Abstract
Ammonium (NH4 +) is an important form of nitrogen nutrient for most plants, yet is also a stressor for many of them. However, the primary events of NH4 + toxicity at the cellular level are still unclear. Here, we showed that NH4 + toxicity can induce the root cell death in a temporal pattern which primarily occurs in the cells of root maturation and elongation zones, and then spreads to the cells in the meristem and root cap. The results from the NH4 +-hypersensitive mutant hsn1 further confirmed our findings. Taken together, NH4 + toxicity inhibits primary root growth by inhibiting cell elongation and division and inducing root cell death.
Keywords: Ammonium toxicity, Root, Cell viability, GDP-mannose pyrophosphorylase, Arabidopsis
1. Introduction
Nitrogen (N) is an important nutrient to plants with two most important nitrogen forms being ammonium (NH4 +) and nitrate (NO3 −). Because NH4 + assimilation requires less energy than that of NO3 − (Reisenauer, 1978), NH4 + is expected to be the preferred form of nutrient to plants. However, this ion has toxic effects on many plant species (Britto and Kronzucker, 2002). Two of the most dramatic symptoms of ammonium toxicity were the chlorosis of leaves and the overall suppression of growth (Britto and Kronzucker, 2002). Ammonium toxicity has also been linked to the damage to agricultural crops under certain ecological conditions (van Breemen and van Dijk, 1988). Several hypotheses to explain the possible mechanisms of NH4 + toxicity in plants have been proposed, including the acidification of external growth environment, displacement of important cations, such as K+ and Mg2+, or excessive energy being wasted due to the abundance of toxic NH4 + requiring removal from the cells (Britto and Kronzucker, 2002). None of these theories have yet been conclusively confirmed.
One characteristic of ammonium syndrome is the inhibition of primary root growth when plants are exposed to higher NH4 + concentrations (Britto and Kronzucker, 2002; Qin et al., 2008). Our previous study demonstrates that guanosine diphosphate (GDP)-mannose pyrophosphorylase (GMPase) is a genetic determinant of ammonium sensitivity in Arabidopsis (Qin et al., 2008). The root growth is severely inhibited in the mutant hsn1 defective in GMPase grown in the growth medium with NH4 +. Defective N-glycosylation of proteins, unfolded protein response, and cell death are likely important downstream molecular events involved in the NH4 +-hypersensitive mutant. The root cellular response to NH4 +, however, is still unclear. Understanding of the root cellular response to NH4 + may help us to elucidate the physiological and cellular mechanisms of the NH4 +-mediated inhibition of root growth.
2. Materials and methods
2.1. Plant materials and growth conditions
The plant materials used in this work include wild type (WT) Arabidopsis thaliana and genetic mutant hsn1 (Col-0 ecotype) (Qin et al., 2008). Seed germination and seedling growth were accomplished using modified Murashige and Skoog (MS) media, which consisted of NO3 −+NH4 + medium (half-strength MS medium, with 10 mmol/L NH4 + and 20 mmol/L NO3 − provided by KNO3 and NH4NO3 as nitrogen sources), NH4 + medium (with 10 mmol/L NH4 + provided by (NH4)2SO4 as sole nitrogen source), and NO3 − medium (with 20 mmol/L NO3 −, provided by KNO3 as sole nitrogen source). The above media were all supplemented with 5 g/L sucrose and 0.5 g/L 2-morpholinoethane sulphonic acid (MES), adjusted to pH 5.7, and solidified with 10 g/L agar-agar (Fisons). The plates grew in a growth cabinet (Percival Scientific, Perry, IA) preset with a 16-h light/8-h dark photoperiod, 300 μmol/(m2∙s) light intensity, and a constant temperature of 20 °C.
To observe marker expression in the hsn1 mutant, we examined F3 seeds from a cross between hsn1 and the marker lines.
2.2. Measurement of root growth
Seeds on plates were kept at 4 °C for 2 d and then were placed vertically in a growth cabinet kept at 20 °C under continuous illumination. Only seedlings that germinated at the same time were used for root growth measurements.
2.3. Histochemical analysis
The histochemical stain for β-glucuronidase (GUS) activity and stain for Starch granules were performed according to prevenient papers (Jefferson et al., 1987; Fukaki et al., 1998). The stained materials were cleared for 10 min in an 8:3:1 (v/v) mixture of chloral hydrate:water:glycerol (Willemsen et al., 1998) and photographed with a photomicroscope (Zeiss AxioCam HRC, Oberkochen, Germany). Starch granules in the columella root cap were visualized by staining with 1% (v/v) Lugol solution (Merck) for 3 min. The samples were rinsed with water, cleared with chloral hydrate, and imaged under a photomicroscope (Zeiss AxioCam HRC, Oberkochen, Germany).
For confocal scanning, roots were stained in 10 µg/ml propidium iodide (PI) solution for 5 min. The images were monitored by the LSM 510 (Carl Zeiss Co., Germany).
2.4. Cell viability assay
Cell viability was examined by detection of the green fluorescent dye fluorescein diacetate (FDA), of which fluorescence decreases as the dye leaks from dead cells (Bais et al., 2003). The roots of seedlings were mounted in 10 ml of 20 µmol/L FDA water solution for 10 min, washed with distilled water, and then imaged for no more than 5 min.
3. Results
3.1. Effect of NH4 + on root cell elongation and cell division
To characterize the effect of NH4 + on root development, we determined the root cell elongation and cell division of WT seedlings grown in the NO3 −+NH4 + and NH4 + media over a 10-d period. When grown in the NH4 + medium, the WT seedlings displayed a typical NH4 + toxicity phenotype, which is distinct from that grown in the NO3 −+NH4 + medium (Fig. 1a). To determine the effect of NH4 + on the root cell elongation, we measured the length of the newly formed mature cortical cells (the cells that are adjacent to the elongation zone). It showed that there was an inhibition of root cell elongation in WT grown in the NH4 + medium. The length of root mature cortical cells adjacent to the elongation zone of WT plants in the NO3 −+NH4 + medium increased 46% (from 120 to 175 µm) 10 d after germination (DAG), while that of WT plants in the NH4 + medium decreased 55% after 10 d culture (from 108 µm at 1 DAG to 49 µm at 10 DAG) (Fig. 1b). As early as 3 DAG, an obvious inhibition of root cell elongation was observed in WT seedlings in the NH4 + medium. Up to 10 DAG, the length of mature cortical cells of WT seedlings in the NH4 + medium was 72% shorter than that in the NO3 −+NH4 + medium (Fig. 1b). These results indicate that NH4 + toxicity affects root cell elongation. To confirm these results, we employed the NH4 +-hypersensitive mutant hsn1 for further characterization. It was reported that NH4 + can severely inhibit primary root of the hsn1 mutant even when grown in the NO3 −+NH4 + medium (Qin et al., 2008) (Fig. 1a). In the hsn1 mutant, as early as 2 DAG, an obvious inhibition of root cell elongation was observed in the NO3 −+NH4 + medium. Up to 10 DAG, the length of mature cortical cells of the hsn1 mutant in the NO3 −+NH4 + medium was 75% shorter than that of WT (Fig. 1b). These results suggest that NH4 + can inhibit root cell elongation and the hsn1 mutant is much more sensitive to the inhibition.
Fig. 1.
Inhibitive effect of NH4 + on root cell elongation and cell division
(a) Comparisons of seedling growth of WT Arabidopsis and hsn1 in the NO3 −+NH4 + and NH4 + media at 10 d after germination (DAG); (b) Comparisons of mature cortical cells between WT Arabidopsis (Col-0) in the NO3 −+NH4 + and NH4 + media, and hsn1 mutant in the NO3 −+NH4 + medium; (c) CYCB1;1::GUS marker expression in primary roots of WT in the NO3 −+NH4 + and NH4 + media and hsn1 mutant plants in the NO3 −+NH4 + medium in a time course up to 10 d. These results have been repeated in three separate experiments
To determine the effect of NH4 + on root cell division, we monitored the temporal expression of the cell cycle marker CYCB1;1::GUS in WT seedlings in both NO3 −+NH4 + and NH4 + media. The expression of CYCB1;1 is a mark for dividing cells (Colon-Carmona et al., 1999). In the NO3 −+NH4 + medium, a typical expression pattern of CYCB1;1::GUS in dividing cells at root meristem was observed (Fig. 1c). However, when grown in the NH4 + medium, the activity of CYCB1;1::GUS was obviously repressed at 3 DAG, and the activity was gradually reduced until nearly no dividing cell was detected at 10 DAG (Fig. 1c). It indicates that the NH4 + can inhibit root cell division in WT seedlings grown in the NH4 + medium. If this is true, we might expect that the root cell division would be inhibited in the hsn1 mutant even if it was grown in the NO3 −+NH4 + medium. Therefore, we introgressed CYCB1;1::GUS in the hsn1 mutant background, and examined the GUS activity of the plants grown in the NO3 −+NH4 + medium. Consistent with our hypothesis, the activity of CYCB1;1::GUS in hsn1 mutant root was repressed at 3 DAG, and the activity was gradually reduced until nearly no cell division was detected at 10 DAG (Fig. 1c). Taken together, it indicated that NH4 + can inhibit root cell division at meristem.
Collectively, we showed that NH4 + can inhibit root cell elongation, and even repress root meristematic cell division.
3.2. Effect of NH4 + on organization and maintenance of the root meristem
To further determine the effect of NH4 + on organization and maintenance of the root meristem, we examined the expression patterns of some cell type-specific markers in WT seedlings grown in the NO3 −+NH4 + and NH4 + media. The expressions of enhancer trap J3612::GFP (marks pericycle initials) and SCRp::GFP (marks the endodermis, the cortex/endodermal initial cells and the quiescent center (QC)) (Wysocka-Diller et al., 2000) showed a typical pattern in WT root grown in the NO3 −+NH4 + medium (Fig. 2). In the NH4 + medium, SCRp::GFP showed a typical expression in WT. Ectopic expressions of J3612::GFP were observed in stele at 10 DAG (Fig. 2). Similar to that, in the NH4 +-hypersensitive mutant hsn1, ectopic expressions of J3612::GFP and SCRp::GFP were all observed in stele of root at 10 DAG even in the NO3 −+NH4 + medium (Fig. 2). These results indicate that the NH4 + can disorder the organization and maintenance of the root meristem, with defects either to specify or to maintain the root radial pattern and cell identity.
Fig. 2.
Green fluorescent protein (GFP) expressions in primary roots of WT in the NO3 −+NH4 + and NH4 + media and hsn1 mutant plants in the NO3 −+NH4 + medium in a time course up to 10 d carrying the enhancer traps J3612::GFP (a) and SCRp::GFP (b)
These results have been repeated in three separate experiments
A failure in root meristem maintenance can be caused by the lack of QC activity. To determine whether NH4 + destroys QC activity, we examined the expression of QC marker, QC25::GUS (van den Berg et al., 1995) in seedlings in the NO3 −+NH4 + and NH4 + media. The WT seedlings grown in the NO3 −+NH4 + medium displayed the typical expression of the QC25::GUS in 2–3 QC cells during a 10-d period (Fig. 3a). When grown in the NH4 + medium, the WT seedlings displayed the typical expression of the QC25::GUS at 4 DAG, but no expression of QC25::GUS was detected in the root at 10 DAG (Fig. 3a). Further observation showed that QC cells were more distally positioned in WT seedlings in the NH4 + medium compared with seedlings in the NO3 −+NH4 + medium at 4 DAG (Fig. 3a). Columella cells have starch granules, which can be stained by Lugol solution (Fukaki et al., 1998). The columella cells in WT seedlings in the NO3 −+NH4 + and NH4 + media were examined using Lugol staining. Columella in WT seedlings in the NO3 −+NH4 + medium showed a typical Lugol staining (Fig. 3b). In the NH4 + medium, Lugol staining signals of columella cells were decreased at 4 DAG (Fig. 3b). These results indicate that NH4 + can alter QC activity and root cap structure. To further confirm it, we examined the expression of QC25::GUS and Lugol staining in the hsn1 mutant grown in the NO3 −+NH4 + medium. At 10 DAG, no expression of QC25::GUS was detected in the hsn1 mutant (Fig. 3a). Lugol staining signals of columella in the hsn1 mutant in the NO3 −+NH4 + medium were decreased at 4 DAG and finally disappeared at 10 DAG (Fig. 3b). These observations corroborate that NH4 + can alter QC activity and root cap structure, and then affect the organization and maintenance of the root meristem.
Fig. 3.
QC25::GUS marker expression (a), staining of starch granule in columella cells (b), DR5::GUS marker expression (c), and IAA2::GUS marker expression (d) in primary roots of WT in the NO3 −+NH4 + and NH4 + media and hsn1 mutant plants in the NO3 −+NH4 + medium in a time course up to 10 d
These results have been repeated in three separate experiments
3.3. Disturbance of the root meristem organization and maintenance in the presence of NH4 + not likely related to auxin signaling, but cell death
It has been shown that the accumulation of auxin at the distal tip of root is required for the correct specification of cell division programs. Alteration of this auxin distal maximum or auxin transport produces defects in the cell fate and formation pattern (Sánchez-Calderón et al., 2005). To determine whether the NH4 + toxicity-mediated alteration in QC cell specification is related to changes of auxin, we visualized the auxin maximum in root tip using DR5::GUS report gene (Sabatini et al., 1999) and IAA2::GUS gene which expressed in the vasculature of the primary root and the root tip (Luschnig et al., 1998). The expression patterns of DR5::GUS and IAA2::GUS in WT seedlings grown in the NO3 −+NH4 + medium were similar to the previous reports (Figs. 3c and 3d) (Luschnig et al., 1998; Sabatini et al., 1999). When grown in the NH4 + medium, the activities of DR5::GUS and IAA2::GUS were lessened at 4 DAG and not present at 10 DAG (Figs. 3c and 3d). Similar trends were also presented in NH4 +-hypersensitive mutant hsn1 even thought it was grown in the NO3 −+NH4 + medium. To determine whether the activity or proper organization of the primary root meristem suffering from ammonium toxicities was due to lack of auxin, we tested the effects of exogenous application of auxin on WT grown in the NH4 + medium. As a result, it showed that the exogenous application of 3-indoleacetic acid (IAA) or 1-naphthylacetic acid (NAA) in the NH4 + medium could not alter the expression patterns of the markers (like CYCB1;1::GUS, J3612::GFP, SCRp::GFP, QC25::GUS, DR5::GUS, IAA2::GUS) and the staining pattern of starch granule in columella cells (data not shown). It suggested that the defects in meristem activities by NH4 + toxicity might not be due to the alteration of auxin signaling.
NH4 + was supposed to affect cell viability (Qin et al., 2008). We also employed fluorescein diacetate (FDA; Sigma, St. Louis, USA) to examine the root cell viability. This dye is retained by living cells but leaks from dead cells, rendering them nonfluorescent (Bais et al., 2003). When grown in the NO3 −+NH4 + medium, the FDA fluorescence of WT primary root was relatively constant during 10 d (Fig. 4). While, in the NH4 + medium, the reductions of the FDA fluorescence in the mature zone and elongation zone were observed at 4 DAG, reflecting the loss of cell viability in these areas (Fig. 4). Subsequently, loss of cell viability in root meristem zone and root cap was detected at 6 and 8 DAG, respectively. Finally, the FDA fluorescence was lost in the whole root at 10 DAG (Fig. 4). A similar result was observed in the hsn1 mutant grown in the NO3 −+NH4 + medium, root cell viability in the different root zones was also lost in a temporal manner during the 10-d growth period (Fig. 4). These results suggested that loss of cell viability was also an important aspect of NH4 + toxicity inhibition of root growth.
Fig. 4.
Analyses of cell death in the roots of WT in the NO3 −+NH4 + and NH4 + media and hsn1 mutant plants in the NO3 −+NH4 + medium in a time course up to 10 d
Cell death was analyzed by FDA staining. This dye is retained by living cells but leaks from dead cells, rendering them non-fluorescent. These results have been repeated in three separate experiments
4. Discussion
Although ammonium is one of the most important nutrients for plants, ammonium toxicity has been linked to the damage to agricultural crops, especially in areas with intensive agriculture. To understand the molecular and cellular mechanisms of plant response to NH4 + toxicity is required to improve crops with higher tolerance to NH4 + and consequently higher nitrogen use efficiency.
One character of ammonium syndrome is inhibition of root growth when plants are exposed to higher NH4 + concentration. High levels of NH4 + have toxic effects on plant cells (Marschner, 1995). Here, we demonstrate that the reduction in root length of plants induced by NH4 + toxicity is because of both reduction in the size of meristem zone and mature cell length. Furthermore, NH4 + can disturb the meristem patterning and maintenance in the root. Our data indicate that NH4 + affects seedlings from early in their development after its entry into root cells and the effect of NH4 + on root cells is through the progress from mature zone to root cap, which is supported by the observation of inhibition of cell length before 3 DAG, the repression of root meristem activity at 3 DAG, and the damage to the root cap after 4 DAG (Figs. 1a, 1b and 3b). Loss of cell viability in the different root zones determined by FDA staining further supports the temporal mode of response of root cells to NH4 + toxicity from mature zone to root cap (Fig. 4).
Most higher plants develop severe toxicity symptoms when grown on ammonium as the sole nitrogen source, and it can be alleviated by co-provision of nitrate (Britto and Kronzucker, 2002). WT seedlings had a toxic symptom when grown in the NH4 + medium, and had a normal phenotype when grown in the NO3 −+NH4 + medium (Figs. 1–4). To further determine that the effects in WT grown in the NH4 + medium were actually due to the presence of NH4 +, rather than a lack of NO3 −, we examined root growth/marker line expressions of WT and hsn1 when grown in the NO3 − medium (using NO3 − as the sole nitrogen source). When WT and hsn1 seedlings were grown in the NO3 − medium, their root growth/marker line expressions were similar as WT seedlings grown in the NO3 −+NH4 + medium (Figs. S1–S4). The repression of WT seedlings in the NH4 + medium was due to ammonium toxicity, and the inhibition of cell elongation in the hsn1 mutant in the NO3 −+NH4 + medium was actually due to the NH4 +, and not a general phenotype of the mutant.
Plant hormones, such as auxin, control most of the characteristics of root systems, including primary root growth. It is suggested that root responses to nutrients may originate from hormonal signals that are triggered by specific nutrient pathways. In this study, we found that the reduction of primary root length in NH4 + toxicity was also caused by a reduction in auxin levels in the meristem, as visualized by the expressions of DR5::GUS and IAA2::GUS (Figs. 3c and 3d). However, the exogenous application of IAA or NAA in the NH4 + medium could not alter the expression patterns of the markers (like CYCB1;1::GUS, J3612::GFP, SCRp::GFP, QC25:: GUS, DR5::GUS, IAA2::GUS) and the staining patterns of starch granules in columella cells as described above. To determine whether the defects in meristem activities in NH4 + toxicity were due to the alteration of auxin signaling or not, further experiments should be performed.
Recent information showed that nutrient-specific signal transduction pathways exist in the root development. Some genes that involved in the nitrate- and phosphate-signaling pathways have been identified (Zhang and Forde, 1998; Rubio et al., 2001; Miura et al., 2005). GMPase, the only component discovered recently, is involved in hypersensitive response to ammonium (Qin et al., 2008). The insights gained in this work may offer clues to aid in the understanding of the mechanism of NH4 + toxicity among different plant families and species. Unraveling the mechanisms of nutrient-induced regulation of root architecture will enable us to increase the crop yield by manipulating the root architecture in field with unevenly distributed nutrients.
List of electronic supplementary materials
Fig. S1 CYCB1;1::GUS marker expression in primary roots of WT and hsn1 mutant plants in NO3 − medium in a time course up to 10 d. These results have been repeated in three separate experiments
Fig. S2 GFP expression in primary roots of WT and hsn1 mutant plants in NO3 − medium in a time course up to 10 d carrying the enhancer traps J3612::GFP (a) and SCRp::GFP (b). These results have been repeated in three separate experiments
Fig. S3 QC25::GUS marker expression (a), staining of starch granule in columella cells (b), DR5::GUS marker expression (c), and IAA2::GUS marker expression (d) in primary roots of WT and hsn1 mutant plants in NO3 − medium in a time course up to 10 d. These results have been repeated in three separate experiments
Fig. S4 Analysis of cell death in the roots of WT and hsn1 mutant plants in NO3 − medium in a time course up to 10 d. These results have been repeated in three separate experiments
Footnotes
Project supported by the National Basic Research Program (973) of China (No. 2005CB120901), the China Postdoctoral Science Foundation (No. 20090451463), and the China Postdoctoral Special Foundation (No. 201003729)
Electronic supplementary materials: The online version of this article (doi:10.1631/jzus.B1000335) contains supplementary materials, which are available to authorized users
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Associated Data
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Supplementary Materials
Fig. S1 CYCB1;1::GUS marker expression in primary roots of WT and hsn1 mutant plants in NO3 − medium in a time course up to 10 d. These results have been repeated in three separate experiments
Fig. S2 GFP expression in primary roots of WT and hsn1 mutant plants in NO3 − medium in a time course up to 10 d carrying the enhancer traps J3612::GFP (a) and SCRp::GFP (b). These results have been repeated in three separate experiments
Fig. S3 QC25::GUS marker expression (a), staining of starch granule in columella cells (b), DR5::GUS marker expression (c), and IAA2::GUS marker expression (d) in primary roots of WT and hsn1 mutant plants in NO3 − medium in a time course up to 10 d. These results have been repeated in three separate experiments
Fig. S4 Analysis of cell death in the roots of WT and hsn1 mutant plants in NO3 − medium in a time course up to 10 d. These results have been repeated in three separate experiments