Auxin is a central hormone that exerts pleiotropic effects on plant growth including the development of roots, shoots, flowers and fruit. The perception and signaling of the plant hormone auxin rely on the cooperative action of several components, among which auxin/indole-3-acetic acid (Aux/IAA) proteins play a pivotal role. In this study, we identified and comprehensively analyzed the entire Aux/IAA gene family in tomato (Solanum lycopersicum), a reference species for Solanaceae plants, and the model plant for fleshy fruit development. Functional characterization using a dedicated single cell system revealed that tomato Aux/IAA proteins function as active repressors of auxin-dependent gene transcription, with, however, different Aux/IAA members displaying varying levels of repression. Phylogenetic analysis indicated that the Aux/IAA gene family is slightly contracted in tomato compared with Arabidopsis, with a lower representation of non-canonical proteins. Sl-IAA genes display distinctive expression pattern in different tomato organs and tissues, and some of them display differential responses to auxin and ethylene, suggesting that Aux/IAAs may play a role in linking both hormone signaling pathways. The data presented here shed more light on Sl-IAA genes and provides new leads towards the elucidation of their function during plant development and in mediating hormone cross-talk.

The nucleotide sequence data from this article can be found in the Genbank/EMBL data libraries under the following accession numbers: JN379431 (Sl-IAA1), JN379432 (Sl-IAA2), JN379433 (Sl-IAA3), JN379434 (Sl-IAA4), JN379435 (Sl-IAA7), JN379436 (Sl-IAA8), JN379437 (Sl-IAA9), JN379438 (Sl-IAA11), JN379439 (Sl-IAA12), JN379440 (Sl-IAA13), JN379441 (Sl-IAA14), JN379442 (Sl-IAA15), JN379443 (Sl-IAA16), JN379444 (Sl-IAA17), JN379445 (Sl-IAA19), JN379446 (Sl-IAA21), JN379447 (Sl-IAA22), JN379448 (Sl-IAA23), JN379449 (Sl-IAA26), JN379450 (Sl-IAA27), JN379451 (Sl-IAA29), JN379452 (Sl-IAA32), JN379453 (Sl-IAA33), JN379454 (Sl-IAA35), JN379455 (Sl-IAA36).

The perception and signaling of the plant hormone auxin involve the cooperative action of several components, among which auxin/indole-3-acetic acid (Aux/IAA) proteins play a pivotal role. Aux/IAA proteins were shown to be a direct target of the auxin transport inhibitor response1 (TIR1) and of its paralogs AUXIN RECEPTOR F-BOX/AFB1 and AFB3F-box receptors (AFBs) (Dharmasiri et al. 2005a, Dharmasiri et al. 2005b, Kepinski and Leyser 2005, Tan et al. 2007). Binding of auxin to its receptors leads to the degradation of Aux/IAA proteins. This auxin-dependent proteolysis releases auxin response factors (ARFs) that otherwise remain trapped via their binding to Aux/IAA partners. The Aux/IAA genes represent a class of primary auxin-responsive genes which were shown to be, in the majority, rapidly induced by auxin (Theologis et al. 1985, Oeller et al. 1993, Yamamoto and Yamamoto 1998). Aux/IAAs are described as short-lived and nuclear-localized proteins (Hagen and Guilfoyle 2002, Liscum and Reed 2002), and biochemical and genetic studies indicated that they generally function as transcriptional repressors of auxin-regulated genes (Tiwari et al. 2001, Tiwari et al. 2004). Canonical Aux/IAA proteins share four conserved amino acid sequence motifs known as domains I, II, III and IV, although several proteins lacking one or more of these domains are also included in the family (Reed 2001). Domain I is a repressor domain that contains a conserved leucine repeat motif (LxLxLx) similar to the so-called EAR (ethylene-responsive element-binding factor-associated amphiphilic repression) domain (Tiwari et al. 2004). Domain I is also required for the recruitment of the transcriptional co-repressor TOPLESS (Szemenyei et al. 2008). Domain II confers protein instability, leading to rapid degradation of Aux/IAA through the interaction with the F-box protein TIR1 (a component of the SCFTIR1 ubiquitin ligase complex) (Dharmasiri et al. 2005a, Dharmasiri et al. 2005b, Kepinski and Leyser 2005, Tan et al. 2007). In fact, mutations in Aux/IAA domain II resulted in increased protein accumulation leading to auxin-related developmental phenotypes (Reed 2001, Liscum and Reed 2002, Uehara et al. 2008). The C-terminal domains III and IV are shared with ARF proteins, and are known to promote homo- and heterodimerization of Aux/IAA polypeptides, as well as interaction between Aux/IAAs and ARFs (Remington et al. 2004, Overvoorde et al. 2005). Aux/IAAs impact the transcriptional activity of target genes through the binding to their ARF partners. ARF proteins are capable of binding to the auxin-responsive cis-element (AuxRE) present upstream of the coding sequence of auxin-responsive genes (Ulmasov et al. 1997). Depending on the amino acid composition of their variable internal region, the ARF proteins can either activate or repress gene transcription (Ulmasov et al. 1999). Most of our understanding of the diverse roles of Aux/IAAs in planta is based on the characterization of gain-of-function mutants in the Arabidopsis model plant, whereas phenotypes associated with loss of function are scarce probably due to important functional redundancy among Aux/IAA family members (Overvoorde et al. 2005). In contrast, down-regulation of various Aux/IAA genes in the Solanaceae species results in visible and distinct phenotypes. Down-regulation of the tomato (Solanum lycopersicum) Sl-IAA9 resulted in pleiotropic phenotypes, consistent with its ubiquitous expression pattern (Wang et al. 2005). Sl-IAA9-inhibited lines also displayed some specific phenotypes such as entire leaves and parthenocarpic fruit, indicating that Sl-IAA9 is a key regulator of fruit set and leaf morphogenesis (Wang et al. 2005, Wang et al. 2009). Down-regulation of another Aux/IAA gene in tomato, Sl-IAA3, results in both auxin- and ethylene-associated phenotypes including altered apical dominance, lower auxin sensitivity, exaggerated apical hook curvature in the dark and reduced petiole epinasty in the light, thus revealing new roles for Aux/IAA genes (Chaabouni et al. 2009a). These data position Sl-IAA3 at the crossroads of auxin and ethylene signaling in tomato (Chaabouni et al. 2009b). More recently, it was shown that Sl-IAA15 is involved in trichome development as Sl-IAA15-down-regulated lines display strong reduction of type I, V and VI trichomes (Deng et al. 2012). Likewise, suppression of St-IAA2 in Solanum tuberosum results in clear phenotypes including increased plant height, petiole hyponasty and curvature of growing leaf primordia in the shoot apex (Kloosterman et al. 2006). These data do not support the functional redundancy among Aux/IAA genes generally described in the plant model Arabidopsis and clearly emphasize the need to widen the functional characterization to other plant species in order to decipher thoroughly the physiological significance of different Aux/IAA family members. To lay the foundation for a better understanding of the Aux/IAA family in the Solanaceae family, the present study identified and comprehensively analysed the entire Aux/IAA gene family in tomato (S. lycopersicum), a reference species for Solanaceae plants. Phylogenetic analysis revealed that some Aux/IAA clades are either expanded or retracted in tomato compared with Arabidopsis. Expression studies revealed a distinctive spatio-temporal pattern of expression for tomato Aux/IAA genes, some of which display differential responsiveness to auxin and ethylene.

Aux/IAA genes belong to a large gene family found in all plant species ranging from 26 members in Sorghum bicolor (S. Wang et al. 2010) to 35 in poplar (Kalluri et al. 2007). In Arabidopsis, this gene family comprises 29 members (Liscum and Reed 2002) while it contains 31 in rice and maize (Jain et al. 2006, Y. Wang et al. 2010). To shed more light on this gene family, structural and functional characterizations of the tomato Aux/IAA genes were carried out. Both BLASTN and TBLASTN search were performed on the whole set of tomato unigenes in the SGN database (Solanaceae Genomics Network, http://www.sgn.cornell.edu/) using either partial tomato Aux/IAA clones (Nebenführ et al. 2000, Jones et al. 2002) or Aux/IAA Arabidopsis protein sequences. This search was further extended taking advantage of the recent sequence information generated by the tomato genome sequencing project (Solanaceae Genomics Network, http://www.sgn.cornell.edu/). In addition, the predicted proteome deduced from the tomato genome was searched against the pfam AUX_IAA hidden-Markov model (PF02309) recognizing both AUX-IAA and ARF protein sequences (Finn et al. 2010) using the HMMER3 software. This HMM-based search identified 24 Aux//IAA genes in the tomato genome annotation (ITAG Release 2.3 predicted CDS). With the exception of Sl-IAA21, all the Aux/IAA genes identified in this work are present in the tomato genome annotation file iTAG2.30. Overall, this in silico search resulted in the identification of 25 tomato genes displaying the conserved features of Aux/IAA (Supplementary Table S1). The coding sequences of these genes were submitted to GenBank/EMBL. The size of the deduced Aux/IAA proteins varies greatly, ranging from 147 amino acids (Sl-IAA33) to 349 amino acids (Sl-IAA9), and the corresponding molecular mass varies from 16 to 37 kDa (Supplementary Table S2). The predicted isoelectric point also varies widely from 5.02 (Sl-IAA32) to 9.08 (Sl-IAA15) (Supplementary Table S2), suggesting that different Aux/IAA proteins might operate in different microenvironments. Pair-wise comparisons of these Sl-IAA protein sequences showed that the identity level ranges from as low as 19% (between Sl-IAA33 and Sl-IAA8/Sl-IAA27) to a highly identical level of 79% (Sl-IAA21 and Sl-IAA23) (Supplementary Table S3). The overall identity among the various proteins is low, even between members of the same phylogenetic branch (Supplementary Fig. S1). Alignment of amino acid sequences of tomato and Arabidopsis Aux/IAAs revealed the typical four highly conserved domains found in canonical Aux/IAA proteins (Reed 2001), with the exception of Sl-IAA32 which lacks domain II and Sl-IAA33 missing domains I and II and containing only a weakly conserved domain III (Fig. 1). Therefore, Sl-IAA32 and Sl-IAA33 can be considered as non-canonical Aux/IAA proteins like their putative orthologs in Arabidopsis (Dreher et al. 2006).

Multiple sequence alignment of the full-length Sl-IAA proteins obtained with ClustalX and manual correction. Conserved domains of Aux/IAA proteins are underlined. Nuclear localization signals (NLSs) are indicated by filled circles. The amino acid position is given on the right of each sequence.

Phylogenetic analysis was conducted to assess the relationship between tomato and Arabidopsis Aux/IAAs. The tomato Aux/IAA genes were renamed to comply with the nomenclature of their closest Arabidopsis homologs. Supplementary Fig. S1 shows that Aux/IAA proteins group into 11 distinct clades named here A–K. Overall, the tomato family is slightly contracted (25 members) compared with the size of that of Arabidopsis (29 members). With reference to Arabidopsis, four clades (D, F, G and I) are contracted in the tomato and two (A and J) are expanded. Clade A includes seven genes in tomato but only four members in Arabidopsis, while clade J is comprised of three genes in tomato and contains a single member in Arabidopsis. The non-canonical clade H lacking the conserved domains II contains three members (AtIAA20, AtIAA30 and AtIAA31) in Arabidopsis but is not represented in tomato. Clade I, which also gathers non-canonical Aux/IAAs lacking either one or two of the conserved domains, is represented by two Aux/IAAs in Arabidopsis (AtIAA32 and AtIAA34) but only by a single member in tomato (Sl-IAA32). Overall, the non-canonical Aux/IAAs are over-represented in Arabidopsis with six genes (AtIAA20, AtIAA30, AtIAA31, AtIAA32, AtIAA33 and AtIAA34), while only two were found in tomato (Sl-IAA32 and Sl-IAA33).

The Sl-IAA sequences were initially mapped on the tomato genome using the introgression line population obtained by crossing and successive back-crossing of cultivated S. lycopersicum with Solanum pennelli (Eshed and Zamir 1995), and the mapping was subsequently refined using the SGN Tomato Whole Genome Scaffolds data (2.40) (http://www.sgn.cornell.edu/tools/blast/; The International Tomato Genome Sequencing Consortium). The 25 tomato Aux/IAA genes are distributed among nine tomato chromosomes (Supplementary Fig. S2), with chromosomes 2, 10 and 11 being devoid of Aux/IAA genes. Six Sl-IAA genes are present on chromosome 6; five on chromosomes 3 and 9; two on chromosomes 4, 7 and 12; and one on chromosomes 1, 5 and 8. The Aux/IAA genes tend to be clustered in preferential genomic regions, with the presence of closely adjacent genes on chromosome 3 (Sl-IAA19, Sl-IAA15, Sl-IAA27 and Sl-IAA26), chromosome 6 (Sl-IAA22, Sl-IAA17 and Sl-IAA7, Sl-IAA4) and chromosome 9 (Sl-IAA1 and Sl-IAA14). Remarkably, the four contiguous tomato Aux/IAA genes mapped on chromosome 3 are located in a region spanning