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First published online June 30, 2006; 10.1104/pp.106.084178 Plant Physiology 141:1425-1435 (2006) © 2006 American Society of Plant Biologists
The hpa1 Mutant of Arabidopsis Reveals a Crucial Role of Histidine Homeostasis in Root Meristem Maintenance1State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou, 310029, People's Republic of China (X.M., Q. Zhu, X.L., J.L., Q. Zeng, P.W.); and Institute of Integrative and Comparative Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom (H.R., H.Z.)
Histidine (His) is an essential ingredient for protein synthesis and is required by all living organisms. In higher plants, although there is considerable evidence that His is essential for plant growth and survival, there is very little information as to whether it plays any specific role in plant development. Here, we present evidence for such a role of this amino acid in root development in Arabidopsis (Arabidopsis thaliana) from the characterization of a novel Arabidopsis mutant, hpa1, which has a very short root system and carries a mutation in one of the two Arabidopsis histidinol-phosphate aminotransferase (HPA) genes, AtHPA1. We have established that AtHPA1 encodes a functional HPA and that its complete knockout is embryo lethal. Biochemical analysis shows that the mutation in hpa1 only resulted in a 30% reduction in free His content and had no significant impact on the total His content. It did not cause any known symptoms of His starvation. However, the mutant displayed a specific developmental defect in root meristem maintenance and was unable to sustain primary root growth 2 d after germination. We have demonstrated that the root meristem failure in the mutant is tightly linked to the reduction in free His content and could be rescued by either exogenous His supplementation or AtHPA1 overexpression. Our results therefore reveal an important role of His homeostasis in plant development.
His is one of the essential amino acids for protein synthesis and is required by all living organisms. Its biosynthetic pathway has been extensively characterized in bacteria and lower eukaryotes and consists of 10 enzymatic steps catalyzed respectively by (in the order of reactions) ATP-phosphoribosyltransferase, phosphoribosyl-ATP pyrophosphohydrolase (PRA-PH), phosphoribosyl-AMP cyclohydrolase (PRA-CH), N'-(5''-phosphoribosyl)-4-imidazolecarboxamide isomerase (BBMII isomerase), Gln amidotransferase, cyclase, imidazoleglycerol-phosphate dehydratase (IGPD), histidinol-phosphate aminotransferase (HPA), histidinol-phosphate phosphatase, and histidinol dehydrogenase (which catalyzes the last two steps; Winkle, 1987  ; Carlomagno et al., 1988; Alifano et al., 1996). In Escherichia coli, these enzymes are encoded by eight genes (the PRA-PH and PRA-CH activities are encoded by a single gene) in a single operon (Carlomagno et al., 1988). However, in Saccharomyces cerevisiae, they are encoded by seven genes scattered throughout the genome. The difference in the number of genes between E. coli and S. cerevisiae is due to the multifunctional properties of two yeast proteins, HIS4 (with PRA-PH, PRA-CH, and histidinol-dehydrogenase activities) and HIS7 (with both Gln amidotransferase and cyclase activities; Mortimer et al., 1994; Alifano et al., 1996). Except for histidinol-phosphate phosphatase, genes encoding for all the other enzymes of the His biosynthetic pathway have been identified in plants (Noutoshi et al., 2005). In Arabidopsis (Arabidopsis thaliana), genomic sequence analysis reveals 10 putative genes in the His biosynthetic pathway (Noutoshi et al., 2005).
It has been well established that His biosynthesis is vital for the survival of plants. Physiological studies using specific inhibitors of the His biosynthetic pathway, such as triazole herbicides that inhibit the activities of IGPD, revealed that a complete blocking of His biosynthesis in plants was lethal (Guyer et al., 1995 However, very little is known about whether His plays any specific role in plant development. This is partly due to the difficulty in experimentally separating the metabolic and regulatory functions of this essential amino acid and the lack of nonlethal (His) deficiency mutants. Our current understanding of the role of His in plants largely comes from studies of plants under severe His starvation, conditions that are usually lethal and can cause a wide range of nonspecific responses. As a result, there is little understanding about the role of this amino acid in plant growth and development under non- or mildly starved conditions.
Here, we describe the characterization of a novel Arabidopsis mutant, hpa1, in the His biosynthetic pathway. This mutant carries a mutation in one of the two Arabidopsis HPA genes, AtHPA1, which encodes the enzyme catalyzing the transamination from L-Glu to 3-(imidazole-4-yl)-2-oxo-propyl phosphate to produce L-histidinol phosphate, the eighth step in the His biosynthetic pathway (Winkle, 1987
Isolation and Morphological Characterization of hpa1 The hpa1 mutant was originally obtained for its extremely short root system (Fig. 1A ). It has no obvious phenotype in the aerial parts and is fully fertile. Genetic analysis indicated that the short root phenotype was caused by a recessive mutation at a single locus (data not shown).
To establish the timing of the primary root growth defect, we monitored the primary root length in the wild-type and mutant seedlings within the first 10 d after germination (DAG; Fig. 1B). At 2 DAG, there was very little difference between the wild-type and mutant seedlings in primary root length, indicating that the root growth defect is probably not due to some developmental disorder during embryogenesis. However, a difference in root length occurred at 2 DAG and increased as the seedlings became older (Fig. 1B).
To characterize the growth defect at the cellular level, we examined a number of root tip-specific markers, including starch granule staining (root cap columella specific), DR5-
The hpa1 Mutant Carries a Mutation in an HPA Gene A map-based strategy was used to clone the HPA1 gene. We first crossed hpa1 (in the Columbia [Col] background) to Landsberg erecta (Ler) wild type and selected progenies with the short root phenotype in the F2 population. Initial mapping analysis revealed a linkage between the short root phenotype and the simple sequence length polymorphism marker NGA249 on chromosome 5. We then developed a series of markers (M322, M326, M331, M339, and M35; for details, see Supplemental Table II) around NGA249 and carried out further mapping analyses. In 1,474 F2 mutant progenies, we observed 2, 1, and 0 recombination events between the hpa1 locus and M322, M331, and M326, respectively (Fig. 3A ). We then cloned (by reverse transcription [RT]-PCR or genomic PCR) and sequenced all the coding regions for the predicted 22 genes between markers M322 and M331. Sequencing analysis revealed only one mutation (G-to-A base substitution), which is in the At5g10330 gene and results in an amino acid change from Ala-69 (GCT) to Thr-69 (ACT; Fig. 3B). At5g10330 encodes a putative HPA with 417 amino acids. The Ala residue affected by the hpa1 mutation is conserved in several organisms, including Arabidopsis, rice (Oryza sativa), and the alga Prototheca wickerhamii (Fig. 3B), but not in tobacco (Nicotiana tabacum; where a Ser residue occupies this position). In the Arabidopsis genome, there are two HPA genes, At5g10330 (designated as AtHPA1) and At1g71920 (designated as AtHPA2), which share 99.1% and 98.8% identity in the nucleotide (Fig. 3C) and amino acid compositions, respectively. However, except for a 394-bp fragment immediately upstream of the ATG codon, the two genes have very little sequence similarity in the 5' promoter regions (Fig. 3C).
The hpa1 Mutant Phenotypes Can Be Rescued by Either Constitutive Expression of the Wild-Type AtHPA1 or Exogenous His Supplementation We carried out several experiments to check whether the root meristem defect of hpa1 is linked to the mutation in AtHPA1 and His biosynthesis. First, we expressed the AtHPA1 cDNA under the 35S promoter in hpa1 and found that root growth in the resultant transgenic lines (subsequently referred to as hpa1/35S-AtHPA1) was partially restored (to about 70% of that of the wild-type seedlings; Fig. 4A ). We confirmed that this partial restoration in root growth is linked to the presence of the transgene (Fig. 4A, insert). Second, we found that hpa1 seedlings grown on media with added His no longer display the mutant phenotypes (Fig. 4B), including the root growth defect and the altered expression of cellular markers (Fig. 2, B, G, L, Q, and V), and that this rescuing effect of the exogenous His is concentration dependent (Supplemental Fig. 1). Within the concentration range (0.001–0.1 mM) tested, 0.01 mM His was optimal for the rescuing (the medium with 0.01 mM His is referred to as the His-containing medium hereafter). The results therefore establish a cause-effect relationship between the altered phenotypes in hpa1 and the mutation in AtHPA1.
AtHPA1 Encodes a Functional HPA Protein To establish whether AtHPA1 encodes a functional HPA protein, we transformed the E. coli HPA (known as hisC in E. coli) defective mutant strain UTH780 with the full-length AtHPA1 cDNA. Due to the lack of HPA activity, the UTH780 strain cannot grow on medium without His supplementation. However, the transformed UTH780 strain expressing the AtHPA1 cDNA grew on a minimal medium without His (Fig. 4, D and E). The ability of AtHPA1 to complement the E. coli HPA defective mutant confirms that AtHPA1 encodes a functional HPA protein.
To establish the impact of the hpa1 mutation on His biosynthesis, we initially measured both the free and total His contents in 10-d-old hpa1 and Col wild-type seedlings. Although no significant difference was found between the mutant and the wild-type control in the total His content (in the hydrolyzed samples; Supplemental Table I), there was a clear reduction in free His content in the mutant compared with the wild-type control. The measurement also indicated that the reduction in concentration of His was similar in both shoots and roots (Table I). This observation prompted an extended measurement of the levels of 17 free amino acids (including His) in hpa1, hpa1/35S-AtHPA1, and Col wild-type seedlings (Table II ). On the His-free medium, the concentration of free His in the mutant was about 30% lower than that in the wild-type seedlings throughout three different developmental time points (3, 7, and 21 DAG), indicating that the reduction is not age dependent. Importantly, the reduction in His content is tightly linked to the mutant phenotype, as the level of free His recovered in hpa1 seedlings when the mutant phenotypes (including the root growth defect and the altered expression of cell patterning markers) were rescued by either AtHPA1 constitutive expression or His supplementation (Table II). The close correlation between the mutant phenotypes and the reduced His level strongly indicates that the mutant phenotypes are linked to the reduction in free His content.
We also noticed changes in other amino acids in the mutant. For example, the levels of free Asp, Lys, Arg, and Glu were also reduced in the mutant. The reduction of Asp and Lys occurred across the three different developmental time points examined, while that of Arg and Glu occurred only at the two early time points (Table II). However, the reduction of Glu, Lys, and Arg in the mutant did not correlate with the mutant phenotypes, as it persisted in hpa1 seedlings no longer displaying the mutant phenotypes, such as hpa1/35S-AtHPA1 seedlings or hpa1 seedlings grown on the His-containing medium. Consistent with this conclusion, supplementation of these amino acids (Glu, Lys, and Arg) in the growth medium did not rescue the root growth defect of hpa1 seedlings (Supplemental Fig. 2). The reduced level of Asp appeared to be correlated with the mutant phenotype (i.e. it was reduced in the mutant seedlings and restored to the wild-type level when the mutation was either complemented by constitutive AtHPA1 expression or rescued by a supplementation of His in the medium). However, as in the case of Glu, Lys, and Arg, addition of Asp to the growth medium also failed to rescue the mutant phenotypes (Supplemental Fig. 2), indicating that the change in this amino acid is not linked to the mutant phenotype. In fact, the addition of a combination of the above-mentioned four amino acids to the medium also failed to rescue the mutant phenotype (Supplemental Fig. 2).
It has been recently reported that in Arabidopsis a proper balance of His is particularly important at early developmental stages (Noutoshi et al., 2005
One possible explanation of the specific impact of the hpa1 on roots is that root tips may have a lower expression of the HPA genes than other parts of the plants and are therefore more sensitive to defects in HPA activity. To check whether this is true, we examined the level of the HPA transcripts (due to the high sequence identity of the two HPA genes, we were unable to design gene-specific markers) in several different organs/tissues and also in different regions of roots in the wild-type plants. The results showed that the level of the HPA transcripts in the root tip region was not lower than that in other parts of roots or the aboveground organs/tissues (Fig. 5 ), indicating that the special sensitivity of the root meristem to the hpa1 mutation is unlikely to be due to a lower expression of the HPA transcripts.
Complete Knockout of AtHPA1 Is Embryo Lethal The moderate impact of the hpa1 mutation on His content raises a question about the relative contribution of AtHPA1 in His biosynthesis and the impact of the hpa1 mutation on the function of the protein. We noticed, on the National Center for Biotechnology Information Web site, the existence of an uncharacterized T-DNA insertional mutant, emb2196 (embryo defective 2196), in AtHPA1 in the Meinke's embryo defective mutant collection (http://www.seedgenes.org/). According to the description on the Web site, the homozygous emb2196 mutant is embryo lethal and carries a mutation in the second exon about 90 bp downstream of the ATG start codon (Fig. 3C). To confirm that the insertion in emb2196 was indeed in AtHPA1, we carried out PCR amplification with (HPA) gene-specific forward primers against the 5' upstream regions of the two HPA genes and a reverse primer specifically against the T-DNA insert. Amplification of a band of the correct size was obtained only when the AtHPA1-specific forward primer was used (Fig. 6C ; in combination with the T-DNA-specific reverse primer). When the AtHPA2-specific primer was used (Fig. 6C), we did not obtain any amplification. This result confirms that the insertion in emb2196 is in AtHPA1. We then screened a segregating plant population from a heterozygous emb2196 plant (homozygous mutant plants could not be obtained due to embryo lethality) using the AtHPA1-specific forward and the T-DNA-specific reverse primers and confirmed that the T-DNA insertion cosegregates with the embryo-lethal phenotype (Fig. 6D). The results therefore demonstrate that AtHPA1 makes a vital contribution to HPA activity and His biosynthesis in Arabidopsis and also indicate that the mutated HPA protein in hpa1 is not functionally inactive and must have some residual activities that are sufficient to maintain the vitality of the hpa1 embryo/plants.
The hpa1 Mutant Is a Novel Plant His-Deficient Mutant and Is Defective in Root Meristem Maintenance
In this article, we describe the characterization of a novel Arabidopsis mutant in the His biosynthesis pathway, and we establish that the mutant carries a mutation in one of the two HPA genes (Fig. 3B). To our knowledge, hpa1 is the first plant mutant with reduced His content. Another Arabidopsis mutant of the His biosynthetic pathway, apg10, has recently been characterized (Noutoshi et al., 2005 At the morphological level, the hpa1 mutant clearly has difficulties in sustaining root growth (Fig. 1). Our results show that the root growth defect is associated with a wide range of alterations in cellular patterning in the root meristematic region (Fig. 2). Because some of the alterations occurred in markers that are known to be linked to meristem functions or maintenance, such as the auxin maximum and QC identity, it would suggest that the root growth defect in the mutant is linked to some impairments in the mechanisms involved in root meristem maintenance.
It has been well established that His is essential for plant survival and that a complete blocking of His biosynthesis is lethal (Guyer et al., 1995
Our results show that hpa1 seedlings had reduced free His content across different stages of seedling development and that this reduction in free His concentration is tightly linked with the mutant phenotypes. The reduction occurred when the mutant displayed the altered phenotypes and disappeared when the mutant phenotypes were either complemented by AtHPA1 overexpression or were rescued by exogenous His supplementation. Interestingly, hpa1 seedlings grown on the His-containing medium also maintained their free His at a similar level as that in the wild type, suggesting that there is an endogenous mechanism to keep His concentration at a certain level. Coincidently, of the different amino acids, the concentration of free His is the lowest in Arabidopsis plants and is maintained at a constant level in both roots and shoots (Table I) and throughout different developmental stages (Table II). Such tight control of free His content may reflect the importance of maintaining this amino acid at the appropriate level.
The existence of two nearly identical HPA genes in Arabidopsis raises a question as to what are the relative contributions of the two genes in the overall His biosynthesis capacity. Our analysis of the emb2196 indicates that AtHPA1 plays an essential role in His biosynthesis and its complete knockout is embryo lethal. The results also indicate that the mutation in hap1 is not functionally null and that the mutated AtHPA1 protein must have some residual activities that are sufficient to supply the vital amount of His required to keep the embryos/plants alive.
Interestingly, both apg10 and hpa1 show some degree of age-related His dependency. In apg10, the mutant phenotype only occurred at the early seedling stage (Noutoshi et al., 2005
Our results have established a link between His homeostasis and root meristem maintenance and illustrates the importance of maintaining the right His content in plants. Interestingly, observations made on the apg10 mutant also support a role for His homeostasis in plant development as it was reported that the albino and pale-green cotyledon and leaf phenotype of the mutant was linked to an increase in His content (Noutoshi et al., 2005 As discussed above, the root meristem failure in hap1 is unlikely to be part of a global His starvation response. Theoretically, there are at least two possible explanations as to why the root meristem fails in the mutant. The first one is that the root meristem may suffer from a localized His shortage. Our current experimental data do not support this argument. For example, the level of free His and also the impact of the hpa1 mutation on the His content seem very constant and uniform within the plant and throughout different developmental stages, indicating that either the biosynthetic capacity is very evenly balanced within a plant and across developmental stages or that some balancing mechanisms may exist to ensure a uniform distribution of this amino acid. In either scenario, a localized starvation could not be established easily. In addition, the lethal phenotype of emb2196 indicates that AtHPA1 is vital for the survival of the whole plant, and our expressional analysis indicated that it is expressed fairly constitutively in different parts of wild-type plants; it is therefore difficult to imagine how the mutation in AtHPA1 could only have a very localized effect on His supply in the root meristem but not in other parts of the mutant plants. Nevertheless, it is not possible to completely rule out the possibility of a localized His starvation in the hpa1 root meristems without a direct His measurement. An alternative explanation of the cause of the root meristem failure in hpa1 is that there are different sensitivities among the different parts, organs, tissues, or cells of plants and that root meristems are particularly sensitive to changes in His homeostasis, probably within a certain range. Such specific high sensitivity of the root meristems could lead to a localized developmental response, such as the root meristem failure. There is some evidence that differential sensitivities to His homeostasis in plants do indeed occur at different developmental stages. For example, in both the apg10 and hpa1 mutants, His dependency changes at different ages, although the level of His content appears constant. Further investigation is needed to establish the true mechanisms of the root meristem defect in hpa1, and this may open the door to His signaling mechanisms in plants.
Marker Lines The DR5-GUS, SCR-GFP, and QC25 marker lines were kindly provided by Ben Scheres (Utrecht University, Netherlands). The enhancer trap lines J0631 and J0481 (generated by Jim Haseloff; http://www.plantsci.cam.ac.uk/Haseloff) were obtained through the Nottingham stock center. The markers were introduced into hpa1 by crossing, and morphological examinations were carried out in the F3 generation.
Wild-type and mutant seeds were surface sterilized with 10% commercial bleach in 100% ethanol for 10 min, followed by several washes with 100% ethanol. The seeds were then dried in a laminar flowhood for 20 to 30 min. All the media used in this study for seed germination and seedling growth were based on a basic medium containing the following ingredients: 200 µM KCl; 80 µM MgSO4; 40 µM CaCl2; 44 µM NaH2PO4; 2.3 µM MnSO4; 1 mM KNO3; 0.18 µM KI; 2 µM H3BO4; 0.28 µM ZnSO4; 4 nM CuSO4; 40 nM Na2MoO4; 4 nM CoCl2; 4 µM Fe-EDTA; 0.5 g L–1 MES, pH 5.7; 1% agar-agar; and 0.5% Suc. For the His-containing medium, His is added before autoclaving. Plates were sealed with parafilm and placed vertically (to allow root growth along the surface of agar) in a growth cabinet (Percival Scientific), with a 15-h-light/9-h-dark cycle, 300 µmol m–2 s–1 photon flux density, and at a constant temperature of 20°C. For the 21-d-old seedlings used for amino acid measurement, seedlings were transferred once at 11 DAG to fresh plates to maintain nutrient supply in the medium.
For mapping analyses, the hpa1 mutant (in the Col background) was crossed to wild-type Ler, and F2 progenies with the short root phenotype were selected. Markers used in the initial mapping analysis were selected from The Arabidopsis Information Resource (TAIR) Web site (http://www.arabidopsis.org). Five new (simple sequence length polymorphism and cleaved amplified polymorphic sequence) markers were developed around the NGA249 marker on chromosome 5 based on differences between the Col and Ler (available at http://www.arabidopsis.org/Cereon/index.jsp) genomic sequences. The sequence information and chromosomal positions of these new markers are provided in Supplemental Table II.
The hisC defective E. coli strain UTH780 (F-, hisC 780, mal A1, lR, xyl-5, mtl-1, rpsL 145, l-; CGSC no. 5954) was obtained from the E. coli Genetic Stock Center and transformed with either the empty expression vector
The overexpression construct was made by inserting a full-length At5g10330 cDNA into the binary plant vector pCAMBIA1301 (35S). The full length of At5g10330 cDNA clone was obtained from RIKEN BRC. This plasmid containing the cDNA was first digested by SfiI, then treated with T4 DNA polymerase to generate one blunt end and then digested by BamHI. The cDNA fragment was inserted into pBSSK vector via the SmaI and BamHI sites, redigested with KpnI and BamHI, and inserted into plant vector pCAMBIA1301 by directional cloning using the KpnI and BamHI sites. Transformation was carried out using the floral-dip method (Clough and Bent, 1998
Genomic DNA was isolated from young leaves of Arabidopsis plants using the cetyl-trimethyl ammonium bromide method (Murray and Thompson 1980
Histochemical GUS staining was performed according to Jefferson et al. (1987) For confocal microscopy, roots were mounted in 10 µM propidium iodide and were imaged using a Carl Zeiss laser scanning system LSM 510.
RNA was extracted from the following tissues: flowers and siliques from 4-week-old plants grown on soil in a growth room at 22°C; and leaves, root tips (the first 0.4 cm from the root tip), the middle (the middle section between 0.4-cm root tip and 1-cm basal root segment), and basal root sections (1-cm root from the stem junction) from 5-DAG seedlings. First-strand cDNA was synthesized from total RNA using a commercial cDNA synthesis kit (Invitrogen) and stored at –20°C before use. The primers used in RT-PCR analyses were as follows: HPA forward 5'-TGATCATGAGATGCGTGTTAGAT-3', HPA reverse 5'-TTGTTGGGAGAAGTTAGGAAT-3', ACTIN forward 5'-TCTCTATGCCAGTGGTCGTA-3', and ACTIN reverse 5'-CCTCAGGACAACGGAATC-3'. Quantitative real-time PCR was performed with an ABI Prism 7000 sequence detection system (Applied Biosystems) using the SYBR green I master mix (Applied Biosystems). Each 20-µL reaction contained 2x SYBR green master mix, 2 µL of cDNA, and 0.1 µL of forward and reverse primers (20 µM). The PCR conditions were set as follows: 95°C for 2 min, followed by 40 cycles of 95°C for 15 s, 58°C for 20 s, and 72°C for 30 s. Fluorescence data were collected during the 72°C step and were analyzed with Sequence Detector version 1.7 software (Applied Biosystems). The quantity of cDNA was calculated from the threshold cycle by interpolation from the standard curve. The amounts of cDNA template in each sample were normalized using the amplification of ACTIN primers.
To establish which of the HPA genes the insertion in emb2196 was located in, we used the following gene-specific forward primers, 5'-TTCCCATAGGCTTCACGTCT-3' (At5g10330 specific) and 5'-AAACTGATTTAGGTATCTCCG-3' (At1g71920 specific), together with a T-DNA-specific reverse primer (5'-TCCCAAATTACCAATACATTACACT-3'), and carried out PCR amplification using genomic DNA from a heterozygous emb2196 plant. The analysis confirmed that the insertion was in AtHPA1. To establish whether the embryo-lethal phenotype cosegregated with the T-DNA insertion, we first obtained a segregating population from a heterozygous (emb2196/EMB2196) plant and assessed the individuals of the population for the production of lethal embryos at the flowering stage to establish whether they were wild type or heterozygous with regard to the embryo-lethal allele. We then extracted genomic DNA from selected wild-type and heterozygous plants and carried out genome-typing analysis using the AtHPA1-specific forward and T-DNA-specific reverse primers to check whether the embryo-lethal phenotype cosegregated with the T-DNA insert.
For measuring amino acid contents, samples (600 mg in fresh weight) of the wild-type and hpa1 seedlings were ground in liquid nitrogen. Free amino acids were extracted with 80% ethanol for 30 min. The extract was filtered into an evaporating dish, concentrated by evaporation at 60°C, and resuspended in 5 mL of citric acid and 1 mL of sulfosalicylic acid. The solution was filtered through Whatman 3MM filter paper and centrifuged at 10,000g for 10 min. The supernatant was analyzed by a Hitachi amino acid analyzer (835-50) for amino acid contents. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number AY117255.
At5g10330 cDNA clones were supplied by RIKEN BRC. We are grateful to Professor Ben Scheres for providing the DR5-GUS, SCR-GFP, and QC25 marker lines, and acknowledge the Nottingham Stock Center for supplying the enhancer trap lines J0631 and J0481. We also thank Dr. David Pilbeam (University of Leeds) for help in reading and editing the manuscript. Received May 25, 2006; returned for revision June 15, 2006; accepted June 16, 2006.
1 This work was supported by the National Natural Science Foundation of China (grant no. 30228003 to H.Z. and P.W.) and by the National Key Basic Research Program of China (grant no. 2005CB120900 to P.W.).  The author 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.plantphysiol.org) is: Ping Wu (clspwu{at}zju.edu.cn). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.084178. * Corresponding author; e-mail clspwu{at}zju.edu.cn; fax 86–571–86971323.
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ABSTRACT
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-glucuronidase (GUS; auxin accumulation marker normally highly expressed in the root cap columella cells and the quiescent center [QC]; Ulmasov et al., 1997
(segment 1), the hisC mutant UTH780 (segment 2), UTH780 transformed with 
FLC-1-B empty vector alone (segment 3), or the 
