High-amylose starch is in great demand by the starch industry for its unique functional properties. However, very few high-amylose crop varieties are commercially available. In this paper we describe the generation of very-high-amylose potato starch by genetic modification. We achieved this by simultaneously inhibiting two isoforms of starch branching enzyme to below 1% of the wild-type activities. Starch granule morphology and composition were noticeably altered. Normal, high-molecular-weight amylopectin was absent, whereas the amylose content was increased to levels comparable to the highest commercially available maize starches. In addition, the phosphorus content of the starch was increased more than fivefold. This unique starch, with its high amylose, low amylopectin, and high phosphorus levels, offers novel properties for food and industrial applications.

Introduction Breast tumors lacking the estrogen receptor-α (ER-α) have increased incidence of resistance to therapy and poorer clinical prognosis.

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SummaryFull length cDNAs encoding a second starch branching enzyme (SBE A) isoform have been isolated from potato tubers. The predicted protein has a molecular mass of 101 kDa including a transit peptide of 48 amino acids. Multiple forms of the SBE A gene exist which differ mainly in the length of a polyglutamic acid repeat at the C-terminus of the protein. Expression of the mature protein in Escherichia coli demonstrates that the gene encodes an active SBE. Northern analysis demonstrates that SBE A mRNA is expressed at very low levels in tubers but is the predominant isoform in leaves. This expression pattern was confirmed by Western analysis using isoform specific polyclonal antibodies raised against E. coli expressed SBE A. SBE A protein is found predominantly in the soluble phase of tuber extracts, indicating a stromal location within the plastid. Transgenic potato plants expressing an antisense SBE A RNA were generated in which almost complete reductions in SBE A were observed. SBE activity in the leaves of these plants was severely reduced, but tuber activity was largely unaffected. Even so, the composition and structure of tuber starch from these plants was greatly altered. The proportion of linear chains was not significantly increased but the average chain length of amylopectin was greater, resulting in an increase in apparent amylose content as judged by iodine binding. In addition, the starch had much higher levels of phosphorous.

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The activity of mitochondria induces, as a byproduct, a variety of post-translational modifications in associated proteins, which have functional downstream consequences for processes such as apoptosis, autophagy, and plasticity; e.g., reactive oxygen species (ROS), which induce N-formyl-kynurenine from oxidized tryptophans in certain mitochondrial proteins which are localized in close spatial proximity to their source. This type of fast molecular changes has profound influence on cell death and survival with implications in a number of pathologies. The quantitative and differential analysis of bovine heart mitochondria by four 2D-PAGE methods, including 2D-PAGE with high-resolution IEF as first dimension, revealed that due to limited resolution, those methods employing blue native-, tricine-urea-, and 16-BAC-PAGE as the first dimension are less applicable for the differential quantitative analysis of redundant protein spots which might give insight into post-translational modifications that are relevant in age- and stress-related changes. Moreover, 2D-PAGE with high resolution IEF was able to resolve a surprisingly large number of membrane proteins from mitochondrial preparations. For aconitase-2, an enzyme playing an important role in mitochondrial aging, a more thorough molecular analysis of all separable isoforms was performed, leading to the identification of two particular N-formylkynurenine modifications. Next to protein redundancy, native protein-protein interactions, with the potential of relating certain post-translational modification patterns to distinct oligomeric states, e.g., oxidative phosphorylation super complexes, might provide novel and (patho-) physiologically relevant information. Among proteins identified, 14 new proteins (GenBank entries), previously not associated with mitochondria, were found.

Transketolases, key enzymes of the reductive and oxidative pentose phosphate pathways, are responsible for the synthesis of sugar phosphate intermediates. Here we report the first molecular analysis of transketolase genes from plants. Three distinct classes of transketolase‐encoding cDNA clones were isolated from the desiccation‐tolerant resurrection plant Craterostigma plantagineum. One class represented by the transcript tkt3 is constitutively expressed in leaves and roots under all physiological conditions tested. By biochemical analysis and protein sequencing of purified transketolase, it was shown that tkt3 is expressed in three enzymatically active isoforms. An intriguing discovery was that accumulation of the two other transketolase transcripts, tkt7 and tkt10, is preferentially associated with the rehydration process of the desiccated plant; whereas tkt10 is only expressed in leaves, tkt7 was detected in leaves and roots. This observation suggests a possible role for these transketolases in the conversion of sugars, which are a major phenomenon in the rehydration process. Despite an abundant level of tkt7 and tkt10 transcripts in rehydrating leaves, proteins could not be isolated. This is due in part to a translational control mechanism acting on the loading of mRNAs to polysomes.

The use of unmodified starches in frozen foods is severely limited by the undesirable textural changes that occur after freezing and thawing. Retrogradation of glucan chains leads to syneresis, a separation of the starch gel and water phases. Stabilization of the starch structure is normally achieved by chemical modification to prevent these changes from occurring. We have now created a freeze-thaw-stable potato starch by alteration of starch composition and structure by genetic modification. An amylose-free starch with short-chain amylopectin was produced by simultaneous antisense downregulation of three starch synthase genes. This starch is extremely freeze-thaw-stable and shows no syneresis even after five freeze-thaw cycles. The use of this starch has potential for environmental and consumer benefits because its production requires no chemical modification.