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.     

Phosphopeptide mapping of Avena phytochrome phosphorylated by protein kinases in vitro

We previously demonstrated that protein kinases are useful probes of conformational changes that occur upon photoconversion of phytochrome [Wong, Y.-S., Cheng, H.-C., Walsh, D. A., & Lagarias, J. C. (1986) J. Biol. Chem. 261, 12089-12097]. Here we present phosphopeptide analyses of oat phytochrome phosphorylated by three mammalian protein kinases and by a polycation-stimulated, phytochrome-associated protein kinase. Phosphorylation of the Pr form by the cAMP-dependent protein kinase occurs predominantly on Ser17 while Ser598 is the preferred phosphorylation site on Pfr. The cGMP-dependent and Ca2(+)-activated, phospholipid-dependent protein kinases, which phosphorylate only the Pr form of phytochrome, recognize the same region on the phytochrome polypeptide as the cAMP-dependent protein kinase. Polycation-stimulated phytochrome phosphorylation reveals that, in contrast to the mammalian enzymes, the plant kinase recognizes the serine-rich, blocked N-terminus of phytochrome. The potential regulatory role of phytochrome phosphorylation, particularly in the structurally conserved serine/threonine-rich N-terminal region of the phytochrome polypeptide, is suggested by these results.     

Two human 90-kDa heat shock proteins are phosphorylated in vivo at conserved serines that are phosphorylated in vitro by casein kinase II

Amino-terminal protein sequence analysis revealed that exponentially growing human HeLa cells at 37 degrees C express two closely related 90-kDa "heat shock" proteins (hsp 90) in nearly equal amounts. Both hsp 90s begin with proline; the initial methionine residue is removed. The alpha protein contains a 9-amino acid segment, TQTQDQPME, from residues 4 to 12, that is replaced by a 4-amino acid segment, VHHG, in the beta form. The purified hsp 90 mixture contains 2 mol of phosphate/mol of polypeptide. Both hsp 90 proteins are phosphorylated at two homologous sites. For the alpha protein, these sites correspond to serine 231 and serine 263. A 5-amino acid segment, ESEDK, between the two phosphorylation sites is absent from the beta protein. The sequence between phosphorylation sites of both hsp 90s is predicted to have alpha helical structure. Dephosphorylated hsp 90 is phosphorylated at both sites by casein kinase II from HeLa cells, calf thymus, or rabbit reticulocytes; no other hsp 90 residues were phosphorylated by casein kinase II in vitro.     

Arabidopsis Aux/IAA genes are involved in brassinosteroid-mediated growth responses in a manner dependent on organ type

We examined whether auxin/indole-3-acetic acid (Aux/IAA) proteins, which are key players in auxin-signal transduction, are involved in brassinosteroid (BR) responses. iaa7/axr2-1 and iaa17/axr3-3 mutants showed aberrant BR sensitivity and aberrant BR-induced gene expression in an organ-dependent manner. Two auxin inhibitors were tested in terms of BR responses. Yokonolide B inhibited BR responses, whereas p-chlorophenoxyisobutyric acid did not inhibit BR responses. DNA microarray analysis revealed that 108 genes were up-regulated, while only eight genes were down-regulated in iaa7. Among the genes that were up- or down-regulated in axr2, 22% were brassinolide-inducible genes, 20% were auxin-inducible genes, and the majority were sensitive neither to BR nor to auxin. An inhibitor of BR biosynthesis, brassinazole, inhibited auxin induction of the DR5-GUS gene, which consists of a synthetic auxin-response element, a minimum promoter, and a beta-glucuronidase. These results suggest that Aux/IAA proteins function in auxin- and BR-signaling pathways, and that IAA proteins function as the signaling components modulating BR sensitivity in a manner dependent on organ type.     

Molecular Characterization of an Aux/IAA of Catharanthus roseus

In Catharanthus roseus cells, auxins are known to negatively regulate the biosynthesis of monoterpenoid indole alkaloids (MIA), a class of valuable secondary metabolites. Despite extensive studies of this regulation, no protein of the auxin signaling pathway has been isolated to date in this plant. We therefore decided to clone and characterize a C. roseus Aux/IAA protein that belongs to a family of gene expression repressors mediating auxin effects. Using PCR, a cDNA encoding the first C. roseus Aux/IAA was cloned and named CrIAA1. The deduced amino acid sequence has four highly conserved domains that are typical of the Aux/IAA protein family and has high homology to the Aux/IAA isoforms of Arabidopsis (>67%). The CrIAA1 gene expression, monitored by real-time PCR, was found to be dramatically induced by auxin treatment in C. roseus cells. Using GFP imagery and a bimolecular fluorescence complementation assay, we found that CrIAA1 can form oligomers in the nucleus. We also found that CrIAA1 is quickly degraded following auxin treatments, suggesting that auxin regulates CrIAA1 availability via a feedback mechanism. These results should help to elucidate the molecular nature of the processes responsible for the auxin-mediated regulation of MIA biosynthesis in C. roseus.     

植物Aux/IAA基因家族生物学功能研究进展

生长素是最重要的植物激素之一,对植物生长发育起着关键调控作用。生长素作用于植物后,早期生长素响应基因家族Aux/IAA、GH3和SAUR等被迅速诱导,基因表达上调。其中Aux/IAA基因家族编码的蛋白一般由4个保守结构域组成,结构域Ⅰ具有抑制生长素信号下游基因表达的作用,结构域Ⅱ在生长素信号转导中主要被TIR1调控进而影响Aux/IAA的稳定性,结构域Ⅲ/Ⅳ通过与生长素响应因子ARF相互作用调控生长素信号。Aux/IAA基因家族在双子叶植物拟南芥(Arabidopsis thaliana)的器官发育、根形成、茎伸长和叶扩张等方面发挥重要作用;在单子叶植物水稻(Oryza sativa)和小麦(Triticum aestivum)中,主要影响根系发育和株型,但大多数Aux/IAA基因的功能尚不清楚。该文主要从Aux/IAA蛋白的结构、功能和生长素信号转导途径方面综述Aux/IAA家族在拟南芥、禾谷类作物及其它植物中的研究进展,以期为全面揭示Aux/IAA家族基因的生物学功能提供线索。     

The Tobacco mosaic virus replicase protein disrupts the localization and function of interacting Aux/IAA proteins

Previously, we identified a correlation between the interaction of the Tobacco mosaic virus (TMV) 126/183-kDa replicase with the auxin response regulator indole acetic acid (IAA)26/PAP1 and the development of disease symptoms. In this study, the TMV replicase protein is shown to colocalize with IAA26 in the cytoplasm and prevent its accumulation within the nucleus. Furthermore, two additional auxin (Aux)/IAA family members, IAA27 and IAA18, were found to interact with the TMV replicase and displayed alterations in their cellular localization or accumulation that corresponded with their ability to interact with the TMV replicase. In contrast, the localization and accumulation of noninteracting Aux/IAA proteins were unaffected by the presence of the viral replicase. To investigate the effects of the replicase interaction on Aux/IAA function, transgenic plants expressing a proteolysis-resistant IAA26-P108L-green fluorescent protein (GFP) protein were created. Transgenic plants accumulating IAA26-P108L-GFP displayed an abnormal developmental phenotype that included severe stunting and leaf epinasty. However, TMV infection blocked the nuclear localization of IAA26-P108L-GFP and attenuated the developmental phenotype displayed by the transgenic plants. Combined, these findings suggest that TMV-induced disease symptoms can be attributed, in part, to the ability of the viral replicase protein to disrupt the localization and subsequent function of interacting Aux/IAA proteins.     

Cyanobacterial thylakoid membrane proteins are reversibly phosphorylated under plastoquinone-reducing conditions in vitro

Reversible, light-dependent protein phosphorylation was observed in isolated thylakoid membranes of the cyanobacterium Synechococcus 6301. A polypeptide of 15 kDa in particular was phosphorylated under plastoquinone-reducing conditions and was not phosphorylated under plastoquinone-oxidising conditions. Phosphorylation and dephosphorylation reactions involving this and several other membrane polypeptides showed sensitivity to inhibitors of protein kinases and phosphatases. Changes in phosphorylation state correlated with changes in low temperature fluorescence emission characteristic of changes in excitation energy distribution between the photosystems. The 15 kDa phosphopolypeptide is likely to be involved directly in light state adaptations in cyanobacteria.     

Auxin Extraction and Purification Based on Recombinant Aux/IAA Proteins

Background

Indole-3-acetic acid (IAA) extraction and purification are of great importance in auxin research, which is a hot topic in the plant growth and development field. Solid-phase extraction (SPE) is frequently used for IAA extraction and purification. However, no IAA-specific SPE columns are commercially available at the moment. Therefore, the development of IAA-specific recognition materials and IAA extraction and purification methods will help researchers meet the need for more precise analytical methods for research on phytohormones.

Results

Since the AUXIN RESISTANT/INDOLE-3-ACETIC ACID INDUCIBLE (Aux/IAA) proteins show higher specific binding capability with auxin, recombinant IAA1, IAA7 and IAA28 proteins were used as sorbents to develop an IAA extraction and purification method. A GST tag was used to solidify the recombinant protein in a column. Aux/IAA proteins solidified in a column have successfully trapped trace IAA in aqueous solutions. The IAA7 protein showed higher IAA binding capability than the other proteins tested. In addition, expression of the IAA7 protein in Drosophila Schneider 2 (S2) cells produced better levels of binding than IAA7 expressed in E. coli.

Conclusion

This work validated the potential of Aux/IAA proteins to extract and purify IAA from crude plant extracts once we refined the techniques for these processes.

     

Genome-wide analysis of Aux/IAA genes in Vitis vinifera: cloning and expression profiling of a grape Aux/IAA gene in response to phytohormone and abiotic stresses

Auxin is one of the most important phytohormones involved in plant growth and development. Here, we identified a total of 26 Aux/IAA genes displaying high sequence identity within the conserved domains I, II, III, and IV by screening the grapevine genome proteome 12× database. The Vitis vinifera Aux/IAA proteins can be classified into two groups (A and B) on the basis of their phylogenetic relationships. A search for cis-regulatory elements in the promoter sequences of VvAux/IAA genes revealed that the majority of these proteins may be regulated by light, phytohormones, and abiotic stresses. We also report the isolation and expression analysis of the cDNA of VvAux/IAA4, the most highly expressed VvAux/IAA gene from V. vinifera cv. Sultanine, according to ESTs in the NCBI database. The VvAux/IAA4 gene contains a full-length open reading frame of 1,080 bp, and its predicted amino acid sequence is highly similar to those of Aux/IAA proteins from other plants, including the presence of an AuxIAA/ARF dimerization motif in the C-terminal region. The expression of VvAux/IAA4 was found to be elevated during berry development, and slowly decrease from the initiation of ripening to the overripening stage. VvAux/IAA4 was highly expressed in young leaves and roots, and expressed at low levels in pollen and tendrils. Finally, the expression of VvAux/IAA4 was rapidly induced in response to NAA treatment, but was decreased by salt, drought, and SA treatments. Our results provide evidence of crosstalk between phytohormone and abiotic stresses, and support a role for auxin in stress responses.     

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.     

AXR2 encodes a member of the Aux/IAA protein family

The dominant gain-of-function axr2-1 mutation of Arabidopsis causes agravitropic root and shoot growth, a short hypocotyl and stem, and auxin-resistant root growth. We have cloned the AXR2 gene using a map-based approach, and find that it is the same as IAA7, a member of the IAA (indole-3-acetic acid) family of auxin-inducible genes. The axr2-1 mutation changes a single amino acid in conserved domain II of AXR2/IAA7. We isolated loss-of-function mutations in AXR2/IAA7 as intragenic suppressors of axr2-1 or in a screen for insertion mutations in IAA genes. A null mutant has a slightly longer hypocotyl than wild-type plants, indicating that AXR2/IAA7 controls development in light-grown seedlings, perhaps in concert with other gene products. Dark-grown axr2-1 mutant plants have short hypocotyls and make leaves, suggesting that activation of AXR2/IAA7 is sufficient to induce morphological responses normally elicited by light. Previously described semidominant mutations in two other Arabidopsis IAA genes cause some of the same phenotypes as axr2-1, but also cause distinct phenotypes. These results illustrate functional differences among members of the Arabidopsis IAA gene family.