Over-expression of the rice OsAMT1-1 gene ... - CSIRO Publishing

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Functional Plant Biology, 2006, 33, 153–163

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Over-expression of the rice OsAMT1-1 gene increases ammonium uptake and content, but impairs growth and development of plants under high ammonium nutrition Mohammad S. HoqueA,B , Josette MasleA , Michael K. UdvardiC , Peter R. RyanB and Narayana M. UpadhyayaB,D A Research

School of Biological Sciences, The Australian National University, Canberra, ACT 2000, Australia. B CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia. C Department of Biochemistry and Molecular Biology, The Australian National University, Canberra, ACT 2000, Australia. Current address: Max Planck Institute for Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany. D Corresponding author. Email: [email protected] Abstract. A transgenic approach was undertaken to investigate the role of a rice ammonium transporter (OsAMT1-1) in ammonium uptake and consequent ammonium assimilation under different nitrogen regimes. Transgenic lines overexpressing OsAMT1-1 were produced by Agrobacterium-mediated transformation of two rice cultivars, Taipei 309 and Jarrah, with an OsAMT1-1 cDNA gene construct driven by the maize ubiquitin promoter. Transcript levels of OsAMT1-1 in both Taipei 309 and Jarrah transgenic lines correlated positively with transgene copy number. Shoot and root biomass of some transgenic lines decreased during seedling and early vegetative stage compared to the wild type, especially when grown under high (2 mM) ammonium nutrition. Transgenic plants, particularly those of cv. Jarrah recovered in the mid-vegetative stage under high ammonium nutrition. Roots of the transgenic plants showed increased ammonium uptake and ammonium content. We conclude that the decreased biomass of the transgenic lines at early stages of growth might be caused by the accumulation of ammonium in the roots owing to the inability of ammonium assimilation to match the greater ammonium uptake. Keywords: ammonium transporter, ammonium uptake, Oryza sativa, OsAMT1-1 over-expression.

Introduction Owing to the introduction of high-yielding rice varieties worldwide, fertiliser use has increased several fold (Bouwman 1997). According to one estimate, 70% of the world’s rice is produced in rainfed lowland or irrigated systems in Asia (IRRI 1997), where nitrogen fertility is a major limiting factor for yield. Consequently, each year large quantities (80–100 million tonnes) of nitrogen fertiliser are used in this system. Most of this nitrogen is applied in the form of urea, which is converted to ammonium before being taken up by plants. The uptake efficiency is known to vary between 30 and 80% (Craswell and Godwin 1984), while the rest of the fertiliser nitrogen escapes from the soil by leaching in the form of NO3 − or by release of gaseous N2 , N2 O and NH3 into the atmosphere (Mengel 1992). Global threat of nitrogen pollution in N-deficient ecosystems and the need for urgent

political and scientific action have been highlighted in recent studies (Beman et al. 2005; Giles 2005). Scientifically, it is important to find ways to improve nitrogen-use efficiency in plants. One plausible way could be to increase nutrient transporter activity in roots. Ammonium is the preferred source of nitrogen for rice plants (Sasakawa and Yamamoto 1978; Goyal and Huffaker 1984). Root hairs, which cover the apical root zone, can contribute 70–80% of the total root surface area and are believed to play a major role in nutrient uptake in general (Marschner 1995). Ammonium transporters are implicated in this uptake and assimilation. Ammonium transporters also appear to be present at the plasma membrane of leaf cells and these transporters probably play a role in N uptake following foliar application of NO3 − or NH4 + (Bowman and Paul 1992; Yin et al. 1996). To date, several ammonium transporter (AMT)

Abbreviations used: AMT, ammonium transporter; GS, glutamine synthase. © CSIRO 2006

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genes have been isolated and partially characterised from bacteria, fungi and plants (for a review see von Wirén and Merrick 2004). Rice apparently contains at least 10 OsAMTlike gene sequences (Suenaga et al. 2003). Using a partial cDNA (GenBank Acc. No D39189 supplied by MAFF DNA Bank, NAIR, Japan) as a probe, we have previously isolated genomic clones containing full-length coding sequences of three members of the AMT1 gene family (highaffinity ammonium transporters): OsAMT1-1 (AF289477), OsAMT1-2 (AF289478) and OsAMT1-3 (AF289479), from the indica rice cv. IR36 (Hoque 2001). The indica OsAMT1-1 was almost identical to the published japonica (variety Nipponbare) OsAMT1-1 (von Wirén et al. 1997). Based on these published sequences (AF289477–AF289479) OsAMT1-1, -2 and -3 have been cloned and their ammonium transport activities confirmed in yeast (Sonoda et al. 2003a). These three OsAMT1s show differential expression. OsAMT1-1 shows constitutive and ammoniumpromoted expression in shoots and roots, OsAMT1-2 shows root-specific and ammonium-inducible expression, and OsAMT1-3 shows root-specific and nitrogen-derepressible expression (Hoque 2001; Sonoda et al. 2003a). Two more genes, OsAMT2-1 and OsAMT3-1 have also been cloned from rice. OsAMT2-1 shows constitutive expression in both roots and shoots and OsAMT3-1 shows very weak expression in roots and shoots (Suenaga et al. 2003). In the past, attempts have been made to study the regulation of ammonium uptake with native gene expression studies under N deprivation and different N regimes (Kumar et al. 2003; Sonoda et al. 2003b). In those studies, up-regulation by N deprivation or down-regulation by NH4 + nutrition were greatest for OsAMT1-1, moderate for OsAMT1-2 and least for OsAMT1-3. However, responses to diurnal changes were highest for OsAMT1-3 (Kumar et al. 2003). Our interest was to study the effect of overexpression of OsAMT1-1 by a transgenic approach, in terms of NH4 + influx in roots and subsequent effect on biomass production under different NH4 + regimes. As OsAMT1-1 appeared to be the most active and / or most N-responsive gene manifesting high-affinity ammonium transporters (HATS) activity, it was selected as the gene to be overexpressed in transgenic lines. In this paper, we report the molecular and physiological characterisation of several OsAMT1-1-overexpressing transgenic lines of two japonica rice cultivars, Taipei 309 and Jarrah. We show that overexpression of OsAMT1-1 in rice increases ammonium uptake and tissue ammonium content, but impairs early growth and development of plants in the presence of millimolar concentration of external ammonium. Materials and methods Rice cultivars and growth conditions Two rice (Oryza sativa L.) cultivars, Taipei 309 and Jarrah were used in this study. Seeds were dehusked, surface sterilised in sodium

M. S. Hoque et al.

hypochlorite (1% v / v) for 15 min and rinsed in sterile water. They were then germinated on filter paper, wetted with sterile water (for wild type plants) or sterile water containing 75 mg L−1 hygromycin (for transgenic plants). After 4 or 5 d, germinated seeds were transferred to hydroponic culture in modified Johnson’s nutrient (MJN) solution (Epstein 1972) containing various nitrogen concentrations as specified for individual experiments. OsAMT1-1 gene construction A DNA fragment carrying the full-length OsAMT1-1 gene (von Wirén et al. 1997) was released from pBluescriptSk (+) by digestion with MluI and NotI. The two ends (MluI and NotI) were end-filled with DNA polymerase I Klenow fragment and cloned into the SmaI site of the plant expression vector pLZUbi1cas, containing the maize Ubi1 promoter (with its own 5 UTR and the first intron) and nos terminator adjacent to the SmaI site (Christensen and Quail 1996). Sense and antisense orientations of the insertion were determined by the sizes of the fragment liberated following BamHI digestion, as there is a BamHI site at the 5 polylinker and another internal BamHI site at 1224 bp down stream. The promoter–OsAMT1-1–terminator cassettes (in sense and antisense orientations) from the resulting recombinant plasmids were recovered as EcoRI(end filled) / HindIII fragments and inserted into XbaI (end filled) / HindIII sites of the binary vector pWBVec8 (Wang et al. 1998) before Agrobacterium-mediated transformation. Tissue culture and transformation The steps involved in the use of mature seeds of cvv. Taipei 309 and Jarrah for callus induction, the Agrobacterium-mediated transformation of embryogenic calli with the OsAMT1-1 binary construct, the subsequent regeneration of transgenic lines and the separation of early events of independent stable transformations within the callus material have been described previously (Upadhyaya et al. 2000). Transformed calli were selected via hygromycin resistance conferred by a CaMV35Spromoter-driven hph gene within the T-DNA. RNA and DNA extraction Nucleic acids were extracted from selected T1 transgenic plants germinated under hygromycin selection and then grown hydroponically in MJN solution (Epstein 1972) with 2.0 mM NO3 − and 1.0 mM of NH4 + for 3 weeks. The seedlings were then transferred to fresh nutrient solution devoid of nitrogen and allowed to grow for 3 d. Total RNA was extracted from the roots and shoots by a modified version of the procedure described previously (Logemann et al. 1987). RNA was quantified by UV spectrophotometry (Sambrook et al. 1989). Genomic DNA was isolated from plants with PureGene reagents (Gentra Systems Inc., Minneapolis, MN) as per manufacturer’s instructions. PCR analysis of transgenic plants Verification of the presence of the hph gene in hygromycinresistant plants was done by PCR with hph-specific primers (Upadhyaya et al. 2002). Primers corresponding to the endogenous rice sucrose synthase gene (RSs1) were used in control reactions (Wang et al. 1992). Southern and northern blot hybridisation Total genomic DNA (2 µg) was digested with BglII (for which there is only one recognition site in the gene construct), blotted onto Hybond N+ nylon filter (Amersham™), and hybridised with a radioactivelylabelled hph gene probe according to the manufacturer’s instructions. For northern blot hybridisation, 25 µg total RNA was separated on 1.5% w / v formaldehyde agarose gel and transferred to nylon membrane (Hybond N; Amersham™) by a standard capillary transfer

Over-expression of OsAMT1-1 in rice

protocol (Sambrook et al. 1989) and hybridised with the OsAMT1-1 probe prepared with the Promega Riboprobe in vitro transcription system according to manufacturer’s protocol (Promega Bioscience, San Luis Obispo, CA). After hybridisation blots were autoradiographed with phosphor screens (Molecular Dynamics, Sunnyvale, CA). Biomass, root ammonium content and N uptake under NH4 + / NO3 − and no N nutrition T1 seedlings of selected transgenic lines were used along with respective wild type plants. Seedlings were grown hydroponically for 16 d in MJN solution with 2.0 mM NO3 − and 1.0 mM NH4 + and split into three sets and each set was subjected to (1) no further treatment, (2) growth for one more day without N, and (3) growth for one more day with N and two more days without N. Five plants from these three treatments (16 d N-fed, 16 d N-fed and 1 d N-deprived, and 17 d N-fed and 2 d N-deprived) were transferred at zero time to a tube containing 75 mL of MJN solution containing 75 µM NH4 + as the sole nitrogen source. A gentle stream of air was bubbled through the solution to ensure uniform distribution of NH4 + and sufficient oxygen during the experiment. Samples of nutrient solution (1 mL) were taken after 10 min, 30 min, 1 h and 3 h. The ammonium uptake rate was determined as the amount of ammonium removed from the nutrient solution per unit time, expressed as µmol g−1 root FW h−1 . After 3 h, plants were harvested and whole-plant weight, root and shoot weight were recorded. Total roots from individual seedlings were homogenised (digested) in 0.3 mM sulfuric acid before measuring the ammonium content. The calorimetric phenol-hypochlorite method (Solorzano 1969) was employed with absorbance of the sample read at 640 nm with a Labsystems Multiskan Plus (Pathtech Diagnostics Ltd, Preston, Vic.) vertical light path filter photometer. Biomass, root ammonium content and N uptake under NH4 + / NO3 − , NO3 − and no N nutrition T2 homozygous seedlings of single-copy (OsAMT1-1) transgenic lines T-40-1 and J-77-1 along with corresponding wild type Taipei 309 and Jarrah, respectively, were grown for 14 d under NH4 + / NO3 − nutrition. One set was maintained under NH4 + / NO3 − nutrition for 7 d. A second set was transferred to NO3 − -only medium and the third set was transferred to a N-free medium. NH4 + feeding was carried out as described above for 3 h. Plant tissues were harvested from each treatment for northern blot analysis and other measurements were taken as described for the first experiment. Biomass accumulation under high and low NH4 + steady-state N-supply Seeds from two homozygous transgenic lines of Taipei 309 (T-38 and T-40) and Jarrah (J-75 and J-77) along with the respective wild types were germinated in water. At the one leaf stage, seedlings were transferred to a continuous flow-through hydroponic nutrient system where roots were exposed to a solution of constant temperature (28◦ C), ionic concentrations, and pH 5.6. For a first batch of plants nitrogen was provided as NH4 + only, in the form of NH4 Cl, at a constant concentration of 2 mM. Other ions were as in standard MJN solution. Plants were grown in a glasshouse at 28–30◦ C / 24◦ C day / night temperature, 14-h photoperiod. The fresh weights of roots and shoots were measured for 6–8 replicate plants on 15, 22, 31 and 38 d after seedling transfer to nutrient solution. A second batch of seeds were then germinated for comparison under similar conditions except for NH4 concentrations, which were set at 10 µM, and the addition of CaCl2 to the solution so that the concentration of chloride ions would remain unchanged. Six to eight plants were harvested on days 15, 22 and 38.


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Results Constructs for altered expression of OsAMT1-1 and production of primary transgenic lines A full-length OsAMT1-1 cDNA clone (containing both 5 and 3 UTR) isolated from the japonica rice variety, Nipponbare, was kindly supplied by von Wirén et al. (1997). For highlevel constitutive expression a maize ubiquitin promoter (with its own 5 UTR and first intron)–nos terminator cassette was used to produce both sense and antisense gene constructs. These gene constructs were inserted into the Agrobacterium binary vector pWBVec8 (Wang et al. 1998), which contained a Cauliflower Mosaic Virus 35S (CaMV 35S) promoter-driven, intron-interrupted (catalase intron from castor bean) hygromycin phosphotransferase (hph) gene as a selectable marker to produce sense and antisense binary vector constructs. Seventeen independent sense transgenic lines (52 plants) and seven antisense transgenic lines (23 plants) of cultivar Taipei 309, and five sense transgenic lines (10 plants) of cultivar Jarrah were produced. No antisense transgenic lines could be obtained for Jarrah. The majority of the sense transgenic Taipei 309 lines were fertile, while more than half of the antisense transgenic Taipei 309 lines were sterile. PCR analyses (with hph gene-specific primers, and rice sucrose synthase-specific primers as internal control) confirmed the transgenic events in these putative primary transgenic lines (Fig. 1A). Transgene and expression analysis in T1 progeny Ten out of 12 Taipei 309 sense transgenic lines, and all five Jarrah sense transgenic lines, showed overexpression of OsAMT1-1 compared with wild type plants (Fig. 2A, C). The transcript levels in Taipei 309 transgenic lines T-38, T-41 and T-46 were at least 4-fold higher than that in the wild type plant and in Jarrah transgenic lines T-47, J-74 and J-75 they were at least 2-fold higher than that in the wild type plant. Transgenic plants containing antisense constructs had OsAMT1-1 expression levels similar to that of wild type plants (Fig. 2B). Three overexpressing lines (T-40, T-38, T-46) and one line (T-35) with no OsAMT1-1 overexpression and all five transgenic lines from Jarrah were used for further analyses. It was interesting to note that wild type Jarrah had an OsAMT1-1 transcript level ∼2-fold higher than that of Taipei 309 (compare first lanes in Fig. 2A and C). Based on the Southern blot hybridisation data, the highest-expressing line, T-46 contained at least six transgene copies and T-38 had at least three copies (Fig. 1B). Lines T-40 and T-35 had a single copy of the transgene (Fig. 1B). A similar pattern was also observed in the case of T0 overexpressing transgenic lines of Jarrah, where multi-copy lines showed higher expression levels than the single-copy lines (Fig. 1C).

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7 C1 8

9 10 11 12 13 14 C2 M

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C

8.5 kb 7.5 kb

WT 71 74

J75 76 77 M

6.11 kb 4.48 kb

6.1 kb 3.59 kb 4.8 kb

3.59 kb

2.81 kb

Fig. 1. Analysis of putative transgenic lines by PCR and Southern blot hybridisation. (A) PCR amplification of a transgene fragment (hph) and / or an endogenous gene fragment (RSs1) from genomic DNA of various transgenic lines (1–14), positive control (C1, plasmids containing hph gene), negative controls (C2, wild type plants). Estimation of OsAMT1-1 transgene copy number in (B) Taipei 309 and (C) Jarrah T1 transgenic lines by Southern blot hybridisation of BglII-digested genomic DNA with 32 P-labelled hph gene probe. Wild type (lane WT) and molecular weight marker (lane M) are included.

The phenotypes of T1 progeny plants of transgenic lines T-38 and T-46 (containing multiple transgene copies) were highly variable compared with those of wild type plants. Plants from line T-46 segregated into several plant types, such as normal height compared with wild type, dwarf, and dwarf-lethal (Fig. 3). The phenotypes of plants from line T-40 (containing a single transgene copy) were uniform and were similar to the wild type plants. In addition to single-copy transgenic lines T-35, T-40, J-71 and J-77, lines J-74, J-75 and J-76 (containing 2–3 transgene copies) showed close to 3 : 1 segregation of hygromycin resistance and sensitivity (Table 1), indicating that they might be transgenic lines containing multiple transgene copies at a single locus. However, multi-copy transgenic lines T-38 and T-46 showed a skewed segregation of less than 2 : 1, indicating

either gene expression problems (gene silencing) or possible adverse effects of OsAMT1-1 overexpression on germination. Biomass, root ammonium accumulation and N uptake under NH4 + / NO3 − and no N nutrition The biomass of all transgenic lines tested (except T-35) were lower than that of wild type Taipei 309 plants under both NH4 + / NO3 − -fed and N-deprived growth conditions (Fig. 4). Root ammonium content, measured (on a root-freshweight basis) after 3 h of uptake in 75 µM ammonium, were higher in transgenic lines T-38 and T-46 compared with wild type plants in all treatments. Root ammonium contents in wild type as well as in transgenic plants were higher in 1-dN-deprived plants compared with those in N-fed and 2-d-Ndeprived plants.


A


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J-76 J-77

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Fig. 2. Northern blot analysis of OsAMT1-1 expression in rice roots. Total RNA (25 µg) isolated from the roots of hydroponically grown Taipei 309 wild type (WT in A and B), Taipei 309 sense-transgenic (T- in A) and antisense-transgenic (B), and Jarrah wild type (WT in C) and transgenic (C) plants, stained with ethidium bromide (lower panel) and hybridised with radioactively-labelled OsAMT1-1 probe. Northern blots were exposed to phosphor screens (Molecular Dynamics) overnight and autoradiographed. The hybridisation signals were normalised against the rRNA signals and expressed relative to that of the wild type (WT) Taipei 309 (boxed values).

Taipei 309

1

2 3 4 T-46 T1 plants

5

1

2 3 T-40 T1 plants

Fig. 3. Phenotypes of transgenic lines overexpressing OsAMT1-1 at mid-vegetative growth stage. T1 plants from transgenic lines T-46 and T-40 along with wild type Taipei 309.

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Ratio

35 40 38 46 71 74 75 76 77

2.3 : 1 1.32 3.6 : 1 0.60 1.7 : 1 11.01 0.8 : 1 40.17 4.5 : 1 1.30 5.6 : 1 5.20 4.6 : 1 0.96 2.4 : 1 0.71 3.5 : 1 0.31

Taipei 309 Taipei 309 Taipei 309 Taipei 309 Jarrah Jarrah Jarrah Jarrah Jarrah

64 75 85 37 36 79 15 66 32

28 21 51 46 8 14 3 27 9

χ2

Ammonium uptake rates in Taipei 309 wild type and transgenic lines (T-35, T-40 and T-38) at different times in these N-feeding experiments are presented in Fig. 5. Measurement of ammonium uptake with ammonium-fed plants was not possible because the ammonium content of the nutrient solution at the early stage of uptake (10 and 30 min) was much higher than the initial ammonium content (presumably because of ammonium efflux from these plants). No ammonium efflux was evident in 1-d- and 2-d-deprived plants. As shown in Fig. 5A the NH4 + uptake rates of 1-d-N-deprived plants were higher in two transgenic lines (T-40 and T-38) than those in control plants. Uptake of ammonium by line T-35 was not significantly different from that of the wild type at any time point. The initial ammonium uptake rate in transgenic lines (T-40 and T-38) was approximately twice that of wild type plants during the first

Fresh wt (g plant–1)

NO3– & NH4+-fed

0.7

7 6

0.6

5

0.5

4

0.4

3

0.3 0.2

2

0.1

1 0

0

LSD (5%)

Whole Shoot Root plant 0.05 0.04 0.02

Root NH4+ 0.6

Biomass and root ammonium content measurements were made on N-deprived (both NH4 + - and NO3 − -deprived), NH4 + -deprived (NO3 − -fed) and N-fed (both NH4 + - and NO3 − -fed) T2 homozygous seedlings of T-40-1 and J-77-1, after 3 h exposure to NH4 + (see Materials and methods). N-fed transgenic seedlings had smaller shoot and root weights than those of wild type Taipei 309 (Fig. 6A). Fresh weight reductions were 45%, 48% and 40% for whole plant, shoot and root fresh weights, respectively, in line T-40-1 from that of wild type plants. In line J-77-1, the reductions were 21% and 25% for shoot and root fresh weight, respectively, compared with those of wild type Jarrah. A similar result was observed in NO3 − -fed plants (Fig. 6C). A 7-d N-deprivation resulted in reduced biomass (mainly due to reduction in shoot weight) in the wild type as well as in transgenic plants compared with N-fed plants. Wild type plants showed reductions of 43% and 14% in shoot and root weight, respectively, compared with N-fed plants. In T-40-1 plants, shoot weights were reduced by 21%, whereas root fresh weights did not change relative to those of N-fed plants (Fig. 6A, B). The same trend was observed in wild type Jarrah plants, but with transgenic Jarrah (J-77-1), shoot weight,

T-40

0.8

B

T-38

1 d N-deprived 7

0.7


A 0.8

T-35 NH4+ (µmol g–1 root FW plant–1)

Taipei 309

Biomass, root ammonium accumulation and N uptake under NH4 + / NO3 − , NO3 − and no N nutrition

6

0.6

5

0.5

4

0.4

3

0.3 0.2

2

0.1

1

0

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0

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2 d N-deprived 7

0.7

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5

0.5 4

0.4 0.3

3

0.2

2

0.1

1 0

0 Whole Shoot Root Plant 0.11 0.07 0.05

Root NH4+ NS

NH4+ (µmol g–1 root FW plant–1)

Genotype used in Hygromycin Hygromycin Line transformation resistant susceptible

10 min. The rate of ammonium uptake decreased with time, although line T-40 and T-38 still took up more ammonium, on a fresh-weight basis, than wild type plants during the first 30 min. A similar trend was observed in 2-d-N-deprived plants (Fig. 5B). This higher NH4 + uptake rate was consistent with the OsAMT1-1 mRNA expression level (Fig. 2A) of these transgenic lines.


Table 1. Segregation analysis in the T1 progeny of selected transgenic lines Table value for χ2 (P=0.05) is 3.84


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Fig. 4. Biomass and root ammonium contents of wild type and transgenic Taipei 309 seedlings under N-fed and N-deprived growth conditions. Whole plant, shoot and root weights and root ammonium contents of (A) 16 d NO3 − / NH4 + -fed, (B) 16 d NO3 − / NH4 + -fed and 1-d-N-deprived and (C) 17 d NO3 − / NH4 + fed and 2-d-N-deprived wild-type and transgenic (T-) Taipei-309 plants harvested after 3 h of NH4 + uptake in 75 µM NH4 + . Error bars indicate the standard error of the mean. Least significant difference (l.s.d. at 5% level) values are indicated.


1 d N (NO3– & NH4+)-deprived

A 70

Taipei 309

60

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10 min LSD (5%)

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2 d N (NO3– & NH4+)-deprived

B

µmol NH4+ g–1 root FW h–1



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25.1

1h

3h

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1h

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NS

3.2

9.5

4.3

4.0

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0 Whole Shoot plant

Root

Root NH4+


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A



1.2


Fig. 5. Ammonium uptake rates of transgenic and wild type (Taipei-309) rice plants during short-term N-feeding after 1- and 2-d N-deprivations. Ammonium uptake (as measured by the depletion of ammonium expressed as µmol g−1 root FW h−1 ) by (A) 16 d NO3 − / NH4 + fed and 1-d-N-deprived and (B) 17 d NO3 − / NH4 + fed and 2d-N-deprived wild type and transgenic (T-) Taipei-309 plants 10 min, 30 min, 1 h and 3 h after of NH4 + feeding. Error bars (from five replicate plants) indicate the standard error of the mean. Least significant difference (l.s.d. at 5% level) values are indicated.

Jarrah

Fig. 6. Biomass and root ammonium contents of wild type and transgenic Taipei 309 and Jarrah plants under NO3 − / NH4 + nutrition, Ndeprivation, NO3 − nutrition. Whole-plant, shoot and root weights and root ammonium contents of (A) 21 d NO3 − / NH4 + fed, (B) 14 d NO3 − / NH4 + fed and 7-d-N-deprived and (C) 14 d NO3 − / NH4 + fed and 7-d-NO3 − fed wild type and transgenic (T-40-1) Taipei 309, wild-type and transgenic (J-77-1) Jarrah plants (five replicates) harvested after 3 h of NH4 + uptake in 75 µM NH4 + . Error bars indicate the standard error of the mean.

as well as root weight, were lower compared with N-fed plants (Fig. 6A, B). Transgenic plants T-40-1 had significantly higher root NH4 + content than wild type Taipei 309 in all treatments. The same trend was observed (although to a lesser extent) with Jarrah J-77 1. The uptake of ammonium was 35% and 27% higher in T-40 1 and J-77 1 compared with their respective wild type controls (Fig. 7). Biomass accumulation under high and low NH4 + steady-state N supply Growth measurements with homozygous plants of transgenic lines T-38, T-40, J-75 and J-77 under high steady-state N

supply are presented in Fig. 8. All sense transgenic lines initially (up to 22 d) grew slower than wild type; by day 38, however Jarrah transgenic lines showed signs of recovery. There was a similar trend in Taipei 309 for the multi-copy line T-38, but growth of the line T-40 remained much slower than wild type. Apart from biomass, there was no apparent difference in root phenotype between lines. Under 10 µM NH4 + the two multiple-copy sense lines T-38 and J-75 grew faster than wild type while the single-copy lines behaved similarly to the wild type. Moreover, the multiple OsAMT1-1 copy line J-75, developed

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this low N concentration, all lines displayed extremely poor growth with clear signs of senescence appearing within 4 weeks.

9


8 7

Discussion

6

The role of the ammonium transporter OsAMT1-1 was studied in transgenic plants from two rice cultivars, Taipei 309 and Jarrah. The introduction of an OsAMT1-1 transgene in both cultivars led to an increase in the accumulation of OsAMT1-1 transcripts and multi-copy primary transgenic lines, in general, had higher transcript levels than singlecopy transgenic lines. Physiological studies, with the overexpressing lines, showed that these transgenic plants had increased ammonium uptake per unit root fresh weight and increased root ammonium content, and decreased biomass compared with wild type when grown under high concentrations of NH4 + during seedling and early vegetative stages. However, under a low concentration of NH4 + , transgenic lines, particularly of cv. Jarrah, increased their biomass accumulation at mid-vegetative stage with some lines recovering to wild type levels by day 38. The effect of suppressing OsAMT1-1 using an antisense approach, however, could not be examined because no transgenic lines with antisense-mediated suppression could be regenerated. Rice cultivars Taipei 309 and Jarrah vary in their transformation efficiency, primarily owing to differences

5 4 3 2 1 0

Taipei 309 T 40-1

J-77-1

Jarrah

Fig. 7. Ammonium uptake measurements in transgenic and wild type rice plants by ammonium depletion and membrane depolarisation. Ammonium uptake (as measured by the depletion of ammonium expressed as µmol g−1 root FW h−1 ) by 14 d NO3 − / NH4 + fed and 7-dN-deprived wild type and transgenic (T-40-1) Taipei-309, and wild type and transgenic (J-77-1) Jarrah plants 3 h after of NH4 + feeding. Error bars indicate the standard error of the mean (of five replicates).

very distinctive roots (data not shown) characterised by reduced elongation, much increased diameter, reduced branching due to stunted lateral roots. However, under 13

Taipei 309

Jarrah

11

T-38

J-77

10

T-40

J-75

Whole-plant fresh weight (g)

12

9 8 7 6 5 4 3 2 1 0

15

22

31

38

Plant age (d) Fig. 8. Growth measurements of wild type and homozygous transgenic plants under steady-state high-N supply. Root, shoot and whole-plant fresh weights of Taipei 309, Jarrah, T-38, T-40, J-75 and J-77 on day 15, 22, 31 and 38. Error bars denote standard errors of the mean.


in the regeneration efficiency of callus tissue (Hiei et al. 1994; Hamid et al. 1996; Upadhyaya et al. 2000). As expected, we obtained more sense transgenic lines from Taipei 309, than from Jarrah, using a well-established Agrobacterium-mediated transformation system (Wang et al. 1997; Upadhyaya et al. 2000). Transgenic lines containing the antisense OsAMT1-1 were successfully obtained with Taipei 309. However, the majority of the antisense Taipei 309 transgenic lines were sterile and those which were fertile showed little, or no, reduction in OsAMT1-1 transcript levels compared to that of wild type plants (Fig. 2B). The expression of antisense RNA is usually accompanied by a decrease in level of target mRNA (Wang and Waterhouse 2000). Previous studies with a T-DNA knockout of Arabidopsis AMT1-1 (AtAMT1-1) showed lethality under high ammonium nutrition (Kaiser et al. 2002). Therefore, it is tempting to suggest that efficient suppression of OsAMT1-1 has a lethal effect on callus proliferation and / or regeneration, particularly under high ammonium nutrition in the culture media. The antisense transgenic lines recovered in this study were probably those with less efficient suppression of ammonium transporters as indicated by the expression analyses. Our results indicate that cultivars Taipei 309 and Jarrah have differences in ammonium assimilation and utilisation as they and their transgenic plants showed differential responses to N-nutrition. The reduction in biomass at the early stages of growth, when plants were grown in high NH4 + was less dramatic in the Jarrah transgenic lines than that of Taipei 309 (data not shown). It is also interesting to note that two Jarrah sense transgenic lines increased their biomass accumulation at the mid-vegetative stage. The same trend was observed with respect to delay in flowering resulting from ammonium nutrition (data not shown) in both wild type and transgenic plants. This is consistent with the fact that cultivar Taipei 309, which is closer to the indica type, has a better ammonium absorption system and a poorer ammonium utilisation system than Jarrah which is a pure japonica type (Augladette 1965). This is perhaps the reason why japonica varieties respond better to fertiliser nitrogen than do the indica varieties. It is not possible to attribute ammonium absorption differences in these two cultivars to OsAMT1-1 activity alone, as we have observed higher OsAMT1-1 transcript levels in Jarrah than in Taipei 309. Overexpression of plant membrane transporters have previously been used to determine whether they are the rate limiting factors in mineral uptake and compartmentalisation. In most cases, such overexpressions have shown short-term increases in the uptake. For example, overexpression of an Arabidopsis zinc transporter in barley resulted in increased uptake initially but had no effect on leaf zinc content or shoot biomass under zinc-sufficient or -deficient conditions (Ramesh et al. 2004). However, such an overexpression increased the seed zinc and iron content (Ramesh et al. 2004).


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In a recent study, Rae et al. (2004) observed no improvement in phosphate uptake under phosphate-sufficient or -deficient growth conditions by overexpression of a phosphate transporter gene in barley. The physiological consequences of OsAMT1-1 overexpression in rice cultivars Taipei 309 and Jarrah under various nitrogen regimes, in general, were increased root ammonium content, increased rates of ammonium uptake and reduced total biomass during early vegetative stages. The simplest explanation for these observed effects is ammonium toxicity due to increased uptake without a subsequent increase in assimilation (Mehrer and Mohr 1989). Transgenic lines would require one or more of the following: higher NH4 + efflux rate; ‘safe’ NH4 + storage mechanisms in intracellular locations, or efficient NH4 + assimilation to avoid any toxic or feed-back regulation effects. Higher efflux would involve ammonium cycling across the plasma membrane, which requires energy. Excessive NH4 + has also been shown to block ATP production and reduce CO2 fixation in the chloroplast (Ikeda and Yamada 1981; Puritch and Baker 1967), and affect starch synthesis (Marwaham and Juliano 1976). High ammonium uptake may also prevent water movement from root to shoot (Anderson et al. 1991) and, as a result, some seedlings may not be viable. This appears to be the case with T2 plants from line T-46 (Fig. 3) with highest OsAMT1-1 expression. Moreover, the accumulation of NH4 + itself is an energy-dependent process, which may explain in part the reduction in root growth (Bowman and Paul 1988). It has been shown that rice can store up to 40 mM ammonium in the root cytoplasm (Wang et al. 1993) but the physiological consequence of such high accumulation is not fully understood. The first enzymes involved in ammonium assimilation are glutamine synthases (GSs). A recent genome-wide transcriptome study showed no coordinated regulation of genes for ammonium transport or assimilation during the first 30 min of N deprivation (Scheible et al. 2004). The effects of variations in external N supply on the expression of GS isoforms have been investigated in several studies with contrasting results from different species. For instance, NH4 + appeared to activate the GS2 promoter of rice (Kozaki et al. 1992), where as GS2 polypeptide and mRNA levels in Phaseolus vulgaris did not change following NH4 + addition (Cock et al. 1990). The observed differential responses may be due to the fact that carbon and nitrogen metabolism are regulated by dynamic changes in C-to-N ratios, and by changes in metabolic status (Lam et al. 1995). Decreases in C or N resources up-regulate genes involved in their acquisition, while abundance of these resources induces expression of genes associated with their use and storage (Lam et al. 1995). In that respect, it is interesting to note the observations of Kumar et al. (2003) that NH4 + deprivation induces and NH4 + nutrition represses OsAMT1-1 expression and that OsAMT1-3 responds to diurnal changes

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with an increase late in the photoperiod. Further studies indicate that it is glutamine, the first product of NH4 + assimilation, rather than NH4 + , that controls the expression of ammonium transporter genes in rice (Sonoda et al. 2003b). Although, overexpression of OsAMT1-1 in our transgenic lines was controlled by a strong constitutive promoter (the maize ubiquitin promoter), we cannot rule out possible feed-back regulation as the gene construct had intact 5 and 3 UTR regions (through which any transcriptional or post-transcriptional regulation is most likely to occur). Nevertheless, we believe that ammonium uptake by transgenic lines overexpressing OsAMT1-1 exceeded their capacity to assimilate the ammonium and / or remove NH4 + by efflux from root cells. Does this mean that increasing GS activity can circumvent the perceived bottleneck? It has been shown that tobacco plants overexpressing pea cytosolic GS1 had a considerable growth advantage over wild type plants. In contrast, plants co-suppressed for both chloroplastic and cytosolic GS had decreased GS activity (Lam et al. 1995) and plants grew poorly. Thus, GS could be a rate-limiting enzyme in plant growth and nitrogen use. By increasing the sink for ammonium, via increases in GS and / or other N-assimilating enzymes, it may be possible to avoid the deleterious effects of AMT1 overexpression in rice. Furthermore, to understand the environmental and developmental regulation of ammonium transport activity and tissue specificity, an AMT1-1 promoter–gus fusion analysis also needs to be carried out. Acknowledgments MS Hoque was supported by a PhD scholarship from the CRC for Plant Sciences, Canberra, Australia and by supplementary funding from the Australian National University (Research of Biological Sciences and Department of Biochemistry and Molecular Biology). Transgenic work was carried out at CSIRO Plant Industry, Canberra, ACT and some of the physiological studies were conducted at the Research School of Biological Sciences, ANU, Canberra, Australia. We thank Drs Brent Kaiser, John Watson, Qian-Hao Zhu and Ramesh Bhat for critical reading of the manuscript. We also thank the technical staff from the Rice Functional Genomics Group, CSIRO Plant Industry for their assistance and Dr SC Wong from the Environmental Biology Group at RSBS for setting up the automatic flow-through nutrient system. References Anderson DS, Teyker RJ, Rayburn AL (1991) Nitrogen form effects on early root morphological and anatomical development. Journal of Plant Nutrition 14, 1255–1266. Augladette A (1965) ‘Nutritional status indicated by plant analysis.’ (John Hopkins Press: Baltimore) Beman MJ, Arrigo KR, Matson PA (2005) Agricultural runoff fuels large phytoplankton blooms in vulnerable areas of the ocean. Nature 434, 211–214. doi: 10.1038/nature03370

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Manuscript received 5 July 2005, accepted 21 September 2005

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