Irrepressible, truncated Auxin Response Factors

Abstract

The molecularly well-characterized auxin signal transduction pathway involves two evolutionarily conserved families interacting through their C-terminal domains III and IV: the Auxin Response Factors (ARFs) and their repressors the Aux/IAAs, to control auxin-responsive genes, among them genes involved in auxin transport.^1,2 We have developed a new genetic tool to study ARF function. Using MONOPTEROS (MP)/ARF5, we have generated a truncated version of MP (MPΔ),³ which has lost the target domains for repression by Aux/IAA proteins. Besides exploring genetic interactions between MP and Aux/IAAs, we used this construct to trace MP’s role in vascular patterning, a previously characterized auxin dependent process.^4,5 Here we summarize examples of naturally occurring truncated ARFs and summarize potential applications of truncated ARFs as analytical tools.

Evolutionarily Conserved ARF Structural Modifications

Auxin facilitates ARF-dependent gene transcription by promoting the degradation of Aux/IAAs, which in turn relieves ARFs from negative regulation.⁶ The foundations for this model lie in at least one basic requirement: the domain structure of the ARFs and Aux/IAAs. While Aux/IAAs do not have the ability to bind DNA, ARFs are capable of binding through their DNA Binding Domain (DBD) to conserved TGTCTC Auxin Response Elements found in the promoters of auxin inducible genes.⁷ The DBD is flanked by the so-called middle region (MR), a variable stretch of amino acids, which determines whether an ARF protein acts as a repressor or activator of transcription.^8,9 Both ARFs and Aux/IAAs share and interact through the C-terminally located protein-protein interaction domains III and IV. Instead of DBD and MR, Aux/IAAs have two other conserved domains: domain I confers their repressor activity,¹⁰ and domain II is crucial for their degradation.^11-13 Interactions through common domains III and IV allow for a very large number of potential ARF-Aux/IAA heterodimers, which may account for a correspondingly large diversity of functions in regulating downstream targets in an auxin–dependent manner (Fig. 1 A and B).

Interestingly, there are naturally occurring truncated ARFs, lacking domains III and IV, which may regulate gene expression in ways that are uncoupled from the influence of auxin¹⁴ (Fig. 1D and E). To date, these have been identified in four species, and it seems that the numbers of C-terminally truncated ARFs increased during evolution, as flowering plants tend to encode more of them than non-flowering plants.^14-16 With one exception, all of the naturally occurring truncated ARFs appear to contain repression middle domains.¹⁶ In at least one case during early angiosperm evolution, in Amborellales and Nymphaeales, mutations leading to apparent C-terminal truncations of ARF3/ETTIN and ARF4 have occurred, but the functional consequences are unknown.¹⁷ When analyzed in Arabidopsis, the ARF3 truncation might be gratuitous, as ARF3′s ability to rescue the ett-1 mutation was only partially affected by the addition of domains III and IV.¹⁷ By contrast, ARF4 does require both domains for its activity. While the native full length ARF4 driven by the ARF3 promoter was able to complement the ett-1 mutation, the artificially generated ARF4 truncations (proETT::ARF4-trunc), failed to complement the ett-1 mutation.¹⁷

Figure 1. C-terminally truncated ARFs, model implications. (A-C) Auxin regulation of full-length ARFs. (A) In low auxin concentrations ARFs form dimers with Aux/IAAs through domains III and IV. As transcriptional repressors, Aux/IAA can interfere with the regulatory properties of ARFs. (B) Auxin promotes ubiquitination through SCF-TIR and degradation of Aux/IAAs, which releases ARFs from repression. (C) ARFs can dimerize with themselves or other ARFs, which may lead to complex and divergent regulatory properties in various cell types. (D-F) Regulatory properties of truncated ARFs. (D, E) In C-terminally truncated ARFs no ARF-Aux/IAA dimer is formed, and thus transcription of auxin dependent genes should be uncoupled from auxin influence. (F) However, C-terminal ARF-ARF interactions are also abolished which may lead to complex and difficult to predict regulatory consequences. (G) Alignment of naturally occurring and artificially generated C-terminally truncated ARFs in Arabidopsis.

In Arabidopsis, domains III and IV are absent or highly abnormal in four out of 22 ARFs¹⁴ (Fig. 1G) and it will be interesting to see whether the targets of those four ARFs are indeed not regulated by auxin. Further, there is a newly identified gain-of-function allele of MP, mp-abn,¹⁸ in which a nonsense codon just upstream of domain IV is expected to result in a truncated protein (Fig. 1G), leading to phenotypes related to those of MPΔ.^5,18 Interestingly, the leaf vasculature in both MPΔ and mp-abn is dramatically increased, opposite to vascular hypotrophy in mp loss-of-function mutant leaves.^5,18,19 Therefore, it will be interesting to see whether truncations in the remaining 17 Arabidopsis ARFs are associated with visible phenotypes and whether those are related to traits observed in other plant species. Given the obvious patterning potential of some ARFs, it is well possible that ARF truncations have important functions in shaping plant morphology.

The Analytical Power of Domain III-IV Deletions

Loss-of-function mutations in MP reduce leaf vasculature to an unparalleled degree and, conversely, MPΔ increases it strongly.^5,19 This is remarkable not only because it presents MP as a sufficient regulator of leaf vasculature, but also because it can be used to trace genetic interactions with other Aux/IAA and ARF proteins. In yeast-two-hybrid studies, Aux/IAA proteins interact readily with MP,²⁰ but genetic tests go further and explore which of these potential interactions are functionally relevant in the plant. Dominant mutations in Aux/IAA degrons render those proteins hyper-stable and constitutively diminish the function of their target ARFs, causing a phenotype similar to loss-of-function mutation in the targeted ARF. For example, leaf vasculature is strongly reduced in dominant iaa12/bodenlos(bdl) mutants,²¹ consistent with MP being a target of repression by IAA12/BDL.²² This previously proposed interaction is supported by the fact that MPΔ is epistatic over bdl, causing increased vascularization in narrow leaves also in the genetic background of bdl.⁵

The removal of domains III and IV from MP not only abolishes repressive interactions with Aux/IAAs, but should also eliminate ARF-ARF homo or heterodimerization, which are mediated by the same domains (Fig. 1Cand F). MP has been shown to interact with itself and with at least one other activating ARF (ARF7) in yeast two-hybrid screens,²³ but one would expect that MPΔ cannot dimerize with itself or its natural interactors and may thus act as a monomer. As such, it might again be used as a genetic probe to search for functionally relevant interactors among the ARFs. For patterning processes, dimerization with ARF7 seems not to be important, as there are no corresponding defects in arf7 mutants.^23-26 But MPΔ provides only partial MP function in mp mutant background and it remains to be seen whether some of the residual defects correspond to defects associated with (possibly multiple) loss-of-function mutations in other ARFs. Should such defects be observed, it would be a testable hypothesis that the respective ARFs need to form heterodimers with MP to regulate the affected processes. To sum, MPΔ, and by extension other truncated ARFs, could be suitable genetic tools to sort out functionally relevant interactions between ARFs and Aux/IAAs as well as within the ARF family from the otherwise unwieldy number of possible combinations.

The Regulatory Relevance of Domain III-IV Deletions in Vascular Patterning

In leaf primordia, pre-procambial cell strands are selected from a field of isodiametrical subepidermal cells guided by a point of high auxin accumulation in the epidermis (an auxin convergence point, or point of internalization of epidermal auxin to subepidermal layers).¹ From the convergence point, auxin is believed to drain basipetally, in canals of cells with enhanced auxin conductivity, specifying vein precursors which will later turn on pre-procambial and procambial molecular markers.²⁷ This auxin-dependent, self-organizing process seems to critically depend on MP as mp mutants show early embryonic defects in cell axialization and, post-embryonically, severely reduced vasculature in lateral organs.¹⁹

Consistent with this view, expression of some of the earliest molecular markers of vein formation are auxin dependent and a key gene required for pre-procambial selection and procambium development, ATHB8, is under direct control of MP.²⁸ Comparison of expression patterns of MP, ATHB8 and PIN1 (schematic in Fig. 2) suggest that narrow strips of ATHB8-expressing cells emerge within a wide MP expression domain (Fig. 2). They may reflect narrow strips of late PIN1 expression, as this precedes ATHB8 expression and PIN1 expression advances from a wider domain toward a slender strip of cells.²⁹ Several mechanisms have been proposed for the selective expression of ATHB8 in a subset of MP expressing cells, including the presence of another procambial regulator.³⁰ Alternatively, auxin itself, channeled into narrowing lines of cells with elevated auxin concentrations, could be this co-factor. In this case, one would expect that MP protein is activated (i.e., not repressed by Aux/IAA proteins) only in this subset of its expression domain. Results with MPΔ provide hints in both directions. The expression domains of PIN1 and ATHB8 are not perfectly congruent with those of MPΔ⁵, supporting the assumption of further regulators, but they are nevertheless clearly expanded in space and duration of expression in MPΔ, which supports that activated MP has a fairly profound and direct impact on ATHB8 and PIN1 expression. Direct simultaneous visualization of all three proteins in natural and experimentally altered conditions will be required to resolve this issue.

Figure 2. Schematic expression patterns of MP, ATHB8 and PIN1 in young and older leaf primordia in WT and MPΔ. (drawn after refs. 5, 28).

In summary, as a tool for investigating developmental processes under MP control, truncated MP seems to be more powerful than semi-ubiquitous MP overexpression.²³ This suggests that similar constructs for other ARFs should reveal new regulatory roles of those proteins that, because of ARF redundancy, would not have been inferred from their loss-of-function phenotypes.

Krogan NT, Ckurshumova W, Marcos D, Caragea AE, Berleth T. Deletion of MP/ARF5 domains III and IV reveals a requirement for Aux/IAA regulation in Arabidopsis leaf vascular patterning. New Phytol. 2012;194:391–401. doi: 10.1111/j.1469-8137.2012.04064.x.