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American Society of Plant Biologists The Arabidopsis Dual-Affinity Nitrate Transporter Gene AtNRT1.1 (CHL1) Is Activated and Functions in Nascent Organ Development during Vegetative and Reproductive GrowthSection of Cell and Developmental Biology, Division of Biology, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093-0116 1 To whom correspondence should be addressed. E-mail ncrawford{at}ucsd.edu; fax 858-534-1637
The AtNRT1.1 (CHL1) transporter provides a primary mechanism for nitrate uptake in Arabidopsis and is expected to localize to the epidermis and cortex of the mature root, where the bulk of nitrate uptake occurs. Using fusions to GFP/GUS marker genes, we found CHL1 expression concentrated in the tips of primary and lateral roots, with very low signals in the epidermis and cortex. A time-course study showed that CHL1 is activated in the primary root tip early in seedling development and at the earliest stages of lateral root formation. Strong CHL1 expression also was found in shoots, concentrated in young leaves and developing flower buds but not in the shoot meristem. These expression patterns were confirmed by immunolocalization and led us to examine CHL1 function specifically in the growth of developing organs. chl1 mutants showed a reduction in the growth of nascent roots, stems, leaves, and flower buds. The growth of nascent primary roots was inhibited in the mutants even in the absence of added nitrate, whereas elongation of lateral root primordia was inhibited specifically at low nitrate and acidic pH. Interestingly, chl1 mutants also displayed a late-flowering phenotype. These results indicate that CHL1 is activated and functions in the growth of nascent organs in both shoots and roots during vegetative and reproductive growth.
The acquisition of inorganic nutrients involves uptake primarily from the soil solution into roots. Root uptake systems use multiple genes and mechanisms that transport ions from the apoplasm of epidermal and cortical cells into the symplasm. For nitrate uptake, three physiological systems have been described: low affinity, inducible high affinity, and constitutive high affinity (reviewed in Crawford and Glass, 1998  ; Forde and Clarkson, 1999; Galvan and Fernandez, 2001; Glass et al., 2001; Williams and Miller, 2001). Two families of nitrate transporter genes, NRT1 and NRT2, have been identified that contribute to these uptake systems (reviewed in Crawford and Glass, 1998; Daniel-Vedele et al., 1998; Forde, 2000; Galvan and Fernandez, 2001; Glass et al., 2001; Williams and Miller, 2001). Members of the NRT2 family are part of the high affinity system; NRT2 mutants (Galvan et al., 1996; Filleur et al., 2001) and antisense suppressed lines (Forde, 2000) have reduced high affinity nitrate uptake. NRT2 expression is nitrate inducible; thus, these transporters are part of the inducible high affinity system (reviewed in Forde, 2000; Galvan and Fernandez, 2001; Glass et al., 2001; Williams and Miller, 2001). These genes also display feedback regulation, because ammonium or glutamine, the end products of the nitrate assimilation pathway, suppress NRT2 expression.
The NRT1 family is more complicated and is composed of genes involved in nitrate and amino acid/peptide uptake (reviewed in Crawford and Glass, 1998
When overall nitrate uptake is examined, CHL1's contribution is found to be dependent on environmental conditions (Huang et al., 1996
Because CHL1 plays an integral role in nitrate uptake, one would expect that CHL1 protein would be found predominantly in the outer cell layers of the mature root, where the bulk of nutrient uptake into the symplasm occurs. This proposal is supported by microelectrode experiments showing that nitrate uptake activity, measured as nitrate-induced depolarization across the plasma membranes of root epidermal cells, is reduced by 50% in chl1 mutants (Wang et al., 1998 To examine the issue of CHL1 localization in greater detail, translational fusions between CHL1 and GFP/GUS marker genes were made and then examined in transgenic Arabidopsis plants. The results presented here show unexpected patterns of expression, indicating that CHL1 is expressed preferentially in rapidly growing regions in both shoots and roots. Experiments to determine the function of CHL1 using the wild type and chl1 mutants also are described. The phenotypes obtained provide new insights into the function of CHL1 during vegetative and reproductive growth.
CHL1 Is Expressed Preferentially in Growing Regions of Roots and Shoots To analyze CHL1 expression, transgenic Arabidopsis plants were generated that carried green fluorescent protein (GFP) or  -glucuronidase (GUS) reporter DNAs fused in frame to the CHL1 coding region at two sites: HaeII and XhoI (Figures 1B and 1C)
. For each construct, >24 transgenic lines were examined. Expression patterns in these lines showed that GFP and GUS signals from the HaeII construct had the same pattern but stronger intensities as those from the XhoI construct (data not shown). For further analysis, transgenic plants from the T2 generation transformed with the HaeII construct were used. Seedlings were grown vertically on plates with 10 mM NH4NO3 as nitrogen source, as described in Methods. Plants were examined by confocal microscopy for GFP expression or by light microscopy for GUS staining.
When 5-day-old seedlings were examined for CHL1-GFP expression in roots, the strongest fluorescence was found in the tips or growing regions of both the primary root (Figure 2A) and lateral roots (Figure 2E). Tissue staining of the same samples with propidium iodide showed the outlines of the roots and some internal cell layers (Figures 2B and 2F), which could be superimposed on the GFP images (Figures 2C and 2G). Closer examination of the GFP images revealed that all cell layers in the root tip fluoresced strongly, including cells in the lateral and columella root cap (Figure 2L). In the elongation zone, the signal was much diminished. In the mature parts of the root, signals were observed in the stele, but at lower levels than in the tip (Figure 2E). In the epidermis or cortex of the mature regions of the root, no significant difference was observed between transgenic and nontransgenic lines.
Next, the time course of CHL1 expression was examined during the first week of seedling growth. Little CHL1 expression was found in seed before germination (data not shown). At day 1, CHL1 expression was activated, with GFP signals highest in the three cell layers of the columella root cap, with weaker signals along the lateral root cap and epidermis (Figure 2D). Almost no expression was detected in the internal layers of the root. At day 3, GFP signals strengthened and expanded into other cell layers, including the root meristematic region (Figure 2H). At day 5, very strong GFP signals were found throughout the root tip (Figure 2L). At day 7, the signal intensities in the root tip began to decrease but continued to extend over the entire tip. These results show that CHL1 expression is activated and expands into the entire root tip during the early stages of seedling growth. The presence of high GFP signals in the tips of roots suggests that CHL1 expression is targeted to cells in rapidly growing regions. To test this possibility further, growing regions in the shoot were examined using CHL1-GFP and CHL1-GUS marker constructs. Strong GFP signals and GUS staining were observed in emerging and immature leaves (Figures 2I to 2K, 2Q, and 2T) and flower buds (Figures 2M to 2O and 2Q to 2S). Close examination of vegetative shoot tips showed high levels of GFP signal and GUS staining throughout emerging leaves and in young stipules but little signal in mature leaves and, significantly, little to no signal in the shoot apical meristem (Figures 2I and 2T and data not shown). In flower buds, signals increased significantly during bud development (Figure 2R) and then, after fertilization and flower opening, GUS staining decreased (data not shown). GUS and GFP signals were not uniform throughout the buds, because the strongest signals were observed in the reproductive organs (i.e., gynoecium and anthers compared with sepals and petals; Figures 2M and 2S). With short GUS staining times, the strongest signals were observed in the style, stigma, and anthers (Figure 2S). As the flowers aged, GUS staining decreased, retaining some signal in the stigma and pollen grains but with almost no staining in sepals, petals, and carpels (data not shown).
Protein Gel Blot Analysis and Immunolocalization of CHL1 Protein
The anti-CHL1 antibody was then used for immunolocalization with whole Arabidopsis seedlings. Images of samples incubated with anti-CHL1 or preimmune sera were visualized by confocal microscopy. The immunofluorescence signals gave patterns very similar to those observed with the GFP/GUS reporter constructs. High levels of immunofluorescence were detected in primary root tips (Figures 4A and 4B) , in lateral root tips (Figures 4E and 4F), in emerging leaves (Figures 4I and 4J), in anthers and the gynoecium (Figures 4M and 4N, showing a bud at stage 12), in sepals (Figures 4Q and 4R), and in stipules (Figures 4S and 4T). The second panel in each pair of images shows a composite of immunofluorescence from the antibody staining and the propidium iodide staining. Little signal was detected in tissues treated with preimmune sera (Figures 4C, 4D, 4G, 4H, 4K, 4L, 4O, and 4P).
CHL1 Expression during Lateral Root Formation We next examined CHL1 expression during the initiation and elongation of lateral roots, critical events that establish the architecture of the root. Lateral root initiation begins with the activation of cell division in pericycle cells, which produces a lateral root primordium (Laskowski et al., 1995 ; Malamy and Benfey, 1997a, 1997b). Primordia emerge from the mature root, forming a new meristem. Eight development stages have been defined during this process (Malamy and Benfey, 1997b). CHL1 expression showed activation at the earliest stage of lateral root initiation (Figures 5A and 5B)
. Strong CHL1-GFP signals were observed in dividing pericycle cells, signals that were higher than in the stele. These strong signals were maintained during the formation of lateral root primordia and emergence. Strong signals were found in the outer and inner cell layers of the primordium at stages II and III (Figure 5C), in the full primordium at stages IV to VII (Figure 5D), in the emerging primordium (Figure 5E), and in the fully emerged lateral root (Figure 5F). These results show that CHL1 expression is activated at the earliest stages of lateral root formation, well before primordia emerge from the mature root.
CHL1 Function Is Required for Lateral Root Formation but Not Initiation at Low Nitrate Concentrations The early activation of CHL1 expression in developing organs of the shoot and root suggests that CHL1 may play a role in nascent organ growth. Our first test of this was to examine the formation and elongation of lateral roots in the wild type (Columbia ecotype) and chl1-5 deletion mutants. We chose conditions in which CHL1 is known to be highly expressed and to make a major contribution to nitrate uptake: growth with NH4NO3 at pH 5.5 (Huang et al., 1996 ; Touraine and Glass, 1997; Wang et al., 1998; Liu et al., 1999). Plants were germinated and grown for 5 days in germination medium (with 10 mM NH4NO3, pH 5.5, as described in Methods) and then transferred for 3 days to test medium [germination medium with NH4NO3 replaced by 2.5 mM (NH4)2SO4 and 50 µM KNO3 at pH 5.5]. With these conditions, lateral root length and number were reduced dramatically in chl1-5 plants compared with wild-type plants (Figures 6A, 7B, and 7C)
; the main root length was little affected (Figure 7A), as was the length of adventitious roots (Figure 6A). An important finding of these experiments is that only the adventitious roots had emerged before the time of transfer to the test medium; no lateral roots were seen.
Next, lateral root growth was tested using alternative test media. With low nitrate (50 µM KNO3) at pH 6.5, lateral root number and length were similar but reduced for wild-type and mutant seedlings (Figures 6B, 7B, and 7C). With higher nitrate concentrations (1 mM KNO3) at pH 5.5, lateral root number and length showed little difference between wild-type and mutant seedlings (Figures 6C, 7B, and 7C). With no nitrate, lateral root number and length were approximately the same for both wild-type and chl1-5 seedlings (Figures 6D, 7B, and 7C). These results show that CHL1's contribution to lateral root formation and growth is dependent on nitrate concentration and pH. We confirmed these results using another allele of chl1, chl1-1, in the Landsberg background (data not shown). The reduction in lateral root number observed for the chl1-5 mutant was examined in more detail to determine whether the defect was in lateral root initiation or in a later stage of lateral root formation. The numbers of emerged lateral roots and of primordia that had not emerged were determined by light microscopy. The chl1 mutant has less than half the number of emerged lateral roots than was the wild type (Figure 7D). However, the mutant had more than three times the number of primordia that had not emerged per seedling than the wild type had. When the total number of lateral organs (emerged roots and primordia that had not emerged) was determined, it was found that chl1 and the wild type had approximately the same number of total organs (Figure 7D). Closer examination showed that most of the primordia in the mutant were arrested at stage VII, when the primordia were about to emerge from the primary root. These results indicate that the reduction in lateral root numbers in the mutant is not attributable to a decrease in lateral root initiation but mainly to an inhibition of lateral root primordia maturation or elongation.
CHL1 Function in Primary Root Growth
CHL1 Function in Vegetative and Reproductive Shoot Growth In the experiments described above, we examined CHL1 function in root development. Because CHL1 also is expressed in the nascent organs of the shoot, it is possible that CHL1 functions in shoot organ development. We examined growth rates of three organs—leaves, primary inflorescence, and flower buds—for plants propagated on peat soil. In all three cases, growth of the nascent organ was reduced in the mutant compared with the wild type for both chl1-5 and chl1-1 alleles. Immature leaves of the mutant expanded more slowly than they did in the wild type, but the fully mature leaves were almost as large (data not shown). When the plants shifted to reproductive development and bolted, the stem of the mutant elongated at a slower rate than did the stem of the wild type (1.4 ± 0.2 cm/day for chl1-5 and 3.0 ± 0.4 cm/day for wild type). After 4 to 5 days, the stem of the mutant elongated at a faster rate, so the final length was about the same as that of the wild type. During floral production, young buds from the mutant expanded at a slower rate than did buds from the wild type (0.4 mm/day for chl1-5 versus 0.65 mm/day for Columbia wild type and 0.25 mm/day for chl1-1 versus 0.6 mm/day for Landsberg wild type), but older buds expanded more rapidly in the mutants (0.55 mm/day for both chl1-5 and chl1-1). These results show that CHL1 function is evident during the early phases of shoot organ growth. We also examined the effect of chl1 mutations on the overall growth of the roots and shoots in young plants. Fresh weights were measured for wild-type and chl1-5 seedlings germinated and grown on agarose plates under different conditions for 8 days. At 8 days, the chl1 mutants had noticeably smaller leaves, but root number and length were similar to those of the wild-type plants. Fresh weights of the shoot were lower in the mutant in all conditions tested (NO3- from 100 µM to 10 mM with or without ammonium at pH 5.5 and 6.5; Figure 9) . Fresh weights of the roots also were lower in the mutant, but the effect was more dependent on the medium conditions, with no ammonium at pH 5.5 showing the most dramatic effects and ammonium at pH 6.5 showing the least difference (Figure 9). Similar overall results were observed for the chl1-1 allele compared with the parental line in the Landsberg ecotype (data not shown).
CHL1 Function in Flower Timing Because chl1 mutants showed reduced stem elongation rates after bolting, we examined flowering time. We expected to see a longer time to the first open flower based on days from germination or number of leaves present. This was observed for both chl1-5 and chl1-1 mutants grown with long days (Figure 10) and 24 hr of light (data not shown). A delay in bolting time also was observed for long day conditions (Figure 10). These results indicate that CHL1 plays an important role in reproductive growth, affecting flower timing and flower bud expansion.
CHL1 Supports the Growth of Nascent Organs in Roots and Shoots Our initial prediction, based on the known function of CHL1 and on NRT1 localization studies in tomato, was that CHL1 expression would occur primarily if not exclusively in root epidermal and cortical cells, where the bulk of nitrate uptake occurs. What we discovered was quite different and surprising. Epidermal and cortical cells in mature regions of the root had the weakest expression. The strongest expression was found in the growing tips of the primary and lateral roots. This observation suggested that CHL1 expression is targeted to regions containing rapidly dividing cells. This idea was further supported when lateral roots were examined in detail. At the earliest stages of lateral root formation, such as during the initial cell divisions in the pericycle, an increase in CHL1 expression was observed that was maintained and enhanced as the lateral root primordia developed and emerged from the primary root body. Activation of CHL1 expression also occurred during the growth of the nascent primary root in the first few days after germination. We then examined the shoot, where little CHL1 expression was expected based on previous RNA gel blot analyses (Tsay et al., 1993 ; Lauter et al., 1996). High levels of expression were found in emerging leaves, stipules, and flower buds. When more mature organs or tissues were examined (mature regions of the root, mature leaves, stems, and flowers), CHL1 expression levels were very low, with the exception of the stele in the root. These results show that the overall pattern of CHL1 expression (activation or enhancement during nascent organ development) is similar in roots and shoot; however, because of the nature of root and shoot development, there are some differences. In the roots, CHL1 expression remains high in the root tips as the root elongates but is much lower in the mature parts of the root (except in the stele). In the shoot, CHL1 expression is high in young developing leaves and flower buds but then decreases to very low levels in mature organs.
The findings from these localization studies guided us to reexamine CHL1 function. Using our findings from the expression studies, we specifically examined the growth of nascent organs in both shoots and roots and found it to be defective in chl1 mutants. For every organ examined (primary roots, lateral roots, leaves, stems, and flower buds), nascent organ growth was inhibited in the mutant compared with that of the wild type. In the case of lateral root development, we could identify the stage that was being affected by chl1. If we simplify lateral root development into four stages—(1) initiation, (2) primordia formation, (3) elongation of the primordia to an emerged lateral root, and (4) activation of the lateral root meristem with subsequent elongation of the lateral root (Laskowski et al., 1995
An interesting but puzzling feature of our findings is that the most dramatic phenotypes in chl1 mutants did not always correlate with the exact timing of maximal CHL1 expression. In the mutants, growth of the primary root was very apparent 2 to 3 days after germination, yet CHL1 expression peaked at 5 days, when little difference in growth was seen for the mutant and wild type. In even more mature roots (7 days and older), no difference in growth rates was observed between wild-type and chl1 roots as measured by length, even though CHL1 was highly expressed in mature root tips. This was true for the primary root and emerged lateral roots (e.g., the adventitious roots in Figure 6). One explanation for these results could be that other transporters, especially NRT2 and NRT1.2, provide enough uptake activity for the mature root that CHL1 is not needed for normal growth at this stage. No studies on NRT2 spatial expression patterns have been reported for Arabidopsis, but in Nicotiana plumbaginifolia, NRT2 transcripts have been found concentrated in mature root tips and in lateral root primordia by in situ hybridization (Krapp et al., 1998
Comparison of CHL1 and CDC, PROLIFERA, and CYCB1 Expression
Role of CHL1 in Nascent Organ Growth
Besides being a source of nitrogen, nitrate serves as a metabolite to buffer acidification from ammonium assimilation and as a signal for growth (reviewed in Crawford, 1995
There are several reports that support this idea. Two show that intracellular pH is affected by CHL1 in the absence of nitrate (Meraviglia et al., 1996
CHL1 Function in Flowering
Plant Material Plants of Arabidopsis thaliana ecotype Columbia were used in all experiments unless specified otherwise. Seed was surface-sterilized, first in 70% ethanol for 5 min and then in 5% bleach for 15 min, washed with water, and plated on germination medium [pH 5.5; 10 mM NH4NO3, 5 mM K2HPO4-KH2PO4, 2 mM MgSO4, 1 mM CaCl2, 0.05 mM FeSO4-EDTA, 50 µM H3BO3, 12 µM MnSO4·H2O, 1 µM ZnCl2, 1 µM CuSO4·5H2O, 0.2 µM Na2MoO4·2H2O, 0.5% sucrose, 1 g/L 2-(N-morpholino)ethanesulfonic acid, 1 mg/L thiamine, 100 mg/L inositol, 0.5 mg/L pyridoxine, 0.5 mg/L nicotinic acid, and 8 g/L agarose]. Plates were kept at 4°C for 2 days and then germinated vertically at 24°C under continuous light.
Plant Transformation CHL1-GUS constructs were made with translational fusions of the 4.9-kb HindIII-XhoI fragment and the 6.1-kb HindIII-SpeI fragment into the HindIII-SalI and HindIII-XbaI sites of the pBI101.2-GUS vector, respectively (Clontech, Palo Alto, CA).
Transgenic Arabidopsis plants were produced by vacuum infiltrating 4-week-old plants in Agrobacterium tumefaciens culture containing the appropriate construct (Bechtold et al., 1993
CHL1 Antibody and Protein Gel Blotting
Microsomal Membrane Protein Isolation
Histochemical Localization of GUS
Whole-Mount Immunolocalization
Confocal Microscopy
We thank Marty Yanofsky for access to his fluorescence microscope; Jeffrey H. Price for assistance using the confocal microscope; Alyson Mack, Mary Galli, and Xiujuan Xing for their technical assistance; and Dong Liu and Zhen-Ming Pei for helpful discussion. This work was supported by Grant GM40672 from the National Institutes of Health. Received March 26, 2001; accepted May 21, 2001.
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TOP
Abstract
INTRODUCTION
4.9 kb of CHL1 promoter sequence and exon 1 (up to amino acid 34 of CHL1) fused in frame at the HaeII site to the coding sequence of GFP or GUS. NOS indicates the 3' termination sequence of the nopaline synthase gene.
