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Lateral root formation, a developmental process under the control of the plant hormone auxin, is a major determinant of root architecture, and defines the ability of a plant to acquire nutrients and water. The LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) proteins play an important role in the lateral organ development of plants, including lateral root formation. However, their downstream components and signalling mechanisms are largely unknown. Here, we show that auxin-responsive LBD18/ASL20 acts as a specific DNA-binding transcriptional activator that directly regulates EXPANSIN14 (EXP14), a gene encoding a cell wall-loosening factor that promotes lateral root emergence in Arabidopsis thaliana. We showed that LBD18 possesses transcription-activating function in both yeast and Arabidopsis protoplasts. We isolated putative LBD18 target genes by microarray analysis, and identified EXP14 as a direct target of LBD18. Dexamethasone-induced expression of LBD18 under the CaMV 35S promoter in transgenic Arabidopsis resulted in enhanced expression of GUS fused to the EXP14 promoter in primordium and overlaying tissues. In contrast, GUS expression under the EXP14 promoter in the lbd18 mutant background was significantly reduced in the same tissues. Experiments using a variety of molecular techniques demonstrated that LBD18 activates EXP14 by directly binding to a specific promoter element in vitro and in vivo. Overexpression of EXP14 in Arabidopsis resulted in the stimulation of emerged lateral roots, but not primordia, whereas EXP14 loss-of-function plants had reduced auxin-stimulated lateral root formation. This study revealed the molecular function of LBD18 as a specific DNA-binding transcription factor that activates EXP14 expression by directly binding to its promoter.
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Root system architecture is critical for the absorption of water and nutrients, and for the anchorage of plants (Hochholdinger and Zimmermann, 2008). Lateral root formation is a major determinant of root system architecture, and is best characterized in Arabidopsis (Péret et al., 2009a). The developmental events associated with lateral root formation in Arabidopsis, which include priming, initiation, primordium development and the emergence of lateral roots, are primarily regulated by auxin (Péret et al., 2009a). The auxin signalling pathway in Arabidopsis involves two large protein families: auxin response factors (ARFs), transcriptional regulators of auxin-regulated gene expression, and Aux/IAA proteins, which are negative regulators of ARFs (Mockaitis and Estelle, 2008). Auxin promotes the interaction between Aux/IAA repressors and the auxin receptor TIR1, an F-box component of a ubiquitin ligase complex for the degradation of Aux/IAA proteins, in turn releasing ARFs from repression. Two auxin response modules, IAA14-ARF7-ARF19 and IAA12-ARF5, control lateral root initiation and the patterning process (Fukaki et al., 2002; Vanneste et al., 2005; De Smet et al., 2010). The GATA23 transcription factor specifies lateral root founder cell identity during IAA28-dependent auxin signalling (De Rybel et al., 2010). ARF7 and ARF19 regulate lateral root formation via the activation of LBD16/ASL18 and LBD29/ASL16 (Okushima et al., 2007). LBD18 regulates lateral root formation in conjunction with LBD16 downstream of ARF7 and ARF19 (Lee et al., 2009a,b). LBD18 along with LBD33/ASL24 mediates lateral root organogenesis through the transcriptional activation of the E2Fa transcription factor that regulates the asymmetric cell division marking lateral root initiation (Berckmans et al., 2011). The results with expression of LBD16-SRDX, a dominant repressor of LBD16 in transgenic Arabidopsis, also indicated that the localized activity of LBD16 is involved in the coordinated asymmetric cell divisions of the primed lateral root founder cells for lateral root initiation (Goh et al., 2012).
The LBD/ASL gene family encodes proteins harbouring a conserved plant-specific lateral organ boundaries (LOB) domain (Iwakawa et al., 2002; Shuai et al., 2002). The LOB domain is approximately 100 amino acids in length and contains a conserved four-Cys motif, a Gly-Ala-Ser block and a Leu-zipper-like coiled-coil motif (Shuai et al., 2002). In Arabidopsis thaliana there are 42 LBD genes that have been assigned to two classes (Shuai et al., 2002). In rice (Oryza sativum) and maize (Zea mays), 35 LBD genes and 43 LBD genes have been identified from genome sequencing data, respectively (Yang et al., 2006; Schnable et al., 2009). A variety of studies have revealed the developmental functions of certain LBD genes in Arabidopsis (Xu et al., 2003; Borghi et al., 2007; Okushima et al., 2007; Soyano et al., 2008; Lee et al., 2009b; Rubin et al., 2009), and their homologues in rice (Inukai et al., 2005; Liu et al., 2005) and maize (Bortiri et al., 2006; Evans, 2007; Taramino et al., 2007). AS2/LBD6 is well characterized. AS2 functions in the establishment of adaxial–abaxial polarity, in conjunction with the AS1 gene encoding a MYB protein (Xu et al., 2003). AS1 and AS2 bind directly, as a complex, to the regulatory motifs present in the target gene promoter and create a loop, forming a repressive chromatin state that blocks enhancer activity during organogenesis (Guo et al., 2008). JAGGED LATERAL ORGAN (JLO)/LBD30/ASL19 is required for coordinated cell division during embryogenesis, and contributes to the auxin-dependent embryonic apical–basal polarity and patterning processes in the embryo (Borghi et al., 2007; Bureau et al., 2010). LBD16, LBD18 and LBD29 function in lateral root development (Okushima et al., 2007; Lee et al., 2009b). LBD18 and LBD30 are involved in regulating tracheary element differentiation (Soyano et al., 2008). LBD37/ASL39, LBD38/ASL40 and LBD39/ASL41 are involved in the regulation of anthocyanin and nitrogen metabolism as repressors (Rubin et al., 2009). A truncated LOB protein containing only the LOB domain, AS2 and LBD4/ASL6 can preferentially bind unique DNA sequences in electrophoretic mobility shift assays (EMSAs; Husbands et al., 2007), indicative of harbouring DNA-binding activity. The LBD homologues in other plants such as rice and maize have been shown to function in lateral organ development. For example, CROWN ROOTLESS 1 (CRL1)/ADVENTITIOUS ROOTLESS 1 (ARL1) is essential for crown root formation in rice, and is regulated by ARF (Inukai et al., 2005; Liu et al., 2005). Although the biological functions of some LBD genes involved in lateral organ development and metabolism from Arabidopsis, rice and maize have been identified, little is known about their molecular function and signalling pathways (Majer and Hochholdinger, 2011).
Here, we show that LBD18 can function as a specific DNA-binding transcriptional activator that regulates the expression of EXP14 encoding a cell wall-loosening factor by directly binding to its promoter, facilitating lateral root formation by promoting the emergence of lateral roots. Our results elucidate the molecular signalling and gene regulatory mechanism by which LBD18 links auxin signalling with lateral root formation, in part by transcriptional activation of a cell wall-remodelling enzyme.
LBD16 and LBD18 have transcription activity in both yeast and Arabidopsis protoplasts
To understand the molecular function of LBD18 in conjunction with LBD16 in lateral root formation, the transcriptional activity of LBD18 was first examined using transient expression assays with protoplasts from Arabidopsis mesophyll cells. We employed a reporter plasmid harbouring three copies of the Gal4-DNA binding element fused to a LUCIFERASE (LUC) reporter gene and an effector plasmid encoding full-length LBD18 fused to the Gal4 DNA-binding domain (Gal4BD). LBD18 conferred higher levels of LUC expression compared with those in the Gal4BD control (Figure 1). Similar results were obtained with LBD16. The transcriptional activity of LBD18 was higher in Arabidopsis root protoplasts than that in mesophyll protoplasts, which confirmed the importance of LBD18 function in roots.
Deletion analyses of LBD16 and LBD18 in both yeast and Arabidopsis protoplasts revealed a minimal transcription-activating domain (Figures S1 and 2). The LBD16 transcription-activating domain was rich in glutamine, and that of LBD18 was rich in glutamine and proline (Figure S2), which are a characteristic of the transactivation domains of many transcription factors (Triezenberg, 1995; Liu et al., 1999). These results indicate that the regions encompassing amino acids 121–193 of LBD16 and 143–216 of LBD18 have transcription-activating function.
LBD18 functions as a transcriptional activator that regulates the expression of EXP14
To investigate the transcriptional response downstream of LBD18, we used dexamethasone (DEX)-induced nuclear localization of LBD18 fused to the glucocorticoid steroid hormone binding domain (GR) (Pro35S:LBD18:GR; Lee et al., 2009b) with the Arabidopsis full-genome array. A 2.5-h time point following DEX treatment was selected to investigate the early transcriptional response. Among numerous genes whose expression was modulated by LBD18 (Tables S1–S3), a few representative genes exhibiting robust expression over sixfold by DEX (Table S4) were selected, including GDSL-MOTIF (At4g30140), EXP14 (At5g56320) and FAD-BD (At1g30760), to further characterize gene expression. As these genes are regulated downstream of LBD18, they should be auxin inducible at a later time point than the induction of LBD18. As predicted, all three genes displayed delayed expression kinetics upon auxin treatment with respect to LBD18 (Figures 3a and S3). DEX treatment of Pro35S:LBD18:GR seedlings induced the expression of these genes, whereas mock treatment of Pro35S:LBD18:GR seedlings did not (Figure 3b). LBD41, previously shown to be upregulated by LBD30 (Borghi et al., 2007), was induced 4 h after DEX treatment. To further show that LBD18 can upregulate the expression of endogenous target genes, transgenic Arabidopsis plants expressing LBD18:GR under its own promoter (ProLBD18:LBD18:GR) were generated, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis of target gene expression was performed before and after DEX treatment (Figure 3c). DEX treatment caused a statistically significant increase in the levels of endogenous target gene transcripts in two different transgenic lines, showing significant LBD18:GR expression out of nine lines generated, compared with those in mock-treated lines, demonstrating that LBD18 upregulates the expression of target genes in tissues where LBD18 is normally expressed. Treatment with the protein synthesis inhibitor cycloheximide prevented DEX-induced expression of GDSL-MOTIF, FAD-BD and LBD41 at both 2 and 8 h (Figure 3d), suggesting that these target genes are secondary response genes requiring new protein synthesis. However, EXP14 induction by DEX treatment was not affected by cycloheximide treatment at 2 and 8 h, demonstrating that EXP14 is a primary response gene that might be directly regulated by LBD18.
To confirm the upregulation of the target genes, including EXP14, by LBD18, protoplast co-transfection assays were performed with a reporter plasmid harbouring the EXP14 promoter fused to LUC (ProEXP14:LUC), and an effector plasmid harbouring LBD18 expressed under the control of the CaMV 35S promoter, with a translational enhancer Ω (Pro35S:Ω:LBD18). LBD18 expression resulted in a fivefold enhancement of LUC expression from ProEXP14:LUC, whereas co-expression of LBD18 with the negative controls, LUC and Pro35Smini:LUC, did not increase LUC expression (Figure 4a). LUC expression was increased twofold by the co-expression of ProFAD-BD:LUC and LBD18. These results demonstrated that LBD18 upregulates the expression of EXP14 and FAD-BD through its own promoter, and that LBD18:GR-induced expression of target genes is not the result of LBD18 translational fusion with GR.
LBD16 does not regulate the expression of EXP14
We next tested if LBD16 could also regulate the expression of LBD18 target genes in Pro35S:LBD16:GR plants (Lee et al., 2009b). LBD16 was expressed within 1 h of auxin treatment, consistent with a previous report (Lee et al., 2009b), and earlier than the expression of GDSL-MOTIF and FAD-BD in response to auxin (Figure S4a). Expression of these two genes was induced by DEX treatment (Figures S4b and S5). However, unlike LBD18, EXP14 expression was not regulated by LBD16 (Figure S4b). GDSL-MOTIF is a secondary response gene regulated by LBD16, as GDSL-MOTIF expression by LBD16 was prevented by cycloheximide treatments (Figure S4c). DEX-inducible expression of FAD-BD by LBD16 was not prevented by cycloheximide treatment after 2 h, but was inhibited after 8 h (Figure S5). This result indicates that FAD-BD might be directly regulated by LBD16 at early time points, but at later time points it might be subject to another layer of regulation, requiring new protein synthesis. Arabidopsis protoplast transient gene expression assays were used to further demonstrate that FAD-BD expression, but not EXP14 expression, is upregulated by LBD16 (Figure 4b).
Loss of function in LBD18 causes reduced GUS expression in primordium and overlaying tissues of ProEXP14:GUS
We showed that EXP14 is a primary target gene of LBD18 (Figure 3). A potential role of EXP14 in cell-wall loosening is consistent with the role of LBD18 in lateral root formation. We therefore focused on EXP14 to understand the molecular mechanism of LBD18 acting as a transcription factor. To demonstrate that LBD18 regulates EXP14 promoter activity in vivo during lateral root formation, transgenic Arabidopsis expressing GFP:GUS reporter proteins under the control of the EXP14 promoter (ProEXP14:GFP:GUS) was constructed, and double transgenic plants were generated, harbouring both ProEXP14:GFP:GUS and Pro35S:LBD18:GR constructs. GUS staining in leaf veins, lateral root primordium and root stele, endodermis and cortex was detected in ProEXP14:GFP:GUS/Pro35S:LBD18:GR double transgenic plants, before DEX treatment (Figures 5a–c), and was then enhanced by DEX treatment (Figures 5d–f). GUS expression in the epidermis of these double transgenic lines was hardly detected before DEX treatment (Figure 5c), but was strongly enhanced after DEX treatment (Figure 5f). These results demonstrate that LBD18 can activate EXP14 expression via the EXP14 promoter in tissues where lateral root development and emergence occur.
To further demonstrate that EXP14 is an endogenous target of LBD18, we generated ProEXP14:GFP:GUS transgenic Arabidopsis in the lbd18-1 mutant background (ProEXP14:GFP:GUS/lbd18-1), and conducted GUS expression analysis. We observed a decrease in GUS staining and expression in roots of lbd18-1 compared with those in the wild-type background (Figure 6). GUS staining in the root stele decreased in lbd18-1 compared with that in the wild type (Figure 6a and e). GUS staining was detected in primordium, endodermis, cortex and epidermis in the wild type (Figures 6b–d), and decreased significantly in lbd18-1 (Figure 6f,g). A cross-section analysis of primary roots also showed weak GUS staining in endodermis, cortex and epidermis, and strong GUS staining in the root stele in the wild type (Figure 6c). GUS staining in all of these tissues in the wild type decreased in lbd18-1 (Figure 6g). These results showed that the LBD18 loss of function reduced GUS expression in primordium and the overlaying tissues such as endodermis, cortex and epidermis, under the control of the EXP14 promoter. Quantitative RT-PCR analysis further confirmed that GUS expression of ProEXP14:GUS decreased significantly by 59% in the root of the lbd18-1 mutant, compared with that in the wild type (Figure 6h). Taken together, these results demonstrate that EXP14 is an endogenous target of LBD18 during lateral root formation.
LBD18 directly binds a specific region of the EXP14 promoter in vitro and in vivo
To determine the EXP14 promoter element required for interacting with LBD18 as a trans-acting factor, a series of constructs containing 5′ to 3′ terminal deletions of the EXP14 promoter fused to LUC were generated, and co-transfection assays were conducted using Arabidopsis mesophyll protoplasts with or without Pro35S:Ω:LBD18. The 100-bp region from –699 to –599 bp of the EXP14 promoter, relative to the start codon, was found to be necessary for transcriptional activation by LBD18 (Figure 7a). A heterologous yeast one-hybrid system was used to determine whether LBD18 binds the 100-bp region of the EXP14 promoter. Two DNA fragments were generated from the EXP14 promoter: one from –799 to –700 bp, designated the A region, served as a negative control; and the other from –699 to –599 bp, designated the B region, comprised the minimal EXP14 promoter element (Figure 7b). The fragments were fused to the HIS3 reporter gene in the pSK1 vector, yielding the pSK1-A region and the pSK1-B region. LBD18, under the control of the GAL1 promoter, was inserted into the pYESTrp2 vector (pYESTrp2-LBD18) to generate a construct in which LBD18 expression could be induced by adding galactose. Whereas yeast strains transformed with pSK1-B region and pYESTrp2-LBD18 could grow on galactose medium lacking histidine, but not on glucose medium (Figure 7c), yeast strains transformed with the pSK1-A region and pYESTrp2-LBD18 did not, indicating that LBD18 activates the HIS3 reporter gene via the B region and that LBD18 probably binds the B region.
To show that LBD18 binds the EXP14 promoter in vivo, we conducted a chromatin immunoprecipitation (ChIP) analysis by overexpressing LBD18 tagged with haemagglutinin (HA) in frame with the N terminus of LBD18 in lbd18-1. Arabidopsis lbd18 transgenic lines harbouring a vector for DEX-inducible LBD18 transcription were generated (Pro35S:DEX-inducible-HA:LBD18/lbd18-1; Figure S6), as constitutive overexpression of LBD18 in Arabidopsis caused severely inhibited growth and eventually death (Soyano et al., 2008; Lee and Kim, 2010). Quantitative PCR assays clearly showed that DEX treatment significantly induced the accumulation of PCR products from the region stretching from –749 to –550 bp in anti-HA antibody immunoprecipitates (Figure 7d), compared with that from the control Arabidopsis EGFP transgenic line (Pro35S:DEX-inducible-HA:EGFP; Figure S7), demonstrating that LBD18 binds to the EXP14 promoter in vivo.
To determine whether LBD18 can directly bind the B region in vitro, an EMSA was conducted by using nine overlapping oligonucleotide probes encompassing the region from –724 to –575 bp of the EXP14 promoter and recombinant proteins (Figure 8a,b). GST-LBD18 bound to probe #6, but not to any of the other probes (Figure 8b). This binding was easily competed out with 5× cold-specific probe #6, but was not affected by 50× non-specific probe #7 (Figure 8c). Taken together, the results demonstrate that LBD18 binds to the 30-bp region from –707 to –674 bp of the EXP14 promoter in a DNA sequence-specific manner.
Overexpression of EXP14 in Arabidopsis stimulates the formation of emerged lateral roots, whereas loss of function in EXP14 reduces auxin-stimulated lateral root formation
Several genes potentially involved in cell-wall remodelling, such as EXP and PG, are expressed in front of the emerging lateral root primordia, which might be crucial for local cell separation and passage of the primordia through the outer cell layers (Swarup et al., 2008; Péret et al., 2009a). The role of EXP14 in lateral root formation was assessed by examining the lateral roots of transgenic Arabidopsis plants expressing EXP14 under the control of the CaMV 35S promoter, compared with the wild type and the exp14-1 knock-out mutant (Figures 9a, S8 and S9). Three different transgenic lines displayed a statistically significant increase in primary root elongation (Figure S9a). The number of emerged lateral roots per primary root length increased in the transgenic lines to some extent, compared with those in the wild type and the exp14-1 mutant (Figure S9b), but did not affect lateral root primordium development (Figure S9c). To further determine whether EXP14 overexpression affects lateral root initiation or not, we counted the number of emerged lateral roots along the portion of the primary root where lateral roots are present (Figure 9b, A′), and the lateral root primordium density, that is, the number of non-emerged lateral root primordia along the portion of the primary root where non-emerged lateral root primordia are present (Figure 9b, portion B′). As shown in Figure 9c and d, we found that lateral root densities in portion A′ in the three different transgenic lines increased to some extent, compared with those in the wild type, whereas lateral root primordium densities were not changed in the transgenic lines, wild type or exp14-1 mutant. The exp14-1 mutant did not show a significant difference in lateral root density compared with that of the wild type. We examined if exogenous auxin treatment could produce a differential effect on lateral root formation in the exp14-1 mutant and the wild type. As shown in Figure 9e, exogenous auxin treatment significantly enhanced lateral root densities in Pro35S:EXP14 transgenics, compared with those in the wild type. However, the exp14-1 mutant exhibited relatively reduced lateral root density compared with that of the wild type at 0.1 μm concentration of auxin 2,4-dichlorophenoxyacetic acid (2,4-D). These results demonstrate that EXP14 is involved in facilitating lateral root formation at the lateral root emergence step.
A variety of the components involved in developmental processes of lateral root formation in Arabidopsis have been identified, and the mechanisms underlying lateral root initiation have been well studied. However, little is known about their gene regulatory mechanisms and molecular signalling during lateral root formation (Péret et al., 2009a,b; De Smet et al., 2010). Here we showed that LBD18 regulates the expression of EXP14, a gene encoding a cell wall-loosening protein, by directly binding to its promoter, contributing to the emergence of lateral roots (Laskowski et al., 2006; Swarup et al., 2008; Péret et al., 2009a).
Several LBD genes have been functionally characterized in Arabidopsis and crop plants (Majer and Hochholdinger, 2011). However, the downstream targets of LBD proteins that link the auxin signalling cascade with developmental processes are largely unknown (Majer and Hochholdinger, 2011). Recently, LBD18 has been shown to transcriptionally regulate E2Fa involved in lateral root initiation through cell cycle reactivation in response to auxin (Berckmans et al., 2011). In the present study, we showed that LBD18 functions as a specific DNA-binding transcriptional activator, and that EXP14 is one of its immediate endogenous target genes. Several lines of evidence obtained in this study support this conclusion. First, LBD18 activated the expression of endogenous EXP14 under the control of the CaMV 35S promoter (Pro35S:LBD18:GR), as well as under the control of the LBD18 promoter (ProLBD18:LBD18:GR) (Figure 3b,c). DEX treatment of ProLBD18:LBD18:GR transgenic Arabidopsis caused a significant increase in EXP14 transcript levels by 40% compared with those in the control (Figure 3c). This relatively low increase in EXP14 expression by DEX treatment of ProLBD18:LBD18:GR plants, compared with that in Pro35S:LBD18:GR plants, might be the result of the limited additional factors in ProLBD18:LBD18:GR plants for the full activation of EXP14 under the native promoter. Nevertheless, our result showed that LBD18 can activate EXP14 expression under its own promoter. Second, LBD18 induced EXP14 expression equally in the presence or absence of cycloheximide, a protein synthesis inhibitor (Figure 3d), indicating that EXP14 is a primary target of LBD18. Third, LBD18 activated LUC reporter gene expression in Arabidopsis protoplasts when the reporter plasmid contained the EXP14 promoter fused upstream of LUC (Figure 4a). Fourth, LBD18 enhanced GUS staining in the primordium and the overlaying tissues of ProEXP14:GFP:GUS in Arabidopsis (Figure 5). Fifth, the LBD18 loss of function reduced GUS staining in the root stele, primordium and the overlaying tissues of ProEXP14:GFP:GUS Arabidopsis (Figure 6). Quantitative RT-PCR analysis further confirmed that the lbd18 mutation significantly decreased GUS expression. Sixth, transient gene expression assays with Arabidopsis protoplasts, using a variety of reporter constructs harbouring the 5′ deletion of the EXP14 promoter, revealed the 100-bp DNA region for LBD18 to trans-activate (Figure 7a). Lastly, yeast one-hybrid, EMSA and ChIP assays demonstrated that LBD18 binds a specific region in the EXP14 promoter in vitro and in vivo (Figures 7b–d and 8).
Consistent with a role of EXP in cell-wall loosening, we found that overexpression of EXP14 in Arabidopsis caused an increase in the number of emerged lateral roots per primary root length, compared with those in the wild type and the exp14 mutant (Figure S9b), but did not affect lateral root primordium development (Figure S9c). We further determined that lateral root densities, where lateral roots are present (Figure 9b, A′), in transgenic lines increased to some extent compared with those in the wild type, whereas lateral root primordium densities (Figure 9b, B′) did not change in transgenic lines, wild type, and the exp14 mutant. We also found that although exogenous auxin treatment significantly enhanced lateral root densities in Pro35S:EXP14 transgenics, compared with those in the wild type (Figure 9e), the exp14 mutant exhibited relatively reduced lateral root density compared with that of the wild type at 0.1 μm concentration of auxin 2,4-D. These results showed that EXP14 is involved in facilitating lateral root formation at lateral root emergence. Enhanced lateral root formation by EXP14 overexpression is also consistent with GUS expression detected in the overlaying tissues, such as the endodermis and cortex, in ProEXP14:GFP:GUS (Figures 4 and 5). However, the exp14 mutant did not show a statistically significant difference in lateral root density compared with that in the wild type. This result may arise from genetic redundancy among 36 expansin and expansin-like genes in Arabidopsis (Sampedro and Cosgrove, 2005). The present microarray analysis showed that other EXPs, such as EXP9 and EXP17, were upregulated by LBD18 (Table S1). Analysis of multiple mutants of EXP14 and those other expansin genes that are upregulated by LBD18 will help clearly elucidate the function of expansin genes during lateral root emergence. Transcriptional regulation of E2Fa by LBD18 (Berckmans et al., 2011) suggests a role of LBD18 in lateral root initiation via auxin-mediated cell cycle reactivation. We have previously shown that the number of primordia of lbd16, lbd18 or lbd16 lbd18 mutants is similar to that observed in the wild type, whereas the number of emerged lateral roots of lbd16 or lbd18 single mutants is reduced significantly, and lbd16 lbd18 double mutants exhibited an additively reduced number of emerged lateral roots compared with the single mutants (Lee et al., 2009b). Therefore, our present results and the previous report (Lee et al., 2009b) together suggest that LBD16 and LBD18 play a role in the emergence of lateral roots, in addition to a role in lateral root initiation.
Swarup et al. (2008) have isolated genes induced by auxin in tissues surrounding the lateral root initiation site, which might be involved in lateral root formation during the emergence of lateral roots, by the analysis of root transcript profiling data sets in order to identify candidate genes involved in cell wall remodelling. We found such 27 candidate genes from the genes that are upregulated by LBD18 (Table S5). It is interesting to note that the expression of EXP17 and a gene encoding subtilisin-like protease (AIR3) in these candidate genes is dependent on LAX3 function (Swarup et al., 2008), because LAX3 encodes the AUX1-like auxin influx carrier that promotes lateral root emergence by affecting the auxin influx of the cortex cells, and is auxin inducible. Moreover, we found that LAX3 is upregulated by LBD18 (Table S5). A gene encoding GDSL lipase/hydrolase family protein is also expressed in the zones of lateral root emergence (Takahashi et al., 2010). However, except for LAX3, none of the 26 candidate genes have been functionally identified for lateral root formation. It remains to be seen whether part of these genes upregulated by LBD18, including LAX3-regulated genes, might contribute to lateral root emergence.
A truncated LOB protein containing only the LOB domain, AS2, and LBD4 had been previously shown to preferentially bind the unique DNA sequences, the LBD motifs, in an EMSA (Husbands et al., 2007). However, we could not find the LBD motifs in the DNA region of the EXP14 promoter that LBD18 can bind in the EMSA or in the other regions of the EXP14 promoter, indicating that LBD18 has a distinct DNA-binding specificity relative to the LOB protein. This observation reflects different biological functions of individual LBD genes through the regulation of different target genes via different DNA-binding specificity. LBD18 directly regulates EXP14 expression by binding its promoter, but LBD16 does not regulate EXP14 expression (Figures 4b and S4). Interestingly, both LBD16 and LBD18 induce the expression of a GDSL lipase/hydrolase family protein found in the lateral root emergence zones (Takahashi et al., 2010), and also the expression of FAD-BD (Figures 3, 4, S4, and S5). Thus, although LBD18 displays distinct DNA-binding specificity relative to LBD16 with regard to the EXP14 promoter, both LBD16 and LBD18 might regulate common targets directly or indirectly. Further biochemical and genetic studies are necessary to prove this hypothesis.
Plant growth and tissue treatment
Arabidopsis thaliana (Col-0) seedlings were grown and treated as described previously (Park et al., 2002). Plants were grown with a 16-h photoperiod on 3MM Whatman filter paper (http://www.whatman.com) on top of agar plates, and the filter paper with the seedlings was then transferred to a plate containing plant hormone or chemicals (20 μm IAA and 10 μm DEX, or 50 μm cycloheximide) and incubated for a given period of time with gentle shaking under light at 23°C.
Plasmid construction and Arabidopsis transformation
The details are described in Appendix S1.
RNA isolation, RT-PCR, qRT-PCR and RNA-gel blot analysis
Following treatment, Arabidopsis plants were immediately frozen in liquid nitrogen and stored at −80°C. Total RNA was isolated from frozen Arabidopsis using TRI Reagent® (Molecular Research Center, Inc., http://www.mrcgene.com). Total RNA was separated on a 1.2% agarose gel, transferred to a nylon membrane, and hybridized with 32P-labelled DNA probes at 68°C for 3 h using 10 ml of QuickHyb solution (Stratagene, now Agilent Technologies, http://www.genomics.agilent.com) and then washed. The blot was then exposed to X-ray film. For RT-PCR analysis, total RNA was isolated using an RNeasy Plant Mini kit (Qiagen, http://www.qiagen.com) and subjected to RT-PCR analysis with the Access RT-PCR System (Promega, http://www.promega.com), according to the manufacturer's instructions. Real-time RT-PCR was carried out using a QuantiTect SYBR Green RT-PCR kit (Qiagen) in a Rotor-Gene 2000 Real-time Thermal Cycling System (Corbett Life Science, http://www.corbettlifescience.com), as described previously (Jeon et al., 2010). All quantitative real-time RT-PCR was conducted in triplicate biological replications and subjected to statistical analysis. The DNA probes for RNA gel-blot analysis were amplified by RT-PCR, subcloned into the pGEM®-T Easy vector (Promega), and verified by DNA sequencing. RT-PCR conditions, primer sequences and DNA probes are shown in Table S6.
Reporter and effector plasmids
Reporter and effector plasmids were transfected into mesophyll or root protoplasts isolated from Arabidopsis using polyethylene glycol. GUS activity was assayed with 1 mm 4-methylumberlliferyl-β-d-glucuronide. LUC activity was normalized to GUS activity. Details are described in Appendix S1.
Transient expression assays with Arabidopsis protoplasts
Protoplasts from 2- to 3-week-old Arabidopsis plants or from root tissues of 8-day-old seedlings grown vertically on MS plates were prepared as described previously (Lee et al., 2008). Isolated mesophyll or root protoplasts were transfected with plasmid DNA by polyethylene glycol (PEG)-mediated protoplast. The details are described in Appendix S1.
Histochemical GUS assays
Histochemical assays for GUS activity were performed with 5-bromo-4-chloro-3-indolyl glucuronide (Duchefa, http://www.duchefa.com), as described previously (Jefferson and Wilson, 1991).
Yeast one-hybrid assays
The LBD18 full-length DNA fragment amplified by PCR was ligated into pYESTrp2 (Invitrogen, http://www.invitrogen.com) at EcoRI and XhoI sites. The A-region and B-region reporter constructs were prepared by inserting the A-region promoter element encompassing the nucleotides from –799 to –700 bp and the B-region from –699 to –599 bp, relative to the AUG initiation codon, into pSK1 (Kim et al., 1997) at NotI and SpeI sites, in front of the HIS3 reporter gene. A yeast one-hybrid assay was performed using the YPH500 yeast strain, as described previously (Choi et al., 2000).
The LBD16 or LBD18 full-length DNA was amplified by PCR and inserted into pGEX 4T-1 vector at SmaI (N terminus) and XhoI (C terminus) sites for LBD16, and at the EcoRI (N terminus) and XhoI (C terminus) sites for LBD18. The GST:LBD16 or GST:LBD18 plasmid was transformed into bacterial strain BL21-CodonPlus(DE3)-RIL competent cells (Stratagene). The transformed cells were grown at 37°C in a shaking incubator until the OD600 of the cell cultures reached 0.8–1.0. After cooling the cultures at 18°C for 30 min, 1% of methanol and 1 mm isopropyl-1-thio-β-d-galactopyranoside (IPTG) were added at the final concentrations into the cultures. The expression of the recombinant GST-fusion proteins was then induced at 18°C overnight in a shaking incubator. The cultured bacterial cells were lysed with lysis buffer (20 mm Tris, pH 8.0, and 150 mm NaCl) by sonication with Vibra-Cell VCX130 (Sonics & Materials Inc., http://www.sonicsandmaterials.com). The GST-fusion proteins were purified using glutathione-Sepharose 4B (GE Healthcare, http://www.gehealthcare.com), according to the manufacturer's instructions. The purified proteins were dialysed against binding buffer (20 mm HEPES-KOH, pH 7.6, 75 mm KCl, 1 mm DTT and 10% glycerol). The EMSA was conducted essentially as described previously (Ausubel et al., 2002). To prepare DNA probes for EMSA, the oligonucleotides (25–37 mers) were denatured by boiling them for 5 min, followed by a slow cooling to 23°C. The annealed oligonucleotides were radio-labelled with [α-32P]dCTP by the standard Klenow fill-in reactions, and then purified on G-50 micro columns (GE Healthcare), according to the manufacturer's instructions. The binding reaction for EMSA was performed in 10 μl of reaction mixture containing 400 fmol DNA probes, binding buffer (20 mm HEPES KOH, pH 7.6, 75 mm KCl, 1 mm DTT and 10% glycerol), 200 ng of poly (dI-dC)• poly (dI-dC), 2 μg of BSA and 100 ng of the purified GST-fusion proteins at room temperature for 30 min. The samples were then loaded on a 4.5% native polyacrylamide gel (29 : 1 acrylamide/bis solution; Bio-Rad, http://www.bio-rad.com) with 0.5× Tris-borate buffer. The gel was subjected to electrophoresis, dried and exposed to X-ray film.
The ChIP assays were conducted essentially as described previously (Jeon and Kim, 2011). Six-day-old seedlings were treated with or without DEX for 2 days. DNA from these seedlings was immunoprecipitated with an anti-HA antibody (Santa Cruz Biotechnology, Inc., http://www.scbt.com), and subjected to PCR analysis (Jeon and Kim, 2011). Quantitative PCR analysis was conducted using SsoFast EvaGreen Supermix (Bio-Rad) on a CFX96 Real-time System machine (Bio-Rad). Details are described in Appendix S1.
Quantitative data were subjected to statistical analysis for every pairwise comparison, using the software for Student's t-test (spss 17.0; SPSS, Chicago, IL, USA).
Microarray and statistical analysis
For Affymetrix GeneChip analysis, Pro35S:LBD18:GR plants were incubated with mock or with DEX for 2.5 h, and total RNAs were isolated with an RNeasy Plant Mini kit (Qiagen). The RNAs were subjected to microarray analysis with triplicate biological replications, followed by statistical analysis to determine the false discovery rates (FDRs). Details are described in Appendix S1.
We thank Dr Guilfoyle for the GD-VP16, GD-ARF5M and Gal4(3X)-GUS vectors, Dr S.Y. Kim for the pSK1 vector and ABRC (http://abrc.osu.edu) for the T-DNA insertion mutants. We also thank Dr Bennet and his lab members for critically reading our manuscript. This work was supported by grants from the World Class University project of the Ministry of Science, Education and Technology of Korea (R31-2009-000-20025-0), and from the Next-Generation BioGreen 21 Program (no. PJ008166), Rural Development Administration, Republic of Korea to J.K.