Publikationen - Molekulare Signalverarbeitung

Although polar transport and the TIR1-dependent signaling pathway of the plant hormone auxin/indole-3-acetic acid (IAA) are well characterized, understanding of the biosynthetic pathway(s) leading to the production of IAA is still limited. Genetic dissection of IAA biosynthetic pathways has been complicated by the metabolic redundancy caused by the apparent existence of several parallel biosynthetic routes leading to IAA production. Valuable complementary tools for genetic as well as biochemical analysis of auxin biosynthesis would be molecular inhibitors capable of acting in vivo on specific or general components of the pathway(s), which unfortunately have been lacking. Several indole derivatives have been previously identified to inhibit tryptophan-dependent IAA biosynthesis in an in vitro system from maize endosperm. We examined the effect of one of them, 6-fluoroindole, on seedling development of Arabidopsis thaliana and tested its ability to inhibit IAA biosynthesis in feeding experiments in vivo. We demonstrated a correlation of severe developmental defects or growth retardation caused by 6-fluoroindole with significant downregulation of de novo synthesized IAA levels, derived from the stable isotope-labeled tryptophan pool, upon treatment. Hence, 6-fluoroindole shows important features of an inhibitor of tryptophan-dependent IAA biosynthesis both in vitro and in vivo and thus may find use as a promising molecular tool for the identification of novel components of the auxin biosynthetic pathway(s).

The history of plant biology is inexorably intertwined with the conception and discovery of auxin, followed by the many decades of research to comprehend its action during growth and development. Growth responses to auxin are complex and require the coordination of auxin production, transport, and perception. In this overview of past auxin research, we limit our discourse to the mechanism of auxin action. We attempt to trace the almost epic voyage from the birth of the hormonal concept in plants to the recent crystallographic studies that resolved the TIR1-auxin receptor complex, the first structural model of a plant hormone receptor. The century-long endeavor is a beautiful illustration of the power of scientific reasoning and human intuition, but it also brings to light the fact that decisive progress is made when new technologies emerge and disciplines unite.

The definition of a plant hormone has not been clearly established, so the compounds classified as plant hormones often vary depending on which definition is considered. In this chapter, auxins, gibberellins (GAs), cytokinins, abscisic acid, brassinosteroids, jasmonic acid-related compounds, and ethylene are described as established plant hormones, while polyamines and phenolic compounds are not included. On the other hand, several peptides that have been proven to play a clear physiological role(s) in plant growth and development, similar to the established plant hormones, are referred. This chapter will focus primarily on the more recent discoveries of plant hormones and their impact on our current understanding of their biological role. In some cases, however, it is critical to place recent work in a proper historical context.

The plant hormone auxin transcriptionally activates early genes. We have isolated a 14-member family of DNA sequences complementary to indoleacetic acid (IAA)-inducible transcripts inArabidopsis thaliana. The corresponding genes, IAA1 and IAA14, are homologs of PS-1AA4/5 and PS-IAA6 from pea, AUX22 and AUX28 from soybean, ARG3 and ARG4from mungbean, and AtAux2-11 and AtAux2-27 from Arabidopsis. The members of the family are differentially expressed in mature Arabidopsis plants. Characterization of IAA gene expression in etiolated seedlings demonstrates specificity for auxin inducibility. The response of most family members to IAA is rapid (within 4 to 30 minutes) and insensitive to cyclohexamide. Cyclohexamide alone induces all the early genes. Auxin-induction of two late genes, IAA7 and IAA8, is inhibited by cyclohexamide, indicating requirement of protein synthesis for their activation. All IAA genes display a biphasic dose response that is optimal at 10 μM IAA. However, individual genes respond differentially between 10 nM and 5μM IAA. Expression of all genes is defective in the Arabidopsis auxin-resistant mutant lines axr1, axr2, and aux1.The encoded polypeptides share four conserved domains, and seven invariant residues in the intervening regions. The spaces vary considerably in length, rendering the calculated molecular mass of IAA proteins to range from 19 kDa to 36 kDa. Overall sequence identity between members of the family is highly variable (36 to 87%). Their most significant structural features are functional nuclear transport signals, and a putative βαα-fold whose modeled three dimensional structure appears to be compatible with the prokaryotic β-ribbon DNA recognition motif. The data suggest that auxin induces in a differential and hierarchical fashion a large family of early genes that encode a structurally diverse class of nuclear proteins. These proteins are proposed to mediate tissue-specific and cell-type restricted responses to the hormone during plant growth and development.

The plant hormone, indoleacetic acid (IAA), transcriptionally activates two early genes in pea, PS‐IAA4/5 and PS‐IAA6 , that encode short‐lived nuclear proteins. The identification of the nuclear localization signals (NLS) in PS‐IAA4 and PS‐IAA6 using progressive deletion analysis and site‐directed mutagenesis is reported. A C‐terminal SV40‐type NLS is sufficient to direct the β‐glucuronidase reporter to the nucleus of transiently transformed tobacco protoplasts, but is dispensible for nuclear localization of both proteins. The dominant and essential NLS in PS‐IAA4 and PS‐IAA6 overlap with a bipartite basic motif which is polymorphic and conserved in related proteins from other plant species, having the consensus sequence (KKNEK)KR‐X(24–71)‐(RSXRK)/(RK/RK). Both basic elements of this motif in PS‐IAA4, (KR‐X41‐RSYRK), function interdependently as a bipartite NLS. However, in PS‐IAA6 (KKNEKKR‐X36‐RKK) the upstream element of the corresponding motif contains additional basic residues which allow its autonomous function as an SV40‐type monopartite NLS. The spacer‐length polymorphism, X(24–70), in respective bipartite NLS peptides of several PS‐IAA4‐like proteins from Arabidopsis thaliana does not affect nuclear targeting function. The structural and functional variation of the bipartite basic motif in PS‐IAA4‐like proteins supports the proposed integrated consensus of NLS.