Will J. Feenstra

Inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs

SummaryGranule-bound starch synthase [GBSS; EC 24.1.21] determines the presence of amylose in reserve starches. Potato plants were transformed to produce antisense RNA from a gene construct containing a full-length granule-bound starch synthase cDNA in reverse orientation, fused between the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator. The construct was integrated into the potato genome by Agrobacterium rhizogenes-mediated transformation. Inhibition of GBSS activity in potato tuber starch was found to vary from 70% to 100%. In those cases where total suppression of GBSS activity was found both GBSS protein and amylose were absent, giving rise to tubers containing amylose-free starch. The variable response of the transformed plants indicates that position effects on the integrated sequences might be important. The results clearly demonstrate that in tubers of potato plants which constitutively synthesize antisense RNA the starch composition is altered.

Sequence of the structural gene for granule-bound starch synthase of potato (Solanum tuberosum L.) and evidence for a single point deletion in the amf allele.

SummaryThe genomic sequence of the potato gene for starch granule-bound starch synthase (GBSS; “waxy protein”) has been determined for the wild-type allele of a monoploid genotype from which an amylose-free (amf) mutant was derived, and for the mutant part of the amf allele. Comparison of the wild-type sequence with a cDNA sequence from the literature and a newly isolated cDNA revealed the presence of 13 introns, the first of which is located in the untranslated leader. The promoter contains a G-box-like sequence. The deduced amino acid sequence of the precursor of GBSS shows a high degree of identity with monocot waxy protein sequences in the region corresponding to the mature form of the enzyme. The transit peptide of 77 amino acids, required for routing of the precursor to the plastids, shows much less identity with the transit peptides of the other waxy preproteins, but resembles the hydropathic distributions of these peptides. Alignment of the amino acid sequences of the four mature starch synthases with the Escherichia coli glgA gene product revealed the presence of at least three conserved boxes; there is no homology with previously proposed starch binding domains of other enzymes involved in starch metabolism. We report the use of chimeric constructs with wild-type and amf sequences to localize, via complementation experiments, the region of the amf allele in which the mutation resides. Direct sequencing of polymerase chain reaction products confirmed that the amf mutation is a deletion of a single AT basepair in the region coding for the transit peptide. Premature termination of translation as a result of this frameshift mutation results in a small peptide. However, a protein reacting with anti-GBSS serum, slightly larger than the wild-type mature GBSS, can be detected in a membrane fraction from amylose-free tubers. A possible explanation for this phenomenon will be discussed.

Studies on a non-fixing mutant of pea (Pisum sativum L.). I. Phenotypical description and bacteriod activity

Abstract In this study a supernodulating non-fixing (AR−) pea mutant FN1 was compared with the supernodulating parent type nod3. Phenotypically the fix− mutant differed from the parent type nod3 by having severe nitrogen deficiency symptoms and an increased nodule mass per plant in N-free hydroculture. The non-fixing character was monogenic and recessive, while grafting experiments showed that the expression was dependent on the genotype of the stock, presumably the roots. The mutated gene was designated nof1. The release of bacteroids from the infection threads into the cortex cells looked normal when viewed under the light microscope. A large accumulation of starch granules in the proximal cortex cells of the mutant indicated that there was an underconsumption of energy in the nodules. Electron microscopy studies indicated an early senescence of the bacteroids and the surrounding peribacteroid membranes. In contrast with bacteroids from the parent type nod3, bacteroids isolated from FN1 nodules were not able to reduce acetylene under micro-aerobic conditions. Bacteroids from both the fix− mutant and the parent type nod3 were able to take up succinate in vitro.

Chimerism as the basis for the occurrence of amylose synthesizing clones derived from an amylose-free potato mutant

Earlier described revertants, obtained after irradiation of an amylose-free (amf) mutant which carries a point deletion in the gene for granule-bound starch synthase, were analysed at the DNA-sequence level. Direct sequencing of fragments amplified by the polymerase chain reaction revealed that all investigated revertants carry the original wildtype sequence. It is argued that mutation as the basis for the re-occurrence of wildtype alleles is highly unlikely. The alternative conclusion is reached that the original amylose-free monoploid clone must have been a chimera. Chimerism with wildtype and mutant tissues was actually found in a plant which at a later stage was obtained from the same mutant clone without the use of X-rays. Wildtype cells could have remained in the L2 layer of the original monoploid mutant, which cannot be analyzed for starch composition.

A putative β-glucanase pseudogene behind the potato GBSS gene

We identified an open reading frame (ORF) which is located closely behind the gene encoding granulebound starch synthase (GBSS) of potato (Solanum tuberosum L.). The ORF ends with a perfect 43 bp direct repeat, which carries the stop triplet precisely at the beginning of the second repeat. The deduced protein shows homology with all known isoforms of plant β-1,3-glucanases and β-1,3-1,4-glucanases. Although the DNA sequence is unique in potato and tomato (Lycopersicon esculentum L.), no expression of the gene was found in these species. Taken together with the unusual codon usage and length of the predicted protein, this sequence could be a pseudogene.

Biosynthesis of starch; identification of potato starch enzymes

Abstract Two important starch enzymes, granule-bound starch synthase and branching enzyme, were purified from potato tubers and characterized by immunological comparison with the corresponding enzymes of other plants. Granule-bound starch synthase was identified as a 60-kd protein homologous to the corresponding enzymes of maize and amaranth; the enzyme was missing in amylose-free potato starch granules. Branching enzyme of potato tubers was purified as a single protein species of 79 kd which appeared to be homologous to maize branching enzyme I, but much less to branching enzymes IIa and IIb.