Contrasting modes of diversification in the Aux/IAA and ARF gene families

The complete genomic sequence for Arabidopsis provides the opportunity to combine phylogenetic and genomic approaches to study the evolution of gene families in plants. The Aux/IAA and ARF gene families, consisting of 29 and 23 loci in Arabidopsis, respectively, encode proteins that interact to mediate auxin responses and regulate various aspects of plant morphological development. We developed scenarios for the genomic proliferation of the Aux/IAA and ARF families by combining phylogenetic analysis with information on the relationship between each locus and the previously identified duplicated genomic segments in Arabidopsis. This analysis shows that both gene families date back at least to the origin of land plants and that the major Aux/IAA and ARF lineages originated before the monocot-eudicot divergence. We found that the extant Aux/IAA loci arose primarily through segmental duplication events, in sharp contrast to the ARF family and to the general pattern of gene family proliferation in Arabidopsis. Possible explanations for the unusual mode of Aux/IAA duplication include evolutionary constraints imposed by complex interactions among proteins and pathways, or the presence of long-distance cis-regulatory sequences. The antiquity of the two gene families and the unusual mode of Aux/IAA diversification have a number of potential implications for understanding both the functional and evolutionary roles of these genes.     

Roles and activities of Aux/IAA proteins in Arabidopsis.

Auxin induces various distinct developmental responses, partly by regulating gene expression. The Aux/IAA genes are a large gene family, many of which are induced by auxin. Work on Arabidopsis Aux/IAA genes has begun to reveal that they can regulate development and auxin-induced gene expression. Furthermore, auxin responses require Aux/IAA protein turnover. Finally, recent evidence suggests that Aux/IAA proteins can mediate light responses. Work in the near future should test whether Aux/IAA proteins are antennae that connect auxin and light signals to endogenous developmental responses.     

Overexpression of the non-canonical Aux/IAA genes causes auxin-related aberrant phenotypes in Arabidopsis

Degradation of Aux/IAA proteins which are triggered by the ubiquitin ligase complex containing the auxin F-box receptors (AFBs), is thought to be the primary reaction of auxin signaling. Upon auxin perception, AFBs bind domain II of Aux/IAA proteins that is conserved in most of the 29 family members in Arabidopsis. However, IAA20 and IAA30 lack domain II. Furthermore, IAA31, which forms a single clade with IAA20 and IAA30 in Aux/IAA protein family, has a partially conserved domain II, which contains an amino acid substitution that would cause a dominant mutation of Aux/IAA genes. It has been shown that the half-lives of these proteins are much longer than those of the canonical Aux/IAA proteins. We generated overexpression lines (OXs) of IAA20 , IAA30 and IAA31 by the use of cauliflower mosaic virus 35S promoter to better understand the molecular function of atypical Aux/IAA proteins in Arabidopsis. OXs of the three genes exhibited similar auxin-related aberrant phenotypes, with IAA20 OX showing the most severe defects: Some of them showed a semi-dwarf phenotype; gravitropic growth orientation was often affected in hypocotyl and root; vasculature of cotyledons was malformed; the primary root stopped growing soon after germination because of collapse of root apical meristem. IAA 20 and IAA30 were early auxin inducible, but IAA31 was not. These results showed that the wild-type genes of the three Aux/IAAs could disturb auxin physiology when ectopically overexpressed.     

Analysis of the genes encoding the largest subunit of RNA polymerase II in Arabidopsis and soybean

We have cloned and sequenced the gene encoding the largest subunit of RNA polymerase II (RPB1) from Arabidopsis thaliana and partially sequenced genes from soybean (Glycine max). We have also determined the nucleotide sequence for a number of cDNA clones which encode the carboxyl terminal domains (CTDs) of RNA polymerase II from both soybean and Arabidopsis. The Arabidopsis RPB1 gene encodes a polypeptide of approximately 205 kDa, consists of 12 exons, and encompasses more than 8 kb. Predicted amino acid sequence shows eight regions of similarity with the largest subunit of other prokaryotic and eukaryotic RNA polymerases, as well as a highly conserved CTD unique to RNA polymerase II.The CTDs in plants, like those in most other eukaryotes, consist of tandem heptapeptide repeats with the consensus amino acid sequence PTSPSYS. The portion of RPB1 which encodes the CTD in plants differs from that of RPB1 of animals and lower eukaryotes. All the plant genes examined contain 2–3 introns within the CTD encoding regions, and at least two plant genes contain an alternatively spliced intron in the 3 untranslated region. Several clustered amino acid substitutions in the CTD are conserved in the two plant species examined, but are not found in other eukaryotes. RPB1 is encoded by a multigene family in soybean, but a single gene encodes this subunit in Arabidopsis and most other eukaryotes.     

A mitochondrial mutator system in maize

Terfestatin A (TrfA), terphenyl-beta-glucoside, was isolated from Streptomyces sp. F40 in a forward screen for compounds that inhibit the expression of auxin-inducible genes in Arabidopsis (Arabidopsis thaliana). TrfA specifically and competitively inhibited the expression of primary auxin-inducible genes in Arabidopsis roots, but did not affect the expression of genes regulated by other plant hormones such as abscisic acid and cytokinin. TrfA also blocked the auxin-enhanced degradation of auxin/indole-3-acetic acid (Aux/IAA) repressor proteins without affecting the auxin-stimulated interaction between Aux/IAAs and the F-box protein TIR1. TrfA treatment antagonized auxin responses in roots, including primary root inhibition, lateral root initiation, root hair promotion, and root gravitropism, but had only limited effects on shoot auxin responses. Taken together, these results indicate that TrfA acts as a modulator of Aux/IAA stability and thus provides a new tool for dissecting auxin signaling.     

The diageotropica mutation alters auxin induction of a subset of the Aux/IAA gene family in tomato

The diageotropica (dgt) mutation has been proposed to affect either auxin perception or responsiveness in tomato plants. It has previously been demonstrated that the expression of one member of the Aux/IAA family of auxin-regulated genes is reduced in dgt plants. Here, we report the cloning of ten new members of the tomato Aux/IAA family by PCR amplification based on conserved protein domains. All of the gene family members except one (LeIAA7) are expressed in etiolated tomato seedlings, although they demonstrate tissue specificity (e.g. increased expression in hypocotyls vs. roots) within the seedling. The wild-type auxin-response characteristics of the expression of these tomato LeIAA genes are similar to those previously described for Aux/IAA family members in Arabidopsis. In dgt seedlings, auxin stimulation of gene expression was reduced in only a subset of LeIAA genes (LeIAA5, 8, 10, and 11), with the greatest reduction associated with those genes with the strongest wild-type response to auxin. The remaining LeIAA genes tested exhibited essentially the same induction levels in response to the hormone in both dgt and wild-type hypocotyls. These results confirm that dgt plants can perceive auxin and suggest that a specific step in early auxin signal transduction is disrupted by the dgt mutation.     

Analysis of the alternative oxidase promoters from soybean

Alternative oxidase (Aox) is a nuclear-encoded mitochondrial protein. In soybean (Glycine max), the three members of the gene family have been shown to be differentially expressed during normal plant development and in response to stresses. To examine the function of the Aox promoters, genomic fragments were obtained for all three soybean genes: Aox1, Aox2a, and Aox2b. The regions of these fragments immediately upstream of the coding regions were used to drive beta-glucuronidase (GUS) expression during transient transformation of soybean suspension culture cells and stable transformation of Arabidopsis. The expression patterns of the GUS reporter genes in soybean cells were in agreement with the presence or absence of the various endogenous Aox proteins, determined by immunoblotting. Deletion of different portions of the upstream regions identified sequences responsible for both positive and negative regulation of Aox gene expression in soybean cells. Reporter gene analysis in Arabidopsis plants showed differential tissue expression patterns driven by the three upstream regions, similar to those reported for the endogenous proteins in soybean. The expression profiles of all five members of the Arabidopsis Aox gene family were examined also, to compare with GUS expression driven by the soybean upstream fragments. Even though the promoter activity of the upstream fragments from soybean Aox2a and Aox2b displayed the same tissue specificity in Arabidopsis as they do in soybean, the most prominently expressed endogenous genes in all tissues of Arabidopsis were of the Aox1 type. Thus although regulation of Aox expression generally appears to involve the same signals in different species, different orthologs of Aox may respond variously to these signals. A comparison of upstream sequences between soybean Aox genes and similarly expressed Arabidopsis Aox genes identified common motifs.