LBD18/ASL20 Regulates Lateral Root Formation in Combination with LBD16/ASL18 Downstream of ARF7 and ARF19 in Arabidopsis

The LATERAL ( LBD/ASL ) genes encode proteins harboring a conserved amino acid domain, referred to as the LOB domain. While recent studies have revealed developmental functions of some LBD genes in Arabidopsis and in crop plants, the biological functions of many other LBD genes remain to be determined. In this study, we have demonstrated that lbd18 mutant evidenced a reduced number of lateral roots and lbd16 lbd18 double mutants exhibited a dramatic reduction in the number of lateral roots compared to lbd16 or lbd18 . Consistent with this observation, significant GUS expression in Pro LBD18 :GUS seedlings was detected in lateral root primordia as well as in the emerged lateral roots. Whereas the numbers of primordia of lbd16 or lbd18 or lbd16 lbd18 mutants were similar to those observed in the wild-type, the numbers of emerged lateral roots of lbd16 and lbd18 single mutants were reduced significantly. lbd16 lbd18 double mutants exhibited additively reduced numbers of emerged lateral roots compared to single mutants. This finding indicates that LBD16 and LBD18 may function in the initiation and the emergence of lateral root formation via a different pathway. LBD18 was shown to be localized into the nucleus. We determined whether LBD18 functions in the nucleus by using a steroid regulator inducible system in which the nuclear translocation of LBD18 can be regulated by dexamethasone (DEX) in wild-type, lbd18 , and lbd16 lbd18 mutant backgrounds. Whereas LBD18 overexpression in wild-type backgrounds induced lateral root formation to some degree, other lines manifested the growth-inhibition phenotype. However, LBD18 overexpression rescued lateral root formation in lbd18 and lbd16 lbd18 mutants without inducing any other phenotypes. Further, we demonstrate that LBD18 overexpression can stimulate lateral root formation in arf7 arf19 mutants with blocked lateral root formation. Taken together, our results suggest that LBD18 functions in the initiation and 35S


INTRODUCTION
The LBD/ASL genes (hereafter referred to as LBD) encode proteins harboring an LOB (lateral organ boundaries) domain, which is a conserved amino acid domain that is detected only in plants, indicative of its function in plant-specific processes (Iwakawa et al., 2002;Shuai et al., 2002). There are 42 Arabidopsis LBD genes, which have been assigned to 2 classes. Class I comprises 36 genes and class II comprises 6 genes (Iwakawa et al., 2002;Shuai et al., 2002). The class I proteins harbor LOB domains similar to those observed in the LOB protein, whereas the class II proteins are less similar to the class I proteins which include the LOB domain, as well as regions outside of the LOB domain. The LOB domain is approximately 100 amino acids in length and harbors a conserved 4-Cys motif with a CX 2 CX 6 CX 3 C spacing, a Gly-Ala-Ser (GAS) block, and a predicted coiled-coil motif with a LX 6 LX 3 LX 6 L spacing, reminiscent of the Leu-zipper found in the majority of class I proteins (Shuai et al., 2002). None of the class II proteins were predicted to form coiled-coil structures.
Although we currently understand very little about the biological roles of the LBD genes, there have been some reports describing the developmental functions of LBD genes in Arabidopsis on the basis of gain-of-function studies. The gain-of-function mutants of LBD36/ ASL1, designated as downwards siliques1 (dsl1-D), showed shorter internodes and downward lateral organs such as flowers (Chalfun-Junior et al., 2005). Although the lbd36 loss-offunction mutants did not show morphological phenotypes, the analysis of lbd36 as2 double mutants showed that these 2 members act redundantly to control cell fate determination in the petals. Another Arabidopsis gain-of-function mutant, jagged lateral organs-D (jlo-D), generates strongly lobed leaves and the shoot apical meristem prematurely arrests organ initiation, terminating in a pin-like structure (Borghi et al., 2007). Thirty-five LBD genes in rice have been identified from the genome sequences of the 2 rice subspecies-a japonica rice (Nippobare) and an indica rice (9311) (Yang et al., 2006).
Analyses of rice mutants have provided evidence of the involvement of a variety of rice LBD genes in lateral organ development. CROWN ROOTLESS1 (CRL1), encoding a LBD protein, is crucial for crown root formation in rice (Inukai et al., 2005). The crl1 mutant showed auxin-related phenotypes, such as decreased lateral root number, auxin insensitivity in lateral root formation, and impaired root gravitropism. A rice AUXIN RESPONSE FACTOR (ARF) appears to directly regulate CRL1 expression in the auxin signaling pathway (Inukai et al., 2005). ADVENTITIOUS ROOTLESS1 (ARL1) encodes an auxin-responsive protein with an LOB domain that controls the initiation of adventitious root primordia in rice, and turned out to be the same gene as CRL1 (Liu et al., 2005).
Lateral roots of Arabidopsis are derived from a subset of the pericycle cells (pericycle founder cells), which are positioned at the xylem poles within the parent root tissues (Casimiro et al., 2003). The mature pericycle cells dedifferentiate to form lateral root primordium (LRP), which undergoes consistent anticlinal and periclinal cell divisions to generate a highly organized LRP (Malamy and Benfey, 1997). The LRP emerges from the parent root via cell expansion, and the activation of the lateral root meristem results in continued growth of the organized lateral root. A growing body of physiological and genetic evidence has been collected to suggest that auxin plays a profound role in lateral root

Phenotype Analysis of lbd16, lbd18, and ld16 lbd18 Mutants
In an earlier study, we constructed a transgenic Arabidopsis that expressed the GR-fused stabilized-iaa1 protein harboring an amino acid change in domain II (Park et al., 2002). DEX treatment of this transgenic Arabidopsis evoked dramatic auxin-related phenotypes and repressed the auxin induction of a variety of Aux/IAA genes (Park et al., 2002;Ku et al., 2009). We previously assessed the effects of DEX-inducible iaa1 on auxin-regulated gene expression, focusing on early genes, with the Affymetrix full genome array, and subsequently identified a transcriptome downstream of iaa1 during the auxin response (Lee et al., 2009). In order to characterize the biological functions of auxin response genes downstream of iaa1, we analyzed a variety of Arabidopsis T-DNA insertion mutants, but none of them evidenced notable morphological phenotypes. Then, we constructed double mutants of similarly classified single mutants and examined auxin-related phenotypes (data not shown). We found that among them, lbd16-1 lbd18-1 double mutants exhibited significantly reduced numbers of lateral roots. Fig. 1, A and B show the schematic view and the results of the PCR analysis of the homozygous T-DNA insertion mutants of lbd16-1 and lbd18-1 single mutants. Fig. 1C shows the results of RT-PCR analysis of homozygous T-DNA insertion mutants of lbd16-1 and lbd18-1, as well as lbd16-1 lbd18-1. We examined the auxin-related phenotypes and morphological changes of these single mutants, but detected no significant phenotypes except for the change in lateral root formation. One example demonstrates the insignificant difference in root growth inhibition between these mutants and the wild-type plants, with varying concentrations of indole-3-acetic acid (IAA) (Supplemental Fig. S1). However, we did note a significant reduction in lateral root number for lbd18-1 as well as lbd16-1 when compared to the wild-type plants at 5 and 8 d after germination (DAG) (Fig. 2, A-C). The lbd16-1 lbd18-1 double mutants showed substantially reduced numbers of lateral roots compared to lbd16-1 and lbd18-1 under a dissecting microscope. The addition of auxin 2,4dichlorophenoxyacetic acid (2,4-D) to the lbd16-1, lbd18-1, and lbd16-1 lbd18-1 mutants rescued the reduced lateral root number to wild-type levels (Fig. 2D). These results indicate that LBD18, in combination with LBD16, is involved in lateral root formation during the auxin response.

GUS Expression Patterns of Pro LBD16 :GUS and Pro LBD18 :GUS Transgenics
It has been previously reported that in Pro LBD16 :GUS seedlings, strong GUS activity was detected in the root stele and the lateral root primordia (Okushima et al., 2007). We additionally assessed the expression of GUS at three different stages of lateral primordium formation (Malamy and Benfey, 1997) and during lateral root emergence in transgenic Arabidopsis harboring the 1.4 kbp LBD16 promoter-GUS fusion construct (Pro LBD16 :GUS) that we generated (Fig. 3, A-D). We noted strong GUS expression not only in the root stele (A-D) and the lateral root primordia (B), but also in the developing (B), emerged (C), and mature (D) lateral roots in 7-d-old light-grown Pro LBD16 :GUS seedlings. Upon treatment with auxin, IAA, strongly enhanced GUS expression was detected in the primary roots and lateral roots ( Fig. 3, A and D, lower panels). We noted intense GUS expression in developing primordia ( Fig. 3B, lower panels). In order to understand the role of LBD18 in lateral root formation, we generated transgenic Arabidopsis harboring the 2.0 kbp LBD18 promoter-GUS fusion construct (Pro LBD18 :GUS) and examined GUS expression during lateral primordium formation and lateral root emergence (Fig. 3, E-H). In 7-d-old light-grown Pro LBD18 :GUS seedlings, significant GUS activity was noted in the root stele (Fig. 3E), similar to what was noted in the Pro LBD16 :GUS seedlings. The GUS staining patterns of Pro LBD18 :GUS seedlings were also assessed for three different stages of lateral root development. GUS activity was observed in the lateral root primordia as well as the emerging lateral root (Fig. 3, F and G). However, no GUS activity was detected in the lateral root stele (Fig. 3H). Treatment with auxin, IAA, resulted in enhanced GUS expression in the primary and lateral roots (Fig. 3, E-H, lower panels). These GUS expression patterns are consistent with the role of LBD18 in lateral root formation during the auxin response. Overlapping GUS expression patterns of Pro LBD16 :GUS and Pro LBD18 :GUS during the formation of lateral root primordium and during the lateral root emergence suggest a common function in lateral root initiation and emergence.

Mutants
Lateral root development can be divided into 4 steps: stimulation and dedifferentiation of pericycle cells, ordered cell division and re-differentiation to generate a highly organized lateral root primordium, emergence of the lateral root primordium via cell expansion, and the activation of the lateral root meristem to permit the continued growth of the organized lateral root (Malamy and Benfey, 1997;Casimiro et al., 2003). In order to determine the step of lateral root formation in which LBD16 and LBD18 act, we enumerated the lateral root primordia and emerged lateral roots of lbd16-1, lbd18-1, and lbd16-1 lbd18-1 mutants by photographing the roots with a light microscope equipped with a camera. As shown in Fig. 4, the numbers of primordia in the lbd16 and lbd18 single or double mutants were similar to the numbers observed in the wild-type. However, the numbers of emerged lateral roots of lbd16 and lbd18 single mutants were significantly reduced as compared to the wild-type. The lbd16 lbd18 double mutants exhibited additively reduced numbers of emerged lateral roots compared to the single mutants. We further analyzed the effects of lbd16 and lbd18 mutations on primordium development by counting the numbers of primordia from stage I to stage VIII on the basis of classification made by Malamy and Benfey (1997), showing that primordium development was not significantly affected at every stage examined (Fig. 4B). These results suggest that LBD16 and LBD18 may be involved in the initiation and the emergence of lateral root formation.

Gain-of-Function Analysis of LBD16 and LBD18
GFP fusion proteins of LBD16 and LBD18 are localized in the nucleus in protoplasts isolated from Arabidopsis mesophyll cells ( These results indicate that appropriate levels of LBD18 expression may be required for the stimulation of lateral root formation, but higher levels of LBD18 expression could result in the inhibition of primary and lateral roots. These results also showed that LBD18, as well as LBD16, function in the nucleus to regulate lateral root formation. In addition, consistent with the observed inhibition of root growth with high levels of LBD18 expression, the hypocotyl lengths of 4 different lines of Pro 35S :LBD18:GR transgenic plants were reduced significantly by DEX treatment (Fig. 7).

Overexpression of LBD18 Rescues Lateral Root Formation of lbd18 Single Mutants and lbd16 lbd18 Double Mutants
Although we have observed statistically significant increases in the numbers of lateral roots in Pro 35S :LBD18:GR transgenic plants (the #28-9 line) with various concentrations of DEX (Fig. 7E), other lines evidenced severe root growth inhibition phenotypes. To address this mixed phenotype problem, we attempted to determine whether LBD18 could rescue the wild-type phenotype in lbd18 and lbd16 lbd18 mutants, using Pro 35S :LBD18:GR. Transgenic significant phenotypes were detected with these transgenic mutants. These results show that LBD18 is responsible for its mutant phenotype, and also that LBD18:GR is functional in lateral root formation. We found that while DEX treatment of Pro 35S :LBD18:GR in lbd18 mutant backgrounds can complement lbd18 at almost a wild-type level, that ofPro 35S :LBD18:GR in lbd16 lbd18 can induce lateral root formation at the same level as can be achieved by DEX treatment of Pro 35S :LBD18:GR in lbd18, but cannot fully complement the lbd16 lbd18 double mutants. These findings suggest that while LBD16 and LBD18 play roles in lateral root formation, they may also perform a distinctive role in a different pathway.

LBD18 Induces the Formation of Lateral Roots in arf7 arf19 Mutants
The results of our earlier study demonstrated that the auxin-upregulated expression of LBD16, LBD18, and LBD29 genes was repressed dramatically by DEX treatment and were inhibited completely by double mutations of ARF7 and ARF19, thereby indicating that these LBD genes might be regulated by ARF7 and ARF19 in auxin signaling (Lee et al., 2009 has also been previously reported that LBD16 and LBD29 overexpression induces lateral root formation in the absence of ARF7 and ARF19, and that dominant repression of LBD16 inhibits lateral root formation (Okushima et al., 2007). To further demonstrate the positive role played by LBD18 in lateral root formation, we constructed DEX-regulated LBD18-

DISCUSSION
Recent studies have demonstrated the functions of some LBD genes in lateral organ development in Arabidopsis and in crop plants, such as rice and maize. For many Arabidopsis LBD genes, loss-of-function mutation phenotypes were not apparent with the exception of LBD6, which performs a role in leaf development (Semiarti et al., 2001;Xu et al., 2003).
However, several loss-of-function mutants of rice and maize LBD genes have been reported to evidence clear phenotypes (Inukai et al., 2005;Liu et al., 2005;Bortiri et al., 2006;Taramino et al., 2007;Evans, 2007). However, the ectopic expression of several LBD genes, as wildtype or as proteins fused to a transcriptional repression domain, yielded morphological phenotypes, providing clues as to the biological functions of these LBD genes (Shuai et al., 2002;Chalfun-Junior et al., 2005;Borghi et al., 2007;Okishima et al., 2007). For example, the overexpression of LBD16 or LBD29 induced lateral root formation in the absence of ARF7 and ARF19 and the dominant repression of LBD16 activity inhibited lateral root formation; these findings suggest that these LBDs function downstream of ARF7-and ARF19-dependent auxin signaling in lateral root formation (Okushima et al., 2007). In this study, we demonstrated that LBD16 and LBD18 are involved in lateral root formation at the emergence step, based on the results of analysis of lbd16 and lbd18 single or double loss-offunction mutants. LBD18 overexpression using a DEX-inducible system was shown to complement lateral root formation in lbd18, as well as the lbd16 lbd18 mutants, without inducing any other phenotypes. LBD18 overexpression also induced lateral root formation in arf7 arf19 mutants with blocked lateral root formation. Collectively, these results suggest that LBD18 performs a function in lateral root formation, particularly during the initiation and emergence steps, in conjunction with LBD16 downstream of ARF7 and ARF19.
The lbd16 lbd18 double mutants exhibited substantially reduced numbers of lateral roots compared to the lbd16 or lbd18 single mutants (Fig. 2), thereby suggesting that LBD16 and LBD18 might function in a combinatorial manner in lateral root formation. Because we failed to detect a protein-protein interaction between LBD16 and LBD18 in a yeast 2-hybrid assay (data not shown), LBD16 and LBD18 might function redundantly in different pathways or might require auxiliary factors for their functional interaction. Some degree of lateral root formation could still be detected in the lbd16 lbd18 double mutants, thereby indicating the existence of additional components for lateral root formation.
In the transgenic line expressing appropriate levels of the LBD18:GR transcripts in the wild-type background, a statistically significant increase in lateral root number was induced by treatment with various concentrations of DEX (Fig. 6E). In contrast, the transgenic lines expressing higher levels of the LBD18:GR transcripts exhibited significantly halted root growth, significantly decreased numbers of lateral roots, and reduced hypocotyl length (Figs. 6 and 7, and Supplemental Figs. S2 and S3). These strong phenotypes may be related to the effects of ectopic tracheary element-like cells in non-vascular cells, as previously reported (Soyano et al., 2008). These results also indicate that appropriate LBD18 expression levels might be necessary for the induction of lateral root formation in wild-type plants. In order to further demonstrate that increased LBD18 expression can stimulate lateral root formation and that LBD18 is responsible for the mutant phenotype, we generated the transgenic mutants, Pro 35S :LBD18:GR in lbd18 mutant background, as well as in lbd16 lbd18 mutant background that express the LBD18:GR transcripts at wild-type level or slightly higher. We noted that DEX treatment can induce lateral root formation in both lbd18 and lbd16 lbd18 mutant backgrounds (Fig. 8, C and D), thus clearly demonstrating that LBD18 plays a role in lateral root formation. GUS expression detected in the developing lateral roots of Pro LBD18 :GUS seedlings (Fig. 3)  be detected in the lateral root primordium (Okushima et al., 2007). We determined that while the GUS expression of Pro LBD16 :GUS seedlings is strong in the root stele and the developing and fully emerged lateral roots, that of Pro LBD18 :GUS is restricted to the primordia and the emerging lateral roots, but is not detected in the lateral root stele. These GUS expression patterns suggest that LBD16 and LBD18 may have an overlapping function in the formation of lateral roots and that LBD16 may play an additional role in the continued growth of lateral roots compared to LBD18. proteins is also required for lateral root formation. Auxin signaling mediated by the Aux/IAA and ARF families of transcriptional regulators has been determined to be required for lateral root formation. Various gain-of-function mutants of Aux/IAA genes, slr-1, shy2/iaa3, msg2/iaa19, axr5/iaa1, iaa28, and crane/iaa18, yielded dramatically reduced numbers of lateral roots (Tian and Reed, 1999;Rogg et al., 2001;Fukaki et al., 2002;Tatematsu et al., 2004;Yang et al., 2004;Uehara et al., 2008). Whereas mutations in ARF19 had a little effect on their own, and ARF7 mutations resulted in impairments in hypocotyl phototropism, a variety of phenotypes that were not detected in these single mutants were observed in arf7 arf19 double mutants, including a reduction in lateral root formation (Harper et al., 2000;Okushima et al., 2005;Wilmoth et al., 2005), suggesting that ARF7 and ARF19 regulate lateral root formation in a redundant fashion. A novel regulator of lateral root primordium development, PUCHI, which encodes AP2/EREBP, has been identified, and has been shown to play a role in lateral root morphogenesis by altering the pattern of cell divisions during the early stages of primordium development (Hirota et al., 2007). It has been suggested that PUCHI functions downstream of auxin signaling.
LBD16 has been previously proposed to be involved in the initiation step of lateral root formation, based on the observation that the dominant repression of LBD16 profoundly inhibited lateral root formation (Okushima et al., 2007). Our present study involving loss-offunction mutant analysis demonstrated that, while the numbers of primordia of lbd16 and lbd18 single or double mutants were similar to those of the wild-type strain, the numbers of emerged lateral roots of the single mutants were reduced significantly compared to the wildtype (Fig. 4A). In the lbd16 lbd18 double mutants, additively reduced numbers of emerged lateral roots were noted as compared to the single mutants. Moreover, the numbers of primordia at stage I to stage VIII were not affected by lbd16 and lbd18 mutations (Fig. 4B).
These findings reveal that LBD16 and LBD18 are likely to be involved in the initiation and the emergence of lateral root formation and function additively in a different pathway. Recent study showed that GUS of Pro LBD18 :GUS transgenics is expressed in immature tracheary elements and that LBD18 functions in differentiation of tracheary elements (Soyano et al., 2008). It is, therefore, possible that the phenotype of lateral root emergence in the lbd16 lbd18 mutant might be related to the vascular differentiation in the lateral root primordium.
The results of microarray analysis demonstrated that the loss of ARF7 and ARF19 function completely suppressed the expression of LBD16, LBD18, and LBD29 in response to auxin (Okushima et al., 2005) mutants induced an increase in lateral root numbers (Okushima et al., 2007). We have also determined that ectopic LBD18 expression in arf7 arf19 mutants as the result of DEX treatment induced a significant increase in lateral root numbers (Fig. 9). Thus, we propose that LBD16, LBD18, and LBD29 might act in a combinatorial fashion to regulate lateral root formation downstream of ARF7 and ARF19 during the auxin response. The results of our previous study demonstrated that the DEX-regulated expression of iaa1 with a domain-II mutation resulted in a severe inhibition of lateral root formation and the suppression of expression was inhibited completely in response to auxin. In P IAA1 :GUS 7-day-old light-grown seedlings, we have detected strong GUS expression in various tissues, including the lateral roots (Lee et al., 2009). In P ARF7 :GUS and P ARF19 :GUS seedlings, GUS expression was also detected in various tissues including the lateral roots, but the expression patterns of these 2 GUS fusion genes are distinct. Partial overlap in the GUS of P ARF7 :GUS seedlings was detected in the root stele and in the developing lateral roots, whereas the GUS of P ARF19 :GUS seedlings was shown to be expressed in the entire region of the primary and lateral roots (Okushima et al., 2005). Collectively, these results indicate that LBD16 and LBD18 might be regulated downstream of the IAA1-ARF7/ARF19 transcriptional regulator system in auxin signaling to modulate lateral root formation at the initiation and the emergence steps. provided in Supplemental Table S1.

Double T-DNA Insertion Mutants
Arabidopsis lbd16-1 (SALK_095791) and lbd18-1 (SALK_038125) T-DNA insertion mutants from ABRC were verified by PCR with primers designed with the T-DNA primer design program, which is available from the Salk Institute Genomic Analysis Laboratory (SIGNAL) (http://signal.salk.edu/). The homozygous T-DNA insertion mutant lines were isolated with the primers shown in Supplemental Table S1. Double lbd16 lbd18 mutants were generated by crossing of lbd16-1 (female) with lbd18-1 (male), and the resultant homozygous lines isolated were verified by PCR. Single copy T-DNA in lbd16 and lbd18 was verified by backcrossing these mutants into wild-type plants and assessing the segregation of T-DNA by PCR. The null mutations of lbd16, lbd18, and lbd16 lbd18 were further verified by RT-PCR analysis. Phenotypic analysis of these T-DNA insertion mutants was conducted for root length, hypocotyl length, number of lateral roots, gravitropic response, and morphological changes, as described previously (Park et al, 2002). For the analysis of the effects of 2,4-D on the lateral root number, we used 5-d-old seedlings grown vertically 4 d after transfer to the media containing the indicated concentrations of 2,4-D (Dharmasiri et al., 2005).  Homozygous lines were isolated on the basis of genotyping and the PCR detection of genomic DNA for the LBD18:GR transgene. Expression levels of the LBD18:GR transcripts in Arabidopsis overexpressing Pro 35S :LBD18:GR in lbd18-1 or lbd16-1 lbd18-1 mutants were determined by RT-PCR analysis.
Arabidopsis overexpressing Pro 35S :LBD18:GR in arf7-1 arf19-1 mutants was generated by crossing arf7-1 arf19-1 (female) with Pro 35S :LBD18:GR (male). Homozygous lines were isolated according to genotype and the lack of the lateral-root phenotype for arf7-1 arf19-1 and also the PCR detection of genomic DNA for the LBD18:GR transgene. Expression levels of the LBD18:GR transcripts in Arabidopsis overexpressing Pro 35S :LBD18:GR in arf7-1 arf19-1 mutants were determined by RT-PCR analysis. The oligonucleotides and PCR conditions utilized are provided in Supplemental Table S1.

RNA Isolation, RNA-Gel Blot Analysis, RT-PCR, and Real-Time RT-PCR
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.). Total RNA was separated on 1.2% agarose gel, transferred to nylon membranes, and hybridized for 3 h with 32 P-labeled DNA probes at 68ºC using 10 ml of QuickHyb solution (Stratagene), then washed. The blots were subsequently exposed to Xray film. The DNA probes for RNA gel-blot analysis were RT-PCR amplified, subcloned into pGEM ® -T Easy vector (Promega), then confirmed by DNA sequencing. For RT-PCR analysis, total RNA was isolated using an RNeasy plant mini kit (Qiagen) and subjected to RT-PCR analysis with an Access RT-PCR System (Promega) according to the manufacturer's    primordia of lateral roots or emerged lateral roots. Plants were grown vertically for 8 d after germination. Primordia or emerged lateral roots were photographed with a camera affixed to a Leica CME Trinocular light microscope at 100-or 400-fold magnification and counted on the basis of Malamy and Benfey (1997). n>10 per column. ** denotes statistical significance with p<0.01. B, Numbers of primordia at given stages before emergence of lateral roots. Stages I to VIII of primordia were based on the classification by Malamy and Benfey (1997).   indicate SE. * and ** denote statistical significance with p<0.05 and p<0.01, respectively. were harvested for genomic DNA, followed by PCR analysis for T-DNA insertion and total RNA was also isolated, followed by RT-PCR analysis using the primers for the LBD18:GR fusion transcripts or the LBD18 transcripts. ACTIN7 mRNA was used as a loading control. B, Quantitative RT-PCR analysis of the LBD18 transcripts in Pro 35S :LBD18:GR/lbd18-1 or Pro 35S :LBD18:GR/lbd16-1 lbd18-1 transgenic mutants compared with lbd18-1 and lbd16-1 lbd18-1 mutants. Relative abundance of the LBD18 transcripts are shown compared to wild type. C, Lateral roots of Pro 35S :LBD18:GR/lbd18-1 or Pro 35S :LBD18:GR/lbd16-1 lbd18-1 transgenic mutants compared with lbd18-1 and lbd16-1 lbd18-1 mutants without or with DEX. Plants were incubated and treated as described in Fig. 7C, but for 12-d. Bars indicate SE. n>24. ** denotes statistical significance with p<0.01. D, Lateral roots of transgenic mutants and wild type. Pictures were taken 12 d after germination.