RESUMEN
Feed supplementation with ß-arginine-aspartate dipeptides (ß-Asp-Arg DP) shows growth promoting effects in feeding trials with fish and might also be beneficial for pig and poultry farming. Currently, these DPs are generated from purified cyanophycin (CGP), with the help of the CGP-degrading enzyme cyanophycinase (CGPase). As alternative to an in vitro production, the DPs might be directly produced in feed crops. We already demonstrated that CGP can be produced in plastids of tobacco and potato, yielding up to 9.4% of the dry weight (DW). We also transiently co-expressed CGPases in the cytosol without degrading CGP in intact cells, while degradation occurs in the homogenized plant tissue. However, transient co-expression is not feasible for field-grown CGP plants, which is necessary for bulk production. In the present study, we proved that stable co-expression of the CGPase CphE241 in CGP-producing tobacco is sufficient to degrade 2.0% CGP/DW nearly completely within 3 h after homogenization of the leaves. In intact senescing leaves, CGP is partially released to the cytosol and degraded into DPs which limits the overall accumulation of CGP but not the level of the stable DPs. Even after 48 h, 54 µmol ß-Asp-Arg DP/g DW could be detected in the extract, which correspond to 1.5% DP/DW and represents 84% of the expected amount. Thus, we developed a system for the production of ß-Asp-Arg DP in field-grown plants.
RESUMEN
Protein glycation is usually referred to as an array of non-enzymatic post-translational modifications formed by reducing sugars and carbonyl products of their degradation. The resulting advanced glycation end products (AGEs) represent a heterogeneous group of covalent adducts, known for their pro-inflammatory effects in mammals, and impacting on pathogenesis of metabolic diseases and ageing. In plants, AGEs are the markers of tissue ageing and response to environmental stressors, the most prominent of which is drought. Although water deficit enhances protein glycation in leaves, its effect on seed glycation profiles is still unknown. Moreover, the effect of drought on biological activities of seed protein in mammalian systems is still unstudied with respect to glycation. Therefore, here we address the effects of a short-term drought on the patterns of seed protein-bound AGEs and accompanying alterations in pro-inflammatory properties of seed protein in the context of seed metabolome dynamics. A short-term drought, simulated as polyethylene glycol-induced osmotic stress and applied at the stage of seed filling, resulted in the dramatic suppression of primary seed metabolism, although the secondary metabolome was minimally affected. This was accompanied with significant suppression of NF-kB activation in human SH-SY5Y neuroblastoma cells after a treatment with protein hydrolyzates, isolated from the mature seeds of drought-treated plants. This effect could not be attributed to formation of known AGEs. Most likely, the prospective anti-inflammatory effect of short-term drought is related to antioxidant effect of unknown secondary metabolite protein adducts, or down-regulation of unknown plant-specific AGEs due to suppression of energy metabolism during seed filling.
