Arbuscular mycorrhizal (AM) fungi contribute to plant nitrogen (N) acquisition. Recent studies demonstrated the transport of N in the form of ammonium during AM symbiosis. Here, we hypothesize that induction of specific ammonium transporter (AMT) genes in Sorghum bicolor during AM colonization might play a key role in the functionality of the symbiosis.
For the first time, combining a split-root experiment and microdissection technology, we were able to assess the precise expression pattern of two AM-inducible AMTs, SbAMT3;1 and SbAMT4. Immunolocalization was used to localize the protein of SbAMT3;1.
The expression of SbAMT3;1 and SbAMT4 was greatly induced locally in root cells containing arbuscules and in adjacent cells. However, a split-root experiment revealed that this induction was not systemic. By contrast, a strictly AM-induced phosphate transporter (SbPt11) was expressed systemically in the split-root experiment. However, a gradient of expression was apparent. Immunolocalization analyses demonstrated that SbAMT3;1 was present only in cells containing developing arbuscules.
Our results show that the SbAMT3;1 and SbAMT4 genes are expressed in root cortical cells, which makes them ready to accommodate arbuscules, a process of considerable importance in view of the short life span of arbuscules. Additionally, SbAMT3;1 might play an important role in N transfer during AM symbiosis.
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Arbuscular mycorrhizal (AM) fungi play a key role in the nutrition of many herbaceous land plants, including crops such as Sorghum bicolor. They take up nutrients from the soil via their extraradical mycelium and translocate them to the plant partner, receiving carbohydrates in return (Smith & Read, 2008). Nutrients, such as phosphorus (P) and nitrogen (N), are then transferred from the fungus to the host plant at the level of a specialized structure, the arbuscule, and the plant takes up these nutrients through a special membrane derived from the plant plasma membrane, the so-called ‘periarbuscular membrane’ (Harrison et al., 2002; Kobae & Hata, 2010).
It is well known that the AM symbiosis modifies the expression of plant transporter genes. For example, some plant P transporters have been shown to be induced or expressed only in arbuscule-containing cells of Lotus japonicus, Medicago truncatula or Oryza sativa (Karandashov et al., 2004; Glassop et al., 2007; Javot et al., 2007; Guether et al., 2009a). In M. truncatula, the P transporter MtPht1;4, which is only expressed during AM symbiosis, has been shown to be localized in the periarbuscular membrane (Harrison et al., 2002).
The role of AM fungi in plant N nutrition has been studied. In the soil, N is present in organic and inorganic forms, but the former is the dominant form. Plants and the extraradical mycelium (ERM) of AM fungi can absorb inorganic N as nitrate or ammonium (Govindarajulu et al., 2005) and some soluble forms of organic N, such as amino acids (Smith & Read, 2008; Leigh et al., 2009). Nitrate and ammonium are relatively mobile in the soil and can be transported to plant roots by mass flow, limiting the depletion zone around the roots. For this reason, the question of the relevance of AM fungi in plant N nutrition was raised (Johnson, 2010; Smith & Smith, 2011). Evidence for inorganic N uptake by AM fungi in the form of ammonium was obtained through the characterization of two ammonium transporters (AMTs) in the AM fungus Glomus intraradices (Lopez-Pedrosa et al., 2006; Pérez-Tienda et al., 2011). Furthermore, Mäder et al. (2000) have shown that the amount of N present in the plant and coming from the AM pathway was up to 42%. In monoxenic cultures of mycorrhizal carrot roots, this amount was c. 30% (Govindarajulu et al., 2005). By contrast, the role of AM fungi in the mineralization of organic forms of N is unclear. Hodge et al. (2001) reported that AM fungi enhance organic N decomposition as well as plant N capture, but other studies have not confirmed these findings (Frey & Schüepp, 1993; Hawkins et al., 2000). Once internalized, N is assimilated and translocated to the intraradical hyphae in the form of amino acids, mainly arginine (Govindarajulu et al., 2005), and is finally transferred to the plant as ammonium (Tian et al., 2010). In a root cell with an arbuscule, this transfer is expected to proceed by the secretion of ammonium into the periarbuscular space through unknown transporters present in the fungal plasma membrane, followed by its uptake through plant AMTs from the periarbuscular membrane.
Plant AMTs have been studied extensively, and phylogenetic analysis has revealed two distinct subfamilies: the AMT1 subfamily and the AMT2 subfamily (Loque & von Wiren, 2004; Supporting Information, Table S1). Here, we focus on the role of AMTs in the mycorrhizal symbiosis of sorghum (S. bicolor), an important crop plant whose genome has been fully sequenced (Paterson et al., 2009). Sorghum, a herbaceous plant belonging to the Poaceae, is the world's fifth biggest crop (after maize, rice, wheat and barley). It can grow under relative arid conditions and is an important source of food, feed and fibers in many developing countries. Bioinformatic analyses of the S. bicolor genome revealed eight genes coding for AMTs. The transcript abundance of these transporters was measured in roots of S. bicolor in the presence or absence of AM fungi under different regimes of N nutrition. Laser microdissection was used to isolate individual root cells with or without mycorrhizal structures (Balestrini et al., 2007; Gomez et al., 2009) to measure cell-specific gene expression. Additionally, a split-root experiment was performed to determine whether the induction process was systemic. Our goal was to identify AM-inducible AMTs in sorghum and to define their pattern of expression during AM symbiosis. This could lead to new insights into the importance of AM-inducible AMTs for AM symbiosis.
Materials and Methods
Plant growth conditions for tissue analysis
Experiments were performed with sorghum (Sorghum bicolor (L.) Moench), cv Pant-5. This cultivar is closely related to BTx623, the sorghum cultivar used for genome sequencing (Paterson et al., 2009). Seeds of cv Pant-5, kindly provided by sorghum breeders of I.G.F.R.I. (CCS Agriculture University of Hissar, Haryana, India) and G. B. Pant University of Agriculture and Technology (Pantanagar, Uttaranchal, India), were surface-sterilized (10 min in 2.5% KClO) and then rinsed with sterile deionized water several times for 1 d and soaked in sterile deionized water overnight. Seeds were pregerminated on autoclaved Terra Green (Oil Dri US-special, American aluminiumoxide, type III/R; Lobbe Umwelttechnik, Iserlohn, Germany) at 25°C for 24 h and then grown in the dark at room temperature for 72 h. The fungal strains G. intraradices BEG-75 and G. mosseae ISCB13 (Botanical Institute, Basel, Switzerland) were propagated by trap cultures as previously described (Oehl et al., 2004). To establish AM symbiosis, pregerminated seeds were individually inoculated in pots containing a mixture of acid-washed Terragreen, sand and loess soil (5 : 4 : 1 w/w/w). About 100 spores were added to the mixture. For the controls (nonmycorrhizal plants), the same amount of autoclaved inoculum was added to the mixture. To correct for possible differences in microbial communities, each pot received 1 ml of filtered washing of AM fungal inoculum (van der Heijden et al., 1998). Plants were grown in a glasshouse with day : night temperatures of c. 28 : 15°C.
Plants were watered twice a week during experiments. From the first week on, 8 ml of modified Hoagland solution was applied weekly. Five different Hoagland solutions, modified after Gamborg & Wetter (1975), were prepared to obtain different N sources or N concentrations (Table S2): ‘−N’, ‘’, ‘’, ‘0.1 × NO3’ and ‘’.
For all experiments, seeds were surface-sterilized and grown in 500 ml pots containing the soil mixture as already described.
Three different AM fungal treatments were applied (+AMF: G. intraradices or G. mosseae, −AMF). Five different Hoagland solutions were applied to the G. mosseae and –AMF treatments, namely ‘−N’, ‘’, ‘’, ‘0.1 × NO3’ and ‘ and three different Hoagland solutions were applied to the G. intraradices treatment, namely ‘−N’, ‘’, and ‘’. All treatments were independently repeated four times. A total of 156 pots were prepared. Shoots and roots of one-third of the plants (52) were harvested separately 5, 8 and 13 wk after inoculation.
Split root experiment
After 2 wk of growth, plantlets were dug out gently and roots were distributed equally between two 350 ml pots fixed together with tape and containing the same mixture as described before. Three different AM fungal treatments were applied: AM fungi on both sides of the split-root system (+AMF: G. mosseae/+AMF: G. mosseae); AM fungi on one side and no AM fungi on the other side of the split-root system (+AMF: G. mosseae/−AMF); and no AM fungi on both sides of the split-root system (−AMF/−AMF). Two different Hoagland solutions were applied, namely ‘−N’ and ‘’. All treatments were independently repeated four times. A total of 40 split-roots systems were prepared. Shoots and roots were harvested separately 13 wk after inoculation.
Laser microdissection experiment
Two different Hoagland solutions were applied, namely ‘−N’ and ‘’. Two different AM fungal treatments were applied (+AMF: G. mosseae, −AMF). All treatments were independently repeated four times. A total of 16 pots were prepared. Shoots and roots were harvested separately 13 wk after inoculation.
Staining of AM fungi in plant roots and quantification of root colonization
From each analyzed plant, one subsample of 100 mg of fresh roots was used to determine the degree of AM fungal colonization, as follows. Root subsamples were stained with trypan blue (0.005% w/v in lactic acid, glycerol, water, 1 : 1 : 1, w/w/w) at 60°C for 10 min in 15 ml tubes in a water bath and destained for 24 h in glycerol : 1% HCl (w/w). Root colonization was quantified according to the grid intersection method as described by Brundrett et al. (1984). Total colonization comprised intersections containing hyphae, vesicles, spores or arbuscules. Differences between means of variables were analysed by ANOVA (P ≤0.05), using SPSS 18.0 (IBM, Chicago, IL, USA).
DNA isolation from field samples and fungal diversity analyses
The diversity of AM fungal species associated with sorghum roots harvested in a field site (northeastern France; 47°62′N, 7°52′E; September 2011) was assessed. For each of the three plants, two subsamples (c. 100 mg) of fresh roots were snap-frozen and stored at −80°C. DNA was extracted using the NucleoSpin tissue KS kit (Macherey-Nagel, D___Germany). The internal transcribed spacer (ITS) region of nuclear ribosomal DNA was amplified on a T3 thermocycler (Biometra, Goettingen, Germany) and the amplified fragments were then subcloned using the TOPO-TA cloning kit (Invitrogen). The full procedure is described in Courty et al. (2011). Sequences were manually corrected using Sequencher 4.2 (Gene Codes, Ann Arbor, MI, USA). To identify fungal species, BLASTN searches were carried out against the sequence databases at the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/).
Identification and characterization of sorghum genes encoding AMT transporters
Sequencing, assembly, and annotation of the S. bicolor genome was described by Paterson et al. (2009). All S. bicolor sequences are available at the Joint Genome Institute (JGI) website (http://genome.jgi-psf.org/Sorbi1/Sorbi1.home.html) and have been deposited at GenBank/European Molecular Biology Laboratory/DNA Data Bank of Japan. Using BLAST search and the INTERPRO domains (IPR018047 and IPR001905) at the JGI website, we identified gene models coding for putative AMTs (AMTs) in the draft genome. Gene prediction at the JGI was performed using gene predictors (FGENESH, and GENEWISE), and gene models were selected by the JGI annotation pipeline (Paterson et al., 2009). Selection of the AMT models was based on expressed sequence tag (EST) support, completeness, and homology to a curated set of proteins. The putative homologs that were detected were characterized based on conserved domains, identities, and E-values in comparison with the use of a range of AMT sequences available from plants at the NCBI GenBank (http://www.ncbi.nlm.nih.gov/) and UNIPROT (http://expasy.org/) (Table S3). All full-length cDNAs were sequenced by cDNA walking (Methods S1). Sequences of the cDNAs described here are available at the NCBI database under accession numbers JX294852 to JX294859.
For phylogenetic analysis, the AMT amino acid sequences were aligned with ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/) using the following multiple alignment parameters: gap opening penalty 15, gap extension penalty 0.3, and delay divergent sequences set to 25%; and the Gonnet series was selected as the protein weight matrix. Neighbour joining trees were constructed using Poisson correction model for distance computation in MEGA4 (Tamura et al., 2007). Bootstrap analysis was carried out with 1000 replicates. Branch lengths (drawn in the horizontal dimension only) are proportional to phylogenetic distances. Gene accession numbers of amino acids sequences are given in the Methods S1.
Samples, RNA isolation and quantitative reverse transcription-PCR
RNA extraction and cDNA synthesis were performed as described by Courty et al. (2009), using the conditions specified in Methods S1. Primers used as controls or for analysis had an efficiency ranging between 90 and 110%. In the split-root and laser microdissection experiments, the transcript abundance of the AM-specific phosphate transporter from S. bicolor SbPt11 was also measured (F. Walder et al., unpublished). In the split-root experiment, the G. mosseae elongation initiation factor (EIF) gene was used as an additional control to confirm the presence/absence of AM fungi. All primers used are listed in Table S4.
From each of the three S. bicolor plants harvested in the field site described, three subsamples (c. 100 mg) of roots, shoots, stem, pistils and stamens were snap-frozen and stored at −80°C for further gene expression analysis.
Concerning the time course, the split root and the laser microdissection experiments, plant roots were carefully washed under tap water to remove all soil adhering to the roots. Three subsamples of 100 mg of fresh roots were snap-frozen and stored at −80°C for further gene expression analysis.
C and N analysis
The remainder of the root samples and the shoot material were dried at 80°C for 72 h and weighed. These samples were ground in 1.5 ml Eppendorf ® tubes using 1.1-mm-diameter tungsten carbide balls (Biospec Products Inc., Bartlesville, OK, USA) in a Retch MM301 vortexer (Retch GmbH & Co., Haan, Germany). Total N and C were measured using an online continuous flow CN analyzer coupled with an isotope ratio mass spectrometer (ANCA-SL MS 20-20 system; Sercon Ltd, Crewe, UK).
Tissue analysis by laser microdissection
Roots were washed with running water to remove the substrate. Pieces of 10–15 mm were cut with a razor blade from differentiated regions of the mycorrhizal and nonmycorrhizal roots. The root segments were embedded in OCT (EMS, Delta Microscopies Aygues-Vives, France) and then frozen at −23°C. Some 40 μm thin sections were cut with a Cryocut (Cryocut 1800 Leica), and the cuts were placed on Fisher Probe-On slides (Fisher Scientific, Ilkirch, France). The sections were washed and fixed as follows: 3 min 70% EtOH, 30 min DEPC H₂O, 2 min 100% EtOH. The slides were then dried for 20 min at 37°C on a warming plate and kept at −80°C before use.
An Arcturus XT microdissection system (Applied Biosystems, Foster City, CA, USA) was used to collect the cells from the mycorrhizal and nonmycorrhizal root sections. The slides with the dissected cells were thawed at 4°C for 15 min and then dried in a dessicator at room temperature for 15 min. Eight replicates of three different cell types were collected: arbuscule-containing cells (ARBs), noncolonized cortical cells from mycorrhizal roots (MNMs) and cortical cells from nonmycorrhizal roots (Cs). A total of 5000–15 000 cells were cut for each sample. RNA from the collected cells was extracted using the Arcturus Pico Pure RNA isolation Kit (Excilone, Applied Biosystems, Foster City, CA, USA), with a DNAse treatment in the column kit following manufacturer's instructions. Quantity and quality of the extracted RNAs were verified using a bioanalyzer with RNA pico chips (Agilent, Santa Clara, CA, USA). Synthesis of cDNA and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis was done as previously described using the iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA), starting with 100 pg RNA.
Heterologous complementation of a yeast mutant defective in ammonium uptake
The full-length SbAMT3;1 and SbAMT4 (Table S2) cDNA was cloned in pDR196 using Gateway technology (Invitrogen), as described earlier (Wipf et al., 2003). The resulting plasmids were called pDR196-SbAMT3;1 and pDR196-SbAMT4. The yeast strain 31019b (MATa ura3 mep1Δ mep2Δ::LEU2 mep3Δ::KanMX2) (Marini et al., 1997) was transformed with pDR196-SbAMT3;1 or pDR196-SbAMT4 according to Dohmen et al. (1991). As a control, we also cloned and transformed similarly the low-affinity transporter AtAMT1;3 from Arabidopsis thaliana described by Gazzarrini et al. (1999).
Western blot analysis
Soluble and cell-wall-adhering proteins were extracted as described in Methods S1. The proteins were separated on SDS/12% polyacrylamide gels and transferred to nitrocellulose membranes in Towbin buffer. Proteins were stained with Ponceau red, indicating equal amounts of protein in each lane. Membranes were washed twice with MilliQ water and then blocked with MTBS (3% milk powder, 150 mM NaCl, 10 mM Tris-HCl, pH 7.5) at 4°C overnight, and then washed twice with MTBS for 5 min each. The blots were incubated at 4°C overnight with a 1 : 1000 dilution of SbAMT3;1 antibodies in MTBS. The membranes were washed three times with MTBS and then incubated with secondary antibody (alkaline phosphatase-conjugated mouse anti-rabbit antibody, 1 : 30 000 dilution; Sigma-Aldrich) at room temperature for 1 h. After two washes with MTBS and one wash with alkaline phosphatase buffer (1 M NaCl, 1 M Tris-HCl pH 9.5), the membranes were incubated in chemiluminescence substrate (CDP-star; Roche).
Antibodies specific for the SbAMT3;1-derived peptides 469-[5′-H2N- WYSDNDTQHNKAPSG-CONH2-3′]-483 and 117-[5′-H2N-QHYYHDSDVVETFEIT-CONH2-3′]-132, corresponding to a nonconserved region in the protein, were produced in rabbits. The antisera and the peptides were obtained from the custom peptide antibody production program (Eurogentec, Seraing, Belgium). Immunolocalization was performed as described by Blancaflor et al. (2001) and Harrison et al. (2002) with some modifications (Methods S1) on nonmycorrhizal and mycorrhizal sorghum roots colonized by G. mosseae. Control images in which the mycorrhizal root sections were treated by replacing primary anti-PtPT10 antibodies with preimmune immunoglobulin at the same concentration showed no specific fluorescence.
An ANOVA was performed on the total biomass, on the C and N content, and on the total and arbuscular colonization for each treatment separately, where the two latter parameters were arcsine-transformed to fit the assumption of normal distribution. The ANOVA was based on N treatments and AMF treatments. Pairwise comparisons between the treatments were done with planned contrast analysis. Independent paired t-tests were performed. A probability of P ≤0.05 was considered to be significant.
Identification of AMT-encoding genes in S. bicolor
Eight genes coding for putative AMTs were identified in the predicted gene catalog resulting from the automated annotation of the S. bicolor genome assembly (v1.0, http://genome.jgi-psf.org/sorghum/sorghum.home.htm). The whole genome assembly was used. We performed a phylogenetic analysis, based on the alignment of the corresponding sequences, in comparison to other plant species (Fig. 1). Two of the AMTs identified in the S. bicolor genome (SbAMT1;1 and SbAMT1;2) are members of the AMT1 subfamily, a well-defined group in both monocots and dicots. According to current knowledge, genes encoding AMT1 proteins contain no intron, with the exception of LjAMT1;1 from L. japonicus (Salvemini et al., 2001). All the other AMTs of S. bicolor (SbAMT2;1, SbAMT2;2, SbAMT3;1, SbAMT3;2, SbAMT3;3 and SbAMT4) belong to a separate clade comprising three clusters, each of which seems to be conserved between monocots and dicots (Fig. 1), with introns roughly conserved in each subclade with regard to size and splicing location (data not shown). The transmembrane prediction programme TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) indicates that each of the eight AMTs has 11 transmembrane domains with an extracellular N-terminus and a cytosolic C-terminus, like other plant AMT members (Marini & Andre, 2000; Thomas et al., 2000). Characteristics of the S. bicolor AMT gene family are summarized in Table S3. Similarity and identity between the AMT amino acid sequences of S. bicolor are summarized in Table S5.
Effect of N source and of mycorrhization
Root colonization rates were c. 20–50% and 25–75% at 5 and 9 wk postinoculation, respectively. The percentage of roots colonized increased up to 80% after 13 wk (Fig. S1). The colonization rate was higher for G. intraradices than for G. mosseae in all the treatments at the second and third harvest (Table 1). N content per plant (Fig. S2) and shoot and root DW (Fig. S3) increased over time and were highest after 13 wk when and were applied. After 13 wk, plant N nutrition had no significant effect on the mycorrhizal colonization with G. mosseae (P =0.4) and G. intraradices (P =0.8) and the plant N content was significantly lower in the ‘−N’ treatment than in the ‘’ and ‘’ treatments for G. intraradices (P =0.008) and G. mosseae (P =0.01), respectively.
Table 1. One-way ANOVA comparing the percentage of colonization and of arbuscules in roots colonized by Glomus intraradices with roots colonized by Glomus mosseae for each of the three sampling dates (5, 8 and 13 wk postinoculation, respectively)
Harvest 1 (5 wk)
Harvest 2 (8 wk)
Harvest 3 (13 wk)
Percentages of root colonization were recorded on four plants for each treatment. P-values are given in each cell. Bold values indicate that roots were significantly (P <0.05) more highlyi colonized by G. intraradices than by G. mosseae.
In the absence of mycorrhizal fungi, there were only minor effects of N nutrition on AMT gene expression in the roots (Table S6). By contrast, SbAMT3;1 and SbAMT4 were highly induced in mycorrhizal compared with nonmycorrhizal plants, independently of the N supply (Fig. 2a,b). SbAMT3;1 and SbAMT4 were similarly up-regulated after 5, 9 and 13 wk although plants were less colonized after 5 wk (Table S7).
Heterologous complementation of a yeast mutant defective in ammonium uptake
A yeast mutant complementation test was used to demonstrate the transport function and to biochemically characterize SbAMT3;1 and SbAMT4. AtAMT1;3 was used as control. All three transporters were expressed through the yeast expression vector pDR196 (Wipf et al., 2003) in a mutant yeast strain, 31019b, which lacked the three endogenous transporter genes (MEP1, MEP2, MEP3) and was unable to grow on a medium containing < 3 mM as the sole N source (Marini et al., 1997). SbAMT3;1cDNA or SbAMT4cDNA functionally complemented the yeast mutant efficiently, when 1 and 2 mM were supplied to the agar medium (Fig. 3). As expected, Arabidopsis AtAMT1;3 complemented as well, but the cells expressing one of the sorghum transporters grew more vigorously.
Tissue-specific expression of the different AMTs
In S. bicolor field samples, SbAMT1;1, SbAMT1;2, SbAMT2;1 and SbAMT3;3 showed a similar expression pattern; they all were expressed at a similar level in the five different tissues (root, shoot, stem, pistils and stamens) (Fig. 4). Additionally, SbAMT3;3 had the highest expression level overall.
SbAMT2;2 and SbAMT3;2 were not expressed in the stamens. SbAMT3;2 was additionally highly expressed in the pistils.
SbAMT3;1 was predominantly expressed in the roots but could also be found in the other tissues. SbAMT4 was exclusively expressed in the roots.
Nonsystemic expression of SbAMT3;1 and SbAMT4 in S. bicolor plants
A split-root experiment was set up to study the expression pattern of the two mycorrhizal-induced genes, SbAMT3;1 and SbAMT4, at the whole-plant level. Plants of S. bicolor were grown with two parts of the root system under different conditions, colonized or not by G. mosseae, and additionally subjected to different N regimes. In all cases, root biomass was equally distributed between the two root compartments (Table S6). There was no mycorrhiza formation in root compartments without AM fungi. In the presence of G. mosseae, colonization of the roots in the root compartments varied between 26 and 48% (Table S8) without any significant differences between the treatments. Transcripts were quantified in each compartment of each split-root system (Fig. 5). In all samples, the genes coding for ubiquitin and for all AMTs were amplified by RT-qPCR (Table 2). In the different AMT genes, no significant differences were observed between the different N treatments.
Table 2. Quantification by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) of the transcript abundance of SbAMT3;1 and of SbAMT4 in split roots either noncolonized or colonized by the arbuscular mycorrhizal fungus Glomus mosseae at 9 wk postinoculation (second harvest) in the different N treatments (−N and )
N treatment AMF treatment
1.7 ± 1.4
1.9 ± 1.6
29.8 ± 9.2
43.7 ± 8.9
2.7 ± 1.5
40.5 ± 15.9
1 ± 0
1 ± 0
46.9 ± 11.6
47.1 ± 11.4
0.5 ± 0.3
0.2 ± 0.2
11.5 ± 5.6
18.1 ± 4.4
1.4 ± 1.3
15.4 ± 3.4
1 ± 0
1 ± 0
19.4 ± 3.1
20.8 ± 4.4
N treatment AMF treatment
The values are the means and SD of four replicates. Ubiquitin was used as the reference transcript. Gene expression was normalized according to the ‘−AMF, ’ treatment (= treatment 4). Differences in relative gene expression between the treatments were performed with a one-way ANOVA (Scheffe's F-test). Bold values indicate a P-value < 0.05.
1.3 ± 1.8
28.6 ± 5.4
0.6 ± 0.4
1.6 ± 1.8
33.7 ± 13.4
44.7 ± 16.8
0.5 ± 0.6
22.2 ± 9.2
1.0 ± 0.7
20.1 ± 6.9
0.9 ± 0.7
21.2 ± 14.6
0.1 ± 0.1
0.0 ± 0.0
14.6 ± 9.5
32.9 ± 18.9
0.1 ± 0.1
39.7 ± 12.9
0.4 ± 0.2
27.8 ± 6.5
Root colonization had no significant effect on gene expression, except for SbAMT3;1 and SbAMT4. The genes coding for SbAMT3;1 and SbAMT4 were only induced in mycorrhizal root halves (Table 2). In the treatment +AMF/−AMF, SbAMT3-1 and SbAMT4 were only induced in the mycorrhizal part of the root system, indicating local but not systemic regulation (Fig. 4). By contrast, a mycorrhiza-induced phosphate transporter, SORbiPht1;11 (SbPt11) from S. bicolor, was also expressed in nonmycorrhizal root halves in the +AMF/−AMF systems. A gradient of expression of SbPt11 was observed (Fig. 5), varying from no expression in both parts of the split-root system of the −AMF/−AMF treatment, to a low but significant expression in the nonmycorrhized part of the root system of the treatment +AMF/−AMF, to a high expression in the mycorrhized part of the root system of the treatment +AMF/−AMF and in both parts of the root system of the +AMF/+AMF treatment. This indicates that this transporter is systematically induced, but the transcript abundances were relatively low in the nonmycorrhizal root halves.
Cell-specific expression of SbAMT3;1 and SbAMT4
The distribution of the SbAMT3;1 and SbAMT4 transcripts was studied after laser-dissection and collection of three types of cells: arbuscule-containing cells, noncolonized cortical cells from mycorrhizal roots, and cortical cells from nonmycorrhizal roots. In all samples, the gene coding for ubiquitin and for all AMTs was amplified after RT-qPCR analysis. Transcripts of SbAMT3;1 and SbAMT4 could be detected in all three cell types but were significantly higher in both arbuscule-containing and noncolonized cortical cells from mycorrhizal roots (Fig. 6). Samples were validated by analyzing the expression of a gene coding for an AM-inducible S. bicolor phosphate transporter, SbPT11, and for a fungal specific gene, GmEIF (elongation factor of G. mosseae): SbPT11 was induced in both arbuscule-containing and noncolonized cortical cells from mycorrhizal roots. Transcripts of GmEIF were only detected in arbuscule-containing cells, where G. mosseae only is present (Fig. 6).
Western blot and immunolocalization of SbAMT3;1
SbAMT3;1 was detected in the insoluble protein fraction prepared from the mycorrhizal plant roots but not in the corresponding fraction from nonmycorrhizal roots (Fig. 7a). No signal was detected in the fractions containing soluble proteins (Fig. 7a). A very low amount of SbAMT3;1 was present in the plant leaves (Fig. 7a).
Confocal laser scanning microscopy was performed to localize SbAMT3;1 in the roots. The cells containing active arbuscules showed a strong red fluorescence arising from the wheat germ agglutinin – AlexaFluor 594 conjugate – and a green fluorescent signal, indicating the presence of SbAMT3;1 in the arbuscule-containing cells. The SbAMT3;1 signal colocalized with the arbuscules surrounding the individual branches (Fig. 8). No signal was detected on hyphae beside the arbuscule or on the vesicules (Fig. 8). Cortical cells with developing arbuscules showed SbAMT3;1 staining, whereas in cortical cells with collapsed arbuscules, no staining was detectable. Our data indicate that the protein SbAMT3;1 occurred only in plant cells containing developing or mature arbuscules, and that it was localized there at the periarbuscular membrane.
Nitrogen is an essential, often limiting element for plant growth. For this reason, the use of N fertilizers in agriculture has greatly increased in the past decades (Tilman et al., 2002). AM fungi may help plants in acquiring N by taking up inorganic and organic N sources (Lopez-Pedrosa et al., 2006) with their extraradical mycelium foraging the soil, and thereby have a great potential in a sustainable agriculture. It has been established that the AM fungus G. intraradices has a functional AMT (Lopez-Pedrosa et al., 2006), allowing the uptake of soil ammonium. Afterwards, AM fungi transfer the absorbed N from the extraradical mycelium to the intraradical mycelium in the form of arginine, and it is thought that their arbuscules deliver N in the form of ammonium to the plant (Tian et al., 2010).
SbAMT3;1 and SbAMT4 are two AM-inducible AMTs
In our study, we identified eight genes coding for AMTs in the genome of S. bicolor. A similar number was described in O. sativa (10) or A. thaliana (six). A higher number was reported for poplar (14) (Couturier et al., 2007). Interestingly, poplar and Arabidopsis have a higher number of AMT genes assigned to the AMT1 subfamily (six and five, respectively) than S. bicolor (two) and O. sativa (three) (Sonoda et al., 2003), indicating a different organization of AMT genes in monocots and dicots.
Sorghum AMT genes had varying expression levels in the different tissues (Fig. 4) of the plant, confirming previous studies showing distinct roles of the different AMTs in the plant (Yao et al., 2008). Sonoda et al. (2003) described that OsAMT1;1 was constitutively expressed in rice shoots and roots, similarly to its sorghum homologous SbAMT1;1. By contrast, OsAMT1;2 from rice was root-specific (Sonoda et al., 2003) compared wth its sorghum ortholog, SbAMT1;2, which is constitutively expressed in the plant with the highest level in the shoots. Transcripts of SbAMT3;1 were found in roots, shoots, stem, pistils and stamens while transcripts of SbAMT4 were exclusively found in roots of the plants (Fig. 4). Additionally, the relative gene expression of SbAMT3;1 and SbAMT4 were significantly (70 and 20 times, respectively) higher in roots colonized by AM fungi than in nonmycorrhized roots (Fig. 2), indicating that SbAMT3;1 and SbAMT4 are AM-inducible AMTs. Functionality of both transporters was confirmed by yeast complementation (Fig. 3). Moreover, growth of the transformed yeast on media with low N concentrations (< 1 mM ) indicate that SbAMT3;1 and SbAMT4 are both high-affinity AMTs compared with AtAMT1;3 from A. thaliana (Gazzarrini et al., 1999), which has been described as a low-affinity transporter. In the phylogenetic analysis (Fig. 1), SbAMT3;1 is clustering with the AM-inducible AMTs GmAMT3;1 and GmAMT4;4 (Kobae et al., 2010) from soybean and belongs to the AMT3 subfamily. The closest homologs are OsAMT3;1 from rice and ZmAMT3;1 from maize; however, to our knowledge, no study has been done on the induction of these genes by AM fungi. SbAMT4 is clustering with the AM-inducible AMTs GmAMT4;1 (Kobae et al., 2010) from soybean and LjAMT2;2 (Guether et al., 2009b) from L. japonicus, and belongs to the AMT4 subfamily. The closest homologs are OsAMT4 from rice and ZmAMT4 from maize. Here, also, the induction of these genes by AM fungi has, to our knowledge, not yet been studied. In contrast to the phosphate transporter gene family in which most AM-inducible transporters are clustering in one group (Nagy et al., 2005), AM-inducible AMTs are distributed among different AMT subfamilies (Fig. 1). Other AM-inducible AMTs were described in the AMT1 subfamily: PtAMT1;2 from poplar (Couturier et al., 2007) and GmAMT1;4 from soybean (Kobae et al., 2010).
No effect of N nutrition on AMT gene expression
Our data show that the percentage of roots colonized with G. mosseae and G. intraradices is not affected by the N nutrition in the time-course experiment (Fig. S1). These data contradict the findings of Blanke et al. (2005), who reported in a field experiment that the colonization of Artemisia vulgaris was higher in N-deficient plots than in N-high plots, but under high P concentration. One potential caveat might be the growth of sorghum plants for up to 13 wk in the relatively small soil-volume of 500 ml. Quantitative PCR analyses on sorghum roots revealed that N nutrition had no significant effect on the abundance of AMT transcripts in the roots. This finding was unexpected, as previous studies on rice (Sonoda et al., 2003), L. japonicus (D'Apuzzo et al., 2004) and citrus (Camanes et al., 2009) showed the effect of N nutrition on AMT gene expression. However, we measured the AMT gene expression after 5, 9 and 13 wk under different N nutrition conditions, in contrast to previous studies where expression was measured in min or h after N exposure. Based on our data, we cannot exclude a fine-tuning of sorghum AMT expression immediately after contact with different N sources. However, we studied the regulation of AMTs when plants had access to different N sources over a long period.
Local but not systemic induction of SbAMT3;1 and SbAMT4 in the root system
The results of the split-root experiment did not show any systemic induction of SbAMT3;1 and SbAMT4 in the whole root system. The expression of both transporters was higher in the AM-colonized parts of the split-root system, but not in the noncolonized parts (Fig. 5). The analysis of the expression level of the AM-induced phosphate transporter SbPt11 (F. Walder et al., unpublished) revealed a different picture: no expression when the two parts of the split-root system were not colonized; high and low levels of expression, respectively, in the mycorrhizal and nonmycorrhizal parts of the split-root system; and a high level of expression when the two parts of the split-root system were colonized. Thus, SbPt11 was slightly, but significantly, induced systemically, indicating that in this case a signal might be transferred to the noncolonized roots, preparing the root for a potential future colonization as described by Gaude et al. (2011).
Root cell-specific expression of SbAMT3;1 and SbAMT4
The exploration of the cell-specific expression pattern of SbAMT3;1 and SbAMT4 revealed that both genes were up-regulated in arbuscule-containing and noncolonized cortical cells from mycorrhizal roots, indicating that specific AMTs might have, like phosphate transporters (Javot et al., 2007), an important role in symbiotic processes. High transcript abundances of both SbAMT3;1 and SbAMT4 were also found in noncolonized cells of M. truncatula roots in the survey of Gaude et al. (2011). As mentioned by Balestrini et al. (2007) in tomato, the fungal structures (senescent or young arbuscules) might be present in the part of the cell which has been removed after sectioning. Using a G. mosseae endogenous gene (elongation initiation factor, EIF), we have shown that we could only detect the AM fungus in arbuscule-containing cells.
Interestingly, using immunolocalization, we found the protein SbAMT3;1 only in the arbuscule-containing cells, although the corresponding mRNA was induced in arbuscule-containing and noncolonized cortical cells from mycorrhizal roots, indicating a degree of post-transcriptional regulation (Fig. 8). This post-transcriptional regulation was also clearly apparent in the difference between transcript and protein accumulation of SbAMT3;1 in shoots (Figs 4, 7a). More precisely, our data indicate that SbAMT3;1 is localized at the periarbuscular membrane, where Harrison et al. (2002) already described the presence of the AM-induced phosphate transporter MtPT4. SbAMT3;1, similarly to MtPT4, was surrounding mature arbuscules, but not young or collapsed arbuscules. It makes sense that the expression of several transporter genes is induced upon AM colonization, as the exchange of mineral nutrients and carbohydrates between host plant and AM fungus is the key element of a functioning AM symbiosis (Smith & Read, 2008). This transfer takes place at the periarbuscular membrane, an extension of the plasma membrane of the cell retaining many of the characteristics of the plasma membrane (Perotto et al., 1994) but where AMTs and phosphate transporters regulated by AM symbiosis are localized (Harrison et al., 2002; Pumplin & Harrison, 2009). The expression pattern of SbAMT3;1 also correlates with a previous report in which H+-ATPase activity staining was noted to disappear from the periarbuscular membrane as the arbuscule aged (Gianinazzi-Pearson et al., 1991), indicating that ammonium transport, like phosphate transport, occurs principally in the mature arbuscule.
Our results support the hypothesis that mycorrhizal genes are activated, induced or overexpressed by a small-scale systemic induction before arbuscule development, which could be part of the prepenetration response as the cell reorganizes and cytoplasmic bridges are built (Genre et al., 2008).
Here, we demonstrate that two AMTs of sorghum, SbAMT3;1 and SbAMT4, are locally but not systemically strongly induced in roots in response to mycorrhizal colonization, in contrast to an AM-inducible phosphate transporter gene (SbPt11), which is induced both locally and systemically. Locally, SbAMT3;1 and SbAMT4 were induced in noncolonized cells neighbouring arbuscule-containing cells, perhaps conditioning those cells to accommodate a future arbuscule (prepenetration response), a process of considerable importance in view of the short life span of arbuscules (c. 6–10 d, depending on the species; Toth & Miller, 1984; Alexander et al., 1989). Using immunolocalization, SbAMT3;1 was found to be present exclusively in arbuscule-containing cells within the periarbuscular membrane, highlighting a degree of post-transcriptional of regulation and a potentially important role of this transporter in the transfer of N from the fungus to the plant in the AM symbiosis. Our observations highlight the need for identifying the functions, substrate specificity and regulation of S. bicolor AMTs, as well as further studies of temporal variations in gene expression on S. bicolor associated with different AM fungal species in agricultural ecosystems or in associations with other plant species connected by a common mycorrhizal network.
We thank Anna Maria Marini (Université Libre de Bruxelles) for help with yeast experiments and Lucy Geay (INRA Dijon) for help with microscopy experiments. This project was supported by the Swiss National Science Foundation (grant nos. 130794 to A.W. and PZ00P3_136651 to P-E.C.). N.A.L., C.A., O.C. and D.W. acknowledge financial support from the Burgundy Regional Council (PARI Agrale 8) and the ANR TRANSMUT (ANR-10-BLAN-1604-0). We are grateful to the ‘Centre de Microscopie INRA/Université de Bourgogne, Plate-Forme DImaCell’ for technical assistance in microscopic analysis. The authors gratefully acknowlege the European Science Foundation and the programm ‘Nitrogen in Europe:Assessment of current problems and future solutions’ for financial support.