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Plant Cell. Jan 2007; 19(1): 118–130.
PMCID: PMC1820965

ARF7 and ARF19 Regulate Lateral Root Formation via Direct Activation of LBD/ASL Genes in Arabidopsis[W]

Abstract

Lateral root formation in Arabidopsis thaliana is regulated by two related AUXIN RESPONSE FACTORs, ARF7 and ARF19, which are transcriptional activators of early auxin response genes. The arf7 arf19 double knockout mutant is severely impaired in lateral root formation. Target-gene analysis in arf7 arf19 transgenic plants harboring inducible forms of ARF7 and ARF19 revealed that ARF7 and ARF19 directly regulate the auxin-mediated transcription of LATERAL ORGAN BOUNDARIES-DOMAIN16/ASYMMETRIC LEAVES2-LIKE18 (LBD16/ASL18) and/or LBD29/ASL16 in roots. Overexpression of LBD16/ASL18 and LBD29/ASL16 induces lateral root formation in the absence of ARF7 and ARF19. These LBD/ASL proteins are localized in the nucleus, and dominant repression of LBD16/ASL18 activity inhibits lateral root formation and auxin-mediated gene expression, strongly suggesting that these LBD/ASLs function downstream of ARF7- and ARF19-dependent auxin signaling in lateral root formation. Our results reveal that ARFs regulate lateral root formation via direct activation of LBD/ASLs in Arabidopsis.

INTRODUCTION

Although the branched root systems of higher plants are hidden underground, they play an essential role in whole plant growth and development. The root system of higher plants consists of an embryonic primary root and postembryonic developed lateral roots and adventitious roots. In dicot plants, lateral root formation is crucial for maximizing a root system's ability to absorb water and nutrients as well as to anchor plants in the soil. The plant hormone auxin, which mediates a variety of physiological processes (Davies, 1995), has long been known to promote lateral root formation (Torrey, 1950; Blakely et al., 1988; Laskowski et al., 1995). However, the molecular details of auxin action in this process remain largely unknown.

In Arabidopsis thaliana, lateral roots are initiated by the auxin-dependent local activation of pericycle cells at the xylem poles (Casimiro et al., 2001, 2003). Delineating the molecular events that lead to lateral root initiation is a prerequisite for a thorough understanding of how auxin modulates developmental functions. Control of these processes by auxin involves the complex regulation of auxin biosynthesis and transport and the ability of cells to respond to auxin in an appropriate manner. Particularly little is known about the auxin signaling cascade during the onset of lateral root initiation.

At the molecular level, auxin rapidly alters transcript levels of numerous genes (Abel and Theologis, 1996). Thus, the effects of auxin, including the promotion of lateral root formation, may be mediated directly through changes in gene expression. Transcriptional regulation of auxin-regulated genes is dependent on two related families of transcriptional regulators, AUXIN RESPONSE FACTORs (ARFs) and AUXIN/INDOLE-3-ACETIC ACIDs (Aux/IAAs). ARFs bind to the auxin response elements (AuxREs) in the promoter region of early auxin response genes and activate or repress their transcription (reviewed in Guilfoyle and Hagen, 2001). Of the 23 ARFs in the Arabidopsis genome (Liscum and Reed, 2002), five members with a Gln-rich sequence in the middle region (ARF5/MONOPTEROS [MP], ARF6, ARF7/NONPHOTOTROPIC HYPOCOTYL4 [NPH4], ARF8, and ARF19) function as transcriptional activators of auxin-responsive genes (Ulmasov et al., 1999; Tiwari et al., 2003; Wang et al., 2005). By contrast, Aux/IAA proteins, which are encoded by 29 genes in Arabidopsis, negatively modulate auxin-regulated gene expression as transcriptional repressors through heterodimerization with the ARF transcription activators (Tiwari et al., 2001, 2003, 2004).

Aux/IAA proteins are labile in planta; they are subject to proteolysis through the ubiquitin-mediated pathway (Gray et al., 2001). Importantly, auxin promotes this degradation pathway. Recent studies have revealed that binding of auxin to the F-box protein TRANSPORT INHIBITOR RESPONSE1 (TIR1) or the related AUXIN RECEPTOR F-BOX PROTEINS (AFBs) accelerates the degradation of Aux/IAAs by promoting their interaction with the SCFTIR1/AFBs ubiquitin-ligase complex (Dharmasiri et al., 2005a, 2005b; Kepinski and Leyser, 2005), thereby derepressing the activity of ARF transcriptional activators. Thus, auxin signaling is largely dependent on the proteolytic regulation of Aux/IAAs. Degradation of Aux/IAAs is dependent on the presence of a conserved domain II, which mediates interaction with the SCFTIR1/AFBs complex. Gain-of-function mutations in domain II of several different Aux/IAAs stabilize the corresponding proteins and cause pleiotropic defects in auxin-regulated growth and development (reviewed in Reed, 2001; Yang et al., 2004).

Given the no (or severely reduced) lateral root phenotype of several Aux/IAA gain-of-function mutants, such as iaa14/solitary root (slr) (Fukaki et al., 2002), iaa3/short hypocotyl or suppressor of hy2 (shy2) (Tian and Reed, 1999), iaa19/massugu2 (msg2) (Tatematsu et al., 2004), and iaa28 (Rogg et al., 2001), it is evident that Aux/IAAs inhibit the activity of specific ARFs required for lateral root formation. Forward and reverse genetic approaches have identified ARF7 and ARF19 as key components in a developmental pathway regulating lateral root formation. arf7 arf19 double mutants exhibit a severely reduced lateral root formation phenotype not observed in arf7 and arf19 single mutants, indicating that lateral root formation is redundantly regulated by these two ARF transcription activators (Okushima et al., 2005; Weijers et al., 2005; Wilmoth et al., 2005; Li et al., 2006). Furthermore, both ARF7 and ARF19 interact with IAA14/SLR, IAA3/SHY2, IAA19/MSG2, and IAA28 in yeast (Tatematsu et al., 2004; Fukaki et al., 2005; Weijers et al., 2005; our unpublished data), and their expression patterns are highly overlapped (Fukaki et al., 2002; Okushima et al., 2005). Thus, the auxin-signaling pathway originating from ARF7 and ARF19 controls lateral root formation together with several Aux/IAA transcriptional repressors, such as IAA14/SLR, IAA3/SHY2, IAA19/MSG2, and IAA28.

Identification of the genes regulated by ARF7 and ARF19 during lateral root formation is essential to reveal the extent and details of the transcriptional network. Because of their well-characterized properties as transcriptional activators of auxin-regulated genes, we were able to take advantage of genome-wide expression analysis to examine alterations of auxin-mediated gene expression of the mutant compared with the wild type. As expected, auxin-induced gene expression is globally and severely impaired in the arf7 arf19 double mutant (Okushima et al., 2005). Based on this microarray data, we have identified >100 ARF7 and ARF19 target gene candidates. However, only a subset of these genes is expected to be direct targets, and it is not certain that all direct target genes are biologically important for lateral root formation.

Here, we describe the identification of LATERAL ORGAN BOUNDARIES-DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) genes as direct regulatory targets of ARF7 and ARF19 in lateral root formation. Our results provide a better framework for understanding how the auxin signal is transmitted by ARF transcription factors during lateral root formation in higher plants.

RESULTS

ARF7 and ARF19 Are Required for the Initiation of Lateral Root Formation

Previous studies have shown that ARF7 and ARF19 play important roles in lateral root formation (Okushima et al., 2005; Wilmoth et al., 2005). However, it is not clear which stage is mediated by ARF7 and ARF19 during lateral root formation. Therefore, we examined the expression of lateral root initiation markers in primary roots of arf7 arf19. Expression of End199, the marker line for stage II and subsequent stages of lateral root primordium development (Malamy and Benfey, 1997), was hardly detected in 4- to 7-d-old arf7 arf19 seedlings, whereas the number of primordia with End199 activity increased in wild-type seedlings as the primary roots lengthened (Figure 1A). These results indicate that lateral root formation is strongly inhibited at the very early stage of initiation in arf7 arf19. Because auxin sensitivity is severely impaired in the roots of the arf7 arf19 double mutant (Okushima et al., 2005; Wilmoth et al., 2005), we next examined the auxin-induced pericycle division. To monitor auxin-induced pericycle cell division during synchronized lateral root initiation, we used the lateral root–inducible system (Himanen et al., 2002) and the G2/M transition–associated ProCycB1;1:CycB1;1(NT)-GUS reporter (Colón-Carmona et al., 1999), which enables the detection of cells undergoing mitosis. No CycB1;1(NT)-GUS expression was observed in the pericycles of either wild-type or arf7 arf19 3-d-old seedlings grown on medium containing 10 μM naphthylphthalamic acid (Figures 1B and 1D). However, strong β-glucuronidase (GUS) activity was detected in the pericycle cells of wild-type seedlings after they were transferred to 10 μM 1-naphthylacetic acid (NAA) for 12 h (Figure 1C), as reported previously (Himanen et al., 2002; Vanneste et al., 2005). By contrast, no GUS activity was induced in pericycle cells of arf7 arf19 seedlings treated with NAA for 12 h (Figure 1E), indicating that 12 h of auxin treatment cannot activate pericycle cell division in arf7 arf19. These observations indicate that auxin-induced pericycle cell division for lateral root initiation is strongly inhibited in the arf7 arf19 double mutant. Thus, ARF7 and ARF19 are required early in lateral root formation, probably at the initiation stage.

Figure 1.
Auxin-Mediated Lateral Root Initiation Is Severely Impaired in the arf7 arf19 Double Mutant.

Consistent with the functions of ARF7 and ARF19 as transcriptional activators (Wang et al., 2005; Wilmoth et al., 2005), ARF7- and ARF19-GFP (for Green Fluorescent Protein) fusion proteins were localized in nuclei (Figures 1F and 1G). ARF7- and ARF19-GFP rescued the phenotype of the arf7 arf19 double mutant when driven by their own promoters (data not shown), strongly suggesting that ARF7 and ARF19 proteins function in the nucleus.

LBD16, LBD29, and LBD33 Genes Are Candidate Targets for ARF7 and ARF19 in Lateral Root Formation

If ARF7 and ARF19 are transcriptional activators, as currently thought (Wang et al., 2005; Wilmoth et al., 2005), they should directly regulate auxin-induced genes rather than auxin-repressed genes. We previously identified ARF7 and ARF19 target gene candidates among the genes that are induced by auxin in wild-type seedlings but not in arf7 arf19 (Okushima et al., 2005). Among these candidates, we focused on a set of closely related genes that encode LBD/ASL protein family members. LBD16/ASL18, LBD17/ASL15, LBD18/ASL20, LBD29/ASL16, and LBD33/ASL24 (hereafter referred to as LBD16, LBD17, LBD18, LBD29, and LBD33, respectively) are induced by auxin in wild-type seedlings but not in arf7 arf19 (Figure 2K) (Okushima et al., 2005). LBD33 was not identified using the original criteria for auxin-induced genes. However, a more lenient significance cutoff (P < 0.01; originally we used P < 0.001) placed it in the gene set of statistically valid auxin-induced genes (data not shown). Interestingly, these five LBD members cluster together within the same clade of the phylogenetic tree (Iwakawa et al., 2002) (Figure 2A), suggesting that they are closely related at the sequence level as well as in terms of auxin-mediated expression properties. In addition, ~2-kb promoter regions of the five candidate LBD genes all have TGTCTC or GAGACA sequences that may function as AuxREs, suggesting that they could be regulated directly by ARF7 and/or ARF19.

Figure 2.
Expression Profiles of Auxin-Responsive LBD/ASL Genes.

Among the five LBD genes, LBD16, LBD29, and LBD33 are the most likely targets of ARF7 and ARF19 during lateral root formation, because they are expressed primarily in roots (Shuai et al., 2002) (see Supplemental Figure 1 online). In 6-d-old ProLBD16:GUS transformant seedlings, strong GUS activity was detected in root stele and lateral root primordia (Figure 2B). A similar but weaker GUS expression pattern in root stele and lateral root primordia was detected in ProLBD29:GUS seedlings (Figure 2C). In ProLBD33:GUS seedlings, GUS activity was localized in lateral root primordia (Figure 2D) and was hardly detected in root vasculature. Expression of all three reporter lines was induced in response to exogenous auxin (Figures 2E to 2J), but their induction profiles were partially distinct. For example, ProLBD16:GUS was strongly expressed in vascular tissues of the mature root region, but auxin-mediated induction was observed in the elongation zone only (Figures 2E and 2F). By contrast, auxin treatment led to increased expression of ProLBD29:GUS and ProLBD33:GUS in the steles of mature regions in primary and lateral roots (Figures 2G to 2J). The previous observation that both ARF7 and ARF19 are expressed in the root stele (Okushima et al., 2005; Wilmoth et al., 2005) supports the observation that auxin-mediated induction of these LBD genes is dependent on ARF7 and ARF19 function.

LBD16 and LBD29 Are Primary Auxin Response Genes

RT-PCR analysis confirmed the auxin-mediated induction (1 μM NAA, 2.5 h) of LBD16, LBD29, and LBD33 and its strong dependence on ARF7 and ARF19 function (Figure 2K). Considering the current model of ARF transcriptional activators as regulators of primary auxin response genes, their direct target genes would be expected to have expression properties similar to those of previously identified primary auxin response genes (e.g., several Aux/IAA genes). As reported previously (Abel et al., 1995), IAA5, one of the typical primary response Aux/IAA genes, was induced significantly by the protein synthesis inhibitor cycloheximide (CHX) (Figure 2K) and responded to exogenous auxin within the first 30 min of treatment (Figure 2L). Interestingly, expression of both LBD16 and LBD29 was strongly induced by CHX in the wild type (Figure 2K) and was induced within 30 min of auxin treatment (Figure 2L). These results qualify LBD16 and LBD29 as primary response genes, which could be regulated directly by ARF7 and ARF19. In addition, mild but significant CHX-mediated induction of LBD16 and LBD29 was still observed in the arf7 arf19 background (Figures 2K and and3B),3B), suggesting the existence of an ARF7- and ARF19-independent pathway probably controlled by an unknown transcription regulator. By contrast, treating wild-type seedlings with CHX did not result in the induction of LBD33 (Figure 2K), and the induction of LBD33 was detectable only after 1 h of auxin treatment (Figure 2L), suggesting that LBD33 belongs to a set of secondary response genes. Although not identified as a candidate target of ARF7 and ARF19 (Okushima et al., 2005), expression of NAC1, a positive regulator of lateral root formation downstream of auxin signaling (Xie et al., 2000, 2002; Guo et al., 2005), was examined. Under our conditions, at least 2.5 h after treatment, neither NAA nor CHX dramatically affected NAC1 expression in either wild-type and arf7 arf19 seedlings (Figure 2K). Thus, NAC1 expression does not appear to be regulated by ARF7 and ARF19.

Figure 3.
LBD16 and LBD29 Are Potential Direct Targets of ARF7.

Direct Activation of LBD16 and LBD29 by ARFs

To examine whether LBD16, LBD29, and LBD33 are regulated directly by ARF7 and ARF19, we produced transgenic plant lines with inducible ARF7 and ARF19 activity. These lines were generated by introducing native promoter-driven ARF7 or ARF19 fused to the steroid binding domain of rat glucocorticoid receptor (GR) (ProARF7:ARF7-GR or ProARF19:ARF19-GR) into the arf7 arf19 double mutant. Both ProARF7:ARF7-GR/arf7 arf19 and ProARF19:ARF19-GR/arf7 arf19 plants showed the typical arf7 arf19–like impaired lateral root formation phenotype unless treated with the steroid hormone dexamethasone (DEX). Continuous DEX treatment resulted in lateral root formation similar to that observed in arf19 and arf7 single mutants (Figure 3A; see Supplemental Figure 2 online), indicating that ARF-GR fusion proteins were biologically functional in a DEX-dependent manner.

With the use of these inducible lines, we examined the expression profiles of LBD16, LBD29, and LBD33. Treatment with DEX plus NAA of ProARF7:ARF7-GR/arf7 arf19 seedlings for 4 h resulted in strong induction of LBD16, LBD29, and LBD33 relative to mock-treated or NAA-treated controls (Figure 3B). A combined treatment with DEX, NAA, and CHX resulted in a further induction of LBD16 and LBD29 (Figure 3B), indicating that LBD16 and LBD29 may be the direct targets of transcriptional activation by ARF7. Induction of LBD16 and LBD29 by DEX plus NAA was detectable within 1 h of treatment, suggesting that induction occurred rapidly after ARF7 activation (data not shown). By contrast, DEX-dependent auxin-mediated induction of LBD33 was completely inhibited by CHX treatment. These results strongly suggest that LBD16 and LBD29 are direct targets of ARF7, whereas LBD33 is not induced directly by ARF7. Similar results were obtained using a ProARF19:ARF19-GR/arf7 arf19 line in the same experimental setup. However, data shown in Supplemental Figure 2B online imply only a minor contribution of ARF19 in the direct induction of LBD16 and LBD29 yet a significant role in the activation of LBD29 transcription. Together, these results strongly suggest that ARF7 directly regulates both LBD16 and LBD29, whereas ARF19 directly regulates both genes, but LBD16 less so. Neither ARF7 nor ARF19 directly regulates LBD33.

As mentioned previously, LBD16 contains one AuxRE in its promoter region, and LBD29 contains two (Figure 3C). To examine whether ARF7 binds to these sequences in vitro, we performed a mobility shift assay using LBD16 and LBD29 promoter fragments containing AuxREs as probes. The N-terminal half of ARF7 that contains a putative DNA binding domain was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli. Recombinant GST-ARF7 protein, but not GST alone, bound to the single LBD16 and to both LBD29 promoter fragments (Figure 3C; see Supplemental Figure 2C online). Similar results were obtained with crude extracts of yeast expressing full-length ARF7 rather than recombinant GST-ARF7 (data not shown). These results suggest that ARF7 binds directly to promoter regions of LBD16 and LBD29 to activate their expression. Similarly, recombinant ARF19 also bound the LBD29 promoter fragments (see Supplemental Figure 2C online). Together, these results indicate that ARF7 and ARF19 regulate the auxin-mediated induction of LBD16 and/or LBD29 through direct binding to the promoter regions of target genes.

Overexpression of LBD16 and LBD29 Rescues Lateral Root Formation of the arf7 arf19 Mutant

To examine the biological function of LBD16 and LBD29 in lateral root formation, LBD16 and LBD29 were overexpressed under the control of the CaMV 35S promoter in the arf7 arf19 mutant background (Pro35S:LBD16/arf7 arf19 and Pro35S:LBD29/arf7 arf19). Multiple independent Pro35S:LBD16 lines displayed increased lateral root formation. The differences in the production of the lateral root phenotype correlate with ectopic LBD16 gene expression (data not shown). A representative line with almost 100 times wild-type expression of LBD16 (97.2- ± 20.2-fold) was selected for detailed studies. Although the number of lateral roots was less than in the wild type, the 12-d-old Pro35S:LBD16/arf7 arf19 seedlings developed lateral roots (Figure 4A), indicating that overexpression of LBD16 partially rescues lateral root formation in the arf7 arf19 line. We also observed that overexpression of LBD16 in a wild-type background causes severely impaired primary root and shoot growth, suggesting the possibility that overexpression of LBD16 could modify ARF7- and ARF19-dependent pathways (our unpublished data).

Figure 4.
Overexpression of LBD16 and LBD29 Partially Suppresses the Lateral Root Phenotype of arf7 arf19.

To address the possibility that LBD16 overexpression affects auxin signaling in the arf7 arf19 mutant, we examined the root lengths and lateral root formation of Pro35S:LBD16/arf7 arf19 transgenic lines grown in the presence or absence of auxin. Although exogenous auxin (1 μM NAA) treatment, which strongly induces lateral root formation in wild-type seedlings, induced very few lateral roots in arf7 arf19, Pro35S:LBD16/arf7 arf19 seedlings produced many lateral roots in response to exogenous auxin (Figure 4B). In addition, primary roots of Pro35S:LBD16/arf7 arf19 were more sensitive to auxin than were those of the auxin-resistant arf7 arf19 mutant (Figure 4C). These data indicate that LBD16 overexpression partially restores the ability of the arf7 arf19 mutant line to respond to exogenous auxin.

Overexpression of LBD29 also restored lateral root formation in arf7 arf19 but was much less effective than LBD16. Only a few of 40 Pro35S:LBD29/arf7 arf19 independent lines showed slightly increased lateral root formation (data not shown), and all of these lines exhibited only ~10-fold wild-type levels of LBD29 expression. We also generated Pro35S:LBD29-GFP/arf7 arf19 lines. Among 24 independent Pro35S:LBD29-GFP/arf7 arf19 lines, the line with the highest transgene expression (58.5- ± 18.7-fold) was selected for detailed studies. At 12 d after germination, the arf7 arf19 mutant did not generate lateral roots, whereas Pro35S:LBD29-GFP/arf7 arf19 seedlings developed a small number of lateral roots (Figure 4A). This observation indicates that overexpression of LBD29 also has the ability to promote lateral root formation in an arf7 arf19 mutant background, but it is much less effective than LBD16. Together, these results indicate that LBD16 and LBD29 induce lateral root formation in the absence of ARF7 and ARF19. We also examined the effects of LBD33 overexpression in arf7 arf19, but none of 20 available transgenic lines had increased lateral root formation (data not shown).

In an attempt to identify the role of LBD16 in wild-type plants, we obtained a T-DNA insertion mutant from the Salk collection (SALK_095791; lbd16-1); there are no LBD29 knockout lines in the public collections. RT-PCR analysis confirmed that lbd16-1 is a knockout allele of LBD16 (data not shown). lbd16-1 plants appeared very similar to wild-type plants at both seedling and adult stages. Root growth was inhibited to almost the same extent in both wild-type and lbd16-1 seedlings in response to exogenous auxin (data not shown). In addition, the lbd16-1 mutation did not affect auxin-regulated gene expression, because auxin induced IAA5, IAA19, LBD29, and LBD33 to a similar degree in lbd16-1 and wild-type seedlings (data not shown). However, the number of lateral roots was reduced slightly in 7-d-old lbd16-1seedlings (8.1 ± 2.8 [mean ± sd] in the wild type [n = 54] and 4.7 ± 2.4 in lbd16-1 [n = 48]; P < 0.001). Although exogenous auxin induced lateral root formation in lbd16-1 as in wild-type seedlings, there were fewer lateral roots in auxin-treated lbd16-1 than in the wild type (63.2 ± 15.8 [mean ± sd] in the wild type [n = 52] and 34.7 ± 8.8 in lbd16-1 [n = 31]; P < 0.001). These observations imply that LBD16 contributes to auxin-mediated lateral root formation in wild-type seedlings. The mild lateral root formation phenotype observed in lbd16-1 may be attributable to the functional redundancy of LBD/ASL gene family members.

Overexpression of LBD16 Fused to a Transcriptional Repressor Domain Inhibits Lateral Root Formation and Auxin-Induced Gene Expression

To examine the subcellular localization of LBD16 and LBD29, we generated transgenic plants that express LBD16-GFP and LBD29-GFP translational fusions under the control of the CaMV 35S promoter in the arf7 arf19 mutant background. Among ~20 independent transgenic lines each for both constructs, three (LBD16-GFP) and one (LBD29-GFP) lines had increased lateral root formation and showed strong nuclear GFP signals in the root epidermal cells (Figures 4A, 5A, and 5B; data not shown). These data strongly suggest that LBD16 and LBD29 act in the nucleus to control lateral root formation.

Figure 5.
Nuclear Localization of LBD-GFP Fusion Proteins, and Dominant Repression Phenotype of LBD16.

The lack of an obvious loss-of-function phenotype of LBD16 may be attributable to redundancy in gene family members. However, transgenic seedlings overexpressing LBD16 fused to the transcriptional suppression domain (SRDX) (Hiratsu et al., 2003) under the control of the CaMV 35S promoter (Pro35S:LBD16-SRDX) exhibited strong morphological defects in roots and aerial organs. Interestingly, lateral root formation in Pro35S:LBD16-SRDX seedlings was strongly impaired in multiple independent lines (11 of 14 independent lines in the T2 generation; Figure 5C). We did not observe any lateral root primordia before emergence in 10-d-old Pro35S:LBD16-SRDX seedlings, suggesting that initiation was severely inhibited in this line (data not shown). In addition, especially in strong lines (line 6; Figure 5C), narrower and upward-curled leaves and strongly impaired root growth were observed in some seedlings (Figure 5E). Although less severe, this phenotype resembles the effects of domain II mutations in Aux/IAA proteins, such as axr3/iaa17 (Leyser et al., 1996; Rouse et al., 1998; Reed, 2001). These observations prompted an examination of auxin-regulated gene expression in Pro35S:LBD16-SRDX seedlings. We used the high LBD16-SRDX expression line 5 for expression analysis (63.0- ± 16.0-fold wild-type native LBD16 level). Auxin treatment (1 μM NAA, 4 h) strongly induced the expression of LBD29, LBD33, and IAA5 in 7-d-old wild-type seedlings but not in the arf7 arf19 double mutant (Figure 5F). Auxin-mediated induction of LBD29, LBD33, and IAA5 were significantly impaired in Pro35S:LBD16-SRDX seedlings (Figure 5F), strongly suggesting that auxin signaling was perturbed in these plants even though they have wild-type ARF7 and ARF19 genes. Therefore, LBD16 may be a positive mediator of auxin signaling in Arabidopsis growth and development, including lateral root formation.

DISCUSSION

Recent molecular genetic studies have identified two members of the ARF transcription activator family, ARF7 and ARF19, that redundantly regulate lateral root formation in Arabidopsis (Okushima et al., 2005; Weijers et al., 2005; Wilmoth et al., 2005; Li et al., 2006). Genes that trigger the lateral root initiation cascade could be regulated directly by ARF7 and ARF19, but little was known of the factors that mediate auxin-mediated lateral root initiation downstream of ARF7 and ARF19. In this study, we identified LBD16 and LBD29 as the direct targets of these ARFs responsible for lateral root initiation from a number of candidate genes that were screened previously by microarray-based analysis (Okushima et al., 2005). Overexpression of LBD16 and LBD29 partially rescues lateral root formation in arf7 arf19, strongly suggesting that these two related LBD/ASLs promote lateral root formation. Target gene analysis using an ARF7- and ARF19-inducible system together with electrophoresis mobility shift assay indicated that ARF7 and ARF19 are direct transcriptional regulators of LBD16 and LBD29.

Thus, in our current auxin signaling cascade model for lateral root initiation (Figure 6), the auxin signal that is captured by TIR1/AFBs accelerates the degradation of Aux/IAA proteins, typified by IAA14/SLR, IAA3/SHY2, IAA19/MSG2, and IAA28, thereby derepressing the function of ARF7 and ARF19 as transcriptional activators. ARF7 and ARF19 activate the transcription of downstream target genes, including LBD16 and LBD29. LBD16 also probably activates a downstream signaling network as a transcriptional regulator. In this pathway, ARF7 makes a major contribution to the activation of the downstream signaling cascade by inducing LBD16 and LBD29. Activation of LBD16 by ARF7 might be particularly important, because LBD16 is much more effective than LBD29 for generating lateral roots in the arf7 arf19 double mutant. On the other hand, ARF19 plays an auxiliary role in the activation of LBDs.

Figure 6.
Model of the ARF7- and ARF19-Dependent Auxin Signaling Cascade for Lateral Root Formation.

Analyses using the ProCycB1;1:CycB1;1(NT)-GUS reporter construct demonstrated that auxin-induced pericycle cell division for lateral root initiation was severely impaired in arf7 arf19 as well as in the gain-of-function mutant iaa14/slr (Fukaki et al., 2002; Vanneste et al., 2005). Consistently, expression of stabilized iaa14/slr mutant protein under the control of either the ARF7 or the ARF19 promoter also blocked lateral root formation, and IAA14/SLR interacts with ARF7 and ARF19 in yeast (Fukaki et al., 2005). These observations strongly suggest that ARF7, ARF19, and IAA14/SLR regulate lateral root initiation at the same stage. We found that dominant repression of LBD16 strongly inhibited lateral root formation, probably at the initiation stage. Induction of auxin-responsive LBDs, including LBD16 and LBD29, is also inhibited in the iaa14/slr mutant that is defective in lateral root initiation (Vanneste et al., 2005). Thus, IAA14/SLR, ARF7, and ARF19 and their target genes, including LBD16, probably act in the initiation stage of lateral root formation. Accordingly, LBD16 and LBD29 are expressed mainly in the root stele and lateral root primordia in wild-type seedlings, and these expression domains overlap the region where ARF7 and ARF19 are expressed. Consistent with the results of microarray and RT-PCR analyses, auxin-mediated expression of ProLBD16:GUS and ProLBD29:GUS was blocked in arf7 arf19 (data not shown), also suggesting that ARF7 and ARF19 regulate the auxin-mediated expression of these LBD/ASL genes. These data imply that a striking induction of LBD16 and LBD29 transcription by ARF7 and/or ARF19 is important to activate the downstream signal cascade for the induction of lateral root initiation. However, transcription of LBD16 and LBD29 is not only regulated by ARF7 and ARF19, because arf7 arf19 roots have basal expression levels of these LBD/ASLs, just like wild-type roots. In addition, mild but significant CHX-mediated induction of LBD16 and LBD29 was still observed in the arf7 arf19 background (Figures 2K and and3B),3B), suggesting the existence of an ARF7- and ARF19-independent pathway, probably controlled by an unknown transcription regulator that is regulated by an unstable repressor. The residual ability to produce lateral roots in the older arf7 arf19 plants implies that other ARFs also contribute to lateral root formation (Okushima et al., 2005). ARF5/MP, an essential regulator for embryonic root development, seems most likely to redundantly regulate lateral root formation via transcriptional activation of downstream target genes of ARF7 and ARF19 (Hardtke, 2006).

LBD16/ASL18 and LBD29/ASL16 are highly related members of the LBD/ASL family, which consists of 42 members in Arabidopsis (Iwakawa et al., 2002; Shuai et al., 2002). Several LBD/ASLs are important for lateral organ development. LBD6/AS2 and LBD36/ASL1, other related members of this family, are required for the differentiation of lateral organs in aerial plant parts, such as leaves and petals (Iwakawa et al., 2002; Chalfun-Junior et al., 2005). Maize (Zea mays) RAMOSA2, a potential ortholog of Arabidopsis LOB/ASL4, is required for the patterning of stem cells in branch meristems (Bortiri et al., 2006). Recent reports show that a rice homolog of LBD16 and LBD29, CROWNROOTLESS1/ADVENTITIOUS ROOTLESS1 (CRL1/ARL1), regulates crown root formation in rice. Mutations in CRL1/ARL1 result in severely impaired crown root formation (Inukai et al., 2005; Liu et al., 2005). In addition, crl1/arl1 mutants show auxin-related phenotypes in roots, including decreased lateral root number and impaired root gravitropism, and CRL1/ARL1 gene expression is induced by auxin (Inukai et al., 2005; Liu et al., 2005). These reports indicate that the function and regulation of LBD16- and LBD29-related genes are partially conserved between monocot and dicot root development.

Several LBD/ASL proteins of Arabidopsis and rice are known to be localized in the nucleus and thus are thought to function in transcriptional control (Iwakawa et al., 2002; Liu et al., 2005). LBD6/AS2 and LBD36/ASL1 negatively regulate several KNOTTED1-like homeobox (KNOX) genes, such as KNAT1/BP and KNAT2, whose products promote stem cell proliferation in leaves (Ori et al., 2000; Semiarti et al., 2001; Lin et al., 2003; Chalfun-Junior et al., 2005). Although it is not known whether these LBD/ASLs directly control the expression of KNOX genes, our data using a LBD16-SRDX overexpressor indicates the possibility that LBD16 could function as a transcriptional regulator. We observed that LBD16-SRDX inhibits the auxin-mediated induction of not only the secondary response gene, LBD33, but also primary response genes, such as LBD29 and IAA5. Furthermore, overexpression of LBD16 in a wild-type background but not in an arf7 arf19 background causes severe developmental defects in both roots and aerial parts, suggesting that LBD16 can modify ARF7- and ARF19-dependent pathways, either directly or indirectly (our unpublished data). Thus, ARF-mediated transcriptional regulation may involve a positive feedback loop through the ARF-mediated induction of LBD16 by auxin. This is in contrast with the negative feedback loop with the ARF-mediated induction of Aux/IAAs. Having proposed that LBD16 has a function in auxin signaling, it would be intriguing to identify its target genes and to dissect the downstream events of LBD16 during lateral root formation. It is possible that LBD33, which is located downstream of ARF7- and ARF19-dependent auxin signaling but is not a direct target, could be one of the downstream genes of LBD16.

Unlike rice crl1/arl1 mutants (Inukai et al., 2005; Liu et al., 2005), T-DNA insertions or RNA interference interruption of the expression of LBD16 and LBD29 did not show any obvious defects in plant development, including lateral root formation (this study; data not shown), whereas the dominant repression of LBD16 function strongly inhibited lateral root formation. The expression domain of LBD16 and LBD29 largely overlaps root stele tissues and lateral root primordia, and both are primary auxin response genes, suggesting a functional redundancy between these two related LBDs. Functional redundancy among LBD16, LBD29, and possibly other closely related family members may preclude genetic analysis using single mutants. LBD/ASLs belong to a large gene family in Arabidopsis, rice, and maize (Iwakawa et al., 2002; Shuai et al., 2002; Bortiri et al., 2006; Yang et al., 2006). As pointed out by Sablowski and Meyerowitz (1998) in the case of floral homeotic genes in the MADS family and their target NAP gene in the NAC family, it is possible that as ARF genes diverged during evolution their target LBD/ASL genes may also have duplicated and specialized.

Since the early 1990s, numerous transcription factors have been identified as master regulators of particular developmental processes in higher plants. Current efforts are now directed toward identifying the direct targets of developmentally important transcription factors to reveal whole regulatory networks, using molecular genetics, genomic, and bioinformatics approaches in both animals and plants (Samach et al., 2000; Ito et al., 2004; William et al., 2004; Levesque et al., 2006; Palaniswamy et al., 2006). Several sets of plant target genes have now been reported, like those of the MADS transcriptional factors that regulate flower development (Ito et al., 2004; de Folter and Angenent, 2006). This work represents an important step in explaining the mechanism of ARF-Aux/IAA pathway control in various auxin-regulated developmental processes of higher plants, because despite the importance of ARFs in plant growth and development, such as ARF5/MP in early embryogenesis (Hardtke and Berleth, 1998), little is known about direct targets important for developmental pathways. Further study with the ARF7- and ARF19-inducible system will enable us to understand the signaling cascade of auxin-mediated lateral root formation and to discover how auxin controls the hidden half of higher plants.

METHODS

Plant Materials and Growth Conditions

Arabidopsis thaliana ecotype Columbia was used throughout this study. The nph4-1 arf19-1 double mutant allele was used as the arf7 arf19 double mutant in this study (Okushima et al., 2005). T-DNA insertion mutant lbd16-1 (SALK_095791) was obtained from the ABRC. Seeds were germinated on Murashige and Skoog medium with 1% sucrose. Plants were grown at 23°C under continuous light conditions. For the root auxin sensitivity assay, 4-d-old seedlings were transferred to vertically oriented agar plates containing NAA. The number of lateral roots and root length were determined after an additional 3 d of growth using a dissecting microscope and ImageJ software.

Vector Construction and Plant Transformation

ProLBD16:GUS, ProLBD29:GUS, and ProLBD33:GUS constructs were generated by fusing promoter fragments (LBD16, 2.5 kb; LBD29 and LBD33, 2.2 kb) in front of the GUS coding sequence in the pBI101.2 vector.

Full-length cDNAs of ARF7, ARF19, LBD16, LBD29, and LBD33 were isolated by RT-PCR from flower or root cDNA libraries and introduced into pDONR221 vector using Gateway BP clonase enzyme mix (Invitrogen). To construct Pro35S:LBD16, Pro35S:LBD29, and Pro35S:LBD:LBD33 plasmids, LBD16, LBD29, and LBD33 coding regions were transferred from the pDONR221 vectors to pGWB2 (GWB vectors were kind gifts from Tsuyoshi Nakagawa) by Gateway LR recombination (Invitrogen). To construct Pro35S:ARF7-GFP, Pro35S:ARF19-GFP, Pro35S:LBD16-GFP, and Pro35S:LBD29-GFP plasmids, ARF7, ARF19, LBD16, and LBD29 coding regions were transferred from the pDONR221 vectors to pGWB5 by Gateway LR recombination. To construct ProARF7:ARF7-GR and ProARF19:ARF19-GR, ARF7 and ARF19 promoter fragments (2.5 kb) were synthesized by PCR and inserted into the binary destination vector pGWB-GR, which contains a Gateway conversion cassette (Invitrogen) in front of the GR coding region (pGWB-GR was constructed by inserting the GR coding region into the XbaI/SacI sites of pGWB1). ARF7 and ARF19 coding regions were subsequently transferred from the pDONR221 vectors by Gateway LR recombination. To generate Pro35S:LBD16-SRDX, the LBD16 coding region with a transcriptional repression domain (SRDX) was generated by PCR using a primer with the SRDX (LDLDLELRLGFA) linker sequence (Hiratsu et al., 2003). This LBD16-SRDX cDNA fragment was introduced into pDONR221 using Gateway BP clonase enzyme mix and was subsequently transferred to pGWB2 by Gateway LR recombination. Details of vector construction are available upon request. The resulting vectors were introduced into Agrobacterium tumefaciens GV3101 (pMP90), and wild-type or arf7 arf19 plants were transformed by floral dipping (Clough and Bent, 1998). Transformants were selected on medium containing 50 mg/L kanamycin or 20 mg/L hygromycin. Homozygous lines were identified in the T3 generation, and T3 or T4 homozygous lines were used for phenotypic and molecular analyses.

Phylogenetic Analysis

Multiple sequence alignment based on the LOB/AS2 domain was done with ClustalW (Thompson et al., 1994) at http://www.ddbj.nig.ac.jp/search/clustalw-j.html using the neighbor-joining tree option with 1000 bootstraps. TreeView was used to generate the graphic output (Page, 1996). The actual alignment is given in Supplemental Figure 3 online.

Histochemical GUS Assay and Microscopy

GUS staining, fixation, and whole-mount clearing preparation of roots were performed essentially as described (Malamy and Benfey, 1997), and roots were inspected with a Nikon Eclipse E800 microscope equipped with Nomarski optics. For confocal microscopy, roots were counterstained with 10 μg/mL propidium iodide and analyzed with an Olympus IX80 confocal microscope.

RNA Extraction and RT-PCR Analysis

Total RNA was isolated from plant tissues using the RNeasy kit (Qiagen). First-strand cDNA was synthesized from 2 μg of total RNA with an oligo(dT)24 primer and SuperScriptII reverse transcriptase (Invitrogen). Transcripts were quantified by RT-PCR or real-time RT-PCR analyses using 1/120th of the resulting cDNA as template. Real-time RT-PCR was performed with the LightCycler system (Roche) with SYBR Premix Ex Taq (TaKaRa). Primers used for analyses are listed in the Supplemental Methods online.

Gel Mobility Shift Assay

To generate recombinant GST-ARF7 and GST-ARF19 proteins, the cDNA fragments encoding the N termini of ARF7 (amino acids 1 to 367) and ARF19 (amino acids 1 to 365) were inserted into the pGEX-2T vector and expressed in BL21 Codon Plus Escherichia coli cells (Stratagene). The empty pGEX-2T vector was used to generate GST alone. The production and purification of GST alone and GST fusion proteins were as described previously (Okushima et al., 2002). DNA probes were prepared by annealing complementary oligonucleotides (see Supplemental Methods online for probe sequences) and then labeled with [32P]dCTP using the Klenow fragment of DNA polymerase I. The binding mixture contained 0.4 fmol of 32P-labeled probe, 0.5 μg of affinity-purified GST fusion protein, 1 μg of poly(dA-dT)·poly(dA-dT), 5 μg of BSA, 20 mM HEPES-KOH, pH 7.9, 50 mM KCl, 15% glycerol, and 1 mM DTT in a 20-μL reaction volume. Reactions were incubated for 30 min at room temperature and then separated on 5% native polyacrylamide gels in 0.25× Tris-borate-EDTA at 100 V for 90 min. After electrophoresis, the gels were dried and autoradiographed. For competition experiments, a 125-fold molar excess of cold double-stranded probe was added to the reaction mixture.

Accession Numbers

Arabidopsis Genome Initiative locus identifiers for the genes mentioned in this article are as follows: ARF7 (At5g20730), ARF19 (At1g19220), LBD16/ASL18 (At2g42430), LBD29/ASL16 (At3g58190), and LBD33/ASL24 (At5g06080).

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure 1. Tissue-Specific Expression of Auxin-Inducible LBDs.
  • Supplemental Figure 2. Regulation of LBDs by ARF19.
  • Supplemental Figure 3. Alignment Used for Phylogenetic Analyses.
  • Supplemental Methods.

Supplementary Material

[Supplemental Data]

Acknowledgments

We thank Tsuyoshi Nakagawa for the pGWB vectors, Philip Benfey for End199 seeds, and Peter Doerner for ProCycB1;1:CycB1;1(NT)-GUS seeds. We gratefully thank Keiko Uno for her excellent technical assistance. We thank Steffen Vanneste and Tom Beeckman for helpful discussion and comments on the manuscript. This research was supported by grants to H.F. for Scientific Research on Priority Areas (Molecular Basis of Axis and Signals in Plant Development; Grants 15031218 and 17027019) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and by a grant to M.T. from the Research for the Future program of the Japan Society for the Promotion of Science. Y.O. was supported by a Japan Society for the Promotion of Science research fellowship for young scientists.

Notes

The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) are: Hidehiro Fukaki (pj.ca.u-ebok.trop@ikakuf-h) and Masao Tasaka (pj.tsian.sb@akasat-m).

[W]Online version contains Web-only data.

www.plantcell.org/cgi/doi/10.1105/tpc.106.047761

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