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Publications - Cell and Metabolic Biology
Publications
Salinity is a serious challenge to global agriculture and threatens human food security. Plant cells can respond to salt stress either by activation of adaptive responses, or by programmed cell death. The mechanisms deciding the respective response are far from understood, but seem to depend on the degree, to which mitochondria can maintain oxidative homeostasis. Using plant PeptoQ, a Trojan Peptoid, as vehicle, it is possible to transport a coenzyme Q10 (CoQ10) derivative into plant mitochondria. We show that salinity stress in tobacco BY-2 cells (Nicotiana tabacum L. cv Bright Yellow-2) can be mitigated by pretreatment with plant PeptoQ with respect to numerous aspects including proliferation, expansion, redox homeostasis, and programmed cell death. We tested the salinity response for transcripts from nine salt-stress related-genes representing different adaptive responses. While most did not show any significant response, the salt response of the transcription factor NtNAC, probably involved in mitochondrial retrograde signaling, was significantly modulated by the plant PeptoQ. Most strikingly, transcripts for the mitochondrial, Mn-dependent Superoxide Dismutase were rapidly and drastically upregulated in presence of the peptoid, and this response was disappearing in presence of salt. The same pattern, albeit at lower amplitude, was seen for the sodium exporter SOS1. The findings are discussed by a model, where plant PeptoQ modulates retrograde signalling to the nucleus leading to a strong expression of mitochondrial SOD, what renders mitochondria more resilient to perturbations of oxidative balance, such that cells escape salt induced cell death and remain viable.
Publications
Site-directed methods for the generation of genetic diversity are essential tools in the field of directed enzyme evolution. The Golden Gate cloning technique has been proven to be an efficient tool for a variety of cloning setups. The utilization of restriction enzymes which cut outside of their recognition domain allows the assembly of multiple gene fragments obtained by PCR amplification without altering the open reading frame of the reconstituted gene. We have developed a protocol, termed Golden Mutagenesis that allows the rapid, straightforward, reliable and inexpensive construction of mutagenesis libraries. One to five amino acid positions within a coding sequence could be altered simultaneously using a protocol which can be performed within one day. To facilitate the implementation of this technique, a software library and web application for automated primer design and for the graphical evaluation of the randomization success based on the sequencing results was developed. This allows facile primer design and application of Golden Mutagenesis also for laboratories, which are not specialized in molecular biology.
Publications
Heterologous screening of a cDNA library from Pinus strobus seedlings identified clones for two chalcone synthase (CHS) related proteins (PStrCHS1 and PStrCHS2, 87.6% identity). Heterologous expression in Escherichia coli showed that PStrCHS1 performed the typical CHS reaction, that it used starter CoA-esters from the phenylpropanoid pathway, and that it performed three condensation reactions with malonyl-CoA, followed by the ring closure to the chalcone. PstrCHS2 was completely inactive with these starters and also with linear CoA-esters. Activity was detected only with a diketide derivative (N-acetylcysteamine thioester of 3-oxo-5-phenylpent-4-enoic acid) that corresponded to the CHS reaction intermediate postulated after the first condensation reaction. PstrCHS2 performed only one condensation, with 6-styryl-4-hydroxy-2-pyrone derivatives as release products. The enzyme preferred methylmalonyl-CoA against malonyl-CoA, if only methylmalonyl-CoA was available. These properties and a comparison with the CHS from Pinussylvestris suggested for PstrCHS2 a special function in the biosynthesis of secondary products. In contrast to P. sylvestris, P. strobus contains C-methylated chalcone derivatives, and the methyl group is at the position predicted from a chain extension with methylmalonyl-CoA in the second condensation of the biosynthetic reaction sequence. We propose that PstrCHS2 specifically contributes the condensing reaction with methylmalonyl-CoA to yield a methylated triketide intermediate. We discuss a model that the biosynthesis of C-methylated chalcones represents the simplest example of a modular polyketide synthase.
This page was last modified on 27 Jan 2025 .
