Session 9: Growth Regulators 1

Chair: Joe Kieber, University of Illinois at Chicago


Several recent advances concerning ethylene and auxin perception and signal transduction were presented in this session. The session began with a discussion of work from my laboratory that focused on CTR1. CTR1 was identified by five recessive mutations that resulted in constitutive ethylene responses. The CTR1 gene was cloned using a T-DNA tagged allele and found to be most similar to the Raf family of Ser/Thr protein kinases. CTR1 was expressed using a baculovirus system and demonstrated to have intrinsic Ser/Thr protein kinase activity. Expression of GUS or luciferase reporter genes fused to CTR1 upstream and downstream sequences was inducible by ethylene. However, CTR1 mRNA was found not to be ethylene-inducible, which suggests that CTR1 may be post-transciptional modified or that the reporters are inappropriately expressed. A second project focused on the characterization of mutants affecting the regulation of ethylene biosynthesis. Five such ethylene overproducing (Eto) loci were identified and characterized. These Eto loci define elements involved in the circuitry regulating the production of ethylene. These mutations specifically affected etiolated seedlings, suggesting that there may be independent elements regulating ethylene biosynthesis in seedlings and adult plants.

Eric Schaller from Tony Bleecker's lab at the University of Wisconsin presented work on the structural analysis of the ETR1. ETR1 was identified as a dominant mutation conferring resistance to exogenous ethylene and shown to encode a protein with homology to prokaryotic two-component regulatory proteins. Yeast expressing wild-type ETR1 bound 14C-ethylene in saturable and reversible manner and with an affinity consistent with the concentration required for a number of Arabidopsis ethylene responses, including the seedling triple response. Yeast expressing a mutant ETR1 protein (etr1-1) failed to display ethylene binding above background. etr1 mutant plants show a strong reduction in ethylene binding (80%) and genetic epistasis experiments suggest that ETR1 acts very early in ethylene signaling. Taken together, these data provide compelling evidence that ETR1 functions as an ethylene receptor and as such is the first definitive identification of a plant hormone receptor.

Vered Raz from Joe Ecker's lab at University of Pennsylvania presented work on differential growth in Arabidopsis seedlings. The plant hormones ethylene and auxin regulate cell elongation and act together, in dark grown seedlings, in the formation of the apical hook. Several mutants were identified that show no differential growth of the hypocotyl, including hookless1 and mutants in auxin response such as axr1 and hls3. The expression of genes that are differentially expressed in the apical hook was studied in the various mutants using GUS fusions as well as whole mount in situ studies. This combination of genetic and molecular analysis should help to understand the roles of ethylene and auxin on apical hook development.

These talks as well as other work suggests that many of the genes involved in ethylene perception have been identified. The challenge of the near future will be to characterize the biochemical activity of the cognate protein products and to understand how these elements interact to perceive ethylene and transduce the signal to bring about the appropriate responses.

The second set of talks focused on auxin signaling. Malcolm Bennett, from the University of Warwick described the molecular characterization of the AUX1 gene. aux1 mutations are recessive and confer resistance to exogenously added auxin as well as to ethylene. aux1 mutants also exhibit agravitropic root growth. It was suggested that AUX1 may be involved in crosstalk between the ethylene and auxin signaling pathways. The AUX1 gene was cloned via a T-DNA tagged allele and found to encode a novel protein. Hydropathy analysis of the predicted protein indicates that there are 7-10 transmembrane spanning domains and some regions of AUX1 show homology to 7-pass serpentine receptors. Southern analysis of Arabidopsis genomic DNA revealed multiple bands, suggesting that AUX1 may be a member of a gene family. When expressed in vitro, the protein runs as an aggregate on SDS PAGE gels. In situ hybridization experiments demonstrated that AUX1 mRNA is expressed in all cells of the root apex with the exception of the columella cells. It was suggested that this pattern indicates that AUX1 may play a role in the transduction or response to gravity stimuli as opposed to gravity perception.

Stephan Abel from the PGEC described work on early auxin-regulated gene expression in Arabidopsis. PS-IAA4 was originally isolated as a gene whose transcription is turned on very early after auxin treatment in peas. A large family of homologs were isolated from Arabidopsis (IAA1-IAA15). All the Arabidopsis genes contain the four conserved and one invariant region present in the pea genes. The proteins all contain nuclear localization signals and similarity to prokaryotic repressors was noted. An extensive description of the kinetics of expression of the genes after auxin treatment was presented. As expected for primary response genes, expression of some genes is induced very early after auxin application and induction is not blocked by cyclohexamide treatment. In fact, cyclohexamide treatment actually induces transcription of some genes by itself, suggesting these are under the control of a short-lived negative repressor. These IAA genes may be responsible for primary events in auxin perception.

Max Ruegger from Mark Estelle's lab at Indiana University discussed the characterization of mutants with altered responses to the auxin transport inhibitors TIBA and NPA. Seven loci were identified that are resistant to these transport inhibitors (tir1-tir7), several of which show defects in auxin-related processes. The TIR1 locus was clone via T-DNA tagging and it appears to encode a novel protein.

Karin Johnson from Dieter Soll's lab at Yale presented work on the rgr1 mutant. This mutant shows reduced gravitropic response and reduced sensitivity towards exogenous auxins and auxin transport inhibitors; molecular analysis of RGR1 is still in progress. The rcn1 mutant was isolated in a screen for resistance to the auxin transport inhibitor NPA. RCN1 shows homology to the A regulatory subunit of protein phosphatase 2A.