:GFP fusion protein were obtained and compared with previously described MP17 transgenic lines. Confocal laser scanning microscopy and immunogold labelling studies revealed an overall affinity of MP17 to Pd in vascular and non-vascular tissue of source leaves, whereas in sink leaves GFP fluorescence was restricted to Pd of trichomes. In source leaves, plasmodesmal size exclusion limits of mesophyll cells were likewise increased by MP17 and MP17:GFP independent from steady-state levels of the protein amount and phenotypic alteration. Conversely, carbohydrate contents in source leaves strictly correlated with quantified MP17 protein levels. Low expression of MP17 and MP17:GFP decreased soluble sugars and starch contents in leaves possibly due to changes in plasmodesmal permeability while increasing MP17 protein levels led to carbohydrate accumulation and a stunted growth. Infection of transgenic lines with the unrelated potato virus Y (PVY)N revealed an expression level-dependent mode of MP17-mediated resistance. Phenotypic changes and carbohydrate-mediated defence responses as indicated by elevated levels of PR-protein transcripts were crucial for increased viral resistance, whereas plasmodesmal targeting and modification by MP17 per se had either no effect or even increased susceptibility to PVY. Thus, our results implicate that the absolute level of expression needs to be critically considered when elucidating the effect of MPs on carbon metabolism, biomass allocation and virus resistance.

In situ localization of the putative movement protein (pr17) from potato leafroll luteovirus (PLRV) in infected and transgenic potato plants.

The potato leafroll virus (PLRV) 17-kDa protein (pr17), the putative movement protein for this phloem-limited luteovirus, was localized on ultrathin sections of leaves from PLRV-infected and transgenic potato plants. The transgenic plants expressed the entire viral genome from a full-length cDNA copy (PLRVfl) or only the gene encoding pr17 (ORF4) under the control of the cauliflower mosaic virus 35S promoter. Virus-infected and PLRVfl-transgenic plants developed symptoms typical of virus infection, whereas pr17-transgenic plants did not display symptoms or ultrastructural alterations. Immunogold electron microscopy using an anti-pr17-serum detected pr17 in plasmodesmata, in virus-induced vesicles, in mitochondria, and in chloroplasts of phloem cells, in PLRV-infected as well as PLRVfl-transgenic plants. In addition, in transgenic plants, pr17 was expressed in mesophyll cells (which are not infected by PLRV under natural conditions) and localized to the same sites as in phloem cells, except in plasmodesmata. In contrast, in pr17-transgenic plants the protein was never observed on organelles, but was almost exclusively associated with plasmodesmata of all leaf cell types, indicating that the targeting of pr17 to plasmodesmata is an intrinsic property of the protein. These results support the role of pr17 in PLRV movement.

In vivo expression of a full-length cDNA copy of potato leafroll virus (PLRV) in protoplasts and transgenic plants.

A full-length cDNA copy (PLRVfl) of potato leafroll virus (PLRV) was constructed and examined in vivo for its biological activities by transient expression experiments with plasmid DNA or in vitro transcribed RNA. In addition, PLRVfl cDNA was stably introduced into the genome of potato plants by Agrobacterium-mediated leaf disc transformation. Both transient and stable expression of PLRVfl resulted in the synthesis of genomic and subgenomic PLRV RNAs. Transgenic plants accumulated the 17-kDa movement protein and displayed the typical symptoms of PLRV infection. This is the first example of the constitutive expression of a phloem-limited virus in planta.

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.

Expression of a luteoviral movement protein in transgenic plants leads to carbohydrate accumulation and reduced photosynthetic capacity in source leaves.

Elucidating the role of viral genes in transgenic plants revealed that the movement protein (MP) from tobacco mosaic virus is responsible for altered carbohydrate allocation in tobacco and potato plants. To study whether this is a general feature of viral MPs, the movement protein MP17 of potato leafroll virus (PLRV), a phloem-restricted luteovirus, was constitutively expressed in tobacco plants. Transgenic lines were strongly reduced in height and developed bleached and sometimes even necrotic areas on their source leaves. Levels of soluble sugars and starch were significantly increased in source leaves. Yet, in leaf laminae the hexose-phosphate content was unaltered and ATP reduced to only a small extent, indicating that these leaves were able to maintain homeostatic conditions by compartmentalization of soluble sugars, probably in the vacuole. On the contrary, midribs contained lower levels of soluble sugars, ATP, hexose-phosphates and UDP-glucose supporting the concept of limited uptake and catabolism of sucrose in the phloem. The accumulation of carbohydrates led to a decreased photosynthetic capacity and carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) probably owing to decreased expression of photosynthetic proteins. In parallel, levels of pathogenesis-related proteins were elevated which may be the reason for the obtained limited resistance against the unrelated potato virus Y (PVY)N in the transgenic tobacco plants. Ultrathin sections of affected leaves harvested from 2-week-old plants revealed plasmodesmal alterations in the phloem tissue while plasmodesmata between mesophyll cells were indistinguishable from wild-type. These data favour the phloem tissue to be the primary site of PLRV MP17 action in altering carbohydrate metabolism.

Genetic engineering of potato for broad-spectrum protection against virus infection.

Transgenic potato plants expressing mutant alleles of PLRV ORF4, the gene for the movement protein pr17 of this luteovirus, were generated for broad-range protection against virus infection. When tested for protection against infection by PLRV, all transgenic lines showed a significant reduction of virus antigen. Potato lines accumulating N- or C-terminally extended PLRV pr17 mutant proteins were resistant to infection by the unrelated potato viruses PVY and PVX. Transgenic lines that did not express protein despite high transcript levels failed to exhibit virus resistance.

Non-canonical translation mechanisms in plants: efficient in vitro and in planta initiation at AUU codons of the tobacco mosaic virus enhancer sequence.

The 5' untranslated leader (Omega sequence) of tobacco mosaic virus (TMV) genomic RNA was utilized as a translational enhancer sequence in expression of the 17 kDa putative movement protein (pr17) of potato leaf roll luteovirus (PLRV). In vitro translation of RNAs transcribed from appropriate chimeric constructs, as well as their expression in transgenic potato plants, resulted in the expected wild-type pr17 protein, as well as in larger translational products recognized by pr17-specific antisera. Mutational analyses revealed that the extra proteins were translated by non-canonical initiation at AUU codons present in the wild-type Omega sequence. In the plant system translation initiated predominantly at the AUU codon at positions 63-65 of the Omega sequence. Additional AUU codons in a different reading frame of the Omega sequence also showed the capacity for efficient translation initiation in vitro. These results extend the previously noted activity of the TMV 5' leader sequence in ribosome binding and translation enhancement in that the TMV translation enhancer can mediate non-canonical translation initiation in vitro and in vivo.

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.

Transposon tagging of the maize Glossy2 locus with the transposable element En/Spm.

The Glossy2 (Gl2) locus of maize is required for the formation of the epicuticular wax layer of young plants. gl2 mutant seedlings can be visually identified because of their glossy leaf surface which is different from the dull surface of wild-type seedlings. The Gl2 locus was isolated by transposon tagging. Seven unstable mutations, gl2-m2 to gl2-m8, were induced in a parental strain carrying an active transposable Activator (Ac) element in the unstable wx-m7 allele. Genetic tests on the gl2-m2 allele indicated that it was not caused by the Ac element but by the insertion of the transposable element Enhancer/Suppressor-Mutator (En/Spm). A Sa/l restriction fragment segregating with the mutant phenotype was identified, by Southern analysis, using sequences from the En/Spm element as a probe. Part of the fragment was cloned and was shown to carry part of the unstable gl2-m2 allele. These gl2 sequences were used to identify a genomic fragment carrying the wild-type allele and to isolate its corresponding cDNA sequence. The predicted Glossy2 protein consists of 426 amino acids. No similar amino acid sequence was found in protein data banks and the biochemical function of the Gl2 gene product is still unknown. The wild-type Gl2 transcript is found predominantly in juvenile leaves. The transcript level in the leaves of seedlings homozygous for a stable recessive gl2-ref allele is hardly detectable.

Mutational analysis of the nucleic acid-binding 17 kDa phosphoprotein of potato leafroll luteovirus identifies an amphipathic alpha-helix as the domain for protein/protein interactions.

The 17 kDa protein (pr17) of potato leafroll luteovirus is translated from a subgenomic PLRV RNA by internal translation initiation and binds to single-stranded nucleic acids (E. Tacke, D. Prüfer, J. Schmitz, and E. Rohde, 1991, J. Gen. Virol. 72, 2035-2038). Chemical crosslinking of in vitro expressed pr17 provided evidence for the preferential formation of pr17 homodimers which were also detected in PLRV-infected potato plants and isolated from potato lines expressing the PLRV pr17 transgene. Mutation analysis identified an amphipathic alpha-helix within the acidic amino-terminus of pr17 which acts as the domain for protein/protein interactions. Pr17 was predominantly associated with subcellular fractions enriched for nuclei, chloroplasts, mitochondria, and membranous structures. In addition it was shown that pr17 was phosphorylated in planta and that this modification did not inhibit binding of the protein to nucleic acids.

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.

The potato leafroll luteovirus 17K protein is a single-stranded nucleic acid-binding protein.

The potato leafroll luteovirus protein of Mr 17K (pr17), which is encoded by an open reading frame on the 3' half of the viral genome, was expressed by using bacterial expression vector systems. Fusion proteins were obtained for the full-length viral protein as well as its N-terminal acidic (GST/pr17N) and C-proximal (GST/pr17C) basic domains and used in nucleic acid-binding studies. Filter-bound as well as soluble pr17 bound to single-stranded RNA or DNA. The binding domain was shown to reside in the basic C-proximal part of the polypeptide, whereas the N-terminal acidic domain did not show any affinity for nucleic acid. These biochemical properties of pr17 together with its structural features suggest a regulatory role for this protein during virus replication.

Characterization of a potato leafroll luteovirus subgenomic RNA: differential expression by internal translation initiation and UAG suppression.

Northern blot analysis of Solanum tuberosum infected with potato leafroll luteovirus revealed the 6 kb genomic RNA and a major 2.3 kb subgenomic RNA. The 5' end of the subgenomic RNA was located at nucleotide 3653 in an intergenic region located at the centre of the viral genome upstream of three open reading frames (ORFs). Transient expression in tobacco and potato protoplasts of the beta-glucuronidase reporter gene fused to various putative regulatory sequences present in the subgenomic RNA was used to study their influence on expression levels. We observed a suppression of the amber stop codon separating the coat protein (CP) gene from a downstream ORF (56K protein), to a level of 0.9% to 1.3%. Translation initiation at the AUG of an ORF (17K protein) which is nested within the CP gene, exceeds translation of the CP gene itself by a factor of 7.

Cloning of the gene for the capsid protein of potato leafroll virus.

DNA complementary to the RNA of purified potato leafroll virus (PLRV) was synthesized and cloned into the lambda insertion vector NM1149. Overlapping PLRV-specific cDNA clones were isolated that represent some 80% of the viral genome. Sequences coding for the capsid protein were identified by subcloning size-selected cDNAs into the lambda expression vector gt11 and screening with PLRV-specific antisera. The gene for the viral capsid protein was shown to reside in the 3' terminal half of the genomic RNA. Sequence comparisons with the recently published genomes of the beet western yellows virus (BWYV) and the barely yellow dwarf virus (BYDV) reveal some 65% protein sequence homology between the capsid proteins of BWYV and PLRV and some 45% homology between BYDV and PLRV. Furthermore, it is evident that the structural organization of the PLRV genome in the CP gene region is similar to that of BWYV and BYDV.

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-m1 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 a1-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 a1-ml.

Cardiac hypertrophy secondary to ACTH treatment in children.

The usefulness of ACTH in the treatment of childhood epilepsy is assessed by improvement in the EEG and in the clinical condition. However, pronounced side effects, even serious ones, must be encountered. The most common complications are Cushing syndrome, infections, and arterial hypertension. We report on seven patients with infantile myoclonic seizures, who exhibited myocardial hypertrophy with increased left ventricular function during ACTH treatment. These changes were detected and followed by serial echocardiographic investigations. Within a period of 5 months after the termination of ACTH therapy the abnormal echocardiographic findings disappeared. We believe that the cardiac hypertrophy is ACTH-induced. Based on the various biological effects of ACTH different explanations are proposed: oedema or deposition of glycogen in the myocardial tissue, hyperinsulinism, arterial hypertension and increased inotropic stimulus. Because of our observations, we suggest careful monitoring of children treated with ACTH by performing serial echocardiographic investigations.

Left ventricular thrombi in three children with dilated cardiomyopathy: diagnostic procedure and clinical course.

In three of nine children with dilated cardiomyopathy (aged 1-9 years), left ventricular thrombi were diagnosed and followed by echocardiography. Thrombi recurred in two patients, in one of them, embolized to the cerebral arteries. Resolution of the thrombi was observed under therapy directed against platelet aggregation. However, this did not prevent thrombus formation.

[Hypertrophic cardiomyopathy during ACTH treatment]. / Hypertrophe kardiomyopathie unter ACTH-Behandlung.

We report on two infants (two and six month old) with infantile myoclonic seizures, who developed signs of hypertrophic cardiomyopathy (HCM), while receiving ACTH treatment (Tetracosactid=Synacthen Depot). The diagnosis of HCM was established by echocardiography. The first patient (R.M. female). showed signs of cardiac insufficiency and was treated with a beta-blocking agent (Propranolol=Dociton). This led to resolution of the clinical symptoms and reversal of myocardial muscle thickness as determined by echocardiography. The second patient (R.S. female) did not develop cardiac symptoms. A correlation between cardiomyopathy and ACTH treatment is discussed. Based on the various biological effects of ACTH different hypothetical explanations for this correlation are proposed: increased deposition of glycogen, enhanced protein synthesis, oedema of the myocardial tissue and systemic hypertension. Because of the correlation observed between ACTH treatment and the development of cardiomyopathy we recommend regular physical and echocardiographic examinations to detect cardiac involvement during treatment with ACTH.