Institut für Biologie

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Heterologe Expression und molekulare Charakterisierung des Dirigentproteins AtDIR6 aus A. thaliana
(2011) Pickel, Benjamin; Schaller, Andreas
Dirigent proteins are involved in the stereo- and regioselective control of plant secondary metabolism. Functionally described dirigent proteins from Forsythia intermedia and Thuja plicata couple coniferyl alcohol radicals to (+)-pinoresinol, a precursor of various lignans including the pharmaceutically relevant podophyllotoxin. The discovery of (?)-lariciresinol in A. thaliana roots and the accumulation of its precursor (?)-pinoresinol in a knock-out mutant lacking two pinoresinol reductases indicated the presence of a novel dirigent activity in A. thaliana which mediates the enantiocomplementary formation of (?)-pinoresinol. In this work AtDIR6 was identified as a candidate for this novel dirigent activity. The protein was cloned and heterologously expressed in a plant cell culture system. The recombinant protein was purified to appearent homogeneity by conventional chromatography methods. The purified protein was functionally active and directed the coupling of coniferyl alcohol radicals to (?)-pinoresinol in vitro. It was further shown that the stereoselectivity of AtDIR6 is opposed to that of known dirigent proteins in F. intermedia and T. plicata, and therefore, AtDIR6 is the first of the long-sought nantiocomplementary dirigent proteins. AtDIR6 was shown to possess a N-terminal signal peptide, which was cleaved during secretion between amino acids 29 and 30. Mature AtDIR6 accumulated extracellularly and remained non-covalently attached to the primary cell wall of suspension cultured cells. The native protein is glycosylated with two complex type and paucimannosidic Nglycans, respectively. It forms homodimers of app. 42 kDa and shows a high content of b-sheets. The functionally described dirigent proteins are small proteins that are characterised by the ability to bind coniferyl alcohol radicals and couple them enantiospecifically without possessing a catalytic activity of their own. Sequence identity between different dirigent proteins may be low. In these aspects dirigent proteins are similiar to lipocalins. A sequence alignment with dirigent proteins and lipocalins shows that the lipocalin-specific sequence motive, which is part of SCR II, is conserved among all functionally described dirigent proteins. Structural and mechanistic features of AtDIR6 suggest that dirigent proteins may belong to the calycin superfamily, which also includes lipocalins, and that their threedimensional structure may be that of a b-barrel.
Regulation von NIMIN- und PR1-Genen aus Arabidopsis thaliana (L.) Heynh. und Nicotiana tabacum (L.) in der Salicylat-abhängigen Pathogenabwehr
(2009) Hermann, Meike; Pfitzner, Artur J. P.
Systemic acquired resistance (SAR) is an important defense mechanism of plants against a broad range of pathogens. NPR1 acts as a central regulator controlling the salicylic acid (SA)-dependent formation of SAR through interaction with TGA transcription factors leading to the induction of ?pathogenesis-related? (PR) proteins. The SA-activated expression of the PR1 genes in Arabidopsis thaliana (At) and Nicotiana tabacum (Nt) depends on cis-acting as-1-like elements with a TGACG sequence. This dissertation studies the functional relevance of NIMIN proteins and SA-dependent PR gene induction using the analysis of gene regulation. Arabidopsis has four NIMIN-genes ? N1, N2, N3 and N1b which interact independently of TGA transcription factors with NPR1. N1 and N2 have a common interaction motif and bind to the C-terminus of AtNPR1, whereas N3 binds to the N-terminus of AtNPR1. The binding site for the TGA transcription factors is located relatively central in the AtNPR1 protein. The analysis of the NIMIN gene expression in the SA-dependent signaling pathway of SAR as well as their possible involvement in the Jasmonic (JA) signaling network ought to offer new aspects for understanding the regulation of plant pathogen defense. The relevance of different as-1-like elements was studied by establishing a yeast one-hybrid system. N1b is likely to be an inactive pseudogene. Neither could transcripts be detected in untreated, SA- or JA-treated Arabidopsis plants nor was the construct N1b[GUS] with the 1135 bp 5?-region able to induce reporter gene expression in transgenic tobacco plants. Expression of N3 occurs constitutively at low levels and independently of NPR1. Treatment with SA or JA does not lead to induction of N3. Likewise, the N3 promoter is not affected by treatment with SA, JA, TMV and phytohormones. Reporter gene expression of the N3 promoter occurs constitutively in transgenic tobacco seedlings. In contrast, N1 and N2 are clearly SA-induced. After SA induction, the expression of N2 is immediate and long-lasting and regulated independently of NPR1. N1 is expressed transiently at a later point in time and is NPR1-dependent. The expression of both, N1 and N2, clearly occurs before PR1 gene expression. The analysis of GUS-reporter gene constructs confirms the early SA-dependent induction of the N1 and N2 promoters before activation of the NtPR1a promoter. Similar to the PR1a promoter, both NIMIN promoters can be induced by thiamine-HCl and show an inhibitory effect of the JA signaling network on the strength of reporter gene expression during simultaneous treatment with SA and JA. At the histological level, the N1 and N2 promoters display SA-dependent activation in leaf and root tissue of young tobacco seedlings. This activity clearly differs from the N3 promoter. The N2 promoter ? just like the AtPR1 and NtPR1a promoters ? contains an as-1-like element with a tandem repeat of TGACG, responsible for the SA sensitivity of the promoter. However the N2 as-1-like element structurally differs from the as-1-like elements in the PR1 promoters. In the N1 promoter a SA-responsive element was located in the region of -436 to -402 with respect to the translation starting point of the N1 gene. However, mutation of a TGATG repeat within this region did not result in a loss of promoter activity. Analysis of chimeric promoter constructs with foreign as-1-like elements showed that the different expression kinetics of PR1 and NIMIN genes are not encoded by the genetic information of the respective as-1-like elements. On the contrary, the as-1-like cis-acting elements affect the promoters? tissue specificity. Due to the integration of the N2-as-1-like element, the NtPR1a promoter, which is solely active in leaf tissue, adopts the typical NIMIN activity in root tissue. The presence of the 35S-as-1 element in the NtPR1a promoter leads to constitutional activation in root tissue. However, the activation in leaf tissue is still SA-dependent. In the yeast one-hybrid system, the interaction of TGA factors with as-1 and the as-1-like elements of the AtPR1, NtPR1a and N2 promoters shows only small differences in binding quality, whereas considerable differences can be detected in quantitative binding strength. Mutation of the as-1-like elements in the NtPR1a and N2 promoters results in the loss of TGA factor binding. The N1 promoter region from -436 to -399 contains a TGA binding site. Mutation of the contained TGATG repeat leads to a total loss of binding of TGA transcription factors. The neighbouring promoter context can exert both positive and negative influence on TGA factor binding. In case of the N1 promoter, the presence of adjacent promoter regions results in increased binding affinity of TGA factors. In contrast, additional NtPR1a promoter context shows a considerable reduction of TGA factor binding to as-1-like elements. Despite independent binding sites, NIMIN proteins and TGA transcription factors compete for binding of AtNPR1 in the yeast three-hybrid system. The presence of N1 and N3 thereby impedes interaction of TGA factors with NPR1, whereas simultaneous binding of N2 and TGA factor is possible without any restrictions. Binding of N1 or N2 simultaneously with N3 at the C- and N-termini of NPR1 also results in reciprocal interference suggesting a spatial folding of NPR1 where the N- and C-termini lie closely together.

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