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.
Morgan, K.E.; Zarembinski, T.I.; Theologis, A.; Abel, S. Biochemical characterization of recombinant polypeptides corresponding to the predicted ßαα-fold in Aux/IAA proteins FEBS Letters 454, 283-287, (1999)
The plant hormone indoleacetic acid (IAA or auxin) transcriptionally activates a select set of early genes. The Auxl IAA class of early auxin-responsive genes encodes a large family of short-lived, nuclear proteins. Aux/IAA polypeptides homo-and heterodimerize, and interact with auxin-response transcription factors (ARFs) via C-terminal regions conserved in both protein families. This shared region contains a predicted βαα motif similar to the prokaryotic β-Ribbon DNA binding domain, which mediates both protein dimerization and DNA recognition. Here, we show by circular dichroism spectroscopy and by chemical cross-linking experiments that recombinant peptides corresponding to the predicted βαα region of three Aux/IAA proteins from Arabidopsis thaliana contain substantial α-helical secondary structure and undergo homo- and heterotypic interactions in vitro. Our results indicate a similar biochemical function of the plant βαα domain and suggest that the βαα fold plays an important role in mediating combinatorial interactions of Aux/IAA and ARF proteins to specifically regulate secondary gene expression in response to auxin.
Wong, L.M.; Abel, S.; Shen, N.; de la Foata, M.; Mal, Y.; Theologis, A. Differential activation of the primary auxin response genes, PS-IAA4/5 and PS-IAA6, during early plant development. Plant Journal 9, 587-599, (1996)
The plant growth hormone auxin typified by indoleacetic acid (IAA) transcriptionally activates early genes in pea, PS-IAA4/5 and PS-IAA6, that are members of a multigene family encoding short-lived nuclear proteins. To gain first insight into the biological role of PS-IAA4/5 and PSIAA6, promoter-β-glucuronidase (GUS) gene fusions were constructed and their expression during early development of transgenic tobacco seedlings was examined. The comparative analysis reveals spatial and temporal expression patterns of both genes that correlate with cells, tissues, and developmental processes known to be affected by auxin. GUS activity in seedlings of both transgenic lines is located in the root meristem, sites of lateral root initiation and in hypocotyls undergoing rapid elongation. In addition, mutually exclusive cell-specific expression is evident. For instance, PS-IAA4/5GUS but not PS-IAA6GUS is expressed in root vascular tissue and in guard cells, whereas only PS-IAA6GUS activity is detectable in glandular trichomes and redistributes to the elongating side of the hypocotyl upon gravitropic stimulation. Expression of PS-IAA4/5 and PS-IAA6 in elongating, dividing, and differentiating cell types indicates multiple functions during development. The common and yet distinct activity patterns of both genes suggest a combinatorial code of spatio-temporal co-expression of the various PS-IAA4/ 5-like gene family members in plant development that may mediate cell-specific responses to auxin.
Abel, S.; Ballas, N.; Wong, L-M.; Theologis, A. DNA elements responsive to auxin Bio Essays 18(8), 647-654, (1996)
Genes induced by the plant hormone auxin are probably involved in the execution of vital cellular functions and developmental processes. Experimental approaches designed to elucidate the molecular mechanisms of auxin action have focused on auxin perception, genetic dissection of the signaling apparatus and specific gene activation. Auxin-responsive promoter elements of early genes provide molecular tools for probing auxin signaling in reverse. Functional analysis of several auxin-specific promoters of unrelated early genes suggests combinatorial utilization of both conserved and variable elements. These elements are arranged into autonomous domains and the combination of such modules generates uniquely composed promoters. Modular promoters allow for auxin-mediated transcriptional responses to be revealed in a tissue- and development-specific manner.
Abel, S.; Theologis, A. Early genes and auxin action Plant Physiology 111, 9-17, (1996)
Abel, S.; Theologis, A. A polymorphic bipartite motif signals nuclear targeting of early auxin- inducible proteins related to PS-IAA4 from pea (Pisum sativum) Plant Journal 8, 87-96, (1995)
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(2471)-(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(2470), 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.
Abel, S.; Nguyen, M.D.; Theologis, A. The PS-IAA4/5-like family of early auxin-inducible mRNAs in Arabidopsis thaliana Journal of Biological Chemistry 270, 19093-19099, (1995)
1-Aminocyclopropane-1-carboxylic acid (ACC) synthase is the key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene. The enzyme is encoded by a divergent multigene family in Arabidopsis thaliana, comprising at least five genes, ACS1-5 (Liang, X., Abel, S., Keller, J. A., Shen, N. F., and Theologis, A.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11046-11050). In etiolated seedlings, ACS4 is specifically induced by indoleacetic acid (IAA). The response to IAA is rapid (within 25 min) and insensitive to protein synthesis inhibition, suggesting that the ACS4 gene expression is a primary response to IAA. The ACS4 mRNA accumulation displays a biphasic dose-response curve which is optimal at 10 μM of IAA. However, IAA concentrations as low as 100 nM are sufficient to enhance the basal level of ACS4 mRNA. The expression of ACS4 is defective in the Arabidopsis auxin-resistant mutant lines axr1-12, axr2-1, and aux1-7. ACS4 mRNA levels are severely reduced in axr1-12 and axr2-1 but are only 1.5-fold lower in aux1-7. IAA inducibility is abolished in axr2-1. The ACS4 gene was isolated and structurally characterized. The promoter contains four sequence motifs reminiscent of functionally defined auxin-responsive cis-elements in the early auxin-inducible genes PS-IAA4/5 from pea and GH3 from soybean. Conceptual translation of the coding region predicts a protein with a molecular mass of 53,795 Da and a theoretical isoelectric point of 8.2. The ACS4 polypeptide contains the 11 invariant amino acid residues conserved between aminotransferases and ACC synthases from various plant species. An ACS4 cDNA was generated by reverse transcriptase-polymerase chain reaction, and the authenticity was confirmed by expression of ACC synthase activity in Escherichia coli.
Abel, S.; Theologis, A. Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression Plant Journal 5, 421-427, (1994)
An improved protocol is reported to isolate and transiently transform mesophyll protoplasts of Arabidopsis thaliana. Transfected leaf protoplasts support high levels of expression of the bacterial reporter gene coding for β-glucuronidase (GUS), under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transient expression of GUS activity was monitored spectrophotometrically and reached a maximum between 18 and 48 h after polyethylene glycol (PEG)-mediated DNA uptake. Histochemical staining for GUS activity revealed reproducible transformation frequencies between 40 and 60%, based on the number of protoplasts survived. To demonstrate the applicability of the transient expression system, the subcellular localization of GUS proteins tagged with different nuclear polypeptides was studied in transfected mesophyll protoplasts, revealing nuclear compartmentalization of the chimeric GUS enzymes. Furthermore, Arabidopsis mesophyll protoplasts support auxin-mediated induction of chloramphenicol acetyl-transferase (CAT) activity when transfected with a transcriptional fusion between the CAT reporter gene and the early auxin-inducible PS-IAA4/5 promoter. Hence, the method allows in vivo analysis of promoter activity and subcellular localization of fusion proteins in a homologous transformation system.
Abel, S.; Oeller, P.W.; Theologis, A. Early auxin-induced genes encode short-lived nuclear proteins PNAS USA 91, 326-330, (1994)
The plant growth hormone indoleacetic acid (IAA) transcriptionally activates gene expression in plants. Some of the genes whose expression is induced by IAA encode a family of proteins in pea (PS-IAA4 and PS-IAA6) and Arabidopsis (IAA1 and IAA2) that contain putative nuclear localization signals that direct a beta-glucuronidase reporter protein into the nucleus. Pulse-chase and immunoprecipitation experiments have defined the t1/2 of the PS-IAA4 and PS-IAA6 proteins to be 8 and 6 min, respectively. Their most prominent feature is the presence of a beta alpha alpha motif similar to the beta-sheet DNA-binding domain found in prokaryotic repressors of the Arc family. Based on these data, we suggest that plant tissues express short-lived nuclear proteins as a primary response to IAA. We propose that these proteins act as activators or repressors of genes responsible for mediating the various auxin responses.