Complex regulation of the TIR1/AFB family of auxin receptors

Abstract

Auxin regulates most aspects of plant growth and development. The hormone is perceived by the TIR1/AFB family of F-box proteins acting in concert with the Aux/IAA transcriptional repressors. Arabidopsis plants that lack members of the TIR1/AFB family are auxin resistant and display a variety of growth defects. However, little is known about the functional differences between individual members of the family. Phylogenetic studies reveal that the TIR1/AFB proteins are conserved across land plant lineages and fall into four clades. Three of these subgroups emerged before separation of angiosperms and gymnosperms whereas the last emerged before the monocot-eudicot split. This evolutionary history suggests that the members of each clade have distinct functions. To explore this possibility in Arabidopsis, we have analyzed a range of mutant genotypes, generated promoter swap transgenic lines, and performed in vitro binding assays between individual TIR1/AFB and Aux/IAA proteins. Our results indicate that the TIR1/AFB proteins have distinct biochemical activities and that TIR1 and AFB2 are the dominant auxin receptors in the seedling root. Further, we demonstrate that TIR1, AFB2, and AFB3, but not AFB1 exhibit significant posttranscriptional regulation. The microRNA miR393 is expressed in a pattern complementary to that of the auxin receptors and appears to regulate TIR1/AFB expression. However our data suggest that this regulation is complex. Our results suggest that differences between members of the auxin receptor family may contribute to the complexity of auxin response.

Fig. 1.

TIR1/AFB gene expression involves posttranscriptional regulation. GUS expression in the cotyledons, young leaves (A–H), primary root tip, and young lateral roots (I–X) of 8-day-old transgenic seedlings. X-Gluc staining is representative of expression levels in seedlings containing pTIR1:GUS (A, I, and J), pTIR1:cTIR1:GUS (B, K, and L), pAFB1:GUS (C, M, and N), pAFB1:cAFB1:GUS (D, O, and P), pAFB2:GUS (E, Q, and R), pAFB2:cAFB2:GUS (F, S, and T), pAFB3:GUS (G, U, and V), and pAFB3:cAFB3:GUS (H, W, and X). Samples A–H, J–L, N–P, and R–T X-Gluc were stained overnight. Samples I, M, Q, and U–X were stained for 5 h.

Fig. 2.

miR393 influences the auxin response in the root. (A) Four-day-old seedlings grown on MS media were transferred to media containing 2,4-D. Root elongation was measured after an additional 4 days and expressed as a proportion of growth in the absence of auxin (Scale bars, SE). (B) Lateral root assay. Four-day-old seedlings grown on MS media were transferred onto media −/+ 2,4-D for an additional 9 days. The number of emerged lateral roots/mm primary root length was measured. (Scale bars, SE.) (C–E) Expression of pmiR393:GUS in the primary root tip (C) and lateral roots (D and E) of 10-day-old seedlings. (F) Expression of pTIR1:cTIR1:GUS in an emerging lateral root in a 10-day-old seedling. (G–I) Expression of pimR393a:GFP in the primary root tip region (G, arrow), root elongation zone (H), and beneath an emerging lateral root (I, asterisk) in 10-day-old seedlings.

Fig. 3.

AFB1 and AFB2 cannot replace TIR1 in tir1–1 plants. (A) Northern blot of RNA isolated from 9-day-old Col-O, tir1–1, tir1 pTIR1:cTIR1:GUS, tir1 pTIR1:cAFB1:GUS, and col pAFB1:cAFB1:GUS seedlings. Expression evaluated using P³²-labeled probes that bind the GUS coding region or the 3′-UTR of the TIR1 gene. rRNA levels are stained using EtBr. (B-G) GUS expression in 8-day-old seedlings containing tir1 TIR1p-cTIR1:GUS line 2 (B), tir1 TIR1p-cTIR1:GUS line 24 (C), tir1 TIR1p-cAFB1:GUS line 8 (D), tir1 TIR1p-cAFB1:GUS line 16 (E), tir1 TIR1p-cAFB2:GUS line 5 (F), and tir1 TIR1p-cAFB2:GUS line 6 (G) transgenes. (H) Root elongation as performed in Fig. 2A with 4-day-old seedling grown in the presence of 2,4-D. Numbers denote independent lines.

Fig. 4.

TIR1 and AFB2 exhibit a stronger interaction with Aux/IAA proteins than AFB1 and AFB3 in vitro. TIR1, AFB1, AFB2, and AFB3 proteins were synthesized in vitro and incubated with GST-IAA3 and GST-IAA7 proteins in the presence or absence of 100 μM of IAA. After pulldown reactions (PD), recovery of the F-box proteins was assessed by autoradiography (Top panels). Middle panel shows the input of in vitro synthethized TIR1, AFB1, AFB2, and AFB3 in the pulldown reactions, whereas Lower panels show the coomassie staining of entire pulldown reactions as loading control for GST-Aux/IAA proteins.