Paul J. Overvoorde

Functional Genomic Analysis of the AUXIN RESPONSE FACTOR Gene Family Members in Arabidopsis thaliana: Unique and Overlapping Functions of ARF7 and ARF19

The AUXIN RESPONSE FACTOR (ARF) gene family products, together with the AUXIN/INDOLE-3-ACETIC ACID proteins, regulate auxin-mediated transcriptional activation/repression. The biological function(s) of most ARFs is poorly understood. Here, we report the identification and characterization of T-DNA insertion lines for 18 of the 23 ARF gene family members in Arabidopsis thaliana. Most of the lines fail to show an obvious growth phenotype except of the previously identified arf2/hss, arf3/ett, arf5/mp, and arf7/nph4 mutants, suggesting that there are functional redundancies among the ARF proteins. Subsequently, we generated double mutants. arf7 arf19 has a strong auxin-related phenotype not observed in the arf7 and arf19 single mutants, including severely impaired lateral root formation and abnormal gravitropism in both hypocotyl and root. Global gene expression analysis revealed that auxin-induced gene expression is severely impaired in the arf7 single and arf7 arf19 double mutants. For example, the expression of several genes, such as those encoding members of LATERAL ORGAN BOUNDARIES domain proteins and AUXIN-REGULATED GENE INVOLVED IN ORGAN SIZE, are disrupted in the double mutant. The data suggest that the ARF7 and ARF19 proteins play essential roles in auxin-mediated plant development by regulating both unique and partially overlapping sets of target genes. These observations provide molecular insight into the unique and overlapping functions of ARF gene family members in Arabidopsis.

Functional genomic analysis of the AUXIN/INDOLE-3-ACETIC ACID gene family members in Arabidopsis thaliana

Auxin regulates various aspects of plant growth and development. The AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) genes encode short-lived transcriptional repressors that are targeted by the TRANSPORT INHIBITOR RESPONSE1/AUXIN RECEPTOR F-BOX proteins. The Aux/IAA proteins regulate auxin-mediated gene expression by interacting with members of the AUXIN RESPONSE FACTOR protein family. Aux/IAA function is poorly understood; herein, we report the identification and characterization of insertion mutants in 12 of the 29 Aux/IAA family members. The mutants show no visible developmental defects compared with the wild type. Double or triple mutants of closely related Aux/IAA genes, such as iaa8-1 iaa9-1 or iaa5-1 iaa6-1 iaa19-1, also exhibit wild-type phenotypes. Global gene expression analysis reveals that the molecular phenotypes of auxin-treated and untreated light-grown seedlings are unaffected in the iaa17-6 and iaa5-1 iaa6-1 iaa19-1 mutants. By contrast, similar analysis with the gain-of-function axr3-1/iaa17-1 mutant seedlings reveals dramatic changes in basal and auxin-induced gene expression compared with the wild type. Expression of several type-A ARABIDOPSIS RESPONSE REGULATOR genes and a number of genes involved in cell wall biosynthesis and degradation is repressed in axr3-1/iaa17-1. The data suggest extensive functional redundancy among Aux/IAA gene family members and that enhanced stability of the AXR3/IAA17 protein severely alters the molecular phenotype, resulting in developmental defects.

IAA17/AXR3: Biochemical Insight into an Auxin Mutant Phenotype

The Aux/IAA genes are rapidly and specifically induced by the plant hormone auxin. The proteins encoded by this gene family are short-lived nuclear proteins that are capable of homodimerizing and heterodimerizing. Molecular, biochemical, and genetic data suggest that these proteins are involved in auxin signaling. The pleiotropic morphological phenotype and altered auxin responses of the semidominant axr3-1 mutant of Arabidopsis result from a single amino acid change in the conserved domain II of the Aux/IAA protein IAA17. Here, we show that the biochemical effect of this gain-of-function mutation is to increase the half-life of the iaa17/axr3-1 protein by sevenfold. Intragenic mutations that suppress the iaa17/axr3-1 phenotype have been described. The iaa17/axr3-1R3 revertant contains a second site mutation in domain I and the iaa17/axr3-1R2 revertant contains a second site mutation in domain III. Transient expression assays show that the mutant forms of IAA17/AXR3 retain the ability to accumulate in the nucleus. Using the yeast two hybrid system, we show that the iaa17/axr3-1 mutation does not affect homodimerization. However, the iaa17/axr3-1 revertants counteract the increased levels of iaa17/axr3-1 protein by decreasing the capacity of the mutant protein to homodimerize. Interestingly, heterodimerization of the revertant forms of IAA17/AXR3 with IAA3/SHY2, another Aux/IAA protein, and ARF1 or ARF5/MP proteins is affected only by changes in domain III. Collectively, the results provide biochemical evidence that the revertant mutations in the IAA17/AXR3 gene affect the capacity of the encoded protein to dimerize with itself, other members of the Aux/IAA protein family, and members of the ARF protein family. By extension, these findings may provide insight into the effects of analogous mutations in other members of the Aux/IAA gene family.

age Mutants of Arabidopsis Exhibit Altered Auxin-Regulated Gene Expression

An Arabidopsis transgenic line was constructed expressing β-glucuronidase (GUS) via the auxin-responsive domains (AuxRDs) A and B (BA–GUS) of the PS-IAA4/5 gene in an indoleacetic acid (IAA)–dependent fashion. GUS expression was preferentially enhanced in the root elongation zone after treatment of young seedlings with 10−7 M IAA. Expression of the BA–GUS gene in the axr1, axr4, and aux1 mutants required 10- to 100-fold higher auxin concentration than that in the wild-type background. GUS expression was nil in the axr2 and axr3 mutants. The transgene was used to isolate mutants exhibiting altered auxin-responsive gene expression (age). Two mutants, age1 and age2, were isolated and characterized. age1 showed enhanced sensitivity to IAA, with strong GUS expression localized in the root elongation zone in the presence of 10−8 M IAA. In contrast, age2 exhibited ectopic GUS expression associated with the root vascular tissue, even in the absence of exogenous IAA. Morphological and molecular analyses indicated that the age1 and age2 alleles are involved in the regulation of gene expression in response to IAA.

A 62-kD sucrose binding protein is expressed and localized in tissues actively engaged in sucrose transport.

Sucrose transport from the apoplasm, across the plasma membrane, and into the symplast is critical for growth and development in most plant species. Phloem loading, the process of transporting sucrose against a concentration gradient into the phloem, is an essential first step in long-distance transport of sucrose and carbon partitioning. We report here that a soybean 62-kD sucrose binding protein is associated with the plasma membrane of several cell types engaged in sucrose transport, including the mesophyll cells of young sink leaves, the companion cells of mature phloem, and the cells of the developing cotyledons. Furthermore, the temporal expression of the gene and the accumulation pattern of the protein closely parallel the rate of sucrose uptake in the cotyledon. Molecular cloning and sequence analysis of a full-length cDNA for this 62-kD sucrose binding protein indicated that the protein is not an invertase, contains a 29-amino acid leader peptide that is absent from the mature protein, and is not an integral membrane protein. We conclude that the 62-kD sucrose binding protein is involved in sucrose transport, but is not performing this function independently.

A soybean sucrose binding protein independently mediates nonsaturable sucrose uptake in yeast.

Heterologous expression of a cDNA encoding a 62-kD soybean sucrose binding protein in yeast demonstrates that this protein, independent of other plant proteins, mediates sucrose uptake across the plasma membrane. Sucrose binding protein-mediated sucrose uptake is nonsaturable up to 30 mM sucrose, is specific for sucrose, and is relatively insensitive to treatment with sulfhydryl-modifying reagents. Alteration of the external pH or pretreatment of the yeast cells with protonophores did not significantly affect the rate of 14C-sucrose uptake. This demonstrates that sucrose binding protein-mediated sucrose uptake is not dependent on H+ movement and delineates it from other plant sucrose transporters. Physiological characterization of sucrose uptake into higher plant cells has shown the presence of both saturable and nonsaturable uptake components. The nonsaturable mechanism is relatively insensitive to external pH, pretreatment with protonophores, and treatment with sulfhydryl-modifying reagents. Sucrose binding protein-mediated sucrose uptake in yeast mimics this physiologically described, but mechanistically undefined, nonsaturable sucrose uptake mechanism in higher plants. Functional characterization of the sucrose binding protein thus defines both a novel component of sucrose uptake and provides important insight into this nonsaturable sucrose uptake mechanism, which has remained enigmatic since its physiological description.

A Plasma Membrane Sucrose-binding Protein That Mediates Sucrose Uptake Shares Structural and Sequence Similarity with Seed Storage Proteins but Remains Functionally Distinct

Photoaffinity labeling of a soybean cotyledon membrane fraction identified a sucrose-binding protein (SBP). Subsequent studies have shown that the SBP is a unique plasma membrane protein that mediates the linear uptake of sucrose in the presence of up to 30 mm external sucrose when ectopically expressed in yeast. Analysis of the SBP-deduced amino acid sequence indicates it lacks sequence similarity with other known transport proteins. Data presented here, however, indicate that the SBP shares significant sequence and structural homology with the vicilin-like seed storage proteins that organize into homotrimers. These similarities include a repeated sequence that forms the basis of the reiterated domain structure characteristic of the vicilin-like protein family. In addition, analytical ultracentrifugation and nonreducing SDS-polyacrylamide gel electrophoresis demonstrate that the SBP appears to be organized into oligomeric complexes with aM r indicative of the existence of SBP homotrimers and homodimers. The structural similarity shared by the SBP and vicilin-like proteins provides a novel framework to explore the mechanistic basis of SBP-mediated sucrose uptake. Expression of the maize Glb protein (a vicilin-like protein closely related to the SBP) in yeast demonstrates that a closely related vicilin-like protein is unable to mediate sucrose uptake. Thus, despite sequence and structural similarities shared by the SBP and the vicilin-like protein family, the SBP is functionally divergent from other members of this group.

Functional characterization of sucrose binding protein-mediated sucrose uptake in yeast

A sucrose binding protein was identified whose temporal and spatial patterns of mRNA expression and localization patterns of protein were tightly correlated with the active uptake of sucrose in several cell types. Heterologous expression of the sucrose binding protein in yeast strains have allowed a functional characterization of sucrose uptake as mediated by this protein. Ectopic expression of the sucrose binding protein in the susy7/ura3 yeast strain restored the ability of this strain to grow on medium containing sucrose as a sole carbon source. Further characterization of the kinetics of [(14)C]-sucrose uptake indicate that the sucrose binding protein mediates the uptake of sucrose in a non-saturable, linear manner and that this uptake is relatively insensitive to pH or treatment with protonophores. These biochemical attributes of sucrose binding protein-mediated sucrose uptake mimic the well characterized linear, non-saturable uptake of sucrose described in higher plants. The sucrose binding protein is a unique plasma membrane protein and shares sequence similarity to the seed storage proteins. The sucrose binding protein may mediate sucrose uptake across the plasma membrane in a plant-specific manner.