Publikationen - Molekulare Signalverarbeitung

Glucosinolates are a class of secondary metabolites with important roles in plant defense and human nutrition. To uncover regulatory mechanisms of glucosinolate production, we screened Arabidopsis thaliana T‐DNA activation‐tagged lines and identified a high‐glucosinolate mutant caused by overexpression of IQD1 (At3g09710). A series of gain‐ and loss‐of‐function IQD1 alleles in different accessions correlates with increased and decreased glucosinolate levels, respectively. IQD1 encodes a novel protein that contains putative nuclear localization signals and several motifs known to mediate calmodulin binding, which are arranged in a plant‐specific segment of 67 amino acids, called the IQ67 domain. We demonstrate that an IQD1‐GFP fusion protein is targeted to the cell nucleus and that recombinant IQD1 binds to calmodulin in a Ca2+‐dependent fashion. Analysis of steady‐state messenger RNA levels of glucosinolate pathway genes indicates that IQD1 affects expression of multiple genes with roles in glucosinolate metabolism. Histochemical analysis of tissue‐specific IQD1 ::GUS expression reveals IQD1 promoter activity mainly in vascular tissues of all organs, consistent with the expression patterns of several glucosinolate‐related genes. Interestingly, overexpression of IQD1 reduces insect herbivory, which we demonstrated in dual‐choice assays with the generalist phloem‐feeding green peach aphid (Myzus persicae ), and in weight‐gain assays with the cabbage looper (Trichoplusia ni ), a generalist‐chewing lepidopteran. As IQD1 is induced by mechanical stimuli, we propose IQD1 to be novel nuclear factor that integrates intracellular Ca2+ signals to fine‐tune glucosinolate accumulation in response to biotic challenge.

Chrysanthemum chlorotic mottle viroid (CChMVd) RNA (398–401 nucleotides) can form hammerhead ribozymes that play a functional role in its replication through a rolling-circle mechanism. In contrast to most other viroids, which adopt rod-like or quasi-rod-like secondary structures of minimal free energy, the computer-predicted conformations of CChMVd and Peach latent mosaic viroid (PLMVd) RNAs are branched. Moreover, the covariations found in a number of natural CChMVd variants support that the same or a closely related conformation exists in vivo. Here we report that the CChMVd natural variability also supports that the branched conformation is additionally stabilized by a kissing-loop interaction resembling another one proposed in PLMVd from in vitro assays. Moreover, site-directed mutagenesis combined with bioassays and progeny analysis showed that: (1) single CChMVd mutants affecting the kissing loops had low or no infectivity at all, whereas infectivity was recovered in double mutants restoring the interaction; (2) mutations affecting the structure of the regions adjacent to the kissing loops reverted to wild type or led to rearranged stems, also supporting their interaction; and (3) the interchange between 4 nucleotides of each of the two kissing loops generated a viable CChMVd variant with eight mutations. PAGE analysis under denaturing and nondenaturing conditions revealed that the kissing-loop interaction determines proper in vitro folding of CChMVd RNA. Preservation of a similar kissing-loop interaction in two hammerhead viroids with an overall low sequence similarity suggests that it facilitates in vivo the adoption and stabilization of a compact folding critical for viroid viability.

Upon a dark/light shift the conditional flu mutant of Arabidopsis starts to generate singlet oxygen (1O2), a non‐radical reactive oxygen species that is restricted to the plastid compartment. Immediately after the shift, plants stop growing and develop necrotic lesions. We have established a protoplast system, which allows detection and characterization of the death response in flu induced by the release of 1O2. Vitamin B6 that quenches 1O2 in fungi was able to protect flu protoplasts from cell death. Blocking ethylene production was sufficient to partially inhibit the death reaction. Similarly, flu mutant seedlings expressing transgenic NahG were partially protected from the death provoked by the release of 1O2, indicating a requirement for salicylic acid (SA) in this process, whereas in cells depleted of both, ethylene and SA, the extent of cell death was reduced to the wild‐type level. The flu mutant was also crossed with the jasmonic acid (JA)‐depleted mutant opr3 , and with the JA, OPDA and dinor OPDA (dnOPDA)‐depleted dde2‐2 mutant. Analysis of the resulting double mutants revealed that in contrast to the JA‐induced suppression of H2O2/superoxide‐dependent cell death reported earlier, JA promotes singlet oxygen‐mediated cell death in flu , whereas other oxylipins such as OPDA and dnOPDA antagonize this death‐inducing activity of JA.

Selective protein degradation by the ubiquitin‐proteasome pathway has emerged as a key regulatory mechanism in a wide variety of cellular processes. The selective components of this pathway are the E3 ubiquitin‐ligases which act downstream of the ubiquitin‐activating and ‐conjugating enzymes to identify specific substrates for ubiquitinylation. SCF‐type ubiquitin‐ligases are the most abundant class of E3 enzymes in Arabidopsis. In a genetic screen for enhancers of the tir1‐1 auxin response defect, we identified eta1 /axr6‐3 , a recessive and temperature‐sensitive mutation in the CUL1 core component of the SCFTIR1 complex. The axr6‐3 mutation interferes with Skp1 binding, thus preventing SCF complex assembly. axr6‐3 displays a pleiotropic phenotype with defects in numerous SCF‐regulated pathways including auxin signaling, jasmonate signaling, flower development, and photomorphogenesis. We used axr6‐3 as a tool for identifying pathways likely to be regulated by SCF‐mediated proteolysis and propose new roles for SCF regulation of the far‐red light/phyA and sugar signaling pathways. The recessive inheritance and the temperature‐sensitive nature of the pleiotropically acting axr6‐3 mutation opens promising possibilities for the identification and investigation of SCF‐regulated pathways in Arabidopsis.