Arabidopsis Plastid AMOS1/EGY1 Integrates Abscisic Acid Signaling to Regulate Global Gene Expression Response to Ammonium Stress^,,

Abstract

Ammonium (NH₄⁺) is a ubiquitous intermediate of nitrogen metabolism but is notorious for its toxic effects on most organisms. Extensive studies of the underlying mechanisms of NH₄⁺ toxicity have been reported in plants, but it is poorly understood how plants acclimate to high levels of NH₄⁺. Here, we identified an Arabidopsis (Arabidopsis thaliana) mutant, ammonium overly sensitive1 (amos1), that displays severe chlorosis under NH₄⁺ stress. Map-based cloning shows amos1 to carry a mutation in EGY1 (for ethylene-dependent, gravitropism-deficient, and yellow-green-like protein1), which encodes a plastid metalloprotease. Transcriptomic analysis reveals that among the genes activated in response to NH₄⁺, 90% are regulated dependent on AMOS1/EGY1. Furthermore, 63% of AMOS1/EGY1-dependent NH₄⁺-activated genes contain an ACGTG motif in their promoter region, a core motif of abscisic acid (ABA)-responsive elements. Consistent with this, our physiological, pharmacological, transcriptomic, and genetic data show that ABA signaling is a critical, but not the sole, downstream component of the AMOS1/EGY1-dependent pathway that regulates the expression of NH₄⁺-responsive genes and maintains chloroplast functionality under NH₄⁺ stress. Importantly, abi4 mutants defective in ABA-dependent and retrograde signaling, but not ABA-deficient mutants, mimic leaf NH₄⁺ hypersensitivity of amos1. In summary, our findings suggest that an NH₄⁺-responsive plastid retrograde pathway, which depends on AMOS1/EGY1 function and integrates with ABA signaling, is required for the regulation of expression of NH₄⁺-responsive genes that maintain chloroplast integrity in the presence of high NH₄⁺ levels.

Ammonium (NH₄⁺) is a key intermediate of nitrogen metabolism in most living organisms (von Wirén and Merrick, 2004). For bacteria, fungi, and plants, NH₄⁺ is also the preferred nitrogen source. However, sensitivity to NH₄⁺ occurs widely in animals, plants, and microorganisms (Britto and Kronzucker, 2002; von Wirén and Merrick, 2004). Extensive studies have been carried out that dissect the underlying mechanisms of NH₄⁺ toxicity in plants, animals, and microorganisms (Britto and Kronzucker, 2002; Hess et al., 2006; Norenberg et al., 2009). The paradox of NH₄⁺ is particularly serious in plants, where stunted growth of roots and chlorosis of leaves are two major symptoms of toxicity (Britto and Kronzucker, 2002). Several physiological responses, including increased proton efflux, deficiency of other cations, carbon shortage, disruptions in hormonal homeostasis, damaged chloroplast ultrastructure, uncoupling of photophosphorylation, disruptions in photosynthesis, and energy depletion linked to futile transmembrane NH₄⁺ cycling, have all been implicated in NH₄⁺ stress in plants (Britto and Kronzucker 2002). Recently, the sites of action of NH₄⁺ and underlying molecular mechanisms in plants have been addressed in more detail. For example, NH₄⁺ inhibition of root growth was shown to target primarily cell elongation and require direct contact of the root tip with NH₄⁺. Furthermore, root growth inhibition was directly linked to excessive NH₄⁺ efflux in the root elongation zone (Li et al., 2010). The sensitivity of root elongation to NH₄⁺ has been shown to be related to impaired functioning of the GDP-Man pyrophosphorylase (GMPase), which controls the synthesis of GDP-Man and is involved in the N-glycosylation of proteins, and, as such, is believed to influence general cellular stability and the regulation of NH₄⁺ transport (Qin et al., 2008; Barth et al., 2010; Li et al., 2010). However, recently, defects in GMPase alone were found to be insufficient to explain root NH₄⁺ sensitivity (Kempinski et al., 2011). For example, NH₄⁺-induced reduction of lateral root formation results from contact of the shoot with NH₄⁺ and decreased transport of shoot-derived auxin to roots, via the auxin influx carrier AUX1 (Li et al., 2011a, 2011b). Interestingly, defects in dolichol phosphate Man synthase1 result in both leaf chlorosis and the inhibition of root elongation in the presence of excess NH₄⁺, not only linking to impaired N-glycosylation but also to a reduction in glycosyl phosphatidylinositol anchor attachment (Jadid et al., 2011). These studies, in combination, provide a significantly improved understanding of the process of NH₄⁺ toxicity in plants.

Previous studies on the NH₄⁺ stress response have focused largely on either a selected single gene or only a few genes. To gain insights into the global effects of NH₄⁺ stress on plant gene expression, and to identify possible key regulators, we combined in this study the screening of mutants hypersensitive to NH₄⁺ using forward genetics approaches with transcriptomics using the full genome microarray, along with bioinformatic analyses and physiological experiments. We identified the ammonium overly sensitive1 (amos1) mutant in Arabidopsis (Arabidopsis thaliana), which displayed severe chlorosis under NH₄⁺ stress and was affected in its global expression of NH₄⁺-responsive genes. Map-based cloning revealed that amos1 was an allelic mutation of the gene encoding ETHYLENE-DEPENDENT GRAVITROPISM-DEFICIENT AND YELLOW-GREEN-LIKE PROTEIN1 (EGY1), which is known as a nucleus-encoded, plastid-localized, membrane-associated, and ATP-independent metalloprotease site-2 protease (S2P; Chen et al., 2005). It is required for normal chloroplast development, including the formation of thylakoid grana, the lamella system, and the accumulation of chlorophyll and chlorophyll a/b-binding proteins in chloroplast membranes. Additionally, EGY1 is required for ethylene-dependent gravitropism of light-grown hypocotyls, a process linked to the regulation of endodermal plastid size and number (Chen et al., 2005; Guo et al., 2008). Our work furthermore explores the role of abscisic acid (ABA) signaling as a downstream component of an AMOS1/EGY1-dependent plastid retrograde signaling pathway, in the context of regulation of the gene expression and protection of chloroplast functionalities from NH₄⁺ stress. Additionally, we examine the involvement of the reactive oxygen species (ROS) response in chloroplasts of guard cells as an upstream component of ABA signaling in the AMOS1/EGY1-dependent pathway during the NH₄⁺ stress response.

Our findings not only document, to our knowledge for the first time, the global profile of NH₄⁺-responsive genes under NH₄⁺ stress but identify the critical role during NH₄⁺ stress for the plastid metalloprotease EGY1-dependent retrograde signaling pathway in the control of nuclear gene expression through recruitment of ABA signaling.

RESULTS

Identification of the amos1 Mutant

Based on enhanced leaf chlorosis on medium containing NH₄⁺, we isolated three independent amos mutants from 5,160 lines of a chemical-inducible activation-tagging T-DNA insertion line library in the ecotype Columbia (Col-0) background on growth medium (GM) supplemented with 30 mm (NH₄)₂SO₄ and 10 μm of the inducer 1,7-β-estradiol (Supplemental Fig. S1, A and B). These mutant lines also displayed the chlorosis phenotype on NH₄⁺ medium in the absence of 1,7-β-estradiol (Supplemental Fig. S1C), suggesting that the mutations in the three lines were a result of the position of insertion of the T-DNA rather than of the activation of adjacent genes. Genetic analyses indicated that the three identified mutants, named amos1-1, amos1-2, and amos1-3, were allelic and that the NH₄⁺-induced leaf chlorosis phenotype in amos1 seedlings is caused by single recessive mutations in a nuclear gene (Supplemental Table S1). In the following experiments, all further phenotypic analyses were based on amos1-1.

The (NH₄)₂SO₄-induced chlorosis in amos1 seedlings was reversible upon transfer to medium without (NH₄)₂SO₄, on which green leaves were formed again (except cotyledons; Supplemental Fig. S1D). This supported the notion that the observed phenotype was indeed a consequence of exposure to high levels of (NH₄)₂SO₄. Because the screening medium contained not only NH₄⁺ but also a relatively high concentration of SO₄²⁻, it was necessary to analyze the specific factor that led to the observed phenotype of amos1. Our data show that leaf chlorosis in amos1 was indeed specific to NH₄⁺, rather than being caused by SO₄²⁻ or ionic stress (Supplemental Fig. S1E).

It was recently suggested that NH₄⁺ accumulation in leaves via direct uptake into shoots from the medium can exceed the accumulation that results from root-to-shoot translocation following uptake into roots (Li et al., 2011a). Therefore, we asked as well whether chlorosis of amos1 was induced by NH₄⁺ contact of the root or the shoot. Our results show that amos1 seedlings were able to grow normally when only the root was in contact with NH₄⁺ following transfer, whereas they exhibited chlorotic leaves when the shoot was in direct contact with NH₄⁺ following transfer (Supplemental Fig. S2). Generally, in addition to foliar application incurred from NH₄⁺-releasing fertilizers, leaves of species such as Arabidopsis easily come into contact with NH₄⁺ when seedlings are germinated and grown directly in soil or sterile medium. Our results show that amos1 indeed displays chlorotic leaves when germinated and grown directly in NH₄⁺ medium (Supplemental Fig. S2). These results, together with previous results (Li et al., 2011a, 2011b), suggest that seedlings in the germination and early developmental stages suffer NH₄⁺ stress more readily because their leaves are able to contact NH₄⁺ in the medium directly.

We further analyzed leaf chlorophyll content in amos1 and wild-type seedlings in response to a range of NH₄⁺ concentrations. In these solid agarose-based nutrient media, chlorophyll concentrations of Col-0 wild-type seedlings increased in the presence of NH₄⁺ up to a concentration of 25 mm and decreased when NH₄⁺ concentrations reached or exceeded 40 mm (Fig. 1A). By contrast, chlorophyll content in amos1 was reduced by approximately 25% and 50%, respectively, when the NH₄⁺ concentration was raised to 20 and 25 mm (Fig. 1A). The most sizable differences in chlorophyll accumulation in amos1 and wild-type seedlings occurred at higher NH₄⁺ concentrations, between 25 and 40 mm (Fig. 1A). We compared chlorophyll accumulation and chloroplast fluorescence between amos1 and wild-type seedlings under identical concentrations of (NH₄)₂SO₄ and K₂SO₄. Chlorophyll accumulation and chloroplast fluorescence were severely affected in amos1 seedlings when exposed to (NH₄)₂SO₄ but not K₂SO₄ (Fig. 1, B and C). Furthermore, we analyzed the phenotypes of amos1 and wild-type seedlings in response to 25 and 40 mm NH₄⁺ over time. In GM without NH₄⁺, the chlorophyll content of amos1 seedlings was between 66.3% and 55.5% of the wild type level (Supplemental Fig. S3, A and B). The difference in chlorophyll content between wild-type and amos1 seedlings remained similar 24 h after NH₄⁺ addition but was clearly exacerbated with prolonged NH₄⁺ treatment. Chlorophyll contents in amos1 were reduced to 24.5% or 12.7% of the wild type, respectively, in the presence of 25 and 40 mm NH₄⁺ treatment for 7 d (Supplemental Fig. S3, A and B). Shoot fresh weight was 10% to 30% lower in amos1 than in the wild type at these time points in GM containing NH₄⁺ (Supplemental Fig. S3, C and D). The rate of shoot biomass production of amos1 seedlings over time was not reduced upon short-term exposure to NH₄⁺, then decreased with longer term NH₄⁺ treatments, although the reduction was similar to the wild type (Supplemental Fig. S3, C and D). Furthermore, inhibition of root growth in response to NH₄⁺, as assessed by primary root length and lateral root number, was not exacerbated in amos1 seedlings when compared with the wild type (Supplemental Fig. S3, E and F). This is distinct from previously described NH₄⁺-hypersensitive mutants of Arabidopsis, in which root morphological changes were the most visible phenotype (Qin et al., 2008; Jadid et al., 2011; Li et al., 2012). Considering these results jointly, amos1 emerges as a novel NH₄⁺-hypersensitivity mutant, displaying severe leaf chlorosis without any impairment of root growth or root development.

Figure 1.

The amos1 mutant is hypersensitive to excess NH₄⁺. A, Relative chlorophyll concentrations in rosettes of 7-d-old wild-type (Col-0) and amos1 seedlings exposed to serial concentrations of NH₄⁺ [provided as (NH₄)₂SO₄] for 7 d, normalized to values for control medium (GM without NH₄⁺) for each genotype. B, Leaf chlorosis (representative images) in 7-d-old wild-type and amos1 seedlings, treated with 25 mm NH₄⁺ or mock (12.5 mm K₂SO₄) for 7 d. Bars = 0.3 cm. C, Representative images of chlorophyll fluorescence of true leaves (top row) and chloroplasts (bottom row) in seedlings as described in B. Bars = 100 μm in the top row and 10 μm in the bottom row. D, NH₄⁺ accumulation in shoots of amos1 and wild-type seedlings. Treatment was as described in B. Values are means ± se (n ≥ 4). No significant differences were detected between the wild type and the mutant (Student’s t test, P > 0.05). FW, Fresh weight.

In addition, we determined the accumulation of NH₄⁺ in shoots, which is considered an important factor contributing to NH₄⁺ sensitivity in plants (Britto and Kronzucker, 2002; Balkos et al., 2010; Li et al., 2011a). Compared with mock treatment, NH₄⁺ contents in shoots of amos1 and wild-type seedlings were increased more than 150-fold upon exposure to 25 mm NH₄⁺, but they were not significantly different between genotypes (Fig. 1D). Thus, the NH₄⁺ sensitivity of amos1 seedlings was not related to excess NH₄⁺ accumulation when compared with the wild type. Given that the gene VITAMIN C-1 (VTC1), encoding the enzyme GMPase, has been implicated as an important genetic locus for NH₄⁺ sensitivity in roots of Arabidopsis (Qin et al., 2008; Barth et al., 2010), we asked whether the chlorosis phenotype under NH₄⁺ exposure is related to this gene. However, amos1/vtc1 double mutants displayed a similar leaf chlorosis response to amos1 seedlings while displaying a root growth response similar to vtc1 seedlings (Supplemental Fig. S4), suggesting that the NH₄⁺-induced leaf chlorosis of amos1 seedlings was not related to the GMPase gene.

Identification of the AMOS1 Locus

Since T-DNA insertion loci isolated by thermal asymmetric interlaced PCR were not associated with the NH₄⁺-hypersensitivity phenotype, a map-based cloning strategy was pursued to isolate the mutated gene. The amos1 allele was located on chromosome 5 (Supplemental Fig. S5A) and was found to correspond to a segmental deletion of 1,071 bp in the gene At5g35220 (Fig. 2A; Supplemental Fig. S5B), which encodes the previously described S2P metalloprotease EGY1. No expression of EGY1 mRNA was detected in amos1 seedlings (Supplemental Fig. S5C). Thus, amos1 was a true null mutant. With exposure to 25 mm NH₄⁺, chlorophyll accumulation in egy1-1 and egy1-2 seedlings was indeed similar to amos1 seedlings (Fig. 2B). Furthermore, complementation of the amos1 mutant with wild-type EGY1 restored wild-type levels of chlorophyll accumulation in the presence of NH₄⁺ (Fig. 2B). Collectively, these results show that the amos1 mutant carries a loss-of-function mutation in the S2P metalloprotease gene EGY1. EGY1 has been identified as the first S2P metalloprotease homolog in plants (Chen et al., 2005; Chen and Zhang, 2010) and has been localized to chloroplasts (likely the thylakoid membrane system), based on the analysis of EGY1-GFP fusion constructs, and the defect arising from expressing the N-terminal transit peptide removed EGY1 in egy1 seedlings (Chen et al., 2005; Zhang et al., 2008).

Figure 2.

AMOS1 identification. A, Diagram illustrating the genomic coding sequence of the Arabidopsis AMOS1/EGY1 gene and the locations of the mutant alleles amos1-1, egy1-1, egy1-2, and egy1-3. UTR, Untranslated region. B, Phenotypes of 7-d-old seedlings of wild-type Col-0, amos1-1, egy1-1, egy1-2, and amos1-1 complemented with AMOS1/EGY1 (T3 generation), treated with 25 mm NH₄⁺ for 7 d. One representative image is shown for each line. The experiments were reproduced at least twice.

Regulation of the Expression of NH₄⁺ Stress-Responsive Genes by AMOS1/EGY1

To elucidate the molecular function of AMOS1/EGY1 in the NH₄⁺ stress response, we used full genomic microarray hybridization to analyze the difference of the Arabidopsis transcriptome between amos1 and the wild type under both NH₄⁺ and mock conditions. First, we analyzed the effect of the AMOS1/EGY1 mutation on the transcriptional responses under both conditions. Only four genes were expressed at higher levels, but 888 genes were expressed at lower levels in amos1 seedlings under both mock and NH₄⁺ conditions, compared with the wild type (q ≤ 5 and fold change ≥ 2 or ≤ 0.5, respectively; Fig. 3A; Supplemental Data Set S1). Of these, 867 (97.2%) were lower in amos1 than in the wild type exclusively under NH₄⁺ stress (Fig. 3A). Surprisingly, the most strongly enriched Gene Ontology (GO) categories in cellular components of these genes were in the nucleus, with only a small representation of chloroplasts (Fig. 3B). GO categories in biological processes indicated that genes in a broad range of pathways known to be associated with biotic and abiotic stress responses, such as “response to chitin,” “response to heat,” “cell redox homeostasis,” “toxin catabolism,” “autophagy,” and “response to oxidative stress,” were expressed at lower levels in the amos1 mutant than in the wild type under NH₄⁺ stress (Fig. 3C). Consistently, among the 10 genes showing the largest alterations in transcript levels in amos1, eight were repressed (Fig. 3D). Additionally, it is noteworthy that three small HEAT SHOCK PROTEINs (HSPs) were among the repressed genes (Fig. 3D). These results indicate that in the presence of NH₄⁺, the full level of expression of nucleus-encoded genes associated with several biotic and abiotic stress-response pathways depends on plastid AMOS1/EGY1.

Figure 3.

Transcriptional profile in amos1 compared with the wild type (WT) under NH₄⁺ stress. A, Total number of genes for which transcript levels were significantly changed in amos1 relative to the wild type after mock treatment or NH₄⁺ stress conditions imposed for 6 h (q ≤ 5 and fold change ≥ 2 or ≤ 0.5). B, Enriched GO categories in cellular components of 867 genes expressed at significantly lower levels in amos1 than in the wild type under NH₄⁺ stress. C, Enriched GO categories for biological processes of 867 genes expressed at significantly lower levels in amos1 than in the wild type under NH₄⁺ stress (P < 0.001). Numbers in B and C indicate the counts of each GO category that appear in the 867 genes. D, The 10 genes exhibiting the largest difference in transcript levels in amos1 (relative to the wild type) under NH₄⁺ conditions (mock conditions given for reference).

To test whether the genes showing lower expression in NH₄⁺-exposed amos1 plants than in the wild type are part of the transcriptional NH₄⁺ response of Arabidopsis, we further analyzed the genome-wide transcriptional response of the wild type and amos1 to NH₄⁺ stress. Eighty-six activated genes and 42 repressed genes were found in the wild type. In amos1, the number of genes activated as a consequence of NH₄⁺ exposure was reduced to 23, whereas the number of repressed genes was increased to 199 (Fig. 4A; Supplemental Data Set S2). Thus, accurate expression of 90% (78) of the NH₄⁺-activated genes and 62% (26) of the NH₄⁺-repressed genes depended upon AMOS1/EGY1 (Fig. 4A). These results support the conclusion that AMOS1/EGY1 is indeed required for regulation of the expression of NH₄⁺-responsive genes, particularly those genes showing higher transcript levels in NH₄⁺-exposed plants. The largest group of 183 genes, however, showed a decrease in transcript levels in response to NH₄⁺ exposure in amos1 without responding to NH₄⁺ in the wild type.

Figure 4.

Genome-wide responses of transcript levels to NH₄⁺ in shoots of amos1 and the wild type (WT). A, Numbers of genes showing significant changes in transcript levels in response to NH₄⁺ in the wild type and amos1 according to the set thresholds (NH₄⁺ relative to mock, q [%] ≤ 5 and fold change ≥ 2 or ≤ 0.5, respectively). B, Enriched GO categories for biological processes of the NH₄⁺-activated (78 in the wild type and 15 in amos1) and NH₄⁺-repressed (26 in the wild type and 183 in amos1) genes depending on AMOS1/EGY1. C, Percentage of promoters containing at least one occurrence of the ACGTG element in wild-type NH₄⁺-activated genes: 86, NH₄⁺-activated genes in the wild type; 78, AMOS1/EGY1-dependent NH₄⁺-activated genes; 43, AMOS1/EGY1-dependent NH₄⁺-activated genes in which transcript levels in amos1 are 50% or lower of those in the wild type during NH₄⁺ stress.

A comparison of GO categories in biological processes of AMOS1/EGY1-dependent NH₄⁺-responsive genes (Fig. 4B) and the 867 genes repressed in amos1 under NH₄⁺ stress (Fig. 3C) revealed that “response to chitin” was the most enriched biological process in the category of NH₄⁺-repressed genes in the wild type and, more pronouncedly, in amos1. However, “response to heat,” “toxin catabolism,” “response to oxidative stress,” and “Suc transport” emerged as NH₄⁺-activated biological processes in the wild type but not in amos1. The biological process “cell redox homeostasis” was only found among NH₄⁺-repressed genes of amos1, but not the wild type. Other biological processes, such as “autophagy,” were not enriched among NH₄⁺ stress-responsive genes. Additionally, “transcription factor import into nucleus,” “ethylene-mediated signaling pathway,” “cotyledon development,” and “response to singlet oxygen” were enriched among NH₄⁺-activated genes in the wild type and also appeared to depend on AMOS1/EGY1 function (Fig. 4B), but they were not identified as significantly less active in amos1 relative to the wild type (Fig. 3C). Unexpectedly, despite the severe leaf chlorosis, there were few known genes involved in chloroplast development or chlorophyll biosynthesis, except coproporphyrinogen III oxidase (hemf2), which was reduced to 22% at the transcript level in amos1 but remained at 75% in the wild type under the NH₄⁺ condition (relative to mock) among the NH₄⁺-repressed genes in amos1 (Fig. 4B; Supplemental Data Set S2).

The Involvement of ABA Signaling as a Downstream Component of the AMOS1/EGY1-Dependent Response to NH₄⁺

To identify the potential signaling pathway involved in the AMOS1/EGY1-dependent NH₄⁺ stress-responsive gene expression, we first analyzed the regulatory elements enriched in 500-bp regions of the sequence upstream of the promoters of the identified AMOS1/EGY1-dependent NH₄⁺-activated and NH₄⁺-repressed genes in the wild type (Fig. 4A). No significantly overrepresented regulatory element was found among NH₄⁺-repressed genes. However, there were two motifs, ACGTG and CCCTCA, that were significantly overrepresented in the promoter regions of these AMOS1/EGY1-dependent activated genes (Supplemental Table S2). Moreover, only ACGTG remained enriched among the 43 AMOS1/EGY1-dependent NH₄⁺-activated genes that were at least 2-fold down-regulated in amos1 relative to the wild type (Supplemental Table S2). Additionally, we analyzed the number of genes with the occurrence of ACGTG at least once in the promoter regions among the 86 NH₄⁺-activated genes, the 78 AMOS1/EGY1-dependent NH₄⁺-activated genes, and the 43 AMOS1/EGY1-dependent NH₄⁺-activated genes that were at least 2-fold down-regulated in amos1 relative to the wild type. The data show that the percentage of genes with the promoter containing ACGTG was as high as 60% in the NH₄⁺-activated genes and approached 70% in the latter category of 43 genes (Fig. 4C). The ACGTG motif is known as the core motif in both the light-responsive G-box (CACGTG; Terzaghi and Cashmore, 1995) and the ABA response element (Hattori et al., 2002; Himmelbach et al., 2003). This finding suggests that ABA, one of the principal phytohormones, might be involved in AMOS1/EGY1-mediated expression of NH₄⁺-responsive genes and enhance NH₄⁺ tolerance during NH₄⁺ stress. Indeed, application of 1 μm ABA in the medium reversed the NH₄⁺-induced suppression of chlorophyll accumulation and chloroplast fluorescence in amos1 seedlings. However, ABA did not improve chlorophyll content in amos1 under mock conditions (Fig. 5, A and B). The recovery of amos1 seedlings by ABA was also confirmed in the other allelic mutants, amos1-2 and egy1-2 (Fig. 5B). These results indicate that ABA indeed rescues the NH₄⁺ hypersensitivity of amos1 seedlings.

Figure 5.

ABA treatment can readjust the expression of NH₄⁺-responsive genes and alleviate NH₄⁺ stress in amos1 seedlings. A, Recovery of chlorophyll accumulation in leaves (left two; bar = 0.3 cm), chlorophyll fluorescence of true leaves (middle; bar = 100 μm), and chloroplasts (right; bar = 10 μm) in amos1 seedlings grown in NH₄⁺ medium through the application of external ABA for 7 d. One representative sample is shown for each type. B, Quantification of chlorophyll accumulation in 7-d-old wild-type (WT), amos1-1, amos1-2, and egy1-2 seedlings with and without ABA treatment. Seedlings in A and B were treated with mock (12.5 mm K₂SO₄), mock + ABA (1 μm), NH₄⁺ [12.5 mm (NH₄)₂SO₄], or NH₄⁺ + ABA (1 μm) for 7 d (n = 3). Different letters indicate significantly different means between treatments within a given genotype (one-way ANOVA with Duncan’s multiple comparison test, P < 0.05). FW, Fresh weight. C, Effects of external ABA on transcript levels of the 78 genes activated in response to NH₄⁺ in an AMOS1/EGY1-dependent manner. Transcript levels of these selected genes are shown in scatterplots of amos1 versus the wild type in medium containing 20 mm (NH₄)₂SO₄ with or without 1 μm ABA for 6 h. Each data point represents the arithmetic mean of three replicate experiments. D, Effects of external ABA on NH₄⁺ accumulation in shoots of amos1 and the wild type under exposure to NH₄⁺ (n = 4; Student’s t test, P < 0.05). Treatments were as in B. E, Endogenous ABA accumulation in shoots of 7-d-old seedlings of the wild type and amos1 treated with mock and NH₄⁺ (25 mm) for 1 d (n = 4). F, Chlorophyll accumulation in abi4, aba2, and aba3 mutants exposed to NH₄⁺. Seven-day-old seedlings of different Arabidopsis genotypes were treated with mock and NH₄⁺ (25 mm) for 7 d. Asterisks indicate significant differences between mock and NH₄⁺ treatment for the given genotype (n = 4; Student’s t test, P < 0.01). All values represent means ± se.

We further asked whether the rescue of NH₄⁺ hypersensitivity of amos1 seedlings by ABA application is the consequence of a readjustment of the expression AMOS1/EGY1-dependent NH₄⁺-responsive genes or of a reduction in NH₄⁺ accumulation in both amos1 and the wild type. To test these two hypotheses, we analyzed the global expression of AMOS1/EGY1-dependent NH₄⁺-responsive genes and the NH₄⁺ content of wild-type and amos1 seedlings cultivated in NH₄⁺ medium with or without ABA. Among the 78 AMOS1/EGY1-dependent NH₄⁺-activated genes in the wild type, the majority of genes recovered to an expression level in amos1 resembling that of the wild type following 1 μm ABA treatment for 6 h (Fig. 5C). Similarly, application of ABA also increased the expression of most AMOS1/EGY1-dependent NH₄⁺-repressed genes (183 genes; Fig. 4A) in amos1 to the level of the wild type (Supplemental Fig. S6A). However, it was interesting to find that there were three small HSP genes among a total of six AMOS1/EGY1-dependent NH₄⁺-activated genes for which transcript levels were still at least 2-fold lower in amos1 than in the wild type even following application of ABA (Supplemental Fig. S6B). In the wild type, the expression of these small HSP genes was almost identical with or without application of ABA under NH₄⁺ stress (Supplemental Fig. S6B). These results indicate that under NH₄⁺ stress, the small HSP response may occur in parallel with ABA signaling. In contrast to gene expression levels, NH₄⁺ accumulation in shoots of amos1 and wild-type seedlings was not affected by the application of 1 μm ABA (Fig. 5D). Collectively, these results show that during NH₄⁺ stress, enhanced ABA signaling can compensate for the loss of the AMOS1/EGY1-dependent protection in amos1 seedlings.

To investigate how ABA signaling is regulated by the AMOS1/EGY1-dependent pathway under NH₄⁺ stress, we analyzed ABA levels in amos1 and wild-type shoots. An approximately 2-fold increase in ABA content was observed in wild-type shoots under NH₄⁺ exposure relative to mock treatment (Fig. 5E), consistent with previous observations (Peuke et al., 1994; Omarov et al., 1998). Unexpectedly, ABA content was about 5-fold higher in amos1 shoots than in the wild type under mock treatment conditions. However, ABA content was dramatically decreased in amos1 shoots upon NH₄⁺ treatment (Fig. 5E), to levels that were 42.6% of those detected in the wild type under the same condition. These results suggest that the NH₄⁺-induced ABA accumulation depends on AMOS1/EGY1, but NH₄⁺ can also decrease ABA levels in amos1 via another pathway. It is alternatively possible that exposure to inhibitory NH₄⁺ exacerbates the chloroplast defect of amos1 to an extent that ABA biosynthesis is compromised, which then leads to NH₄⁺-induced chlorosis in this genotype. We proceeded to test whether a defect of ABA signaling alone can bring about hypersensitivity of chloroplasts to NH₄⁺ in the wild type. We analyzed chlorophyll accumulation in the ABA-insensitive abi4 mutant and the ABA-deficient aba2 and aba3 mutants, in the Col-0 background, under NH₄⁺ stress. Chlorophyll accumulation in abi4 was decreased under NH₄⁺ stress, as was found in amos1. By contrast, NH₄⁺ stress did not result in a reduction of chlorophyll contents in the two ABA-deficient mutants (Fig. 5F). It has previously been reported that the ABA-insensitive mutant abi4 is defective in retrograde signaling from plastid to nucleus, while the other ABA-insensitive or -deficient mutants are not impaired (Koussevitzky et al., 2007). These results indicated that a defect in ABA signaling that is possibly connected to plastid retrograde signaling, and not a defect in ABA biosynthesis, is responsible for the hypersensitivity of amos1 to NH₄⁺.

Recently, mild production of ROS in chloroplasts has been proposed as an important plastid retrograde signal triggering processes that protect the plant from moderate environmental stress (Galvez-Valdivieso and Mullineaux, 2010). Therefore, we tested whether the ROS response in chloroplasts is affected in amos1 during NH₄⁺ stress. To quantify the ROS response in amos1 seedlings under NH₄⁺ stress, we analyzed the accumulation of hydrogen peroxide (H₂O₂) using 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) fluorescence. The signal of H₂DCFDA fluorescence in leaves was indeed enhanced in wild-type and amos1 seedlings when exposed to high levels of NH₄⁺ for 6 h. It was interesting to find that the accumulation of H₂O₂ in guard cell chloroplasts of amos1 seedlings was markedly lower than in the wild type upon exposure to high NH₄⁺ (Fig. 6). Stomatal density was almost identical between the wild type and amos1 (Supplemental Fig. S7), which suggests that the reduced H₂O₂ formation is not a consequence of reduced stomatal density. Furthermore, the H₂O₂ response in amos1 seedlings could not be restored by the application of ABA (Fig. 6). This indicated that the ROS signaling capacity is compromised in chloroplasts of amos1 seedlings under NH₄⁺ stress and might play a role as a component of AMOS1/EGY1-dependent ABA signaling upstream, but not downstream, of ABA.

Figure 6.

H₂O₂ response to NH₄⁺ challenge and ABA treatment in guard cell chloroplasts of amos1 and wild-type (WT) seedlings. Seven-day-old amos1 and wild-type seedlings were treated with mock (12.5 mm K₂SO₄), NH₄⁺ [12.5 mm (NH₄)₂SO₄], or NH₄⁺ + ABA (1 μm) for 6 h, then H₂O₂ fluorescence (green, top row) and chloroplast fluorescence (red, bottom row) were observed. The insets show a single guard cell. A representative image from six individual leaves for each treatment is shown. The experiments were reproduced twice. Bars = 100 μm.

DISCUSSION

In the work presented here, we identified the Arabidopsis amos1 mutant, characterized by hypersensitivity to excess NH₄⁺ in leaves, manifest as severe chlorosis, whereas chlorophyll content remained normal in wild-type seedlings. We further revealed that the nuclear AMOS1 locus is identical to EGY1, which encodes a membrane-bound and ATP-independent metalloprotease localized to plastids that is required for chloroplast biogenesis (Chen et al., 2005). The allelic amos1 mutation did not disturb tissue NH₄⁺ distribution or cause NH₄⁺ hyperaccumulation in shoots, different from the previously characterized mutant amos2 (Li et al., 2012). The analysis of the amos1/vtc1 double mutant indicates that the NH₄⁺-induced chlorosis in amos1 is also not related to the GMPase gene, which has been implicated in the sensitivity of root development to NH₄⁺ (Qin et al., 2008). Transcriptomic analysis identified 86 NH₄⁺-activated genes, the expression of 90% of which depended on the function of EGY1. Furthermore, the number of NH₄⁺-activated genes was decreased 3.7 times in amos1 compared with the wild type, whereas the number of NH₄⁺-repressed genes was increased almost five times in amos1 relative to the wild type. Therefore, the metalloprotease EGY1 in chloroplasts is an important component required for the correct regulation of NH₄⁺-responsive transcript levels of nuclear genes.

ABA is a key hormone in the orchestration of stress signal transduction and defensive responses in plants (Xiong et al., 2002; Hubbard et al., 2010). ABA signaling may be involved in the NH₄⁺ stress signal indicated by the enhanced ABA content in plants under the NH₄⁺ condition (Peuke et al., 1994; Omarov et al., 1998). However, how exactly ABA signaling regulates NH₄⁺ stress remains largely to be investigated. In this study, we have presented bioinformatic, pharmacological, physiological, genomic, and genetic data to show that ABA signaling acts as an important downstream component of the AMOS1/EGY1-dependent plastid retrograde signaling pathway to regulate the expression of NH₄⁺ stress-responsive genes and to enhance chloroplast functionality under NH₄⁺ stress. Furthermore, our results show that ABA signaling is not the sole downstream component of the AMOS1/EGY1-dependent NH₄⁺ responses, because a very small portion of AMOS1/EGY1-dependent genes, such as small HSP genes, are still defective in amos1 seedlings following the application of ABA. Recently, these small HSP genes have been shown to be required for the targeting of chloroplast outer membrane proteins (Kim et al., 2011). Furthermore, these HSP genes are activated by a chloroplast-ribosomal-protein-S1-dependent retrograde signaling during heat stress responses (Yu et al., 2012). These combined results indicate that, in addition to ABA-mediated signaling, an ABA-independent response (particularly small HSP genes) may act as another downstream component of the plastid AMOS1/EGY1-dependent retrograde pathway to maintain chloroplast functionality under NH₄⁺ stress. Is defective ABA signaling alone sufficient to cause the hypersensitivity of chloroplasts to NH₄⁺ in Arabidopsis? Our data show that ABA-deficient mutants were not hypersensitive to NH₄⁺ stress, whereas abi4 was sensitive to NH₄⁺ stress. Considering that ABI4 is implicated in both ABA and retrograde signaling (Koussevitzky et al., 2007), the sensitivity of the abi4 mutant to NH₄⁺ stress may be due to defects in both ABA and ABI4-mediated retrograde signaling. Therefore, a defect in ABA signaling that is possibly connected to plastid retrograde signaling, and not a defect in ABA biosynthesis, is responsible for the hypersensitivity of amos1 to NH₄⁺. The observations described here differ from those on ABA signaling in other abiotic stresses such as salinity or osmotic stress, in which the sensitivities of both ABA-insensitive and -deficient mutants are altered (Xiong et al., 2002; Hubbard et al., 2010).

The plastid-specific localization of the AMOS1/EGY1 protein, the specifically impaired chloroplast functionality, the defect in the H₂O₂ response of guard cell plastids, and the reduced expression of stress-associated nuclear genes occurring in amos1 seedlings point to the importance of chloroplasts in the resistance to NH₄⁺ stress and to their importance as a source of signals regulating nuclear gene expression during NH₄⁺ stress. These results thus suggest the existence of a retrograde pathway in the regulation of cellular NH₄⁺ stress responses. Plastid retrograde signaling is known to regulate nuclear gene expression, which serves not only to coordinate nucleus-encoded chloroplast protein levels but also to mediate plant stress responses such as those to high-light and drought stress (Fernández and Strand, 2008; Estavillo et al., 2011). ROS production in chloroplasts has frequently been proposed to act in plastid retrograde signaling (Kleine et al., 2009; Galvez-Valdivieso and Mullineaux, 2010). Recently, H₂O₂ in chloroplasts has been shown to trigger retrograde signaling, thus regulating the expression of nucleus-encoded genes involved in both biotic and abiotic stress responses, for example to pathogen attack, chilling, and high light (Maruta et al., 2012). Additionally, H₂O₂ in chloroplasts has been proposed to regulate the expression of nucleus-encoded HSP genes under heat stress (Yu et al., 2012). Therefore, H₂O₂ produced in guard cell chloroplasts may constitute an AMOS1/EGY1-dependent plastid retrograde signal, because the H₂O₂ response is defective in guard cell chloroplasts in amos1 during NH₄⁺ stress. As the H₂O₂ response in guard cell chloroplasts was not restored by ABA, it may be the upstream event of AMOS1/EGY1-dependent ABA signaling during the cellular NH₄⁺ stress response. Interestingly, ABA signaling has been implicated in the H₂O₂-dependent plastid signal to activate high-light-responsive gene expression in leaves (Galvez-Valdivieso et al., 2009) and in the ¹O₂-dependent plastid signal during late embryogenesis to predetermine plastid differentiation by reactivating relevant nucleus-encoded genes (Kim et al., 2009), although ABA is not regarded as a direct or actual candidate of the plastid signal (Koussevitzky et al., 2007). Thus, ABA signaling is proposed to integrate ROS-dependent plastid signaling (Kleine et al., 2009; Galvez-Valdivieso and Mullineaux, 2010). Therefore, this study provides evidence that ABA signaling is integrated into an AMOS1/EGY1-dependent chloroplast signal to regulate the expression of NH₄⁺-responsive genes.

There have been two recent reports on primary retrograde signaling in plastids: first, a Mg-protoporphyrin IX/heme-dependent plastid retrograde signaling mechanism has been proposed to regulate nucleus-encoded chloroplast proteins (Koussevitzky et al., 2007); second, ¹O₂-dependent plastid retrograde signaling has been proposed to play a central role in the activation of a stress-related signaling cascade rather than direct control of chloroplast biogenesis (Lee et al., 2007). Our work indicates that the primary function of the AMOS1/EGY1-mediated NH₄⁺-related retrograde signaling lies in the activation of a stress-response signaling cascade during NH₄⁺ stress that is more similar to ¹O₂-dependent plastid retrograde signaling than to Mg-protoporphyrin IX/heme-dependent plastid retrograde signaling. Therefore, ROS release in chloroplasts is proposed to participate in AMOS1/EGY1-dependent plastid retrograde signaling. The H₂O₂ response defect in guard cell chloroplasts during NH₄⁺ stress also supports this hypothesis. However, we cannot exclude the involvement of other ROS signals or precursors of chlorophyll in plastid signaling in the amos1 seedling response to NH₄⁺ stress. Clearly, the role of the dampened H₂O₂ response in AMOS1/EGY1-dependent chloroplast retrograde signaling and NH₄⁺ hypersensitivity of amos1 warrants future experimentation.

A further interesting question that arises from this study is how the NH₄⁺-sensitive response and decrease in ABA and H₂O₂ accumulation are related to defective EGY1 metalloprotease activity in chloroplasts. The membrane-bound protease EGY1 is probably involved in chloroplast membrane protein metabolism, such as that of chlorophyll a/b-binding proteins, although the detailed mechanisms for this are as yet unknown (Chen et al., 2005). Excess NH₄⁺ is well known for its harmful effects on chloroplast development and photosynthesis (Britto and Kronzucker, 2002; Drath et al., 2008). Therefore, considering EGY1’s role as a metalloprotease S2P homolog in plants (Chen et al., 2005; Chen and Zhang, 2010), the enzyme may be expected to be involved in cleaving chloroplast membrane proteins that suffered structural integrity in the process of NH₄⁺ treatment and, thus, maintaining the turnover and assembly of the membrane-associated components of PSI and PSII. A key precursor for ABA synthesis is derived from carotenoids in chloroplasts (Cutler and Krochko, 1999), and H₂O₂ is generated by photoreduction of oxygen in PSI under many stress conditions (Asada 1999). Our results show that the NH₄⁺-induced ABA and H₂O₂ accumulation depend on AMOS1/EGY1, suggesting that NH₄⁺-induced ABA and H₂O₂ biogenesis regulate normal chloroplast functionality. However, NH₄⁺ can also decrease ABA levels in amos1 shoots. It is alternatively possible that exposure to inhibitory NH₄⁺ exacerbates the chloroplast defect of amos1 to an extent that ABA biosynthesis is compromised. Thus, the defect in chloroplast functionality in the amos1 mutant may impair the regulation of ABA metabolism and H₂O₂ production under NH₄⁺ stress. However, important points to note in studies of this kind are that care must be taken with the interpretation of data based on even ostensibly highly specific mutations, as pleiotropic effects are common, especially in alterations of aspects as profound as nitrogen metabolism and response to nitrogen toxicity (for a recent case where pleiotropies have been documented, see that of the role of GMPase in root NH₄⁺ sensitivity; Qin et al., 2008; Barth et al., 2010; Kempinski et al., 2011). Further investigation of the substrates of AMOS1/EGY1 metalloprotease may reveal the underlying relationship clearly.

Additionally, we should point out that future studies will need to be designed to address the effects of variable nitrogen source, especially the copresence of nitrate, and Suc level, both of which can have fundamental shift effects on thresholds of NH₄⁺ toxicity (Kronzucker et al., 1999; Britto and Kronzucker 2002; Qin et al., 2008; Li et al., 2011b). It is reasonable to speculate that nitrate metabolism and Suc level/signaling might be altered in amos1 mutants as a secondary effect of the impairment of chloroplast function. In this study, we focused on the frequently used combination of nitrate and NH₄⁺ in the Arabidopsis nutrient medium and a constant level of Suc (Hirsch et al., 1998; Rawat et al., 1999; Li et al., 2010, 2011b), conditions for which NH₄⁺ toxicity development is nevertheless clearly seen (Fig. 1). In summary, we have identified a previously unrecognized molecular connection between the function of plastid AMOS1/EGY1 and the activation of NH₄⁺-responsive genes in the nucleus, which is required for NH₄⁺ tolerance in Arabidopsis. Furthermore, we found that ABA signaling acts as an important downstream component of an AMOS1/EGY1-dependent plastid retrograde signaling pathway to regulate the expression of NH₄⁺ stress-responsive genes and to enhance chloroplast functionality under NH₄⁺ stress. The work reported here identifies a new component of the physiological adaptation of plants to high NH₄⁺ levels in the soil.

MATERIALS AND METHODS

Arabidopsis Lines and Growth Conditions

Arabidopsis (Arabidopsis thaliana) mutants amos1-1, amos1-2, and amos1-3 were isolated in this study (Supplemental Materials and Methods S1); egy1-1 and egy1-2 were reported previously (Chen et al., 2005) and provided by Dr. Ning Li; vtc1-1, abi4-1, aba3-1, and aba2-3 were obtained from the Arabidopsis Biological Resource Center at Ohio State University. All mutants are in Col-0 background, except egy1-1 (ecotype Wassilewskija background). The amos1/vtc1 double mutant lines were generated by crossing amos1-1 and vtc1-1 mutants. Seeds were surface sterilized for 5 min in 10% (v/v) chlorine bleach and 0.1% (w/v) SDS, followed by five rinses with sterile distilled water. Seeds were stored in an Eppendorf tube in the dark at 4°C for 2 to 3 d before sowing on GM on 13- × 13-cm square plates. The GM was composed of 2 mm KH₂PO₄, 5 mm NaNO₃, 2 mm MgSO₄, 1 mm CaCl₂, 0.1 mm Fe-EDTA, 50 μm H₃BO₃, 12 μm MnSO₄, 1 μm ZnC1₂, 1 μm CuSO₄, 0.2 μm Na₂MoO₄, 1% (w/v) Suc, 0.5 g L⁻¹ MES, and 0.8% (w/v) agar (pH 5.7 adjusted with 1 m NaOH). Seedlings were kept in a growth chamber at 23°C ± 1°C under a light intensity of 100 μmol photons m⁻² s⁻¹ and a photoperiod of 16 h of light and 8 h of dark. Unless otherwise stated, NH₄⁺ and mock treatments were in GM supplemented with 12.5 mm (NH₄)₂SO₄ and 12.5 mm K₂SO₄, respectively. Other chemical compound treatments were also provided as additions to GM, as indicated.

amos1 Mapping and Complementation

The amos1 mutant was crossed with the Landsberg erecta ecotype to generate the mapping population. Mapping was performed as described previously (Peters et al., 2003; Li et al., 2012). In brief, DNA was extracted from 300 mutant F2 individual seedlings, 30 individuals as a mixed DNA pool for preliminary mapping and others for further mapping, and genotyped with known insertion/deletion (InDel) molecular markers (Salathia et al., 2007; Supplemental Table S3). The candidate gene was mapped between the InDel marker C5P71 and the centromere. Known mutants in the identified region and the egy1 mutants were tested in NH₄⁺ medium (Fig. 2B). Then, amplification of the genomic sequences for EGY1 and an analysis by reverse transcription-PCR of the levels of EGY1 mRNA were carried out in the amos1 mutant (Supplemental Materials and Methods S1; Supplemental Fig. S5). Finally, the pCAMBIA 35S::EGY1-GFP construct, identified previously (Chen et al., 2005) and provided by Dr. Ning Li, was introduced into the amos1 seedlings to complement the amos1 mutant function (Fig. 2B).

Chlorophyll Quantification

Chlorophyll content was assayed according to Wintermans and de Mots (1965). Shoots were extracted with 96% (v/v) ethanol at 23°C for 16 h. Absorbance was measured at 665 and 649 nm, and chlorophyll concentration was calculated as milligrams per gram of fresh weight.

Determination of H₂O₂

H₂DCFDA fluorescence was used to detect the H₂O₂ response. Seven-old-day amos1 and Col-0 seedlings were treated with 12.5 mm (NH₄)₂SO₄ or 12.5 mm K₂SO₄ for 6 h. The leaves were transferred into 25 μm H₂DCFDA in 50 mm potassium phosphate buffer (pH 7.4) solution for 30 min in the dark at 23°C and washed with the potassium phosphate buffer for 15 min (Behnke et al., 2010). The leaves were observed with a Zeiss LSM710 confocal microscope as described below.

Fluorescence and Image Analysis

Images of whole leaves were taken using a Canon G7 camera. Chlorophyll fluorescence and H₂DCFDA fluorescence were observed by a Zeiss LSM710 confocal microscope with the following settings: 561- and 488-nm excitation wavelengths; 600- to 700-nm and 505- to 520-nm emission wavelengths, respectively. The images were captured and analyzed using Zeiss 2009 software, using representatives of at least 10 individual plants from each treatment. Experiments were repeated at least twice. Graphs were prepared using SigmaPlot 10.0 software.

Determination of NH₄⁺

Shoots (30–50 mg fresh weight) of each sample were washed with 10 mm CaSO₄, frozen in liquid nitrogen, and then extracted with 1 mL of 10 mm formic acid for the NH₄⁺ content assay by HPLC, following derivatization with o-phthaldialdehyde as described previously (Husted et al., 2000; Balkos et al., 2010).

Endogenous ABA Assay

ABA was quantified using a HPLC-electrospray ionization-tandem mass spectrometry method (Chen et al., 2011) in three independent biological replicates and two technical repeats. In brief, shoots (120–150 mg fresh biomass) of each sample were collected, frozen in liquid nitrogen, and then finely ground, followed by extraction with 1.0 mL of methanol containing 20% water (v/v) at 4°C for 12 h. [²H₆]ABA (50 ng g⁻¹) was added to plant samples as the internal standard prior to grinding. After centrifugation (10,000 rpm, 4°C, 20 min), the supernatant was collected and passed through a C-18 (100 mg) SPE cartridge, which was preconditioned with 8 mL of water, 8 mL of methanol, and 8 mL of methanol containing water (20%, v/v). Eluates were pooled and evaporated under a stream of nitrogen gas and reconstituted in 1 mL of water. The solution was acidified with 120 μL of 0.1 mol L⁻¹ HCl and extracted with ethyl ether (4 × 1 mL). The ether phases were combined, dried under nitrogen gas, and reconstituted in 80 μL of acetonitrile. To the resulting solution, 10 μL of trimethylamine (20 μmol mL⁻¹) and 10 μL of 3-bromoactonyltrimethylammonium bromide (20 μmol mL⁻¹) were added. The reaction solution was vortex mixed for 30 min and evaporated under nitrogen gas, reconstituted with 200 μL of acetonitrile containing 20% (v/v) water, and 10 μL of the solution was subjected to HPLC-electrospray ionization-tandem mass spectrometry analysis.

Microarray Analysis and Data Mining

Seven-day-old amos1 and Col-0 seedlings were treated with 20 mm K₂SO₄ (mock), 20 mm (NH₄)₂SO₄, and 20 mm (NH₄)₂SO₄ with 1 μm ABA for 6 h. Total RNA was extracted from shoots using Trizol reagent (Invitrogen) and was further purified using NucleoSpin RNA clean-up (Macherey-Nagel) according to the manufacturer’s instructions. The RNA was used to prepare Cy₃-dCTP-labled cDNA. Three independent biological replicate experiments were carried out, composed of approximately 80 pooled seedlings for each treatment. The NimbleGen Arabidopsis thaliana Gene Expression 12×135K array was used in these microarray experiments, which were carried out according to the NimbleGen Expression user’s guide and performed at CapitalBio in Beijing, China. Unless stated otherwise, the differentially expressed genes were determined by the microarray software (SAM, version 3.02), with a selection threshold of false discovery rate (q [%] ≤ 5; Storey and Tibshirani, 2003) and fold change ≥ 2.0 in the SAM output result. The differentially expressed genes were evaluated for enrichment of biological functions and cellular components in GO categories by use of the CapitalBio Molecule Annotation System (http://bioinfo.capitalbio.com/mas3/). The results were converted into corresponding graphs with SigmaPlot 10.0 software, based on statistical significance (P value) and count (the total number of times the GO category occurred in these genes). Promoter analysis (http://element.cgrb.oregonstate.edu/) was performed as described previously (Nemhauser et al., 2004).

Statistical and Graphical Analyses

For all experiments, statistical analyses of data and graph production were carried out using SPSS 13.0 and SigmaPlot 10.0 software. Details are presented in the figure legends. All the images and graphs were arranged with Adobe Photoshop 7.0 software.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AMOS1/EGY1 (At5G35220) and CBP20 (At5G44200).

Supplemental Data

The following materials are available in the online version of this article.

• Supplemental Figure S1. Isolation and characterization of the amos1 mutant.

• Supplemental Figure S2. NH₄⁺-induced chlorosis in leaves of amos1 seedlings dependent on the direct contact between NH₄⁺-containing medium and shoot tissues.

• Supplemental Figure S3. Concentration dependence of chlorophyll concentrations and morphological parameters in amos1 and the wild type in response to NH₄⁺ stress.

• Supplemental Figure S4. Phenotypes of amos1/vtc1 double mutants exposed to NH₄⁺.

• Supplemental Figure S5. Map-based cloning of AMOS1 and confirmation.

• Supplemental Figure S6. Effect of external ABA on the levels of transcripts responsive to NH₄⁺ in amos1.

• Supplemental Figure S7. Stomatal density of wild-type and amos1 seedlings.

• Supplemental Table S1. Genetic analysis of the amos1 mutants.

• Supplemental Table S2. The regulatory elements enriched in 500-bp promoter regions upstream of AMOS1/EGY1-dependent NH₄⁺-activated genes.

• Supplemental Table S3. InDel markers selected from the database (Salathia et al., 2007) used for map-based cloning as described in Supplemental Figure S5.

• Supplemental Data Set S1. Comparison of genes differentially expressed in amos1 relative to Col-0 under NH₄⁺ and mock treatment.

• Supplemental Data Set S2. Comparison of genes differentially expressed in NH₄⁺ relative to mock treatment in amos1 and Col-0.

• Supplemental Materials and Methods S1.

Glossary

NH₄⁺

ammonium

ABA

abscisic acid

S2P

site-2 protease

ROS

reactive oxygen species

Col-0

ecotype Columbia

GM

growth medium

GO

Gene Ontology

H₂O₂

hydrogen peroxide

H₂DCFDA

2′,7′-dichlorodihydrofluorescein diacetate

InDel

insertion/deletion