Journal of Experimental Botany, Vol. 53, No. 370, pp. 835-844,
April 15, 2002
© 2002 Oxford University Press
Original Papers |
The Arabidopsis dual-affinity nitrate transporter gene AtNRT1.1 (CHL1) is regulated by auxin in both shoots and roots
Section of Cell and Developmental Biology, Division of Biology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0116, USA
Received 18 July 2001; Accepted 22 November 2001
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Abstract |
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The AtNRT1.1 (CHL1) gene of Arabidopsis encodes a dual-affinity nitrate transporter and contributes to both low and high affinity nitrate uptake. Localization studies have shown that CHL1 expression is preferentially targeted to nascent organs and growing regions of roots and shoots in Arabidopsis. In roots, CHL1 expression is concentrated in the tips of primary and lateral roots and is activated during lateral root initiation. In shoots, strong CHL1 expression is found in young leaves and developing flower buds. These findings suggest that CHL1 expression might be regulated by a growth signal such as the phytohormone auxin. To test this, auxin regulation of CHL1 was examined. Using transgenic Arabidopsis plants containing CHL1::GUS/GFP DNA constructs, it was found that treatment with exogenous auxin or introduction of the auxin overproducing mutations (yucca and rooty) resulted in a strong increase in CHL1::GUS/GFP signals in roots and leaves. When mature roots were treated with auxin to induce lateral root formation, CHL1::GFP signals were dramatically enhanced in dividing pericycle cells and throughout primordia development. RNA blot analysis showed that CHL1 mRNA levels in whole seedlings increase within 30 min of auxin treatment. The distribution of CHL1 expression in Arabidopsis roots and shoots was found to be similar to that of DR5::GUS, a synthetic, auxin-responsive gene. These results indicate that auxin acts as an important signal regulating CHL1 expression and contributes to the targeting of CHL1 expression to nascent organs and root tips in Arabidopsis.
Key words: Arabidopsis, AtNRT1.1, auxin, CHL1, nitrate transport.
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Introduction |
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Nitrate is one form of nitrogen available to plants to support growth and development. Nitrate is taken up by active transport processes in the roots and then is reduced to ammonium by nitrate reductase and nitrite reductase, enzymes present in both roots and shoots. Ammonium is then incorporated into amino acids via glutamine synthetase and glutamate synthase. Large amounts of reductant and carbon skeletons are needed for these steps as nitrate assimilation consumes extensive metabolic resources. Nitrate can be a significant factor limiting plant growth and productivity because soil nitrate concentrations can vary by several orders of magnitude. Several recent reviews provide comprehensive coverage of nitrate assimilation (Forde and Clarkson, 1999
; Tischner, 2000; Campbell, 2001; Crawford and Forde, 2001).
Besides serving as a nutrient, nitrate plays an important signalling role for plants (reviewed by Koch, 1997; Forde and Clarkson, 1999; Stitt, 1999; Zhang and Forde, 2000; Coruzzi and Bush, 2001; Coruzzi and Zhou, 2001; Crawford and Forde, 2001). Nitrate reprogrammes metabolism to favour organic acid over starch metabolism and to provide energy for nitrate/nitrite reduction (Scheible et al., 1997a; Stitt, 1999). Nitrate also rapidly induces the expression of many genes especially those devoted to nitrite reduction (Stitt, 1999; Wang et al., 2000). Many of these genes are also regulated by carbon and amino acid signals so that nitrate assimilation can be integrated into overall nitrogen and carbon metabolism (Koch, 1997; Stitt, 1999; Coruzzi and Bush, 2001; Coruzzi and Zhou, 2001). These responses result in differential organ growth with high nitrate levels inhibiting root growth, stimulating shoot growth and delaying the onset of flowering (Lambers et al., 1990; Scheible et al., 1997b; Stitt, 1999). Nitrate also affects root architecture, with high levels in the soil producing a systemic inhibitory effect on lateral root growth while localized zones of high nitrate stimulate root branching (Drew and Saker, 1975; Zhang et al., 1999).
The transport systems responsible for nitrate uptake have been well characterized physiologically (reviewed in Glass and Siddiqi, 1995; Crawford and Glass, 1998; Forde and Clarkson, 1999; Crawford and Forde, 2001). Nitrate is unique among inorganic nutrients in that a cationic form (ammonium) is also available for uptake so that loss of nitrate does not necessarily lead to nitrogen deprivation. Studies primarily with barley, maize and Lemna, indicate that there are three primary transport systems: a nitrate-inducible high affinity system (iHATS), a nitrate-constitutive high affinity system (cHATS) and a low affinity system (LATS) (reviewed in Glass and Siddiqi, 1995; Crawford and Glass, 1998; Forde and Clarkson, 1999). The high affinity systems show saturable kinetics with Km values in the range of 10–100 µM, while the low affinity system shows linear, non-saturable kinetics.
So far, two gene families, NRT1 and NRT2, have been identified in plants that encode nitrate transporters (reviewed in Crawford and Glass, 1998; Daniel-Vedele et al., 1998; Forde, 2000; Crawford and Forde, 2001; Galvan and Fernandez, 2001; Glass et al., 2001; Williams and Miller, 2001). Several members of the NRT2 family encode iHATS transporters (Forde, 2000; Cerezo et al., 2001; Filleur et al., 2001; Glass et al., 2001). The NRT1 family encodes an eclectic mix of nitrate transporters including the low affinity transporter AtNRT1.2 (Huang et al., 1999), the nitrate/histidine bispecific transporter BnNRT1.2 (Zhou et al., 1998), and the dual affinity transporter AtNRT1.1 (CHL1) (Tsay et al., 1993; Huang et al., 1996; Touraine and Glass, 1997; Wang et al., 1998; Liu et al., 1999). This paper focuses on the CHL1 gene.
The chl1 locus was identified in a genetic screen using chlorate, the chlorine analogue of nitrate (Braaksma and Feenstra, 1973; Doddema and Telkamp, 1979). The CHL1 gene was isolated from a T-DNA-tagged, chlorate-resistant mutant and found to encode a hydrophobic protein with 12 membrane spanning regions indicative of a cotransporter (Tsay et al., 1993). Expression of CHL1 in Xenopus oocytes showed that it encodes an electrogenic nitrate transporter whose activity is enhanced by acidifying the external medium (Tsay et al., 1993). A most interesting property of CHL1 is that it contributes to both low affinity (Doddema and Telkamp, 1979; Huang et al., 1996; Touraine and Glass, 1997) and high affinity nitrate uptake (Wang et al., 1998; Liu et al., 1999) in plants. In Xenopus oocytes, it displays dual affinity activity with two saturable components (Liu et al., 1999). CHL1's contribution to nitrate uptake in plants depends on the growth conditions with a key factor being ammonium (Huang et al., 1996; Touraine and Glass, 1997). When plants are grown on ammonium nitrate, chl1 mutants take up much less nitrate than the wild type; however, there is little difference when plants are grown without ammonium (i.e. on potassium nitrate) (Touraine and Glass, 1997). Another key factor is the external pH. CHL1 mRNA levels are higher at acidic pH (Tsay et al., 1993), and, interestingly, chl1 mutants have altered intracellular pH levels even in the absence of added nitrate (Meraviglia et al., 1996; Romani et al., 1996).
Given that CHL1 is part of the nitrate uptake systems of Arabidopsis, one would expect that the protein would be targeted to epidermal and cortical cells of the root. Several members of the NRT1 family do conform to this prediction. AtNRT1.2, a component of LATS, is highly expressed in root hairs and epidermal cells of Arabidopsis (Huang et al., 1999). In tomato, NRT1 genes were found to be expressed in root hairs (LeNRT1.2) or throughout the root (LeNRT1.1) (Lauter et al., 1996). Initial studies with CHL1, however, revealed a surprising result. In situ hybridization experiments showed that, distal to the root tip, CHL1 mRNA is found primarily in the internal layers of the root (Huang et al., 1996).
To investigate the localization of CHL1 expression more thoroughly, plants were examined with CHL1::GFP/GUS fusion constructs (Guo et al., 2001). The resulting expression patterns in transgenic plants showed that CHL1 is preferentially expressed in rapidly growing tissues in both shoots and roots. The strongest GFP signals were found in the tips or growing regions of both the primary and lateral roots in 5 or 7-d-old transgenic seedlings. Closer examination of the GFP images revealed that all cell layers in the root tip express strongly, including cells in the lateral and columella root cap. In the elongation zone, the signal is much diminished. In the mature parts of the root, weak but significant signals were observed in the stele, but little signal above background was observed in the epidermal and cortical cells. During root development CHL1 expression is activated in the tip of primary roots during the first day of seedling growth and expands into the entire tip by day 5. During lateral root development, CHL1 expression is enhanced at the earliest stages of root initiation. Strong GFP signals are observed in dividing pericycle cells and remain high during the formation and emergence of lateral root primordia. These results show that CHL1 expression is activated and is sustained during the growth and development of nascent roots. These expression patterns were confirmed by immunolocalization experiments.
Because there is a correlation between CHL1 expression and growth of nascent organs in roots, it seemed possible that a similar correlation would be found in shoots. When shoots containing the CHL1::GFP/GUS fusions were examined, strong GFP signals and GUS staining were observed in developing leaves and flower buds, but little signal was found in mature organs or in the shoot apical meristem. Thus, CHL1 is preferentially expressed in nascent organs of the shoot.
One explanation for the preferential expression of CHL1 is that CHL1 is regulated by a growth signal. The phytohormone auxin is a good candidate as it is a key growth signal inducing cell division, expansion and differentiation, and as well as morphogenesis and oncogenesis in plants (Davies, 1995; Hooykaas et al., 1999; Zhao et al., 2001). For example, auxin plays a key role in lateral root initiation with IAA and its transport being required to establish a population of rapidly dividing pericycle cells that give rise to lateral root primordia (Celenza et al., 1995; Laskowski et al., 1995; Casimiro et al., 2001; Rashotte et al., 2001). At the molecular level auxin rapidly activates transcription of a set of early genes that are thought to mediate, at least in part, the various effects of auxin (reviewed in Abel and Theologis, 1996; Walker and Estelle, 1998; Hagen and Guilfoyle, 2001; Reed, 2001). The role of auxin in regulating and targeting CHL1 expression using auxin treatments, auxin over-producing mutants and a synthetic auxin-responsive gene was investigated. The results from these experiments are presented below and provide a mechanism for targeting transporter gene function.
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Materials and methods |
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Plant materials and growth conditions
Plants of Arabidopsis thaliana ecotype Columbia were used in all experiments unless otherwise specified. For auxin induction experiments, seeds were surface-sterilized and germinated in liquid culture in 10 ml flasks with growth medium (as described in Guo et al., 2001
) which includes pH 5.5 and 0.5% sucrose, except that 10 mM ammonium succinate was the sole nitrogen source. For experiments involving lateral root formation in response to added auxin, seeds were surface-sterilized and plated onto growth medium with 0.8% agarose except 10 mM NH4NO3 was the nitrogen source. All plants were grown in constant white light at 24 °C.
Plant transformation
CHL1::GFP/GUS constructs containing the 4.9 kb HindIII-HaeII promoter region of CHL1 were as described earlier (Guo et al., 2001). Transgenic Arabidopsis plants were produced by vacuum infiltrating 4-week-old plants in Agrobacterium culture containing the appropriate construct (Bechtold et al., 1993). Seeds from treated plants were collected and screened for kanamycin resistance. Transgenic plants identified at this generation were classified as T1 plants. CHL1::GFP homozygous plants were used to pollinate yucca and rooty mutants; mutants were selected in the F2 generation and analysed.
Auxin treatment of Arabidopsis seedlings
Seedlings analysed for CHL1::GUS/GFP expression were grown for 7 d in liquid culture in germination media and then treated with IAA at the final concentration of 1 or 10 µM for 6 h or 12 h. Seedlings treated with auxin to induce lateral root initiation, were grown for 5 d in germination media (pH 5.5, 10 mM NH4NO3 as nitrogen source) and then transferred to the same medium supplemented with 10 µM IAA. All seedlings were grown vertically on agarose plates before and after transferring under continuous light at 24 °C.
RNA blot analysis
Total RNA was extracted from whole seedlings treated with IAA and 5 µg total RNA was loaded onto 1.5% agarose gels containing formaldehyde and processed as described previously (Wang et al., 2000). RNA gel blots were autoradiographed, the film was scanned, and hybridization signals were quantified using Adobe Photoshop 4.0 (Adobe Systems, Mountain View, CA).
Histochemical staining of GUS activity
Whole seedlings or organs from transgenic plants were submerged in staining solution consisting of 25 mM sodium phosphate buffer, pH 7.0, 2 mM 5-bromo-4-choro-3-indolyl-ß-D-glucuronide cyclohexylamine salt (X-gluc), 0.5 mM ferricyanide, 0.5 mM ferrocyanide and 10 mM EDTA at 37 °C for 2–6 h. The GUS staining solution was replaced with 70% ethanol for bleaching the tissues.
Confocal microscopy
Whole seedlings were stained with 10 µg ml-1 propidium iodide (PI) (Sigma) and mounted in water under glass coverslips for GFP fluorescent signal analysis using a confocal laser scanning microscope (MRC-600, Bio-Rad) equipped with a krypton-argon laser and an inverted microscope (Nikon). GFP images were collected using a 530±15 nm emission filter, and a 620±15 nm emission filter for PI images.
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Results |
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CHL1 expression is enhanced by elevated auxin levels
The effect of auxin on CHL1 gene expression was examined by treating transgenic Arabidopsis plants with auxin and then monitoring the expression of CHL1 using a GFP/GUS fusion construct containing 4.8 kb of upstream CHL1 sequence and 180 bp of transcribed sequence including 96 bp of the CHL1 coding region (HaeII construct; Fig. 1
). This construct was used in previous studies showing that CHL1 is preferentially expressed in nascent organs and root tips of Arabidopsis (Guo et al., 2001). Seven-day-old seedlings containing this construct were grown in liquid culture without nitrate, treated with IAA (1 or 10 µM) for 6 or 12 h and then examined for GUS activity using histochemical staining as described in Materials and methods. In these experiments the incubation time with the histochemical stain was reduced from the usual 6–10 h to 2 h to decrease the background signal from untreated plants.
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The results from these experiments show that IAA treatment enhances the expression of the CHL1::GUS marker gene. In primary roots, GUS staining increased in root tips in proportion to the amount of auxin added or the time of treatment (Fig. 2A
–D). The strongest signals were observed in the seedlings treated with 10 µM IAA for 12 h (Fig. 2D), but increased expression was observed with 1 µM IAA after 6 h (Fig. 2B). A similar response was found in lateral root tips treated with IAA (Fig. 2E–H) while a weaker but enhanced GUS staining was found in the mature regions of roots especially in the stele (data not shown). In shoots of liquid-grown plants, GUS activity was enhanced by IAA treatment but the response was less dramatic than in the roots (data not shown). Auxin treatment of detached leaves of 14-d-old seedlings grown in peat soil (containing nitrate) showed increased, but non-uniform, GUS staining in leaves depending on the concentration of IAA and the duration of the treatment (Fig. 2I–L). In these experiments, the strongest GUS staining was observed along the edges of the blade and in the petioles.
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Next, CHL1::GFP expression was examined in two auxin over-producing mutants: yucca and rooty/superroot. YUCCA encodes a flavin mono-oxygenase-like enzyme that is involved in auxin biosynthesis, and plants overexpressing this gene contain elevated levels of free auxin (Zhao et al., 2001
). The ethyl methane sulphonate (EMS)-induced mutant rooty/superroot exhibits extreme proliferation of adventitious and lateral roots, and its free endogenous indole-3-acetic acid concentrations are 1.5–3.7 times higher than in the wild type, depending on seedling age (Boerjan et al., 1995; King et al., 1995). CHL1::GFP constructs were introduced into these two mutants by genetic crosses. Mutant plants carrying CHL::GFP reporter constructs showed elevated GFP signals relative to wild-type transgenic plants (Fig. 2M
–X). In primary root tips of 5-d-old seedlings, the GFP signals in the yucca mutant (Fig. 2N) and in the rooty mutant (Fig. 2O) were stronger, especially in the elongation zone compared with wild-type plants (Fig. 2M). A similar pattern was observed for young lateral roots with stronger signals in the mutants that extended beyond the root tip (data not shown). In the mature part of the root, the mutants showed higher levels of GFP in the stele (Fig. 2R, S) than in the wild type (Fig. 2Q). In the rooty mutant, strong signals were also found in the endodermal cells (Fig. 2S). To show the outlines of the roots, organs were also stained with propidium iodide (PI) and composite images were taken with both GFP and PI signals (Fig. 2U–W, outlines of root are also drawn in Fig. 2Q, R). When shoots were examined, little difference in GFP signals was found between the rooty mutant (Fig. 2X) and the wild type (Fig. 2P); however, a significant increase was observed in cotyledons of the yucca mutant (Fig. 2T). Taken together, these results show that overall CHL1 expression level is significantly enhanced in auxin over-producing mutants.
To confirm that CHL1 responds to auxin, RNA blot analyses were performed. Total RNA was isolated from whole seedlings treated with IAA (10 µM) at different time points and then analysed using a CHL1 DNA probe. The results show a 50% increase of CHL1 mRNA levels after 30 min of treatment, a peak after 1 h with a slight decrease from the peak after 3 h (Fig. 3). These results confirm that CHL1 responds to auxin and show that the response at the RNA level is rapid.
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Comparison of CHL1 and DR5::GUS expression
Because CHL1 is responsive to auxin, it would be interesting to compare the distribution of CHL1 expression within a plant to that of another auxin-responsive gene. This was done by comparing the expression patterns of CHL1::GUS and DR5::GUS. The synthetic DR5 promoter contains seven tandem repeats of an auxin-responsive element fused to the minimal 35S CaMV promoter (Ulmasov et al., 1997). The DR5 construct has been used to examine the effect of auxin treatments, polar auxin transport mutations and inhibitors on auxin-responsive gene expression (Ulmasov et al., 1997; Sabatini et al., 1999; Casimiro et al., 2001; Rashotte et al., 2001).
The overall GUS staining patterns for DR5::GUS (Fig. 4A–D) and CHL1::GUS (Fig. 4E–H) are similar, showing strong expression in primary root tips, lateral root primordia, young leaves, and tips of cotyledons. Signals from the DR5::GUS construct were weaker than from the CHL1::GUS construct so longer staining times were required for DR5::GUS plants (16 h for DR5 versus 6 h for CHL1). Some differences in staining patterns were found between DR5::GUS and CHL1::GUS plants. There was little GUS staining in the stele of DR5::GUS plants compared with CHL1::GUS plants except around the lateral root buds (Fig. 4B). Beyond the quiescent region of the root meristem, DR5::GUS expression is found in the internal cell files destined to become the stele (Fig. 4A), while CHL1 expression disappears in these internal cell files until the mature stele is reached (Fig. 4E). When roots of DR5::GUS plants are treated with auxin, expression increases dramatically in all cell files (Ulmasov et al., 1997; and data not shown), while that of CHL1::GFP shows the strongest increase in dividing pericycle cells (see below). The effect of the auxin transport inhibitor TIBA was tested; and it was found that TIBA treatment results in an expansion of CHL1::GFP expression in root tips (data not shown) similar to what has been reported for DR5::GUS (Sabatini et al., 1999; Casimiro et al., 2001). It is also noteworthy that both the DR5::GUS (Ulmasov et al., 1997) and CHL1::GUS (see above) show greater auxin induction in roots versus shoots. These results show that the overall distribution of CHL1 expression is similar to that of DR5::GUS and indicate that auxin is an important signal for establishing the pattern of CHL1 expression in Arabidopsis.
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CHL1 expression is activated during auxin-induced lateral root initiation
Previous results indicated that CHL1 expression is activated during the initiation of lateral roots (Guo et al., 2001). One way to induce lateral root formation is by the application of IAA. Transgenic plants carrying the CHL1::GFP/GUS constructs were examined to determine how CHL1 responds during auxin-activated, lateral root formation. Five-day-old seedlings were treated with 10 µM IAA, and a time-course of CHL1 expression was determined during the early stages of lateral root formation (Fig. 4I–X). At time zero, weak GFP signals are observed in the stele in mature regions of the root (Fig. 4I; Fig. 4M shows corresponding GFP/PI composite). After 2 h of auxin treatment, a small enhancement of GFP signals occurred in the stele, but not in the epidermis and cortex (Fig. 4J, N). By 5 h, GFP signals increased significantly and expanded into the epidermis and cortex with the strongest signals found in patches of dividing pericycle cells (Fig. 4K, O). At 8 h, the GFP signal continued to increase with patches of dividing pericycle cells becoming more noticeable and enlarging in a radial direction adjacent to and just outside of the xylem poles (Fig. 4L, P, note: xylem poles appear as red lines in the stele of the composite image; Fig. 4P). At 12 h, strong GFP signals were found in all of the xylem-adjacent pericycle cells undergoing cell division and result in the formation of two continuous columns of dividing, pericycle-derived tissues (Fig. 4Q, U). A similar structure was reported for radish roots treated with IAA (Laskowski et al., 1995). At 16 h, high intensity GFP signals were observed in lateral root primordia (LRP) which had penetrated the endodermal layer (Fig. 4R, V). After 24 h, the primordia began to emerge from the root body, and very strong GFP signals were found in LRP (Fig. 4S, W). Over the course of 2 d, the LRP gave rise to a large number of closely spaced lateral roots which show strong GUS staining (Fig. 4X). Interestingly, the GFP signals declined in the primary root tip including the root cap and meristem region when numerous LRP arise from the mature roots treated with auxin for 24 h (Fig. 4T). These results indicate that CHL1 expression is activated at the earliest stage of lateral root formation when induced by exogenous auxin application and is then maintained during lateral root development.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The results described above indicate that CHL1 is an auxin-regulated gene. This finding can account for the preferential expression of CHL1 to nascent organs and regions of rapid cell proliferation and for the activation of CHL1 in dividing cells forming new organs such as in young primary roots, leaves and flower buds, and in pericycle cells giving rise to lateral root primordia. Several lines of evidence support this conclusion including induction by auxin treatment, overexpression in auxin-overproducing mutants, and overlapping expression patterns with DR5::GUS reporter constructs. Thus, auxin becomes another of the many signals (including nitrate, sugar, diurnal cycles, pH, N-starvation) that affect CHL1 expression.
Initially, it was surprising to find that auxin is such a key signal for CHL1. The original model for CHL1 function was that it serves as a component of the nitrate uptake systems in roots (reviewed in Crawford and Glass, 1998). Such signals as pH, sucrose and nitrate have all been shown to influence CHL1 expression (Tsay et al., 1993; Crawford and Glass, 1998; Lejay et al., 1999; Forde, 2000; Galvan and Fernandez, 2001; Glass et al., 2001), which makes sense for a component of the nitrate uptake systems. One would not predict that auxin would play much of a role in controlling such a gene. However, the discovery that CHL1 is preferentially expressed in root tips, lateral root primordia, young leaves, and flower buds revealed a new facet of CHL1 function: supporting the growth of nascent organs (Guo et al., 2001). This new picture definitely includes a role for auxin. In fact, finding that CHL1 is responsive to auxin explains why CHL1 expression is targeted to specific regions of rapid cell proliferation. This linkage was most clearly shown during auxin-induced, lateral root initiation. Initiation of lateral roots is an auxin-induced response as IAA and its transport are initially required to establish a population of rapidly dividing pericycle cells (Boerjan et al., 1995; Celenza et al., 1995; King et al., 1995; Laskowski et al., 1995; Casimiro et al., 2001; Rogg et al., 2001). The application of auxin to Arabidopsis roots leads to activation of both lateral root formation and CHL1 expression (Fig. 4), insuring the presence of CHL1 in rapidly dividing cells.
An interesting question that arises from these results is what is the role of nitrate in the auxin induction of CHL1. The initial experiments (Fig. 2) were performed on plants grown without nitrate (i.e. with ammonium succinate) and showed clear auxin induction of CHL1::GUS, indicating that nitrate is not necessary for auxin induction. Another set of experiments that included nitrate in the growth media showed induction of CHL1::GFP/GUS expression during auxin-activated formation of lateral roots (Fig. 4I–X), indicating that nitrate does not obscure or block in the induction. It is important to note that no evidence for nitrate induction of the CHL1 HaeII constructs was found in roots (data not shown). Thus, at this point nothing can be said about the relationship between nitrate and auxin regulation of CHL1.
One thing these results suggest is that auxin might influence nitrogen assimilation to support rapid growth of nascent roots and root tips. Such a linkage has been suggested for chicory roots based on studies of the nitrate reductase gene, which is induced by auxin and is expressed in newly initiated lateral root meristems in this system (Vuylsteker et al., 1998). This linkage is also supported by results from several auxin-responsive mutants. The enhancement of lateral root elongation by localized supply of nitrate is reversed by the auxin resistance mutation axr4, but not aux1 or axr2 (Zhang et al., 1999; Zhang and Forde, 2000). Also, inhibition of primary root growth by ammonium in the absence of potassium is reversed by the auxin resistance mutations aux1, axr1 and axr2 (Cao et al., 1993). Care must be taken in interpreting these results, however, in that some of these mutants have altered responses to other hormones as well (Pickett et al., 1990; Rogg et al., 2001). Though these results are suggestive, further work will be needed to establish a direct link between auxin and nitrate metabolism and responses.
The regulation of the dual-affinity nitrate transporter gene CHL1 has turned out to be more complex than initially expected. Multiple signals affect its expression including auxin, which appears to target CHL1 expression to regions of rapid cell division and organ growth. CHL1 supports the growth of these cells, but its precise role is still a mystery.
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Acknowledgments |
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We thank Joann Chory for the yucca mutant seeds; Tom Guilfoyle for the DR5::GUS construct and seeds; the Arabidopsis Biological Resource Center for rooty mutant seeds; Marty Yanofsky for access to his microscope; Jeffrey H Price for assistance using the confocal microscope and Mingsheng Chen for his contribution to these projects. This work was supported by Grant No. GM40672 from the National Institutes of Health.
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Notes |
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1 To whom correspondence should be addressed. Fax: +18585341637. E-mail: ncrawford{at}ucsd.edu
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References |
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