SummaryThe original aim of this work was to increase starch accumulation in potato tubers by enhancing their capacity to metabolise sucrose. We previously reported that specific expression of a yeast invertase in the cytosol of tubers led to a 95% reduction in sucrose content, but that this was accompanied by a larger accumulation of glucose and a reduction in starch. In the present paper we introduced a bacterial glucokinase from Zymomonas mobilis into an invertase-expressing transgenic line, with the intention of bringing the glucose into metabolism. Transgenic lines were obtained with up to threefold more glucokinase activity than in the parent invertase line and which did not accumulate glucose. Unexpectedly, there was a further dramatic reduction in starch content, down to 35% of wild-type levels. Biochemical analysis of growing tuber tissue revealed large increases in the metabolic intermediates of glycolysis, organic acids and amino acids, two-to threefold increases in the maximum catalytic activities of key enzymes in the respiratory pathways, and three-to fivefold increases in carbon dioxide production. These changes occur in the lines expressing invertase, and are accentuated following introduction of the second transgene, glucokinase. We conclude that the expression of invertase in potato tubers leads to an increased flux through the glycolytic pathway at the expense of starch synthesis and that heterologous overexpression of glucokinase enhances this change in partitioning.

Plant cysteine (Cys) synthesis can occur in three cellular compartments: the chloroplast, cytoplasm, and mitochondrion. Cys formation is catalyzed by the enzyme O-acetylserine(thiol)lyase (OASTL) using O-acetylserine (OAS) and sulfide as substrates. To unravel the function of different isoforms of OASTL in cellular metabolism, a transgenic approach was used to downregulate specifically the plastidial and cytosolic isoforms in potato (Solanum tuberosum). This approach resulted in decreased RNA, protein, and enzymatic activity levels. Intriguingly, H 2 S-releasing capacity was also reduced in these lines. Unexpectedly, the thiol levels in the transgenic lines were, regardless of the selected OASTL isoform, significantly elevated. Furthermore, levels of metabolites such as serine, OAS, methionine, threonine, isoleucine, and lysine also increased in the investigated transgenic lines. This indicates that higher Cys levels might influence methionine synthesis and subsequently pathway-related amino acids. The increase of serine and OAS points to suboptimal Cys synthesis in transgenic plants. Taking these findings together, it can be assumed that excess OASTL activity regulates not only Cys de novo synthesis but also its homeostasis. A model for the regulation of Cys levels in plants is proposed.

Macrodomains are conserved protein interaction modules that can be found in all domains of life including in certain viruses. Macrodomains mediate recognition of sequence motifs harboring adenosine diphosphate ribose (ADPR) modifications, thereby regulating a variety of cellular processes. Due to their role in cancer or viral pathogenesis, macrodomains have emerged as potential therapeutic targets, but the unavailability of small molecule inhibitors has hampered target validation studies so far. Here, we describe an efficient screening strategy for identification of small molecule inhibitors that displace ADPR from macrodomains. We report the discovery and characterization of a macrodomain inhibitor, GeA-69, selectively targeting macrodomain 2 (MD2) of PARP14 with low micromolar affinity. Co-crystallization of a GeA-69 analogue with PARP14 MD2 revealed an allosteric binding mechanism explaining its selectivity over other human macrodomains. We show that GeA-69 engages PARP14 MD2 in intact cells and prevents its localization to sites of DNA damage.

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