Publikationen - Molekulare Signalverarbeitung

We present a comprehensive analysis of ADP-glucose pyrophosphorylase (AGP)-repressed pea (Pisum sativum) seeds using transcript and metabolite profiling to monitor the effects that reduced carbon flow into starch has on carbon-nitrogen metabolism and related pathways. Changed patterns of transcripts and metabolites suggest that AGP repression causes sugar accumulation and stimulates carbohydrate oxidation via glycolysis, tricarboxylic acid cycle, and mitochondrial respiration. Enhanced provision of precursors such as acetyl-coenzyme A and organic acids apparently support other pathways and activate amino acid and storage protein biosynthesis as well as pathways fed by cytosolic acetyl-coenzyme A, such as cysteine biosynthesis and fatty acid elongation/metabolism. As a consequence, the resulting higher nitrogen (N) demand depletes transient N storage pools, specifically asparagine and arginine, and leads to N limitation. Moreover, increased sugar accumulation appears to stimulate cytokinin-mediated cell proliferation pathways. In addition, the deregulation of starch biosynthesis resulted in indirect changes, such as increased mitochondrial metabolism and osmotic stress. The combined effect of these changes is an enhanced generation of reactive oxygen species coupled with an up-regulation of energy-dissipating, reactive oxygen species protection, and defense genes. Transcriptional activation of mitogen-activated protein kinase pathways and oxylipin synthesis indicates an additional activation of stress signaling pathways. AGP-repressed embryos contain higher levels of jasmonate derivatives; however, this increase is preferentially in nonactive forms. The results suggest that, although metabolic/osmotic alterations in iAGP pea seeds result in multiple stress responses, pea seeds have effective mechanisms to circumvent stress signaling under conditions in which excessive stress responses and/or cellular damage could prematurely initiate senescence or apoptosis.

In a screen for enhancers of tir1-1 auxin resistance, we identified two novel alleles of the putative mitochondrial pyruvate dehydrogenase E1α-subunit, IAA-Alanine Resistant4 (IAR4). In addition to enhancing the auxin response defects of tir1-1, iar4 single mutants exhibit numerous auxin-related phenotypes including auxin-resistant root growth and reduced lateral root development, as well as defects in primary root growth, root hair initiation, and root hair elongation. Remarkably, all of these iar4 mutant phenotypes were rescued when endogenous indole-3-acetic acid (IAA) levels were increased by growth at high temperature or overexpression of the YUCCA1 IAA biosynthetic enzyme, suggesting that iar4 mutations may alter IAA homeostasis rather than auxin response. Consistent with this possibility, iar4 mutants exhibit increased Aux/IAA stability compared to wild type under basal conditions, but not in response to an auxin treatment. Measurements of free IAA levels detected no significant difference between iar4-3 and wild-type controls. However, we consistently observed significantly higher levels of IAA-amino acid conjugates in the iar4-3 mutant. Furthermore, using stable isotope-labeled IAA precursors, we observed a significant increase in the relative utilization of the Trp-independent IAA biosynthetic pathway in iar4-3. We therefore suggest that the auxin phenotypes of iar4 mutants are the result of altered IAA homeostasis.

Viroids, due to their small size and lack of protein-coding capacity, must rely essentially on their hosts for replication. Intriguingly, viroids have evolved the ability to replicate in two cellular organella, the nucleus (family Pospiviroidae) and the chloroplast (family Avsunviroidae). Viroid replication proceeds through an RNA-based rolling-circle mechanism with three steps that, with some variations, operate in both polarity strands: i) synthesis of longer-than-unit strands catalyzed by either the nuclear RNA polymerase II or a nuclear-encoded chloroplastic RNA polymerase, in both instances redirected to transcribe RNA templates, ii) cleavage to unit-length, which in the family Avsunviroidae is mediated by hammerhead ribozymes embedded in both polarity strands, while in the family Pospiviroidae the oligomeric RNAs provide the proper conformation but not the catalytic activity, and iii) circularization. The host RNA polymerases, most likely assisted by additional host proteins, start transcription from specific sites, thus implying the existence of viroid promoters. Cleavage and ligation in the family Pospiviroidae is probably catalyzed by an RNase III-like enzyme and an RNA ligase able to circularize the resulting 5’ and 3’ termini. Whether a chloroplastic RNA ligase mediates circularization in the family Avsunviroidae, or this reaction is autocatalytic, remains an open issue.

To test whether the response to electrical current and heat treatment is due to the same signaling pathway that mediates mechanical wounding, we analyzed the effect of electric-current application and localized burning on proteinase inhibitor II (Pin2) gene expression in both wild-type and abscisic acid (ABA)-deficient tomato (Lycopersicon esculentum Mill.) and potato (Solanum phureja) plants. Electric-current application and localized burning led to the accumulation of Pin2 mRNA in potato and tomato wild-type plants. Among the treatments tested, only localized burning of the leaves led to an accumulation of Pin2 mRNA in the ABA-deficient plants. Electric-current application, like mechanical injury, was able to initiate ABA and jasmonic acid (JA) accumulation in wild-type but not in ABA-deficient plants. In contrast, heat treatment led to an accumulation of JA in both wild-type and ABA-deficient plants. Inhibition of JA biosynthesis by aspirin blocked the heat-induced Pin2 gene expression in tomato wild-type leaves. These results suggest that electric current, similar to mechanical wounding, requires the presence of ABA to induce Pin2 gene expression. Conversely, burning of the leaves activates Pin2 gene expression by directly triggering the biosynthesis of JA by an alternative pathway that is independent of endogenous ABA levels.

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