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

^a Plant Gene Expression Center, Albany, California 94710
^b Salk Institute for Biological Studies, La Jolla, California 92037

⁶ To whom correspondence should be addressed. E-mail theo{at}nature.berkeley.edu; fax 510-559-5678.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
The plant hormone auxin, typified by indole-3-acetic acid (IAA), regulates a variety of physiological processes, including apical dominance, tropic responses, lateral root formation, vascular differentiation, embryo patterning, and shoot elongation (Davies, 1995). At the molecular level, auxin rapidly induces various genes (Abel and Theologis, 1996). Several classes of early auxin-responsive genes have been identified including the Aux/IAA, GH3, and SAUR-like genes (Abel and Theologis, 1996; Guilfoyle et al., 1998). The GH3-like genes encode acyl adenylate–forming isozymes (Staswick et al., 2002). Several GH3-like proteins covalently modify IAA, jasmonic acid, or salicylic acid, indicating that they play a global role in various hormone signaling pathways. The function of the SAUR-like genes is still unknown, but it has been suggested that they may encode short-lived nuclear proteins involved in auxin signaling by interacting with calmodulin (Yang and Poovaiah, 2000; Knauss et al., 2003).

The Aux/IAAs have been among the first auxin-regulated genes to be isolated and are the most characterized among early auxin-responsive genes. They are encoded by a large gene family in Arabidopsis thaliana with 29 members (Abel et al., 1995; Reed, 2001; Liscum and Reed, 2002; Remington et al., 2004). They encode short-lived nuclear proteins, and most of them contain four highly conserved domains (I to IV) (Abel et al., 1994; Reed, 2001). Each domain contributes to the functional properties of the protein. Domain II confers instability of the protein (Worley et al., 2000; Ouellet et al., 2001). Domains III and IV serve for homodimerization and heterodimerization with other Aux/IAA gene family members as well as for heterodimerization with the Auxin Response Factors (ARFs) (Kim et al., 1997; Ulmasov et al., 1997, 1999a, 1999b). Domain I is responsible for the transcriptional repressing activity of the proteins (Tiwari et al., 2004).

The ARF proteins are also encoded by a large gene family in Arabidopsis (23 members). A typical ARF protein contains a B3-like DNA binding domain in the N-terminal region, and domains III and IV are similar to those found in the C terminus of Aux/IAAs. An ARF binds to auxin-responsive cis-acting elements (AuxREs) found in the promoter region of auxin-responsive genes through its DNA binding domain (Abel et al., 1996; Ulmasov et al., 1997, 1999a). The amino acid composition of the middle region between the DNA binding domain and domains III/IV determines whether an ARF protein functions as an activator or repressor (Ulmasov et al., 1999b; Tiwari et al., 2003). The Aux/IAA proteins regulate auxin-gene expression through interaction with the ARF proteins. The Aux/IAAs are targets for degradation by the SCF^TIR1 complex, and most importantly, auxin mediates their interaction with the proteolytic machinery (Gray et al., 1999, 2001; Ward and Estelle, 2001; Dharmasiri and Estelle, 2004). Aux/IAA protein stability is a central regulator in auxin signaling.

Several gain-of-function Aux/IAA mutants, including shy2/iaa3 (Tian and Reed, 1999), axr2/iaa7 (Nagpal et al., 2000), bdl/iaa12 (Hamann et al., 2002), slr/iaa14 (Fukaki et al., 2002), arx3/iaa17 (Rouse et al., 1998), msg2/iaa19 (Tatematsu et al., 2004), and iaa28-1 (Rogg et al., 2001), have been isolated by forward genetics. These mutants have amino acid substitutions in highly conserved residues of domain II, resulting in enhanced protein stability that causes altered auxin response and dramatic defects in growth and development. Loss-of-function mutations of AUX/IAAs do not show an obvious visible growth phenotype (Rouse et al., 1998; Tian and Reed, 1999; Nagpal et al., 2000; P.J. Overvoorde and Y. Okushima, unpublished data). Loss-of-function mutants in five ARF genes have been previously isolated. Mutations in the ARF3/ETT affect gynoecium patterning (Sessions et al., 1997; Nemhauser et al., 2000). Loss-of-function mutations of ARF7/NPH4/MSG1/TIR5 result in impaired hypocotyl response to blue light and other differential growth responses associated with changes in auxin sensitivity (Watahiki and Yamamoto, 1997; Stowe-Evans et al., 1998; Harper et al., 2000). Mutations in ARF5/MP interfere with the formation of vascular strands and the initiation of the body axis in the early embryo (Hardtke and Berleth, 1998). Mutations in ARF2/HSS have been identified as suppressors of the hookless phenotype (Li et al., 2004). ARF2 acts as a communication link between the ethylene and the auxin signaling pathways for regulating hypocotyl bending. Lastly, ARF8 functions in hypocotyl elongation, and it is involved in auxin homeostasis (Tian et al., 2004). The biological functions, however, of the remaining ARF gene family members are unknown.

Here, we have employed a functional genomic strategy that involves the identification of T-DNA insertion in the ARF gene family members to elucidate some of the biological functions of the ARF transcription factors. Most of the single arf T-DNA insertion mutants fail to show an obvious growth phenotype. However, double mutants, such as arf7 arf19, show a strong auxin phenotype that results in the absence of lateral root formation than neither the arf7 nor arf19 single mutant expresses. The results suggest that there are unique and overlapping functions among related ARF gene family members in Arabidopsis.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
The Arabidopsis ARF Gene Family
The Arabidopsis genome contains 23 ARF genes scattered among the five chromosomes (Arabidopsis Genome Initiative, 2000; annotation version V5.0, Figure 1A). The locations of the four previously described loss-of-function mutations, arf3/ett (Sessions and Zambryski, 1995), arf5/mp (Hardtke and Berleth, 1998), arf7/nph4 (Harper et al., 2000), and arf2/hss (Li et al., 2004), are highlighted in Figure 1A. A cluster of ARF genes, ARF12, 13, 14, 15, 20, 21, and 22, is present in the upper arm of chromosome I (Figure 1A). These genes share a high degree of similarity among their amino acid and nucleotide sequences (see Supplemental Figure 1 and Table 1 online). ARF23 is a pseudogene (see Supplemental Figure 1 online; Guilfoyle and Hagen, 2001). Phylogenetic analysis reveals that the genes fall into three branches (marked with different colors in Figure 1B). Class I has the most members (15) that can be subdivided into three subclasses, Ia (five members, shaded brown), Ib (eight members, shaded blue), and Ic (two members, shaded green). Their middle region is rich in Pro, Ser, Gly, or Leu (Guilfoyle and Hagen, 2001; see Supplemental Figure 1 online), and some of them function as repressors (Ulmasov et al., 1999b; Tiwari et al., 2003). Class II (shaded pink) has five members, and some of them function as activators. Their middle region is rich in Glu (Ulmasov et al., 1999a; Guilfoyle and Hagen, 2001). Class III (shaded yellow) also contains three members that are the most divergent compared with those encoded by the other two classes. ARF3 and ARF17, which are considered to lack the C-terminal domains III and IV (Guilfoyle and Hagen, 2001), may potentially contain highly divergent domains III and IV (see Supplemental Figure 1 online). Furthermore, ARF13 does not have domains III and IV in this new alignment (see Supplemental Figure 1 online). The ARF polypeptides vary in size ranging from 57 (ARF13) to 129 kD (ARF7) (see Supplemental Table 2 online). This size variation is primarily attributable to the different amino acid content in the middle region (see Supplemental Figure 1 online).

View larger version (30K): [in this window] [in a new window]   Figure 1. The ARF Gene Family of Arabidopsis. (A) Chromosomal location of ARF genes. The locations of 23 putative ARF genes on the Arabidopsis chromosomes (I to V) are shown according to version 5.0 of the Arabidopsis Genome annotation submitted to GenBank. Mutants that have been isolated in the ARF gene are shown on the left side of the chromosomes. The ARF genes clustered on chromosome I are boxed. (B) Phylogenetic analysis. An unrooted dendogram was generated using ClustalW (Thompson et al., 1994). TreeView was used to generate the graphical output (Page, 1996). The numbers at the branching points indicate the percentage of times that each branch topology was found during bootstrap analysis (n = 1000). The gene names, accession numbers, protein identifier, and the accession numbers of the full-length open reading frames (ORFs) used for this analysis are also shown. Predicted ORFs from the genomic annotation were used for ARF14, ARF15, ARF21, and ARF23 (pseudogene) genes. The full-length ORFs of ARF2, ARF6, ARF7, ARF8, ARF11, ARF12, ARF13, ARF19, ARF20, and ARF22 were constructed during this study. A differential spliced form of ARF13 has been cloned recently (accession number AY680406).

Isolation of ARF T-DNA Insertion Mutants
We initiated this project using a PCR-based screening approach to identify T-DNA insertion mutants for a large number of ARF genes. A total of 80,000 T-DNA insertion line populations in the Columbia ecotype were initially screened, and eight lines were identified (Alonso et al., 2003). Subsequently, the laboratory participated in generating the garlic lines in collaboration with the former Torrey Mesa Research Institute, and 10 additional lines were isolated (Sessions et al., 2002). More recently, we obtained another nine T-DNA insertional lines from the Salk T-DNA express line collection (http://signal.salk.edu/cgi-bin/tdnaexpress). Taken together during the last 6 years, we identified 27 T-DNA insertion lines located in the coding region of 18 ARF genes. Figure 2 and Supplemental Table 3 online provide a summary of all the mutants isolated and characterized during the course of this study. All the lines have been backcrossed at least once and partially characterized phenotypically. We plan to deposit all the lines in the Arabidopsis Biological Resource Center (http://www.biosci.ohio-state.edu/plantbio/Facilities/abrc/abrchome.htm) for further molecular and phenotypic characterization by the community.

View larger version (28K): [in this window] [in a new window]   Figure 2. Location of T-DNA Insertions in the ARF Gene Family Members. Boxes represent exons. T-DNA insertions with gray triangles denote lines whose characterization has been completed. T-DNA insertions with white triangles denote lines not yet characterized.

View larger version (80K): [in this window] [in a new window]   Figure 4. Phenotypes of the arf7 arf19 Double Mutant. (A) Four-week-old soil-grown plants of the wild type, arf19-1, nph4-1, msg1-2, and arf7-1 (top) and the wild type, nph4-1 arf19-1, msg1-2 arf19-1, arf7-1 arf19-1, and slr-1 (bottom). (B) Seventeen-day-old seedlings of wild type, arf19-1, nph4-1, msg1-2, arf7-1, nph4-1 arf19-1, msg1-2 arf19-1, arf7-1 arf19-1, and slr-1. (C) Twenty-two-day-old seedlings of the wild type, nph4-1 arf19-1, and slr-1 grown on agar plates vertically. (D) Gravitropic response of 3-d-old dark-grown seedlings of the wild type, arf19-1, nph4-1, msg1-2, arf7-1, nph4-1 arf19-1, msg1-2 arf19-1, arf7-1 arf19-1, and slr-1. (E) Root hair formations of the wild type, arf19-1, nph4-1, and nph4-1 arf19-1. (F) Phototropism of 3-d-old dark-grown seedlings of the wild type, arf19-1, nph4-1, msg1-2, arf7-1, nph4-1 arf19-1, and arf7-1 arf19-1. Seedlings were exposed to unilateral blue light from the right for 8 h.

View larger version (92K): [in this window] [in a new window]   Figure 3. Phenotype of Mature Mutant Plants. Three wild-type (left) and three mutant plants (right) are shown. The plants were grown at the same time. White dots indicate the boundaries between the wild-type and the mutant plants.

Isolation and Characterization of arf7 arf19 Double Mutants
ARF7 and ARF19 are phylogenetically related (Figure 1B; Liscum and Reed, 2002; Remington et al., 2004). Given the close relationship of ARF7 and ARF19, we tested whether the arf19 mutant had an altered phototropic response similar to that reported for nph4/arf7 (Liscum and Briggs, 1995). We found that the arf19-1 mutant hypocotyl responded to blue light in a wild type–like manner (Figure 4F). Mature arf7 mutant plants (nph4-1, arf7-1, and msg1-2/nph4-102) do not show any gross developmental defects, except that they have epinastic rosette leaves and the length of the inflorescence stems is slightly shorter than that of the wild-type plants (Figure 3; data not shown; Watahiki and Yamamoto, 1997). These characteristics are more pronounced in the arf7 arf19 double mutant. The appearance of mature arf19 plants is identical to that of the wild type (Figures 3 and 4A). The results suggest that the expression of ARF7 functionally compensates for the loss of ARF19 expression responsible for differential hypocotyl growth, but not vice versa.

We initially used the nph4-1 mutant (Liscum and Briggs, 1995) as the arf7 allele for crossing into arf19-1 to generate the arf7 arf19 double mutant. Among the F2 population, approximately one out of 16 plants had short and thin inflorescence stem and small leaves. PCR analysis confirmed that these small plants were double homozygous for both mutations. Because the original nph4-1 line was screened from fast neutron-mutagenized seeds carrying the homozygous recessive glabrous1 (gl1) mutation (Liscum and Briggs, 1995), we backcrossed the nph4-1 and nph4-1 arf19-1 to Columbia (Col) wild-type plants. The nph4-1 and nph4-1 arf19-1 mutant lines without the gl1 mutation were used for further analysis.

The nph4-1 arf19-1 double mutant exhibits much stronger auxin-related phenotypes than those of nph4-1 and arf19-1 single mutants. Adult nph4-1 arf19-1 mutant plants have thin and short inflorescence stems, and their rosette leaves are small and epinastic (Figures 4A to 4C; see Supplemental Figure 4 online; data not shown). In addition, nph4-1 arf19-1 has reduced numbers of inflorescence stems, suggesting enhanced apical dominance. By contrast, the flowers of nph4-1 arf19-1 appear to be normal, and they fertilize normally (data not shown). The phenotype of nph4-1 arf19-1 is the most obvious at its seedling stage, with its most prominent phenotype being severely impaired lateral root formation (Figure 4B, Table 1). The primary roots of arf19-1 produce as many lateral roots as the wild type, whereas the arf7 mutant produces fewer lateral roots compared with the wild type (Figure 4B, Table 1). The primary roots of the nph4-1 arf19-1 seedlings fail to produce lateral roots in 2-week-old seedlings. However, nph4-1 arf19-1 seedlings start to generate several lateral roots after 2 weeks of growth, and their morphological appearance is normal (Figure 4C; data not shown). The nph4-1 arf19-1 mutant also displays agravitropic responses in both hypocotyls and roots (Figure 4D). When seedlings are grown vertically under dark conditions, the hypocotyl growth orientation of arf7 is significantly skewed compared with the wild type, whereas the arf19-1 mutant has a normal gravitropic response (Figure 4D; Harper et al., 2000). Interestingly, in the nph4-1 arf19-1 seedlings, regulation of growth orientation is disrupted in both hypocotyls and roots, with the hypocotyls occasionally growing downward and the roots upward (Figure 4D). Also, the roots and hypocotyls of nph4-1 arf19-1 show reduced gravitropic curvatures compared with the wild type when vertically dark-grown seedlings are reoriented by 90° (data not shown). The phototropic response toward blue light in hypocotyls of nph4-1 arf19-1 seedlings is disrupted as in the arf7 single mutants (Figure 4F). We generated additional combinations of arf7 arf19 double mutants using other alleles of arf7 and arf19 to confirm the phenotypes of nph4-1 arf19-1. We used msg1-2/nph4-102 (Watahiki and Yamamoto, 1997) and arf7-1 as the arf7 alleles for crosses with arf19-1 and arf19-2. All five additional arf7 arf19 double mutant alleles, msg1-2 arf19-1, arf7-1 arf19-1, nph4-1 arf19-2, msg1-2 arf19-2, and arf7-1 arf19-2 (Figures 4A, 4B, and 4D, Table 1; data not shown), display the same phenotypes as nph4-1 arf19-1: smaller plant size, impaired lateral root formation, and agravitropic response. These results confirm that the phenotypes of nph4-1 arf19-1 are caused by the loss of ARF7 and ARF19 function.

Auxin Sensitivity of arf7 arf19
The arf7 single mutants display reduced auxin sensitivity in hypocotyl growth, whereas they show normal auxin response in the roots (Figures 5A and 5B; Watahiki and Yamamoto, 1997; Stowe-Evans et al., 1998). By contrast, arf19-1 shows normal auxin sensitivity in the hypocotyls and a mild but significant resistance to exogenous auxin in the roots (Figures 5A and 5B). The same level of auxin resistance is also observed in the roots of arf19-2 (data not shown), suggesting that the auxin response is slightly impaired in the roots of the arf19 single mutants. Interestingly, the arf7 arf19 double mutants display severely reduced auxin sensitivity in both roots and hypocotyls (Figures 5A and 5B). The root auxin sensitivity is impaired in arf7 arf19 to the same degree as in slr-1. The data suggest that the hypocotyl auxin sensitivity is impaired in the arf7 single mutants, the root auxin sensitivity is impaired in the arf19 single mutants, and both are severely impaired in the arf7 arf19 double mutant. Surprisingly, the slr-1 hypocotyls fail to elongate after transfer to dark conditions, and exogenous auxin application does not affect their hypocotyl growth (Figures 5B and 5C).

View larger version (16K): [in this window] [in a new window]   Figure 5. Auxin Sensitivity of the Wild Type, arf7, arf19, arf7 arf19, and slr Mutants. (A) Inhibition of root growth by exogenous auxin. Each value represents the average of more than 10 seedlings. Bars represent SE of the average. (B) and (C) Inhibition of hypocotyl elongation by exogenous auxin. Data represent the mean of hypocotyl length as a percent of controls (B) or of actual measurements (C). Bars represent SE of the average. See Methods for experimental details.

View larger version (80K): [in this window] [in a new window]   Figure 6. Expression of GUS in ProARF7:GUS and ProARF19:GUS Transgenics. (A) GUS expression in a 6-d-old light-grown ProARF7:GUS seedling. (B) GUS expression in a 6-d-old light-grown ProARF19:GUS seedling. (C) Root apex of a ProARF7:GUS seedling primary root. (D) Root apex of a ProARF19:GUS seedling primary root. (E) to (I) ProARF7:GUS expression in the vascular tissue of mature primary root, lateral root primordia ([E] and [F], arrowhead), and developing lateral roots ([G] to [I]). (J) to (L) ProARF19:GUS expression in entire tissue of primary root and developing lateral roots.

View larger version (55K): [in this window] [in a new window]   Figure 7. Developmental Defects by ARF19 Overexpression. (A) to (C) Growth inhibition in 5-week-old plants (A), first true leaves (B), and 12-d-old light-grown seedlings (C). (D) Alteration of root architecture in 10-d-old seedlings. (E) Expression of ARF19 in overexpressing lines from 7-d-old light-grown seedlings. ARF gene expression was assessed by RT-PCR as described in Methods. The lanes are as follows: 1, the wild type; 2, arf19-1; 3, Pro35s:ARF19 line 1; 4, Pro35s:ARF19 line 2; 5, genomic DNA. Accumulation of the ACT8 transcript was used as an internal control. White and black arrowheads indicate the size of genomic and cDNA fragments, respectively.

Figure 8 shows the scatter plots representing the auxin-regulated transcriptional profiles of wild-type, arf19-1, nph4-1, and nph4-1 arf19-1 mutants. A cursory examination of these scatter plots demonstrates that the loss of ARF7 and ARF19 causes gross changes in auxin-induced gene expression. The wild-type scatter plot shows that the gene expression profile is globally altered by exogenous auxin treatment. The scatter plot of arf19-1 shows a similar degree of distribution as with the wild type, suggesting that almost normal auxin-regulated gene expression is maintained in the arf19 single mutant (Figure 8). However, the scatter plots of nph4-1 and nph4-1 arf19-1 display a smaller degree of distribution than that of the wild type, indicating that the auxin-mediated transcriptional regulation is globally repressed in these mutants (Figure 8). We extracted the auxin-regulated genes using the log₂ expression values from the robust multichip analysis (RMA) output file (Irizarry et al., 2003) and established rigorous statistical criteria based on a variance measurement to generate auxin-regulated gene lists (see Methods). Among the 22,800 genes, only 203 met the criteria for more than twofold auxin induction (I, induced genes), and 68 genes met the criteria for more than twofold repression (R, repressed genes). A complete list of all the auxin-regulated genes and how they are affected by the mutants can be found in the Supplemental Tables 4 and 5 online. These gene lists include various classes of known auxin-regulated genes, such as Aux/IAA, GH3, SAUR, and ACS, consistent with similar studies reported previously (Tian et al., 2002; Ullah et al., 2003; Redman et al., 2004). The genes identified as auxin-regulated (induced or repressed) were functionally categorized to examine the auxin-regulated cellular and metabolic processes affected by either or both loss-of-function mutations of ARF7 and ARF19. Supplemental Figure 6 online shows their functional classification. Approximately 80% of the auxin-regulated genes is currently annotated as encoding proteins of known or putative function.

View larger version (23K): [in this window] [in a new window]   Figure 8. Global Gene Expression Profiling. MA plots (Dudiot et al., 2002) showing changes of auxin-regulated gene expression levels in the wild type, arf19-1, nph4-1, and nph4-1 arf19-1. Each plot represents the log ratio of the average of the auxin-treated samples (I) to the control samples (C) [M = log2 (I/C)] versus overall average intensity [A = log2(I*C)]. The genes induced by auxin treatment (M > 1) are highlighted in red, and the genes repressed by auxin treatment (M < –1) are highlighted in green. The data were further analyzed for variance to extract statistically valid auxin-regulated genes (see Methods).

View larger version (58K): [in this window] [in a new window]   Figure 9. Comparative Analysis of Genes Differentially Regulated by Auxin in nph4-1, arf19-1, and nph4-1 arf19-1. Differentially regulated genes in mutants among auxin-induced (A) and repressed (B) genes are shown. Each circle within the Venn diagram indicates numbers and percentages (in parentheses) of genes with repressed induction or repression levels. Only those genes with greater than twofold fold change ratio (FCR) in nph4-1, arf19-1, and nph4-1 arf19-1 were analyzed (see Methods). We defined each area of the Venn diagram from A to H, and each class was further divided into two subgroups based on their auxin-induced expression profiles in the wild type. The genes classified into class D are considered to be preferentially regulated by ARF7, and those classified into class F are considered to be preferentially regulated by ARF19. The genes classified into classes E and G are considered to be redundantly regulated by ARF7 and ARF19. The class A genes have similar expression profiles to class D genes. Likewise, class C genes have similar expression profiles to class F genes. The expression profiles of the representative genes from each class are shown in Supplemental Figure 5 online.

View larger version (52K): [in this window] [in a new window]   Figure 10. The Expression Profiles of Representative Auxin-Regulated Genes in the Wild Type, nph4-1, arf19-1, and nph4-1 arf19-1. The data represent the average relative intensity expression level of control (open bar) or auxin-treated (blue bar) samples from triplicate experiments. Bars represent SD of the average. Boxes next to gene names indicate classification color codes according to Figure 9.

View larger version (46K): [in this window] [in a new window]   Figure 11. Effect of the nph4-1, arf19-1, and nph4-1 arf19-1 Mutations on Global Gene Expression in Untreated Control Samples. (A) Induced genes in the mutants under control conditions. (B) Repressed genes in the mutants under control conditions. Each circle within the Venn diagram indicates the number of genes with greater than twofold induction or repression. (C) Expression profiles of induced classes of genes. (D) Expression profiles of repressed classes of genes. Data represent the average relative intensity expression levels of control (open bar) or auxin-treated (blue bar) samples from triplicate experiments. Bars represent SD of the average. Boxes next to gene names indicate classification color codes according to (A) and (B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Unique and Overlapping Developmental Functions of ARF7 and ARF19
Considering the phenotypes of arf7 and arf19 single mutants, ARF7 appears to regulate auxin-dependent differential growth in the hypocotyls, and ARF19 partially mediates auxin signaling in the roots. The severity of their phenotypes is greatly enhanced in the double mutant compared with the single mutations, demonstrating redundant functions between ARF7 and ARF19. The arf7 arf19 mutant exhibits strong auxin-related phenotypes, including severely impaired lateral root formation, agravitropic hypocotyls and roots, and small organs and enhanced apical dominance in aerial portions. These phenotypes are observed only in the arf7 arf19 double mutant, but not in the single mutants, indicating that these developmental events are redundantly regulated by ARF7 and ARF19. Expression of one ARF allows for functional compensation for the loss of the other in arf7 and arf19 single mutants. This may be because of the high similarity of these two proteins. The analysis of promoter-GUS transgenic plants demonstrated that there is a significant agreement between the expression patterns and the developmental defects in the single and double mutants. Pro_ARF7:GUS is strongly expressed in the hypocotyls, whereas Pro_ARF19:GUS is strongly expressed in the roots. Furthermore, expression of Pro_ARF7:GUS is detected throughout the hypocotyl, whereas the expression of Pro_ARF19:GUS is restricted to the vascular tissue of the hypocotyls (Figures 6A and 6B). However, despite the global Pro_ARF19:GUS expression and an altered auxin sensitivity in arf19 root, only the arf7 mutants have slightly reduced numbers of lateral roots (Table 1), suggesting that the ARF7 has a regulatory function in lateral root initiation. The microarray experiments show that the auxin-dependent induction of ARF19 is impaired in the nph4-1 mutant (Figure 10A). Interestingly, the promoter region of ARF19 contains two AuxREs (data not shown), suggesting that ARF7 may directly modulate the expression of ARF19. This may provide an alternative explanation for the apparent phenotype of the arf7 mutants. The inadequate auxin-mediated induction of ARF19 expression may have an additive effect on the loss of ARF7 function, yielding an obvious phenotype. We have not tested yet whether the ARF7 and ARF19 proteins can complement the loss of each other. Promoter-swapping experiments using transgenic arf7 and arf19 single or double mutants harboring ARF7 promoter:ARF19 and ARF19 promoter:ARF7 gene constructs have the potential to clarify this issue.

ARF7 and ARF19 Regulate Both Unique and Partially Overlapping Sets of Target Genes
The microarray data provide clear evidence for the unique and redundant functions of ARF7 and ARF19 on auxin-mediated gene expression. The almost complete lack of auxin-mediated transcriptional regulation in the arf7 arf19 mutant is puzzling (Figure 9). It implies that ARF7 and ARF19 are the only ARF factors that are necessary and sufficient for auxin signaling in 7-d-old light-grown seedlings. Are the rest of the ARFs dispensable? The possibility exists that the majority of auxin-regulated gene expression during this stage of development is mediated by the ARF7/ARF19 pair. It should be noted that the adult arf7 arf19 plants, although smaller in size, have a normal appearance with normal flowers and fertility, suggesting that the ARF7/ARF19 pair may not be critical for auxin-mediated transcriptional regulation during the development of aerial organs. Such a proposition is supported by the phenotypes of two other ARF mutants, arf5/mp and arf3/ett; they control auxin-mediated gene regulation responsible for axial cell and gynoecium patterning during organogenesis, respectively, indicating that ARF5 and ARF3 may also act in a particular developmental window. In addition, several single and double arf mutants, including arf2, arf1 arf2, arf3, and arf6 arf8, have flowers with abnormal morphology and/or poor fertility, suggesting that these ARFs may act redundantly in auxin-mediated gene regulation responsible for flower development. Comparative microarray analysis with different double mutants at different developmental stages has the potential to clarify this view. Alternatively, the remaining ARFs may regulate genes that are not auxin regulated at that particular developmental stage. The current prevailing view that all ARFs regulate auxin-mediated gene expression has not been tested experimentally with vigor. Finally, the remaining ARFs may regulate genes in a cell-specific manner (distinct cell types) that the microarray analysis fails to detect. This last possibility points to the necessity of conducting global expression studies in specific cell types (Birnbaum et al., 2003).

Comparative analysis of the gene sets in which auxin-mediated regulation was suppressed in nph4-1, arf19-1, and nph4-1 arf19-1 mutants allowed us to classify the auxin-regulated genes into gene sets preferentially regulated by ARF7 and ARF19 alone or redundantly regulated by both ARF7 and ARF19 (Figure 9). The data suggest that the ARF7 and ARF19 regulate both distinct and partially overlapping sets of target genes (Figure 9). ARF7 appears to regulate many more auxin-induced genes (47%) than ARF19 (4%), and 30% of the auxin-induced genes are redundantly regulated by ARF7 and ARF19. It is of a great interest that 90% of the auxin-induced or -repressed genes contain at least one AuxRE (TGTCnC or GnGACA) in their 2-kb promoter region (data not shown), suggesting that they are directly regulated by these ARFs. This suggests that the ARF7 and ARF19 proteins have the capacity to act as transcriptional activators or repressors of various auxin-regulated genes. The current assignment of ARF7 and ARF19 solely as transcriptional activators is not warranted. Although microarray analysis provides useful and a vast amount of information regarding the genes regulated by the ARF7/ARF19 pair, more direct global technologies, such as chromatin immunoprecipitation and DNA CHIP (ChIP:CHIP), have the potential to identify target genes that are regulated by this and other ARF pairs (Ren et al., 2000; Iyer et al., 2001).

The lists of auxin-regulated genes in which expression is inhibited in the mutants contain putative downstream targets of ARF7 and ARF19. LATERAL ROOT PRIMORDIUM1 (LRP1) is one such candidate gene. The expression level of LRP1 is induced by auxin treatment in the wild type (Figure 10F; Ullah et al., 2003), and its auxin-mediated induction is inhibited in nph4-1 arf19-1 (Figure 10F). LRP1 is expressed during the early stage of lateral root primordia (Smith and Fedoroff, 1995), and its inhibition is consistent with impaired lateral root formation in the nph4-1 arf19-1 mutant. Another potential candidate is the AUXIN-REGULATED GENE INVOLVED IN ORGAN SIZE (ARGOS) gene, which is inhibited in auxin-treated and -untreated nph4-1 and nph4-1 arf19-1 mutants (Figure 10F). Loss-of-function and gain-of-function mutants of ARGOS result in smaller and larger plant sizes, respectively (Hu et al., 2003). The small plant size of arf7 arf19 may be related to the low expression level of ARGOS. Other potential targets of ARF7 and ARF19 are the genes encoding LATERAL ORGAN BOUNDARIES (LOB) domain (LBD) family members (Iwakawa et al., 2002; Shuai et al., 2002). The current analysis reveals that four LBD genes, LBD16, LBD17, LBD18, and LBD29, are induced by auxin, and their auxin-dependent induction is severely impaired in nph4-1 and nph4-1 arf19-1 mutants (Figure 10F). All four highly similar auxin-inducible LBD genes contain potential AuxREs in their regulatory regions (data not shown). Although the function of these LBD genes is still unclear, LOB is considered to participate in boundary establishment or communication links between the meristems and initiating lateral organs (Shuai et al., 2002). Overexpression of several LBD gene family members results in strong morphological changes (Nakazawa et al., 2003). The root-specific expression of LBD16 and LBD29 (Shuai et al., 2002) suggests that these two LBDs may be involved in lateral root formation. Overexpression of LBD16 rescues the lateral root phenotype of the arf7 arf19 double mutant (Y. Okushima and H. Fukaki, unpublished data). Finally, multiple classes of genes encoding auxin conjugating or auxin synthesis enzymes, cell wall–related proteins, metabolic enzymes, and transcription regulators are potential targets of the ARF7/ARF19 pair (Figure 10; see Supplemental Tables 4 and 5 online).

Regulation of ARF7 and ARF19 by IAA14 and Other Aux/IAAs
The phenotypes of the arf7 arf19 mutants are quite similar to those observed in the iaa14/slr mutant. Enhanced IAA14 protein level and the loss of both ARF7 and ARF19 functions have similar effects, indicating that all three proteins act on the same developmental pathway. Promoter-GUS expression analysis has revealed that the ARF7, ARF19, and IAA14 have overlapping expression patterns at least in the root tissue (Fukaki et al., 2002). This raises the prospect that IAA14 may be a molecular partner of ARF7 and ARF19 by forming heterodimers in planta, thereby repressing the activity of these two ARFs. This interaction may inhibit ARF7- and ARF19-mediated transcriptional activation/repression. Division of pericycle cells is blocked during lateral root initiation in the iaa14/slr-1 mutant (Fukaki et al., 2002). The stronger phenotype of iaa14/slr compared with that observed in arf7 arf19 (i.e., complete lack of lateral roots and few root hairs) may be attributable to the inhibition of other ARFs by the stabilized IAA14 protein. In addition to the iaa14/slr mutant, iaa3/shy2 (Tian and Reed, 1999), iaa19/msg2 (Tatematsu et al., 2004), and iaa28-1 (Rogg et al., 2001) also have reduced numbers of lateral roots, whereas the iaa14 T-DNA insertion mutant (loss of function) has a normal root phenotype (Y. Okushima and A. Theologis, unpublished data). These data suggest that the function of ARF7 and ARF19 may be negatively regulated by multiple Aux/IAA proteins. Similar functional interactions have been proposed between ARF5 and IAA12 (Hamann et al., 2002; Vogler and Kuhlemeier, 2003), IAA19/MSG2 and ARF7 (Tatematsu et al., 2004), and ARF7 and IAA12 (Hardtke et al., 2004). In planta heterodimerization studies using bimolecular fluorescence complementation have the potential to elucidate the heterodimeric interactions among the Aux/IAA and ARF gene family products (Hu et al., 2002; Tsuchisaka and Theologis, 2004).

	METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Materials
The pBI101 vector was purchased from Clontech (Palo Alto, CA). All chemicals used for this study were American Chemical Society reagent grade or molecular biology grade. Oligonucleotides were purchased from Operon Technologies (Alameda, CA) or synthesized in house with a Polyplex oligonucleotide synthesizer (GeneMachines, San Carlos, CA).

Molecular Biology
Standard protocols were followed for DNA manipulations described by Sambrook et al. (1989). Standard protocols for DNA sequencing were used to confirm the accuracy of the DNA constructs.

Plant Growth Conditions
Arabidopsis thaliana ecotype Col was used throughout this study. Seeds were surface sterilized for 8 min in 5% sodium hypochlorite + 0.15% Tween-20, excessively rinsed in distilled water and plated on 0.8% agar plates containing 0.5x MS salts (Life Technologies, Rockville, MD) + 0.5 mM Mes, pH 5.7, + 1% sucrose + 1x vitamin B5. The plates were incubated in the dark at 4°C for 2 d and were subsequently transferred to a 16-h-light/8-h-dark cycle at 22°C for light-grown seedlings or in the dark for etiolated seedlings. Mature plants were also grown under the light conditions mentioned above. The root auxin sensitivity assay was performed as follows: 4-d-old light-grown seedlings were transferred to vertically oriented agar plates containing appropriate concentrations of IAA. The root length was determined after an additional 5 d of growth. The auxin sensitivity assay for hypocotyl elongation was performed with 3-d-old seedlings grown on plates lacking auxin and then was transferred to the plates containing various concentrations of IAA and grown for an additional 5 d in the dark. The root and hypocotyl lengths were determined using the NIH Image 1.63 program (http://rsb.info.nih.gov/nih-image/download.html). The phototropic response of etiolated seedlings to blue light was performed as previously described by Liscum and Briggs (1995). Three-day-old etiolated seedlings were exposed to unilateral blue light (1 µmol m^–2 s^–1) for 8 h and then photographed.

Identification and Characterization of T-DNA Insertion Alleles
Screening for T-DNA Insertions
The identification of insertional mutants was performed using a PCR-based screen. For each gene, a forward (F) primer annealing to 100 to 150 bp 5' of the ATG and a reverse (R) primer annealing to 100 to 150 bp 3' of the translation stop codon were designed. The size of the genomic products ranged from 6 to 3.2 kb. Eight sets of DNA template derived from 10,000 plants each (80,000 lines total) were screened. Each set of template contained 40 tubes of DNA (10 each of DNA combined from column, row, plate, and individual superpools). Identification of an individual requires a PCR product in each of the four superpools. Using all combinations of F and R primers with primers annealing to the left border and right border of the T-DNA, PCRs were run (4 x 40 x 8 = 1280 reactions per gene). All operations were adapted to a 384-well format and handling of samples performed with a BioMek robot (Beckman, Palo Alto, CA). The products were analyzed by DNA gel blotting to allow increased sensitivity of detection and assess the specificity of screening. Subsequent to this screen, two large databases containing sequence of DNA flanking T-DNA inserts in 100,000 and 20,000 independent lines have been screened in silico. Data for the 100,000 lines were generated in a collaboration of the University of California, Berkeley, with the Torrey Mesa Research Institute, and the 20,000 lines have been obtained by SIGNAL (http://signal.salk.edu/cgi-bin/tdnaexpress).

Confirmation of T-DNA Lines
The nature and location of the T-DNA insertion is confirmed by sequencing PCR products. Once the location of the T-DNA insertion was confirmed, we designed gene-specific PCR primers that flank the T-DNA for use in a codominant genotyping analysis. By performing two sets of PCR, one using the gene-specific primer pair and the other using a gene-specific primer and the T-DNA border primer, we could determine whether the individual is homozygous for no T-DNA insertion, heterozygous for the T-DNA insertion, or homozygous for the T-DNA insertion.

Molecular Characterization of the T-DNA Lines
To determine the number of T-DNA inserts present in the lines, we compared the DNA gel blot hybridization patterns arising from sibling plants that were either homozygous for the T-DNA insertion or homozygous for no T-DNA. To remove additional T-DNA loci from the lines of interest, backcrosses to wild-type Col were performed, and plants homozygous for the T-DNA insertion were again identified.

Construction of Promoter-GUS Fusions
The following primers were used to amplify the ARF promoter fragments: ARF7, F 5'-CTAAGCTTGTCGACAGTACGTAGATTATTTTCCACAACTCTCTC-3' and R 5'-GAGGATCCATGATCACTCAACTTTACTTTCTCTGAAG-3'; ARF12, F 5'-GGAGGTCGACACAAACAACATGATTGAATAAG-3' and R 5'-GATCGGATCCCCAAAATATGTTATCTCAAC-3'; ARF19, F 5'-ACTGAAGCTTTGGGCTAGATTCATCCGTATCTGGGT-3' and R 5'-CCCGGGAATTCTCATGATGGTTTGGTGCAGGGAAG-3'; ARF22, F 5'-GAAGAAGAGTGAAATCCAGTGACC-3' and R 5'-AGGATCCATAAGCTCGTATCTAAAGCTCGG-3'.

Promoter fragments (ARF12 and ARF22, 2 kb; ARF7, 2.5 kb; ARF19, 3.2 kb) upstream of the translation initiation codon were synthesized by PCR using wild-type (Col) genomic DNA and the primers listed above. The fragments were sequenced and subcloned into the pBI101.2 (ARF7, ARF12, and ARF22) or pZP121 (ARF19; Hajdukiewicz et al., 1994) vectors as SalI/BamHI (ARF7 and ARF12), HindIII/BamHI (ARF22), and SalI/BspHI (ARF19) fragments. The pZP121 vector was modified by introducing the GUS gene as an NcoI/SacI fragment. Among the four promoter GUS constructs, Pro_ARF12:GUS, Pro_ARF22:GUS, Pro_ARF7:GUS, and Pro_ARF19:GUS, the Pro_ARF19:GUS promoter also contains 889 bp of the 3' region of the ARF19 gene (from the 41-bp 5' of the ARF19 translation stop codon to the 848-bp 3' of the translation stop codon). It was amplified by PCR with the primers, F 5'-ACTGGAGCTCGTACACTATGAAGACACTTCTGCTGCAGCT-3' and R 5'-TGACGAATTCAAGACGCGATTGAACCAACCCGGTATGA-3', using BAC T29M8 DNA as a template. It was subcloned as a SacI/EcoRI fragment into a pZP121-Pro_ARF19-GUS construct. With the SacI site present in the forward primer and the EcoRI site located in the reverse primer, the PCR product was cloned into pNcoI-GUS to create pGUS-3A11.

These constructs were introduced into Agrobacterium tumefaciens strain GV3101, and wild-type Col plants were transformed by dipping (Clough and Bent, 1998). Kanamycin-resistant plants in the T2 (Pro_ARF7:GUS) and T3 (Pro_ARF12:GUS, Pro_ARF19:GUS, and Pro_ARF22:GUS) generations were histochemically stained to detect GUS activity by incubating seedlings or tissues in 100 mM sodium phosphate buffer, pH 7.5, containing 1 mM 5-bromo-4-chloro-3-indolyl-ß-D-glucuronic acid, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, and 0.1% Triton X-100 for 5 h at 37°C followed by dechlorophylation in 70% ethanol. Several independent lines were examined for GUS staining.

Overexpression of ARF19
Transgenic plants overexpressing the ARF19 protein (Pro_35S:ARF19) under the control of the 35S promoter were generated by subcloning the 35S-ARF DNA (pS-A11) as a XhoI fragment into the binary vector pKF111.XL (Ni et al., 1998) and transforming plants as described (Clough and Bent, 1998). Fifty-two T1 transformants were selected in soil based on resistance to Finale (Farnam Companies, Phoenix, AZ) diluted 1:1,000 (final concentration 0.05% glufosinate ammonium) in 0.005% Silwet, and sprayed on the germinating seedlings. Two lines (line 1 and line 2) were examined in detail.

RT-PCR Analysis