Do some IAA proteins have two repression domains?

Abstract

The plant hormone auxin regulates the transcription of specific genes through the interplay of Auxin Response Factors (ARFs) and Aux/IAA (IAA) repressors. We have recently shown that stabilized IAA repressors with identical amino acid substitutions in their conserved repression domains (i.e., domain I) confer either “low auxin” or “high auxin” phenotypes when the IAA proteins are constitutively expressed in transformed Arabidopsis plants. We have suggested that when domain I loses its capacity to repress, “high auxin” phenotypes generally result, but a subset of IAA proteins (e.g., IAA17) appear to contain a second repression domain resulting in the maintenance of “low auxin” phenotypes. Here we provide evidence for a second repression domain that lies between domains I and II in IAA7, an IAA repressor within the same clade as IAA17.

Aux/IAA (IAA) repressors interact with ARF activators on promoters of auxin response genes to bring about repression of gene expression when auxin concentrations in cells are below a threshold level.¹ When auxin levels increase, the IAA repressors are destroyed by the ubiquitin-proteasome pathway,² and auxin-response genes become derepressed/activated. Most IAA proteins contain an LxLxL motif (i.e., where L is leucine and x is another amino acid) in conserved domain I that functions as part of a EAR-like repression domain, although this motif may be more extensive in some IAA repressors (i.e., LxLxLxL or LxLxLxLxL).³

When the first leucine in an LxLxL motif is substituted with an alanine in domain I of IAA repressors such as IAA3, IAA6 or IAA19, the IAA proteins lose their ability to repress auxin response genes. When these stabilized proteins are constitutively expressed in transformed Arabidopsis plants, “high auxin” phenotypes are observed, and we have suggested a mechanism for how “high auxin” phenotypes arise in these plants.⁴ In contrast, when an identical substitution is made in domain I of IAA17, plants expressing a stabilized version of this protein have “low auxin” phenotypes. We have suggested that these “low auxin” phenotypes are observed because IAA17 and proteins within the same clade have a second LxLxL repression domain, which continues to function when the LxLxL motif in domain I is rendered nonfunctional.

Here, we have analyzed plants that constitutively express a stabilized IAA7 protein with an alanine substituted for the first leucine in the LxLxL motif of domain I. IAA7 is a close relative of IAA17, and the two proteins are found in the same clade within a phylogenetic tree of Arabidopsis IAA proteins.⁵ Figure 1 shows that both IAA7 and IAA17 contain an LxLxL motif in domain I as well as an LxLxL motif located between domains I and II. IAA14 and IAA16 also contain a second repression domain between domains I and II. This contrasts with IAA19 (see Fig. 1) and other Arabidopsis IAA proteins, which contains an LxLxL motif only in domain I.

Figure 1.

Diagrams of IAA proteins with positions of LxLxL motifs. IAA7, IAA17 and portions of IAA7 are colored in green, and IAA19 and portions of IAA19 are colored in orange. Amino acids within LxLxL motifs and conserved domain II are indicated in IAA7, IAA17 and IAA19. Amino acid substitutions in domains I and II are shown for IAA19mIA/IAA7mII, III, IV and IAA19mIB/IAA7mII, III, IV. Conserved domains I, II, III and IV are labeled within the ovals. IAAmImII mutations were made as described previously in reference ⁴ and ⁶. For domain swaps, IAA19mIA/IAA7mII, III, IV was constructed by replacing the first 19 amino acids of IAA7mImII with the first 19 amino acids of IAA19 containing a leucine to alanine substitution in domain I, and IAA19mIB/IAA7 mll, III, IV was constructed by replacing the first 76 amino acids of IAA7mImII with the first 64 amino acids of IAA19 containing a leucine to alanine substitution in domain I.

Constitutive expression of a stabilized IAA7 protein with a leucine to alanine substitution in domain I (i.e., IAA7mImII-1) results in stably transformed Arabidopsis plants with “low auxin” phenotypes similar to those observed previously for IAA17mImII-1 (Fig. 2).⁴ The “low auxin” phenotypes include seedlings with short hypocotyls and petioles, agravitropic roots, reduced numbers of lateral roots and reduced DR5rev:GFP expression in root tips. Adult plants were dwarfish and similar to plants previously described for IAA17mImII-1. These results indicate IAA7mImII-1, which contains two LxLxL motifs, behaves like IAA17mImII-1 in conferring “low auxin” phenotypes to plants constitutively expressing the transgene.

Figure 2.

Phenotypes of wild-type plants and plants expressing 35S:IAA7mImII-1, 35S:IAA19mImII-1, 35S:IAA19mIA/IAA7mII, III, IV and 35S:IAA19mIB/IAA7mII, III, IV. (A) Pictures of 7-day-old wild-type (Wt) and transformed seedlings. (B) DR5 reporter gene expression in wild-type and transformed seedlings. Transgenic plants expressing 35S:IAA19mImI-1, 35S:IAA19mIB/IAA7mII, III, IV were in a DR5:GUS background. 35S:IAA7mImI-1 and 35S:IAA19mIA/IAA7mII, III, IV were in a DR5rev:GFP background. (C) Five-week-old wild-type plant and plants expressing the 35S transgenes. The bar equals 2 cm.

To provide support for IAA7 containing a second repression domain located between domains I and II, we carried out domain swap experiments using IAA7mImII-1 and IAA19mImII-1. Figure 1 shows diagrams of these swaps where domain I of IAA19mI was substituted for domain I of IAA7mI (IAA19mIA/IAA7mII, III, IV) or the amino terminus of IAA19mI up to, but not including domain II replaced the equivalent portion of IAA7mI (IAA19mIB/IAA7mII, III, IV). With IAA19mIA/IAA7mII, III, IV the second LxLxL motif located between domains I and II of IAA7 was preserved, and Arabidopsis seedlings constitutively expressing a 35S:IAA19mIA/IAA7mII, III, IV transgene had “low auxin” phenotypes similar to plants expressing 35S:IAA7mImII-1 (Fig. 2). With IAA19mIB/IAA7mII, III, IV, the second LxLxL motif of IAA7 was eliminated, and seedlings expressing a 35S:IAA19mIB/IAA7mII, III, IV transgene had “high auxin” phenotypes, with long hypocotyls, epinastic cotyledons, short primary roots, and enhanced DR5:GUS expression compared to wild-type. These “high auxin” phenotypes of seedlings expressing the 35S:IAA19mIB/IAA7mII, III, IV transgene resembled those of seedlings expressing the 35S:IAA19mImII-1 transgene (Fig. 2A); however, the DR5:GUS staining in roots was much reduced in 35S:IAA19mIB/IAA7mII, III, IV seedlings compared to 35S:IAA19mImII-1 seedlings (Fig. 2B). The DR5:GUS results suggest that the IAA19mImII-1 “high auxin” phenotype is not entirely duplicated with IAA19mIB/IAA7mII, III, IV, and further investigation will be required to determine why the difference in root staining is observed when these modified IAA proteins are expressed in Arabidopsis seedlings.

The two domain swap experiments provide support for IAA7 having two repression domains, with one of these in domain I, and a second in a region between domains I and II. Although the domain swap results do not provide definitive evidence that the LxLxL motif between domains I and II is a functional repression domain, the swap experiments do indicate that the nonconserved region between domains I and II is a determinant in conferring the “low auxin” phenotyes observed in plants that express 35S:IAA7mImII-1. Furthermore, the domain swap experiments argue against domain II or a region carboxyl-terminal to domain II (e.g., domains III and IV) in IAA7 and IAA19 being the determinant that confers the “low auxin” and “high auxin” phenotypes with plants expressing 35S:IAA7mImII-1 and 35S:IAA19mImII-1, respectively. Thus, it is unlikely that protein stability or selectivity of interactions between the IAA proteins and ARFs plays a major role in conferring the “low auxin” and “high auxin” phenotypes that we have observed here and in previous exeriments.⁴

Future work will focus on carrying out similar domain swap experiments between IAA19mImII-1 and IAA17mImII-1 as well as experiments where both LxLxL motifs in IAA7 and IAA17 are mutated. Plants constitutively expressing stabilized versions of IAA7 and IAA17 with mutations in both repression domains would be expected to have “high auxin” phenotypes. The presence of two repression domains within a subclass of IAA proteins may increase the repressive capacity of these proteins in regulating the expression of auxin response genes during specific stages of plant growth and development.

Addendum to: Li H, Tiwari SB, Hagen G, Guilfoyle TJ. Identical amino acid substitutions in the repression domain of Aux/IAA proteins have contrasting effects on auxin signaling. Plant Physiol. 2011;55:1252–1263. doi: 10.1104/pp.110.171322.