Publikationen - Molekulare Signalverarbeitung

Auxin coordinates plant development largely via hierarchical control of gene expression. During the past decades, the study of early auxin genes paired with the power of Arabidopsis genetics have unraveled key nuclear components and molecular interactions that perceive the hormone and activate primary response genes. Recent research in the realm of structural biology allowed unprecedented insight into: (i) the recognition of auxin-responsive DNA elements by auxin transcription factors; (ii) the inactivation of those auxin response factors by early auxin-inducible repressors; and (iii) the activation of target genes by auxin-triggered repressor degradation. The biophysical studies reviewed here provide an impetus for elucidating the molecular determinants of the intricate interactions between core components of the nuclear auxin response module.

The plant hormone auxin activates primary response genes by facilitating proteolytic removal of AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA)-inducible repressors, which directly bind to transcriptional AUXIN RESPONSE FACTORS (ARF). Most AUX/IAA and ARF proteins share highly conserved C-termini mediating homotypic and heterotypic interactions within and between both protein families. The high-resolution NMR structure of C-terminal domains III and IV of the AUX/IAA protein PsIAA4 from pea (Pisum sativum) revealed a globular ubiquitin-like β-grasp fold with homologies to the Phox and Bem1p (PB1) domain. The PB1 domain of wild-type PsIAA4 features two distinct surface patches of oppositely charged amino acid residues, mediating front-to-back multimerization via electrostatic interactions. Mutations of conserved basic or acidic residues on either face suppressed PsIAA4 PB1 homo-oligomerization in vitro and confirmed directional interaction of full-length PsIAA4 in vivo (yeast two-hybrid system). Mixing of oppositely mutated PsIAA4 PB1 monomers enabled NMR mapping of the negatively charged interface of the reconstituted PsIAA4 PB1 homodimer variant, whose stoichiometry (1:1) and equilibrium binding constant (KD ∼6.4 μM) were determined by isothermal titration calorimetry. In silico protein–protein docking studies based on NMR and yeast interaction data derived a model of the PsIAA4 PB1 homodimer, which is comparable with other PB1 domain dimers, but indicated considerable differences between the homodimeric interfaces of AUX/IAA and ARF PB1 domains. Our study provides an impetus for elucidating the molecular determinants that confer specificity to complex protein–protein interaction circuits between members of the two central families of transcription factors important to the regulation of auxin-responsive gene expression.

Calcium (Ca2+) is a key second messenger in eukaryotes and regulates diverse cellular processes, most notably via calmodulin (CaM). In Arabidopsis thaliana, IQD1 (IQ67 domain 1) is the founding member of the IQD family of putative CaM targets. The 33 predicted IQD proteins share a conserved domain of 67 amino acids that is characterized by a unique arrangement of multiple CaM recruitment motifs, including so-called IQ motifs. Whereas IQD1 has been implicated in the regulation of defense metabolism, the biochemical functions of IQD proteins remain to be elucidated. In this study we show that IQD1 binds to multiple Arabidopsis CaM and CaM-like (CML) proteins in vitro and in yeast two-hybrid interaction assays. CaM overlay assays revealed moderate affinity of IQD1 to CaM2 (Kd ∼ 0.6 μm). Deletion mapping of IQD1 demonstrated the importance of the IQ67 domain for CaM2 binding in vitro, which is corroborated by interaction of the shortest IQD member, IQD20, with Arabidopsis CaM/CMLs in yeast. A genetic screen of a cDNA library identified Arabidopsis kinesin light chain-related protein-1 (KLCR1) as an IQD1 interactor. The subcellular localization of GFP-tagged IQD1 proteins to microtubules and the cell nucleus in transiently and stably transformed plant tissues (tobacco leaves and Arabidopsis seedlings) suggests direct interaction of IQD1 and KLCR1 in planta that is supported by GFP∼IQD1-dependent recruitment of RFP∼KLCR1 and RFP∼CaM2 to microtubules. Collectively, the prospect arises that IQD1 and related proteins provide Ca2+/CaM-regulated scaffolds for facilitating cellular transport of specific cargo along microtubular tracks via kinesin motor proteins.

Racemic jasmonic acid (3R,7R/3S,7S)-(±)-JA) was chemically conjugated with different biogenic amines originating from aliphatic and aromatic α-amino acids by decarboxylation. The resulting isomeric compounds were subjected to reversed-phase high-performance liquid chromatography (HPLC) and to HPLC on the chiral stationary phases Chiralpak AS and Nucleodex β-PM. Under reversed-phase conditions, all the homologous amine derivatives tested could be separated from each other except the JA-conjugates containing 2-phenyl-ethylamine and 3-methylbutylamine. On both chiral supports the (3R,7R)-(−)-JA conjugates eluted earlier than those of the enantiomeric counterpart (3S,7S)-(+)-JA. On Chiralpak AS all the isomers studied could be separated to baseline with a mobile phase containingn-hexane and 2-propanol. The calculated resolution factors were between 1.80 and 4.17. The pairs of isomers were also chromatographed on the cyclodextrin stationary phase Nucleodex β-PM with methanol-triethylammonium acetate buffer as mobile phase. Under these conditions resolution factors were between 0.74 and 1.29. The individual isomers were chiroptically characterized by measurement of their circular dichroism.

The chiral separation of peptide-like conjugates of jasmonic acid and of cucurbic acid isomers was investigated by liquid chromatography on Chiralpak AS and Nucleodex β-PM. The retention sequences reflect distinct chromatographic properties with respect to the chirality of the jasmonic acid part or of the cucurbic acid isomers. The chromatographic behaviour of the amide conjugates on a reversed-phase C18 column provides evidence for the resolution of diastereomeric conjugates depending on the chirality of both constituents of the conjugate molecule. The chromatographic procedures are suitable for the analytical and preparative separation of such conjugates.

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Synthetic amide conjugates of (−)-jasmonic acid and its (+)-enantiomer were resolved by means of chiral liquid chromatography. The diastereomeric pairs prepared by chemical reaction of (±)-jasmonic acid with a series of (S)- or (R)-amino acids and with some (S)-amino acid alcohols were completely separated on Chiralpak AS using a mixture of n-hexane/2-propanal as mobile phase. The retention data indicate that the (−)-jasmonic acid conjugates eluted faster than those of the (+)-enantiomer, independent on the configuration of the bound amino acid. Likewise, enantiomeric derivatives of (±)-jasmonic acid and non-chiral amino acids were completely separated on the chiral stationary phase and showed the same elution sequence. The resolution factors,Rs, were found to range between 1.13 and 6.64. The separated compounds were chiropatically analyzed by measurement of the circular dichroism.

The effect of osmotically active substances on the alteration of endogenous jasmonates was studied in barley (Hordeum vulgare L. cv. Salome) leaf tissue. Leaf segments were subjected to solutions of d-sorbitol, d-mannitol, polyethylene glycol 6000, sodium chloride, or water as a control. Alterations of endogenous jasmonates were monitored qualitatively and quantitatively using immunoassays. The structures of jasmonates isolated were determined on the basis of authentic substances by capillary gas chromatography-mass spectrometry. The stereochemistry of the conjugates was confirmed by high performance liquid chromatography with diastereoisomeric references. In barley leaves, jasmonic acid and its amino acid conjugates, for example, with valine, leucine, and isoleucine, are naturally occurring jasmonates. In untreated leaf segments, only low levels of these native jasmonates were found. After treatment of the leaf tissues with sorbitol, mannitol, as well as with polyethylene glycol, an increase of both jasmonic acid and its conjugates could be observed, depending on the stress conditions used. In contrast, salt stress was without any stimulating effect on the levels of endogenous jasmonates. From barley leaf segments exposed to sorbitol (1m) for 24 h, jasmonic acid was identified as the major accumulating compound. Jasmonic acid-amino acid conjugates increased likewise upon stress treatment.