Author for correspondence: Stephen Rolfe Tel: +44 (0)114 2220039 Fax: +44 (0)114 2220022 Email: firstname.lastname@example.org
• Physiological and molecular responses to phosphorus (P) supply and mycorrhizal infection by Glomus intraradices were compared in European (River) and African (H511) maize (Zea mays) cultivars to examine the extent to which these responses differed between plants developed for use in high- and low-nutrient-input agricultural systems.
• Biomass, photosynthetic rates, nutrient and carbohydrate contents, mycorrhizal colonization and nutrient-responsive phosphate transporter gene expression were measured in nonmycorrhizal and mycorrhizal plants grown at different inorganic phosphorus (Pi) supply rates.
• Nonmycorrhizal River plants grew poorly at low Pi but were highly responsive to mycorrhizal infection; there were large increases in biomass, tissue P content and the rate of photosynthesis and a decline in the expression of phosphate transporter genes. Nonmycorrhizal H511 plants grew better than River plants at low Pi, and had a higher root : shoot ratio. However, the responses of H511 plants to higher Pi supplies and mycorrhizal infection were much more limited than those of River plants.
• The adaptations that allowed nonmycorrhizal H511 plants to perform well in low-P soils limited their ability to respond to higher nutrient supply rates and mycorrhizal infection. The European variety had not lost the ability to respond to mycorrhizas and may have traits useful for low-nutrient agriculture where mycorrhizal symbioses are established.
Of the food crops upon which humans depend for their survival, maize (Zea mays) is produced globally in the highest quantities (FAO, 1999). While yields of the plant can be sustained in the developed world by application of fertilizers, in those parts of the world where populations are most directly dependent upon harvests of this crop, production is determined by the effectiveness of natural processes of capture of growth-limiting nutrients from the soil. In many sandy soils in the semiarid tropics and in the acid, weathered soils of the semihumid and humid tropics, phosphorus (P) is the limiting nutrient (Buresh et al., 1997). Soil P availability therefore represents a major limiting factor for crop production in the low-input, subsistence agricultural systems which dominate developing countries in tropical Africa and Asia, in contrast to the high-input agricultural systems of Western Europe and North America.
While there are intrinsic differences between cultivars of maize in the effectiveness with which they take up inorganic phosphorus (Pi) when grown axenically (Da Silva & Gabelman, 1992), in nature, under conditions of low Pi supply, the ability of cereal roots to capture P from the soil is augmented by the foraging activities of fungal symbionts with which they form arbuscular mycorrhizas (AMs) (Smith & Read, 1997). The AM fungal hyphae increase the volume of the soil prospected and extend beyond the rhizosphere P depletion zone, improving access to mineral nutrients. For Z. mays growing in acidic, tropical soils up to 60% of the total P requirement of the plant can be supplied by the AM fungus Glomus intraradices (Nurlaeny et al., 1996).
Mycorrhizal responsiveness, usually defined in terms of improved growth or Pi uptake, is often negatively correlated with root morphological traits which improve the ability of the nonmycorrhizal plant to acquire Pi directly from the soil (reviewed in Tawaraya, 2003) and has often been shown to vary between modern and older cultivars or wild accessions of the same species (Krishna et al., 1985; Koide et al., 1988; Bryla & Koide, 1990; Mercy et al., 1990; Rao et al., 1990). For example, Manske (1989) found that landraces of mycorrhizal wheat grown in low-Pi soils gave a higher yield than that obtained with more modern varieties grown under the same conditions, whilst Hetrick et al. (1992, 1993) found that older cultivated wheats, developed before 1950 and thus before the widespread use of inorganic fertilizers on breeding stations, were more reliant on mycorrhizal symbiosis than either wheat ancestors or modern wheats. This is in agreement with Zhu et al. (2001), who found that the mycorrhizal responsiveness of modern wheat cultivars, measured in terms of shoot P, was generally lower than that of older cultivars, indicating that this trait may have been reduced during modern breeding programmes. However, this correlation between cultivar age and mycorrhizal responsiveness is not universal. Khalil et al. (1994) found that, whilst some unimproved varieties of maize were unresponsive to mycorrhizal infection, others exhibited a 400% growth increase.
Both the ability of plants to grow at low Pi and mycorrhizal responsiveness are strongly influenced by the genotype of the host (Toth et al., 1990; Khalil et al., 1994; Hetrick et al., 1995). These characteristics were examined in a population of recombinant inbred lines of maize by Kaeppler et al. (2000), who identified three quantitative trait loci (QTL) which reflected improved growth in low-Pi soil and an additional QTL associated with mycorrhizal responsiveness, indicating that considerable scope exists for the development of new maize lines adapted for growth in low-nutrient-input systems.
In the present study, we carried out an intensive comparative analysis of the responses of two maize cultivars to colonization by a single species of AM fungus, G. intraradices, to determine whether cultivars of diverse origins respond differently to mycorrhizal colonization. The two cultivars chosen were maize cv. River, which is grown intensively in high-input agricultural systems of Western Europe, and maize cv. H511, a cultivar grown extensively in subsistence agricultural systems in African countries, notably Kenya and Tanzania (Karanja, 1996). The growth responses, the rates of photosynthesis, and the carbohydrate concentrations of the leaves and roots of mycorrhizal and nonmycorrhizal plants of both cultivars were determined as they were grown at six different soluble Pi concentrations ranging from 0 to 65 µm. In addition, we isolated four putative phosphate and four monosaccharide transporter genes from maize to investigate how the expression of such genes varies in these two cultivars in response to Pi supply and mycorrhizal colonization. These genes provide a means to examine the responses of the cultivars at the molecular level. We hypothesize that mycorrhizal responsiveness to colonization by G. intraradices and the underlying alterations in the expression of genes potentially involved in nutrient transport may be different in these two maize cultivars, developed for production in very different agricultural systems.
Materials and Methods
Plant growth conditions
Nonmycorrhizal and mycorrhizal plants of two commercial maize (Zea mays L.) cultivars were grown. One of the cultivars (cv. River) is used widely in Europe and the other (cv. H511) in Africa. Seeds were germinated on moist filter paper in Petri dishes for 3 d and then individual seedlings were transferred into cylindrical pots (length 25 cm; diameter 7.5 cm; volume 1105 ml) containing autoclaved dune sand. Seedlings were grown in nonmycorrhizal conditions or were colonized by the arbuscular mycorrhizal fungus Glomus intraradices (Schenck & Smith). Mycorrhizal colonization was induced by layering throughout the pot a total of 80 g of a commercial inoculum consisting of spores of G. intraradices in a calcined clay aggregate suspension (Biorize Sarl, Dijon, France). Plants were maintained in a glasshouse (August–September) under natural irradiance. Mean maximum daytime temperature was 32 ± 2°C and mean minimum nighttime temperature was 23 ± 2°C during the experiment. Each pot was irrigated to excess each day with a basal Long Ashton nutrient solution [1 mm NH4NO3, 0.8 mm CaCl2, 0.4 mm K2SO4, 0.3 mm MgSO4·7H2O, 40 µm Fe-ethylenediaminetetraacetic acid (EDTA), 20 µm H3BO3, 4 µm MnSO4·4H2O, 0.4 µm ZnSO4·7H2O, 0.4 µm CuSO4·5H2O, 0.2 µm Na2MoO4·2H2O and 0.1 µm CoCl2·6H2O] which was supplemented with six different concentrations of soluble Pi (0, 0.1, 1, 5, 20 and 65 µm), supplied as Na2HPO4·12H2O. The sodium ion concentration (130 µm) was maintained by the addition of NaCl. The dune sand contained 1–2 µg g−1 Pi which could be extracted with 0.5 m sodium bicarbonate (Olsen et al., 1954) but was relatively rich in fragmented mollusc shells containing insoluble, polymeric CaPO4 (Francis, 1985).
For experiments in which soluble Pi was withheld and subsequently resupplied, plants were grown with nutrient medium containing 65 µm soluble Pi for 17 d. At this point, half the plants were switched to irrigation with nutrient solution containing no P (the sodium concentration was maintained by the addition of NaCl). After 7 d, soluble Pi was resupplied at 65 µm. Control plants received 65 µm soluble Pi throughout.
Analyses of biomass accumulation, rate of photosynthesis and soluble sugar content
The rate of photosynthesis (measured at 380 µl l−1 CO2; 2100 µmol m−2 s−1 irradiance) of the upper portion of the youngest, fully expanded leaf blade in four mycorrhizal and four nonmycorrhizal maize plants from each Pi treatment was measured 32–34 d after germination using an LCA4 infrared gas analyser (Analytical Development Company, Hoddesdon, UK). Actinic light (300–700 nm) was supplied to the leaf by a Schott KL1500T lamp (Schott, Mainz, Germany) and the leaf was allowed to reach steady-state photosynthesis (∼20 min), at which point the net rate of photosynthesis was recorded.
At the end of the experiment (day 34), discs from the youngest fully expanded leaves of four nonmycorrhizal and four mycorrhizal plants were taken at noon and immediately frozen in liquid nitrogen for the analysis of foliar soluble sugars. The plants were then harvested and divided into root, stem and leaf material. A subsample of fresh root material was taken for the analysis of soluble sugars, the isolation of total RNA and determination of mycorrhizal colonization. The rest of the plant material was dried at 70°C for biomass analysis. The fresh weight : dry weight ratio of leaf discs and root tissue from each plant was determined to allow the total biomass to be corrected for material retained for other analyses. Soluble sugar content was measured as described previously (Wright et al., 1998a).
Measurement of total phosphorus and nitrogen
One hundred milligrams of dried material was suspended in 2 ml concentrated sulphuric acid containing salicylic acid (33 g l−1). A lithium sulphate:copper sulphate [10 : 1 by weight (w/w)] catalyst was added and the sample heated to 340°C for 9 h. After cooling, the extract was made up to a volume of 50 ml using nanopure distilled water. Total nitrogen (N) and total P were determined by flow injection analysis (Pittwell, 1990) using a flow injection analyser (Tecator Analyser, Perstorp Analytical, Maidenhead, UK). Recovery of a certified reference hay powder (Office of Reference Materials, Laboratory of the Government Chemist, Teddington, UK) was 94% for both N and P. Specific P uptake was calculated as total P uptake (mg P) per gram of dry root mass [total P in plant (mg)/root dry weight (g)] according to Zhu et al. (2001).
Determination of mycorrhizal colonization of maize roots
Roots were cleared in 10%[weight/volume (w/v)] KOH overnight, rinsed in 10%[by volume (v/v)] HCl for 20 min and then placed in 0.05% (w/v) trypan blue in lactoglycerol (lactic acid:glycerol:water 1 : 1 : 1 v/v) for 48 h to stain fungal structures. Roots were destained using several changes of 50% (v/v) glycerol and mounted in glycerol on a microscope slide. Percentage colonization of roots by mycorrhizal fungi was determined using the grid line intersect method (Giovannetti & Mosse, 1980) at the end of the experiment.
Extraction of nucleic acid and cDNA synthesis
High-molecular-weight genomic DNA was isolated from mature leaves of 14-d-old nonmycorrhizal maize plants and from isolated spores of G. intraradices (obtained from Biorize Sarl, Dijon, France) using the hexadecyltrimethylammonium bromide (CTAB) extraction procedure (Reiter et al., 1992). The extraction buffer contained 100 mm Tris-HCl (pH 8.0), 1.4 m NaCl, 2% (w/v) CTAB, 20 mm EDTA and 1% (w/v) polyvinylpyrollidone.
Total RNA was extracted from leaf and root tissues of maize plants following the procedure of Loening (1969). Messenger RNA was isolated from total RNA using the Dynabead Oligo (dT)25 mRNA Direct system [Dynal (UK) Ltd, Bromborough, UK] and then converted to cDNA by reverse transcription using a Superscript Preamplification Kit for First Strand cDNA Synthesis (Invitrogen Ltd, Paisley, UK).
Amplification and sequencing of genes encoding transporter proteins and internal transcribed spacer (ITS) regions
Degenerate oligonucleotide primers were designed against conserved peptide sequences in published phosphate transporter genes (GenBank) and used to amplify maize genes encoding phosphate transporters. The primers used were:
Monosaccharide transporter genes were amplified with primers ATH1 and ATH3 (Weig et al., 1994) and T1 and T2 (Harrison, 1996). For amplification of the internal transcribed spacer (ITS) regions of maize and G. intraradices, primers ITS1 and ITS4 were used (White et al., 1990).
Maize genomic DNA or cDNA was used as the template in combination with degenerate primers in both standard and touchdown polymerase chain reactions (PCRs). Standard PCR (40 cycles: 94°C for 30 s; 50°C for 30 s; 72°C for 60 s, then 72°C for 30 min) or touchdown PCR (94°C for 30 s; 65°C for 30 s; 72°C for 60 s with the annealing temperature decreasing by 1°C every two cycles to 94°C for 30 s; 55°C for 30 s; 72°C for 60 s then 20 cycles of 94°C for 30 s; 55°C for 30 s; 72°C for 60 s followed by 72°C for 30 min) was carried out using a thermal cycler [PHC-3 Techne (Cambridge) Ltd, Duxford, Cambridge, UK] in a volume of 50 µl. Taq DNA polymerase was added after heating the PCR mix to 94°C to avoid nonspecific amplification of gene products. PCR products were separated on 1% (w/v) agarose gels, isolated and ligated into the plasmid vector pGEM-T Easy (Promega UK, Southampton, UK) then transformed into Escherichia coli DH5alpha. For each amplified PCR product, 12 colonies containing inserts were identified using a blue/white screen and plasmid DNA was prepared. Plasmids containing inserts of the correct size were digested with the frequent-cutting restriction enzymes RsaI and NlaIII and the products separated on a 5% (w/v) polyacrylamide gel. DNA banding patterns were visualized by staining with ethidium bromide. Duplicate representatives of each distinct banding pattern were sequenced in both directions by the Protein and Nucleic Acid Chemistry Laboratory (PNACL), University of Leicester, Leicester, UK.
To obtain full-length clones of selected monosaccharide and P transporter genes, the sequences 5′ and 3′ to the initial amplified product were obtained using the Firstchoice RLM RACE kit (Ambion (Europe) Ltd, Huntingdon, UK) according to the manufacturer's instructions. Oligonucleotide primers were then designed complementary to the 5′ and 3′ ends of the mRNA and full-length cDNA was amplified by PCR. Full-length clones were sequenced in their entirety in both directions to confirm their identity. Homology searches of sequence data with libraries of known gene sequences were carried out using the ncbi blast program (Altschul et al., 1997).
Northern blot analysis
Membranes for northern analysis were prepared using standard procedures (Sambrook et al., 1989). Total RNA was electrophoresed through a denaturing formaldehyde agarose gel, transferred to nylon membrane (Zetaprobe; Bio-Rad, Hemel Hempstead, UK) by capillary blotting overnight and immobilized by UV cross-linking.
A modified pGEM-3Z vector (Promega) was used for the preparation of antisense riboprobes of maize monosaccharide and phosphate transporter gene fragments. The pGEM-3Z vector was cut with SacI and SphI, treated with T4 DNA polymerase to create blunt ends and then re-circularized using T4 DNA ligase, thus removing the majority of the multiple cloning site. Gene fragments were then ligated into the EcoRI site of the modified vector and plasmids containing inserts in the appropriate orientation were selected. The vectors were linearized by digestion with HindIII and used as the template for the synthesis of antisense radiolabelled riboprobes using T7 RNA polymerase in the presence of uridine 5-[α32P] triphosphate (1.85 MBq at 370 MBq ml−1). After transcription, the riboprobe was incubated at 37°C for 30 min with 3 units RQ1 RNase-free DNase to remove template DNA, treated with phenol-chloroform to remove protein and then ethanol-precipitated (Sambrook et al., 1989).
Hybridizations were performed in 120 mm sodium phosphate (pH 7.2), 250 mm NaCl, 50% (v/v) formamide, 10% (w/v) polyethylene glycol 8000 and 7% (w/v) sodium dodecyl sulphate (SDS) at 55°C for 16 h. After hybridization, blots were washed as follows: 2 × saline sodium citrate (SSC), 2 × 5 min washes; 2 × SSC, 0.1% (w/v) SDS; 0.5 × SSC, 0.1% (w/v) SDS; 0.2 × SSC, 0.1% (w/v) SDS each for 30 min at 55°C) to remove nonspecific binding of radiolabelled probe. SSC contained 150 mm NaCl and 15 mm tri-sodium citrate, pH 7.0. Blots were then treated with RNase [ pre-blocked with 2 × SSC and sheared herring sperm DNA (∼1.5 kb, 0.4 mg ml−1) at 22°C for 40 min, followed by 2 × SSC, herring sperm DNA (0.4 mg ml−1) and RNase (1 µg ml−1) at 22°C for 60 min, then washed in 0.2 × SSC and 0.1% (w/v) SDS at 55°C for 30 min] to ensure all nonspecific binding had been removed (Sambrook et al., 1989). Further washes at higher stringency (0.1 × SSC and 0.1% (w/v) SDS) and higher temperatures (55–70°C) were carried out on individual blots as required. Signals were visualised by exposure to Kodak Biomax film (Sigma-Aldrich Company Ltd, Gillingham, UK) at −80°C.
Maize or G. intraradices ITS DNA fragments were radiolabelled with deoxycytidine 5-[α32P] triphosphate (1.85 MBq at 370 MBq ml−1) using a Megaprime DNA labelling kit (Amersham Biosciences UK Ltd, Chalfont St Giles, UK) and purified using a QIAquick nucleotide removal kit (Qiagen Ltd, Crawley, UK). Hybridizations were performed in 500 mm sodium phosphate (pH 7.2), 7% (w/v) SDS at 65°C for 16 h. The blots were washed at 65°C in two changes (each 30 min) of 80 mm sodium phosphate (pH 7.2) and 5% (w/v) SDS followed by two changes of 80 mm sodium phosphate (pH 7.2) and 1% (w/v) SDS to remove nonspecifically bound probe, and then visualized by exposure to Kodak Biomax film at room temperature.
The signal intensities of bands on northern blots were quantified using an InstantImager electronic autoradiography unit (Packard Instruments, Pangbourne, UK). Transporter gene expression was normalized using the ITS signal as a loading control.
Southern blot analysis
Southern membranes were prepared using standard procedures (Sambrook et al., 1989). Fifteen micrograms of high-molecular-weight DNA from mature maize leaves was digested with EcoRI, BamHI or HindIII and separated by electrophoresis through a 1% (w/v) agarose gel. After depurination and denaturation, the DNA was transferred to Zetaprobe nylon membrane (Bio-Rad) by capillary blotting overnight. DNA was bound to the membrane by UV cross-linking.
Maize hexose and phosphate transport gene DNA fragments were radiolabelled as described above. Southern hybridizations were performed in 50% (v/v) formamide containing 120 mm sodium phosphate (pH 7.2), 250 mm NaCl and 7% (w/v) SDS at high (65°C) and low (37°C) stringency for 16 h. High-stringency blots were washed twice at 65°C for 30 min with 80 mm sodium phosphate (pH 7.2) and 5% (w/v) SDS, and then twice with 80 mm sodium phosphate (pH 7.2) and 1% (w/v) SDS. Low-stringency blots were subjected to the following series of washes: 2 × SSC, 2 × 5 min washes, 25°C; 2 × SSC and 0.1% SDS, 15 min, 25°C; 0.5 × SSC and 0.1% SDS, 15 min, 37°C; 0.5 × SSC and 0.1% SDS, 15 min, 50°C. Blots were visualized between washes by exposure to Kodak Biomax film (Sigma) at −80°C for varying periods of time.
Analysis of variance (ANOVA) was used to determine statistically significant differences between measurements (Minitab 13.3; Minitab Inc., State College, PA, USA).
The effect of soluble Pi supply and mycorrhizal colonization on African (H511) and European (River) maize varieties
Mycorrhizal colonization and plant growth To compare the growth of nonmycorrhizal and mycorrhizal River and H511 maize cultivars under different soluble Pi supply regimes, plants were grown in the presence and absence of mycorrhizal inoculum with varying concentrations of Pi in the nutrient medium. Over the range of soluble Pi concentrations tested, the roots of both River and H511 cultivars were extensively colonized by G. intraradices, with between 76 and 94% of the root containing fungal structures. There was no significant difference in colonization of the two cultivars, or in colonization between Pi treatments (P < 0.05; data not shown). No colonization of noninoculated maize roots was observed at any time. Under these conditions, nonmycorrhizal maize plants were completely reliant on seed reserves and Pi supplied in the nutrient medium, whereas mycorrhizal plants could access additional insoluble Pi in the sand substrate.
Soluble Pi supply and mycorrhizal colonization had a marked effect on the growth of both River and H511; however, the responses of the two cultivars to these abiotic and biotic signals were quantitatively different. The cultivar River grew poorly at low to intermediate Pi supply rates (0–5 µm), achieving a total biomass of only half that of plants grown at higher supply rates (20 and 65 µm). In contrast, the mycorrhizal River plants grew well at all soluble Pi supply rates – Pi in the nutrient medium had only a small effect on total biomass, with mycorrhizal plants grown at 0 µm Pi achieving a biomass of 84% of that of plants grown at 65 µm Pi, the highest concentration examined (Fig. 1a). The mycorrhizal responsiveness of River (i.e. the percentage growth response that could be attributed to mycorrhizal infection; see Materials and Methods) was greatest below 5 µm Pi and close to zero at 20 µm Pi and above.
The response of H511 to soluble Pi and mycorrhizal colonization was more complicated. As with River, growth of nonmycorrhizal H511 plants was poor at the lowest Pi concentrations (0 and 0.1 µm) but, in contrast to River, growth improved at 1 µm Pi and above (Fig. 1b). However, the improved growth of nonmycorrhizal H511 plants relative to River plants was not maintained at the highest Pi concentrations. Although the biomass of nonmycorrhizal and mycorrhizal H511 plants did not differ significantly at intermediate soluble Pi concentrations (1 and 5 µm; P = 0.44), mycorrhizal colonization led to a stimulation of the growth of H511 at the lowest (0 and 0.1 µm) and highest (20 and 65 µm) soluble Pi concentrations. The differences in Pi sensitivity and mycorrhizal responsiveness of the two cultivars resulted in nonmycorrhizal H511 outperforming nonmycorrhizal River at intermediate Pi (1 and 5 µm) but nonmycorrhizal River outperforming nonmycorrhizal H511 at higher Pi (20 and 65 µm), using total biomass as a performance indicator. When mycorrhizal, River outperformed H511 at 0–5 µm Pi concentrations whilst performance was similar at 20 and 65 µm Pi.
The allocation of biomass to the root and shoot (Fig. 1c and d), and hence the root : shoot ratio (RSR) (Fig. 1e and f), were markedly different between the two cultivars. The RSR of River plants was not significantly affected by mycorrhizal colonization or Pi supply, except for the 0 µm Pi treatment, where the RSR of nonmycorrhizal plants was greater than that in the other treatments (Fig. 1e). Although the total biomass of the River cultivar differed markedly between treatments, a balanced alteration in root and shoot biomass occurred. H511 plants allocated a greater proportion of the total biomass to roots than River plants under all conditions tested (P < 0.05) (Fig. 1d and f); this difference was most marked when plants were nonmycorrhizal, but was still apparent when they were mycorrhizal. The RSR of the nonmycorrhizal H511 plants was significantly greater than that of the mycorrhizal plants (P < 0.05) (Fig. 1f). This difference was most marked at lower Pi concentrations, indicating that the RSR of H511, in contrast to River, responded strongly to both Pi supply and mycorrhizal status.
To examine the impact of Pi supply and mycorrhizal status on the nutrient content of the shoots and roots, the total N and P contents of these tissues were determined at the end of the experiment. The N content of the supplied nutrient medium was constant in all treatments, and this was reflected in the tissue N content which was unaffected by alterations in Pi supply. The foliar N content of the nonmycorrhizal River cultivar was somewhat higher than that of H511 (33.4 ± 0.8 mg g−1 vs 25.4 ± 0.5 mg g−1) whilst the N content of the roots did not differ significantly (13.1 ± 0.6 mg g−1 vs 13.2 ± 0.5 mg g−1). These values were not affected significantly by mycorrhizal colonization.
Figure 2a and b show the effect of soluble Pi supply and mycorrhizal colonization on the total P contents of leaves and roots of both cultivars. The P contents (expressed on a dry weight basis) of leaves of nonmycorrhizal River and H511 plants were similar (P = 0.808) and unaffected by Pi supply, with the exception of the 65 µm Pi treatment in which the foliar P content was approximately twice that of the other treatments. The P content of nonmycorrhizal roots of both cultivars was lower than that of the leaves and affected little by Pi supply rate. In both cultivars, mycorrhizal colonization led to a marked increase in leaf (∼2-fold greater), and particularly root (∼4-fold greater), P content, although again this was not affected by soluble Pi supply rate. Figure 2c and d show the specific P uptake (SPU) of the two cultivars calculated as the total P content of the plant expressed per gram dry weight of root. This calculation provides a measure of the efficiency with which the roots acquire P, correcting for differences in root size. The SPU of River was significantly greater than that of H511 when both nonmycorrhizal and mycorrhizal (P < 0.001). For both cultivars, mycorrhizal colonization led to a marked increase (∼3-fold) in SPU.
Photosynthesis and carbohydrate content
The maximum, light-saturated rate of photosynthesis of the youngest fully expanded leaves of River and H511 plants was strongly influenced by leaf P content (Fig. 3a) and soluble Pi supply rate (Fig. 3b). The rate of photosynthesis of mycorrhizal plants was similar in both cultivars and largely independent of both soluble Pi supply and leaf P content, indicating that photosynthesis was not P limited in these plants (Fig. 3a). Only the nonmycorrhizal plants grown at the highest soluble Pi concentration (65 µm) attained a similar rate of photosynthesis (Fig. 3a and b) and had a leaf P content which approached that of mycorrhizal plants [1.1–2.2 mg P g−1 leaf dry weight (d. wt)]. The leaf P content of nonmycorrhizal plants grown at soluble Pi concentrations of 20 µm or less was uniformly low (0.5–0.7 mg P g−1 leaf d. wt) and photosynthetic rates were approximately half that of P-sufficient plants. However, over this range of soluble Pi concentrations, the photosynthetic rates of leaves of nonmycorrhizal H511 were significantly greater than those of River (Fig. 3b). This difference was most marked at 0 µm soluble Pi, where the photosynthetic rate of H511 was 64% greater than that of River, but was also consistently greater (8–38%) for plants grown at higher soluble Pi concentrations.
In the leaves of both nonmycorrhizal and mycorrhizal plants, increasing soluble Pi supply was associated with an increase in total leaf soluble carbohydrate (sucrose, glucose and fructose) (Fig. 4). The soluble carbohydrate content of nonmycorrhizal roots was not influenced by soluble Pi concentration in either cultivar. However, the soluble carbohydrate content of mycorrhizal roots was consistently greater than that of their nonmycorrhizal counterparts.
Isolation of putative phosphate and hexose transporter genes of maize
The uptake of inorganic phosphate from the soil is facilitated by specific membrane-localized, high-affinity Pi transport proteins. Genes homologous to these transport proteins were isolated from maize cv. River and their expression in response to soluble Pi and mycorrhizal infection determined in both cultivars. In addition, genes encoding monosaccharide transport proteins were isolated, as hexose sugars are the principle form of carbohydrate taken up by mycorrhizal fungi.
Fragments of four putative phosphate (ZmPT1–4) and monosaccharide (ZmMST1–4) transporter genes were isolated by PCR amplification of genomic DNA and cDNA, using degenerate oligonucleotide primers complementary to conserved amino acid motifs. Full-length cDNAs were subsequently obtained for ZmPT1 and ZmMST1. DNA sequences have been deposited in GenBank under the accession numbers AY639019–AY369022 (ZmPT1–4) and AY683004–AY683007 (ZmMST1–4). The full-length ZmPT1 cDNA was 1770 bp long and predicted to encode a protein containing 539 amino acids forming 12 membrane-spanning helices. The gene fragments of ZmPT2–4 were between 836 and 1070 bp in length. An alignment of the predicted amino acid sequences encoded by ZmPT1–4 is shown in Fig. 5. ZmPT1 showed closest homology to ZmPT2 (85% identity at the amino acid level) followed by ZmPT3 (70%), then ZmPT4 (57%). Alignments of these sequences with sequences in the GenBank database indicated that all were highly homologous to the phosphate transporter 1 (Pht1) group of high-affinity proton-coupled Pi transporters. Within this group, ZmPT1–3 aligned with Pht1 class I transporters (as defined by Davies et al., 2002) which contains members from rice, wheat and barley. ZmPT4 fell within class II, a group of less related proteins from both monocots (wheat and rice) and dicots (Arabidopsis thaliana and Medicago truncatula). The ZmMST1 cDNA was 1865 bp long and was predicted to encode a protein containing 523 amino acids forming 11 membrane-spanning helices. The gene fragments of ZmMST2–4 ranged in length from 248 to 339 bp and encoded proteins homologous to other plant monosaccharide transporters (data not shown).
Southern blots of maize River and H511 genomic DNA hybridized at low stringency with radioactively labelled ZmPT1 or ZmMST1 revealed multiple bands, indicating that each was a member of a small, multigene family. High-stringency hybridizations with ZmPT1–4 and ZmMST1–4 produced single bands in each case (except where the genomic sequence indicated the presence of a restriction enzyme site within the probe), indicating that each gene was present as a single copy within the maize genome, and that the hybridization conditions produced a specific signal for each family member (Fig. S1, available online as supplementary material). The hybridization patterns observed with DNA isolated from the European cultivar, River, and the African cultivar, H511, were identical (data not shown).
The expression of the monosaccharide and phosphate transporter genes was measured in different tissues of young maize plants (River) by northern blot analysis (Fig. S2, available online as supplementary material) using riboprobes of high specific activity. All the transcripts were of low abundance. Of the four monosaccharide transporters, ZmMST1 was by far the most abundant. Expression was greatest in roots and lower in green tissue (although noticeably higher in the prophyll than in sink and source leaves). The expression of ZmMST2–4 was much lower than that of ZmMST1 in all tissues examined. The expression patterns of ZmPT1–3 were similar to each other, again with highest expression in the roots and much lower expression in green tissues. No expression of ZmPT4 was detectable in the tissues examined, or in response to other treatments reported in this study.
Expression of phosphate transporter genes in response to Pi and mycorrhizal infection
To examine the responsiveness of the putative phosphate transporter genes to soluble Pi, gene expression was measured in the roots of nonmycorrhizal River plants subjected to a period of Pi deprivation followed by resupply. Control plants received Pi continuously. The expression of ZmPT1–3 was quantified and expressed relative to that of the ribosomal ITS (Fig. 6). In addition, the expression of ZmMST1, which was not predicted to be P responsive, was determined in the same manner. When soluble Pi was withdrawn from the nutrient medium, the expression of ZmPT1–3 remained constant for 12 h, but then increased from 24 h onwards. When Pi was resupplied, a further increase in the expression of ZmPT1–3 was observed which was maintained for 3 d, followed by a gradual decline (Fig. 6a). No Pi-induced changes in gene expression were apparent for ZmMST1 (Fig. 6b), and the expression of all genes was constant in control plants supplied with Pi continuously.
To determine the effect of the interaction between soluble Pi and mycorrhizal infection on transporter gene expression in the River and H511 cultivars, RNA was prepared from the roots of nonmycorrhizal and mycorrhizal plants grown at soluble Pi concentrations between 0 and 65 µm and used for high-stringency northern blots. The same quantity of total RNA was loaded in each lane, but as the RNA extracted from mycorrhizal plants contained both fungal and plant RNA, the blots were also probed with Z. mays- and G. intraradices-specific ITS probes to allow direct comparisons to be made between samples.
The expression of each P transporter gene (ZmPT1–3) declined in the roots of nonmycorrhizal maize as the concentration of soluble Pi in the nutrient medium increased (Fig. 7), such that expression in the highest treatment (65 µm) was barely detectable. Although the overall pattern was the same in both cultivars, expression was maintained at higher Pi concentrations in H511 compared to River (compare expression in the 20 µm treatment in nonmycorrhizal roots). In mycorrhizal roots of River, ZmPT1–3 expression was very low (less than 10% of that observed in nonmycorrhizal roots grown in the absence of soluble Pi). Although expression was also repressed with increasing soluble Pi concentrations in H511, expression was maintained somewhat in the mycorrhizal roots of H511 grown with soluble Pi concentrations of 1 µm or less – expression levels in mycorrhizal roots grown in the absence of added soluble Pi were 34–51% of that of nonmycorrhizal roots grown under the same conditions. In addition, there was a marked stimulation of ZmMST1 expression in these samples compared with other treatments.
Figure 8 shows the expression values of ZmPT1–3 plotted as a function of root P content (although some P in mycorrhizal samples will be contained in fungal structures). In both cultivars, phosphate transporter gene expression was repressed as the root P content increased. However, it is apparent that ZmPT1–3 expression was maintained at higher levels in the roots of H511 than in River as root P content increased, both in the mycorrhizal and nonmycorrhizal conditions.
In this study, we describe significant differences in the physiological and molecular responses of two maize cultivars, one commonly grown in high-input European agriculture (River) and one grown in low-input African agricultural systems (H511), to mycorrhizal colonization at varying soluble Pi concentrations.
In the absence of mycorrhizal infection, the growth of maize cv. River was only promoted once the external Pi supply was 20 µm or greater – at lower Pi supply rates plants accumulated a total of ∼1.5 g dry matter over the course of the experiment, whilst plants grown in the presence of higher Pi concentrations accumulated twice as much. Whilst the growth of nonmycorrhizal H511 was similar to that of River at the two lowest Pi supply rates (0 and 0.1 µm), a promotion of growth was observed over a much wider range of Pi concentrations. Nonmycorrhizal H511 plants grown at 1 to 65 µm Pi accumulated ∼2 g of dry matter. Clearly the sensitivity of the two cultivars to soluble Pi supply differed. Nonmycorrhizal H511 grew better than River under low-nutrient conditions, but was unable to make use of the highest Pi supplies to increase biomass. Hence, in the absence of mycorrhizal infection, H511 outperformed River when grown with a low Pi supply but River outperformed H511 when grown with high Pi supply, perhaps reflecting adaptations of the two cultivars to different soil nutrient conditions. These differences in cultivar performance illustrate the importance of determining plant responses over a range of soluble Pi supply rates. A comparison of River and H511 at 0.1 µm soluble Pi would indicate that nonmycorrhizal River was as well adapted to growth at low Pi as nonmycorrhizal H511, and that River outperformed H511 at high soluble Pi. In contrast, comparisons of plants grown at 1 µm soluble Pi would give a different picture, with nonmycorrhizal H511 outperforming nonmycorrhizal River. These quantitative responses probably reflect the polygenic nature of maize responses to low Pi (Kaeppler et al., 2000).
The differences in biomass accumulation were attributable, in part, to a marked difference in the RSRs of the two cultivars. Whilst the RSR of nonmycorrhizal River plants was largely unaffected by soluble Pi, the RSR of nonmycorrhizal H511 was significantly greater at lower Pi supply rates, indicating a much greater phenotypic plasticity in this cultivar, a response that is characteristic of plants adapted for growth in impoverished nutritional conditions (Chapin, 1980; Koide et al., 1988; Marschner, 1995). The difference in RSR between cultivars was most noticeable at 1 and 5 µm Pi supply rates, where nonmycorrhizal H511 plants produced a much larger root system and significantly outperformed (in terms of total biomass accumulation) nonmycorrhizal River. However, at higher soluble Pi supply rates, where nonmycorrhizal River produced an extensive root system, the European variety could support a larger shoot biomass than H511.
Both cultivars formed extensive and effective mycorrhizal associations with G. intraradices, with over 75% of the root colonized by fungal mycelium over the range of soluble Pi concentrations tested. The growth of River was strongly promoted by mycorrhizal infection; even in the absence of soluble Pi, the biomass accumulation of mycorrhizal River plants approached that of nonmycorrhizal plants grown at 65 µm Pi, the highest concentration examined, and hence the growth of mycorrhizal River plants was relatively unaffected by the supply of soluble Pi supply. Although H511 plants were also responsive to mycorrhizal infection, the growth of mycorrhizal H511 plants was still influenced by soluble Pi. Mycorrhizal H511 plants grown at 20 and 65 µm soluble Pi attained the same biomass as River plants, indicating that the two cultivars had similar potential for growth under these conditions. However, at lower soluble Pi supply rates, mycorrhizal H511 plants did not grow as well as mycorrhizal River plants, a difference most noticeable for plants grown at 1 and 5 µm soluble Pi. At these soluble Pi concentrations the growth of H511 plants was not stimulated by mycorrhizal colonization, in marked contrast to the growth promotion observed in River.
Reports of increased biomass production upon mycorrhizal colonization are numerous (Smith & Read, 1997) and well documented in maize (Kothari et al., 1990a; Khalil et al., 1994; Clark & Zeto, 1996a; Nurlaeny et al., 1996; Kaeppler et al., 2000; Kelly et al., 2001; Liu et al., 2003). Wide variability in mycorrhizal responsiveness among improved and unimproved maize cultivars has been observed (Khalil et al., 1994; Nurlaeny et al., 1996; Kaeppler et al., 2000). It has been proposed that mycorrhizal responsiveness is governed to a large extent by the ability of the nonmycorrhizal plant to develop a root system in low-Pi soils; hence plants that are poorly adapted to growth in low-Pi soils show a greater mycorrhizal responsiveness than those that are well adapted. Our data support this view, as River plants showed a high mycorrhizal dependence at 1 and 5 µm soluble Pi, whereas H511 plants did not. River was better able to exploit mycorrhizally acquired Pi over a much wider range of soil Pi than H511. The RSR of H511 was more sensitive to soil Pi than that of River irrespective of mycorrhizal colonization. When H511 was nonmycorrhizal, this improved performance in low-Pi soils, but the maintenance of the larger root system restricted its ability to fully exploit higher soil Pi or mycorrhizally derived Pi. H511 only achieved a total biomass equivalent to that of River when a combination of high soil Pi and mycorrhizally derived Pi resulted in the two cultivars having similar RSRs.
Despite the marked differences between cultivars in both the RSR and the responsiveness of the RSR to soluble Pi supply, the tissue P contents of the two cultivars behaved in a similar manner across all treatments. As a consequence, the specific P uptake (SPU) of nonmycorrhizal River was significantly greater than that of nonmycorrhizal H511, indicating that the P uptake efficiency of nonmycorrhizal River roots was greater. The greater efficiency of River roots at acquiring nutrients was not restricted to P, as specific N uptake was also significantly greater, although N was not limiting in these experiments.
Colonization by G. intraradices significantly improved the tissue P concentrations and P contents of both cultivars – in both cultivars, the SPU of mycorrhizal plants was approximately 3-fold greater than that of nonmycorrhizal plants. These results are in agreement with those of previous studies that reported improved P concentration and P content in maize plants following mycorrhizal colonization (Kothari et al., 1990b; Khalil et al., 1994; Clark & Zeto, 1996b; Nurlaeny et al., 1996; Kelly et al., 2001; Liu et al., 2003). This improvement in P nutrition can be attributed to the well-reported ability of mycorrhizal fungal hyphae to better exploit a given soil volume than plant roots and to extend beyond the P depletion zone surrounding the plant roots (Smith & Read, 1997). The increase in P content of mycorrhizal plants compared to nonmycorrhizal plants in the absence of added soluble Pi indicates that plants infected with G. intraradices had access to the other forms of P present in the sand substrate. As the P concentration of mycorrhizal plants was relatively unaffected by soluble Pi supply, uptake of P in these plants was either saturated or regulated. The SPU of mycorrhizal River plants remained greater than that of mycorrhizal H511, indicating that the more efficient P uptake of nonmycorrhizal River plants was maintained in the mycorrhizal conditions. Zhu et al. (2003) observed increased SPU in a modern barley Hordeum vulgare variety, Clipper, compared with an African landrace, Sahara, when each was colonized by G. intraradices and suggested that this AM fungus played a larger role in P uptake in the modern cultivar.
The photosynthetic capacities of mycorrhizal plants of the two cultivars were similar and largely independent of both soluble P supply rate and total leaf P content, indicating that photosynthesis was not P limited in these plants. Only nonmycorrhizal plants grown at the highest soluble P concentration attained a similar rate of photosynthesis to mycorrhizal plants. Nonmycorrhizal plants grown at soluble P concentrations of 20 µm or less maintained a photosynthetic capacity of approximately half that of mycorrhizal plants. However, nonmycorrhizal H511 plants maintained a small but significant increase in assimilatory capacity over nonmycorrhizal River plants. Whilst this could result from genotypic variations, it may reflect an increased sink demand for carbon (C) to maintain the higher root biomass of nonmycorrhizal H511 plants. At the lower P supply rates used in this study, the superior assimilatory capacity of mycorrhizal plants was sufficient to support both the C requirements of the fungal partner and improved growth of the plant partner. Indeed, our results suggest that, at these suboptimal P supply rates, mycorrhizal colonization may significantly improve the growth and yield of maize plants in low-input agricultural systems. At higher P supply rates, a growth depression can be observed in mycorrhizal plants if the cost of the fungal symbiont to the C economy of the plant exceeds the benefit of improved nutrient acquisition (Peng et al., 1993; Johnson et al., 1997) although the increased assimilatory capacity of mycorrhizal plants has the potential to compensate for this (Wright et al., 1998a,b, 2000). Although the C assimilatory capacities of nonmycorrhizal and mycorrhizal plants of both cultivars grown at the highest P supply rate were similar, no growth depression was observed in mycorrhizal plants, suggesting that the transfer of C to the fungal partner may have been restricted or that a longer growth period was required before such a depression became apparent. Despite the large differences in photosynthetic capacity observed in this study, mycorrhizal and nonmycorrhizal plants of both cultivars maintained similar leaf total soluble carbohydrate pools. However, the pools of sucrose, glucose and fructose were substantially increased in mycorrhizal compared with nonmycorrhizal roots. We suggest that the partitioning of C was altered within mycorrhizal plants such that an increased proportion of assimilated C was allocated to the mycorrhizal roots to support the additional C requirement of the fungal partner.
In this study we isolated fragments of four monosaccharide transporter genes, ZmMST1–4. For one, ZmMST1, we obtained a full-length sequence which shows high homology to other energy-dependent proton-coupled hexose transporters. Southern analysis confirmed that ZmMST1–4 belong to a multigene family, consistent with multigene families already described in other species (Lalonde et al., 1999). ZmMST1 showed highest expression in root tissues, consistent with the majority of hexose transporters which exhibit higher expression in sink tissues (Sauer & Stadler, 1993; Weig et al., 1994; Truernit et al., 1996, 1999; Ylstra et al., 1998; Fillion et al., 1999; Buttner et al., 2000; Sherson et al., 2000; Toyofuku et al., 2000). Interestingly, the expression of ZmMST1 increased in colonized H511 roots, particularly those grown at submicromolar P supply rates. We have also shown that the soluble carbohydrate pool was increased in these roots. Under conditions of severe P stress it may be expected that the C flow to the root system and fungal partner may be high in support of nutrient acquisition. Our results indicate that ZmMST1 may play a role in C movement within mycorrhizal root systems, although immunolocalization studies are required to determine its precise role. In the only other study to examine the expression of plant monosaccharide transporters in AM root systems, Harrison (1996) observed increased expression of the monosaccharide transporter Mtst1 in cortical cells within the roots of M. truncatula that were highly colonized by Glomus versiforme.
We also isolated fragments of four P transporter genes, ZmPT1–4, from maize, each of which shows homology to proton-coupled P transporters from fungi and other higher plants. ZmPT1–3 showed high expression in roots, much lower expression in green tissues and regulation of expression in response to P starvation and resupply. These observation are consistent with other studies which indicate that, whilst expression of Pi transporters has been observed in tissues such as leaves, stems, tubers and flowers (Leggewie et al., 1997; Liu et al., 1998a; Kai et al., 2002), Pi transporters are usually strongly expressed in roots and show regulation of expression in response to phosphate starvation (Smith et al., 2003; Rausch & Bucher, 2002).
We further investigated P uptake characteristics in cultivars River and H511 by examining the expression of maize P transporter genes, ZmPT1–3, in response to the soluble Pi supply rate and mycorrhizal colonization. The expression of each increased substantially in nonmycorrhizal root tissue as the external soluble Pi concentration was decreased. This is comparable to the regulation of Pi transporters in response to P starvation observed in the roots of other plants (Daram et al., 1998; Liu et al., 1998a,b). Although the overall expression patterns were similar in nonmycorrhizal roots of the two cultivars, expression was higher in H511 than River for a given root Pi concentration. Following mycorrhizal colonization, a significant decrease in the expression at all external soluble Pi concentrations was observed. A similar decline in the expression of plant genes encoding high-affinity P transporters, localized in the epidermis and root hairs, upon mycorrhizal colonization has been reported in several species (Liu et al., 1998b; Rosewarne et al., 1999; Chiou et al., 2001). However, in this study, the impact of mycorrhizal colonization upon the expression of ZmPT1–3 differed between the two cultivars. Expression levels were particularly low in mycorrhizal River roots at all external Pi supply rates. In mycorrhizal H511 roots, expression was maintained when the external soluble Pi concentration was 1 µm or less. As in nonmycorrhizal roots of River and H511, the expression of ZmPT1–3 was maintained at a higher level in the roots of H511 compared to River as the root P concentration increased. We propose that the decline in expression of ZmPT1–3 results from the improved P nutrition of mycorrhizal roots and operates in a manner analogous to that observed in nonmycorrhizal roots relieved from P starvation (Raghothama, 1999, 2000; Bucher et al., 2001; Mukatira et al., 2001; Abel et al., 2002; Franco-Zorrilla et al., 2004). However, repression of P transporter gene expression in H511 was inherently less sensitive to internal or external P signals compared with River, indicating a general alteration in P responsiveness.
Two distinct pathways of P uptake are possible in mycorrhizal roots. Uptake can occur directly via high-affinity P transporters at the root/soil interface or via the mycorrhizal hyphae in the soil. It may be that the contribution of the direct P uptake pathway and that occurring via the mycorrhizal hyphal pathway varies with the exact plant cultivar–AM fungus association and the conditions under which they are grown. Certainly the expression of plant genes involved in the P-starvation response varied in the roots of one cultivar of M. trunculata or Lycopersicon esculentum when colonized by seven different AM fungi (Burleigh et al., 2002). We expect that ZmPT1–3 may be expressed as part of the direct P uptake pathway at the root/soil interface (Daram et al., 1998; Liu et al., 1998a; Muchhal & Raghothama, 1999; Chiou et al., 2001) and that other P transporters, specifically induced and operational in the mycorrhizal hyphal P uptake pathway, characterized recently (Rausch et al., 2001; Harrison et al., 2002; Paszkowski et al., 2002), are present and expressed in maize. Recently, Smith et al. (2003) reported that, when flax (Linum usitatissimum), medic and tomato were colonized by G. intraradices, all of the P was delivered to the plant via the mycorrhizal hyphal P uptake pathway and that there was loss of function of the direct P uptake pathway, mediated by plant high-affinity P transporters. The extremely low expression levels of ZmPT1–3, observed in roots of mycorrhizal River plants, would be consistent with P uptake occurring predominantly via G. intraradices hyphae. The mycorrhizal hyphal uptake pathway may also dominate in colonized H511 roots, but the expression of plant P transporters is clearly also increased and may contribute to P uptake under some growth conditions. Interestingly, increased ZmPT1–3 expression occurred when the SPU of mycorrhizal H511 plants was lowest, providing an independent indication that the contribution of G. intraradices to P uptake may be reduced.
We investigated the responses of two maize cultivars to soil P supply rate and mycorrhizal infection. Although the European cultivar River has been developed for growth in high-nutrient-input agriculture, and as such will normally obtain little benefit from mycorrhizal associations, it still retained the capacity to form efficient symbiotic associations with the AM fungus G. intraradices and, in fact, performed better (in terms of biomass accumulation) than the African variety H511 when mycorrhizal. The specific P uptake of River was always significantly greater than that of H511, and P transporter gene expression was more sensitive to external Pi supply under all conditions tested. The H511 cultivar only outperformed River when nonmycorrhizal at intermediate Pi (1–5 µm) supply rates. A major difference between these cultivars was the responsiveness of the RSR to Pi supply and mycorrhizal infection. Clearly, a large root system, as found in H511, will have a beneficial effect on water acquisition, as well as nutrient foraging, which is likely to be important in African agriculture. However, a smaller, mycorrhizal root system may provide a more effective strategy for nutrient acquisition from low-nutrient soils.
This work was supported by The Leverhulme Trust (Grant number: F/118/AT).