IN THIS ISSUE

Auxin Signaling: Homing in with Targeted Genetics

Auxins influence numerous events during plant growth and development, including cell division and elongation, differentiation, apical dominance, tropisms, senescence, abscision, and flowering. With such a diverse range of auxin-mediated effects, and given the strong interactions between auxins and other plant hormones, unraveling auxin biology was always going to be difficult. Even so, it is still something of a surprise that after several decades of research, we have achieved only a rudimentary understanding of how auxins are synthesized and transported around the plant, and how they act at the cellular level to influence plant growth and development.

Two approaches have yielded the most useful information—a search that began almost 20 years ago for genes that are upregulated by auxin (Abel and Theologis 1996 ), and the more recent generation and characterization of Arabidopsis mutants with defects in their auxin responses (Hobbie 1998 ). These strategies now appear to be converging upon a complex control system for auxin signaling in which the stability of target proteins is governed by a ubiquitin/ubiquitin-like protein degradation pathway.

Complexity is a hallmark of plant hormone signaling mechanisms, and although the first wave of genetic analyses generated significant progress, it is becoming increasingly clear that there is now a need to develop and use more refined experimental tools. On pages 1649–1662 of this issue, Theologis and his colleagues (Oono et al. 1998 ) take an important step along this path. They describe the use of a potentially very precise genetic approach to focus in on specific components of the auxin signaling pathway that act in trans on cis elements in the promoters of auxin-responsive genes.

Early Auxin–Responsive Genes and Auxin Signaling
Changes in gene expression are among the earliest responses to applied auxin. Some genes are induced within minutes and without the need for de novo protein synthesis (Abel and Theologis 1996 ). In fact, because early auxin–responsive genes can also be induced by the protein synthesis inhibitor cycloheximide, it is thought that a rapidly turning over pool of repressors of transcription may be involved in regulating the expression of these genes. But what do the products of these rapidly induced/derepressed genes do?

The best-studied family of early auxin–responsive genes consists of the Aux/IAA genes, some 25 of which have been identified in Arabidopsis. Aux/IAA proteins contain nuclear localization signals and four conserved domains, one of which, with some adjacent amino acids, is predicted to form a structure similar to a DNA binding domain found in the prokaryotic transcription repressors Arc and MetJ. Moreover, nuclear targeting of some Aux/IAA proteins has been demonstrated; some are very low in abundance; and some have half-lives of 5 to 10 min. All of these features suggest that at least some of the Aux/IAA proteins are involved in controlling transcription (Guilfoyle 1998 ).

Where and how they may function, however, are less clear. The expression of some Aux/IAA family members is induced within 5 min of auxin treatment, whereas that of others is induced only after 20 min or more. Aux/IAA genes show different induction kinetics, dose responses, and tissue-specific patterns of expression. Furthermore, Aux/IAA proteins can form homodimers and heterodimers with one another, and they can also form heterodimers with a family of auxin response factors (ARFs) that are known to bind to auxin response elements in the promoters of early auxin–responsive genes. This apparent functional diversity may be essential for regulation of the secondary phase of auxin-responsive gene expression that is associated with the diverse downstream biological responses.

Auxin Response Mutants
Until now, screens for Arabidopsis mutants that exhibit aberrant responses to auxin have been based largely on simple morphological assessment of mutagenized populations. For example, auxin-resistant mutants at eight loci (aux1; axr1, 2, 4, 5, and 6; Dwf; and tir1) have been isolated by their ability to elongate roots on normally prohibitive concentrations of auxin (Hobbie 1998 ). Some of the corresponding genes have been cloned. AUX1 encodes a putative auxin influx carrier (Bennett et al. 1996 ); AXR1 encodes a protein similar to yeast ENR2, which is a component in a pathway that ensures efficient ubiquination of the cyclin-dependent kinase inhibitor Sic1p (Leyser 1998 ); and TIR1 encodes an F-box protein that is similar to another component of the Sic1p ubiquination pathway (Ruegger et al. 1998 ). Taken together, it is therefore likely that AXR1 and TIR1 may possess related functions in regulating the turnover of proteins in an auxin-dependent manner. Possible targets for such turnover include proteins involved in auxin-mediated regulation of the cell cycle, the Aux/IAA proteins, and other unidentified auxin-signaling proteins.

Mutations at a single locus, axr3, confer auxin overresponsiveness in a range of bioassays, and they trigger ectopic expression from the auxin-inducible SAUR-AC1 promoter (Leyser et al. 1996 ). AXR3 corresponds to one of the Aux/IAA genes, IAA17, and the axr3 mutations occur in one of the domains conserved in this family of proteins (Rouse et al. 1998 ). This important finding provides further strong evidence implying that Aux/IAA proteins can act as mediators of auxin signaling. Indeed, the pictures emerging from these genetic and molecular studies suggest that auxin regulates the expression of a network of transcription factors that are components of the signaling pathway, and that an additional point of control involves auxin regulation of protein turnover.

Targeted Genetics: A New Wave of Mutants
It has been recognized for some time that increasingly sophisticated screens will be required to identify some components of plant hormone signaling networks. Oono et al. report the results of a different kind of targeted genetic screen aimed at further elaborating the role and regulation of early auxin–responsive genes. The authors' strategy was to screen an ethane methylsulfonate–mutagenized population of Arabidopsis containing an auxin-responsive promoter–ß-glucuronidase (GUS) construct. Oono et al. chose the auxin-responsive domains A and B (AuxRD A and AuxRD B; Ballas et al. 1995 ) from an early auxin–induced pea gene, PS-IAA4/5. This choice was made on the basis of their initial tests, which demonstrated that a construct containing a single copy of each of these domains (the BA–GUS construct) would allow a robust screen for mutants with defects in auxin-mediated GUS expression in roots. Oono et al. went to considerable lengths to confirm the fidelity of the reporter construct in a chosen transgenic line that showed strong and consistent auxin-induced GUS expression (i.e., two- to 15-fold auxin-mediated stimulation of GUS expression in the elongating zone of the root). Others contemplating similar screens might be well advised to take the same care, if only to avoid unnecessary tangents.

An elegant, nondestructive screen yielded the age1 and age2 lines. Preliminary histochemical analysis of GUS activity in age1 revealed higher levels in the elongating zone of the root in response to 10^-8 M IAA compared with the corresponding "wild-type" (i.e., BA–GUS) plants. This tissue-specific enhanced auxin sensitivity was also accompanied by higher GUS activity in other parts of the seedling.

In contrast, GUS activity in the elongating zone of roots of the age2 mutant was no different from that of the wild type when treated with 10^-8 or 10^-7 M IAA. However, age2 seedlings had elevated levels of GUS in the vascular tissue of the upper root and in the hypocotyl that were independent of applied auxin. The targeted approach therefore appears to have worked beautifully, yielding mutants with GUS activity characteristics that fit well with current understanding of the complex auxin-mediated and tissue-specific regulation of early auxin–responsive genes.

The prediction that the AGE1 and AGE2 genes are involved in the regulation of early auxin–responsive genes begs the question, do the mutations affect expression of the endogenous Aux/IAA genes? The answer is that they do, but in a complex way. Again, this is probably to be expected. In age1 seedlings, transcript levels of three early auxin–responsive genes, IAA1, IAA4, and IAA5, increase after auxin application but seemingly not to the same extent that they do in wild-type plants. This apparent reduction in response capacity will doubtless be the subject of further studies, and it highlights the urgent need to fully define the Arabidopsis Aux/IAA gene promoters.

In the absence of auxin, age2 mutants accumulate higher levels of IAA1 and IAA12 transcripts compared with those that accumulate in the wild type, but similar levels of IAA4 transcripts. Thus, age2 mutants exhibit an overexpession phenotype with respect to two early auxin–inducible genes and a normal expression phenotype for a third.

The age mutants possess morphological phenotypes that are characteristic of specific defects in auxin signaling. Both are short and bushy, but age1 plants also have altered root and leaf morphology, and they exhibit alterations in flowering. Significantly, it is clear that neither of the age mutants would have been detected in screens for auxin inhibition of root growth. The identity of the AGE1 and AGE2 genes awaits further investigation. However, with their map locations already defined, it is clear that they do not correspond to other known auxin-related mutants, and the Theologis laboratory is no doubt hot on their trail.

In this exciting development, Oono et al. have extended their earlier studies on early auxin–responsive genes by targeting a mutant screen on two important elements in the promoter of one of them. The two mutants characterized so far look intriguing, and the prospects that other mutants will be identified by using this approach and derivatives of it are seemingly good. Indeed, these targeted approaches already are being pursued by other investigators studying different aspects of plant hormone signaling, and the indications are that we can expect a range of new plant hormone signaling mutants to emerge from these experiments. Time and the molecular characterization of the affected genes will tell us just how precise the new targeted genetics approach really is.

REFERENCES

Abel, S., and Theologis, A. (1996) Early genes and auxin action. Plant Physiol. 111:9-17[CrossRef][ISI][Medline].

Ballas, N., Wong, L.M., Ke, M., and Theologis, A. (1995) Two auxin-responsive domains interact positively to induce expression of the early indoleacetic acid–inducible gene PS-IAA4/5. Proc. Natl. Acad. Sci. USA 92:3483-3487[Abstract/Free Full Text].

Bennett, M.J., Marchant, A., Green, H.G., May, S.T., Ward, S.P., Millner, P.A., Walker, A.R., Schulz, B., and Feldmann, K.A. (1996) Arabidopsis AUX1 gene: A permease-like regulator of root gravitropism. Science 273:948-950[Abstract].

Guilfoyle, T.J. (1998) Aux/IAA proteins and auxin signal transduction. Trends Plant Sci. 3:205-207.

Hobbie, L.J. (1998) Auxin: Molecular genetic approaches in Arabidopsis.. Plant Physiol. Biochem. 36:91-102[CrossRef][ISI].

Leyser, O. (1998) Auxin signalling: Protein stability as a versatile control target. Curr. Biol. 8:R305-R307[CrossRef][Medline].

Leyser, H.M.O., Pickett, F.B., Dharmasiri, S., and Estelle, M. (1996) Mutations in the AXR3 gene of Arabidopsis result in altered auxin response including ectopic expression from the SAUR-AC1 promoter. Plant J. 10:403-413[CrossRef][ISI][Medline].

Oono, Y., Chen, Q.G., Overvoorde, P.J., Köhler, C., and Theologis, A. (1998) age mutants of Arabidopsis exhibit altered auxin-regulated gene expression. Plant Cell 10:1649-1662[Abstract/Free Full Text].

Rouse, D., Mackay, P., Stirnberg, P., Estelle, M., and Leyser, O. (1998) Changes in auxin response from mutations in an AUX/IAA gene. Science 279:1371-1373[Abstract/Free Full Text].

Ruegger, M., Dewey, E., Gray, W.M., Hobbie, L., Turner, J., and Estelle, M. (1998) The TIR1 protein of Arabidopsis functions in auxin response and is related to human SKP2 and yeast Grr1p. Genes Dev. 12:198-207[Abstract/Free Full Text].

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