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Keywords:

  • cytosolic glutamine synthetase;
  • knockout mutant;
  • nitrogen remobilization;
  • retrotransposon Tos17;
  • rice

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Rice (Oryza sativa L.) plants possess three homologous but distinct genes for cytosolic glutamine synthetase (GS1): these are OsGS1;1, OsGS1;2, and OsGS1;3. OsGS1;1 was expressed in all organs tested with higher expression in leaf blades, while OsGS1;2, and OsGS1;3 were expressed mainly in roots and spikelets, respectively. We characterized knockout mutants caused by insertion of endogenous retrotransposon Tos17 into the exon-8 (lines ND8037 and ND9801) or the exon-10 (line NC2327) of OsGS1;1. Mendelian segregation occurred in each progeny. Homozygously inserted mutants showed severe retardation in growth rate and grain filling when grown at normal nitrogen concentrations. Abnormal mRNA for GS1;1 was transcribed, and the GS1 protein and its activity in the leaf blades were barely detectable in these mutants. The glutamine pool in the roots and leaf blades of the mutants was lower than that of the wild type. Re-introduction of OsGS1;1 cDNA under the control of its own promoter into the mutants successfully complemented these phenotypes. Progeny where Tos17 was heterozygously inserted or deleted during segregation showed normal phenotypes. The results indicate that GS1;1 is important for normal growth and grain filling in rice; GS1;2 and GS1;3 were not able to compensate for GS1;1 function.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In rice (Oryza sativa L.) plants, approximately 80% of the total nitrogen in the ear arise from remobilization through the phloem from senescing organs (Mae and Ohira, 1981). The major forms of nitrogen in the phloem sap are glutamine and asparagine (Hayashi and Chino, 1990). Synthesis of glutamine in senescing organs is the essential step for nitrogen recycling in rice because asparagine is synthesized from glutamine (Ireland and Lea, 1999). Glutamine synthesis occurs via glutamine synthetase (GS, EC 6.3.1.2) which catalyzes an ATP-dependent conversion of glutamate to glutamine using NHinline image derived from fertilizer, nitrate reduction, photorespiration, and numerous other sources including catabolic release of NHinline image during senescence (Ireland and Lea, 1999). Leaves of most plants contain two isoenzymes of GS. These are termed as GS1 and GS2 from their order of elution from an ion-exchange column (McNally et al., 1983). GS1 is localized in the cytosol whereas GS2 has been found in the chloroplasts/plastids in a range of species (Branjeon et al., 1989; Carvalho et al., 1992; Peat and Tobin, 1996; Pereira et al., 1992). GS2 is the major form in green leaves. It is important in the reassimilation of NHinline image released during photorespiration (Blackwell et al., 1987; Wallsgrove et al., 1987). Because barley mutants lacking GS2 were able to grow normally under non-photorespiratory conditions (Blackwell et al., 1987; Wallsgrove et al., 1987), this indicates that GS1 might be important for the synthesis of glutamine for normal growth and development. In general, there is only one gene that encodes GS2, whereas a small multigene family encodes GS1 proteins, which vary from two to six genes in various monocotyledonous and dicotyledonous plants (Ireland and Lea, 1999). Each GS1 gene product has a distinct function (Edwards et al., 1990; Kamachi et al., 1992; Lam et al., 1996; Tingey et al., 1987). Four GS1 genes were found to functionally complement each other in Arabidopsis roots (Ishiyama et al., 2004a).

Rice plants were thought to possess just two GS1 genes: OsGS1 (highly expressed in leaves) and OsGSr (mainly expressed in roots) (Sakamoto et al., 1989). However, the recent successful completion of sequencing of the rice genome has led to the discovery of another gene, OsGS1;3 (Ishiyama et al., 2004b). Consequently, we re-named the previous two genes as OsGS1;1 for OsGS1 and OsGS1;2 for OsGSr. These three genes are highly homologous, and their gene products were indistinguishable at the protein levels with a GS1-specific antibody produced in our laboratory (Kamachi et al., 1992). Our cellular localization studies using the GS1-specific antibody have shown that the GS1 protein is mainly localized in phloem companion cells and related vascular cells of senescing rice leaf blades (Kamachi et al., 1992; Sakurai et al., 1996). In tobacco, one of two GS1 genes is expressed in the vascular tissues of the stem and leaf midrib (Dubois et al., 1996). Promoter analyses also revealed that one of three GS1 genes in pea (Edwards et al., 1990) and kidney bean (Watson and Cullimore, 1996) directed vascular-tissue specific expression in the heterologous transformation system. Localization studies suggest that GS1 is important for glutamine synthesis, which is the major form of nitrogen in phloem sap in rice (Hayashi and Chino, 1990), and for export from mature and senescing leaves (Tobin and Yamaya, 2001). In contrast to the vascular-specific localization of GS1 in the aerial tissues, GS1 protein has been detected in all cell types in rice roots (Ishiyama et al., 1998). Given the recent genetic information, indicating multiple genes for GS1 [i.e., four in Arabidopsis roots (Ishiyama et al., 2004a) and two in rice roots (Ishiyama et al., 2004b)] it is important to reconsider these earlier localization studies in order to determine the function of these distinct GS1 isoenzymes. In rice, the OsGS1;1 transcript was accumulated in the dermatogen, epidermis, and exodermis under nitrogen-limited conditions, whereas the OsGS1;2 transcript was abundantly expressed in the same cell layers under nitrogen-sufficient conditions, replenishing the loss of the OsGS1;1 transcript following ammonium supply (Ishiyama et al., 2004b).

An important approach to gaining an understanding of the precise functions of the GS1 gene products is to use mutants or genetically modified plants. However, unlike GS2, there have been no studies of mutants lacking any GS1 species, even in Arabidopsis thaliana. We have produced transgenic rice with either over-expressed or antisense-inhibited GS1;1 protein in leaves under the control of the native promoter, but we have observed no clear phenotypic characteristics in the T0 generation (Hanzawa et al., 2002). Previous studies illustrate that over-expression of GS isoenzymes is not uniformly linked to the growth of plants. For example, transgenic Lotus corniculatus plants over-expressing soybean GS1 under the control of the 35S promoter exhibited both an accelerated growth rate and early leaf senescence (Vincent et al., 1997). Growth improvements have been reported for poplar trees expressing a conifer 35S::GS1 (Gallardo et al., 1999) and for tobacco expressing 35S::GS1 (Fuentes et al., 2001; Oliveira et al., 2002). In contrast, transgenic alfalfa expressing 35S::GS1 (Ortega et al., 2001) exhibited no difference in growth.

Reverse genetic approaches are powerful for elucidating gene function. For rice plants, seeds of various lines mutated by the random insertion of an endogenous retrotransposon Tos17 (Hirochika et al., 1996) into the rice genome are now available from the Project for Rice Genome Research, the Ministry of Agriculture, Forestry and Fisheries of Japan (mutant panel: http://tos.nias.affrc.go.jp/miyao/pub/tos17/) upon request. The current paper describes the characteristics of OsGS1;1-knockout mutants that were successfully isolated from the mutant pool. The possible function of GS1;1 and genetic evidence for its fundamental role in productivity of rice plants are discussed.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Severe reduction in growth rate and productivity in OsGS1;1 knockout mutants of rice

A line of OsGS1;1 (formally OsGS1; Sakamoto et al., 1989)-knockout mutant (line ND8037) was screened by searching the flanking sequence database (Miyao et al., 2003) of the mutant panel (http://tos.nias.affrc.go.jp/miyao/pub/tos17/) of the Project for Rice Genome Research, where mutant lines, caused by the random insertion of endogenous retrotransposon Tos17 into rice genome (Hirochika et al., 1996), were collected. Another four independent lines were also isolated using PCR screening against the mutant pool. In lines ND8037 and ND9801, Tos17 was inserted into exon-8 (from +2435 to +2431 when the translation start is +1) (Figure 1a). The apparent abnormal order of nucleotide number was caused by the addition of identical 5 bp sequences (5′-GGTTG-3′) at the right and left borders of Tos17 in both lines. In line NC2327, it was inserted into exon-10 with the addition of a 2-bp sequence 5′-AC-3′ (from +2755 to +2754) of OsGS1;1. In two other lines (NF1130 and NC0866), Tos17 was inserted into an intron of GS1;1. These two lines are not used for further analysis in the current study. Mendelian segregation occurred in each progeny.

image

Figure 1. Phenotypic characteristics of OsGS1;1-knockout mutants. (a) Diagram of insertion position of retrotransposon Tos17 in OsGS1;1. Exons are indicated as boxed regions whereas lines represent introns and the 5′- and 3′-untranscribed regions. Open boxes correspond to untranslated sequences. Arrows indicate locations of primers that were designed for PCR screening. (b) Phenotype of the knockout line ND8037 [Tos17 was homozygously inserted into OsGS1;1 (−/−): four pots from the right], wild type (WT), heterozygote (+/−), and no-insertion null plants (+/+) at 54 days after germination. (c) Phenotype of another knockout line NC2373 at 41 days after germination. Symbols for genotypes for Tos17 in OsGS1;1 are the same as in (b). (d) Phenotype at harvest. The knockout line ND8037 (−/−) and the wild type were grown for 140 and 132 days after germination, respectively. (e) Phenotype of panicle and spikelet on a main stem of the knockout line ND8037 (−/−) and wild type. The red and white areas of the scale bar in (b)–(d) are 10 cm, respectively.

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Homozygously inserted mutants from these three lines (ND8037, ND9801, and NC2327) showed severely retarded growth throughout their lifespan. Phenotypic characteristics were apparent at the vegetative stage in ND8037 and NC2327 (Figure 1b,c), and at the ripening stage in ND8037 (Figure 1d). The tiller number was identical between the mutant and wild type at the seedling stage, but leaf-blade elongation was inhibited severely when the mutant plants were grown with a normal supply of nitrogen (Figure 1b,c). The plant height of the mutants at the harvest stage was markedly lower than the wild type; moreover, ripening was retarded, panicle size was very small, and grain filling was severely inhibited (Figure 1d,e, Table 1). In comparison, the null line, in which Tos17 had segregated out (+/+), showed the normal phenotype (Figure 1b,c). Because the phenotypes of the three knockout mutants were identical, one line, ND8037, was chosen as the main material for further analyses.

Table 1.  Reduction of productivity in the OsGS1;1-knockout mutant
LineSpikelet number on a main stem (number/panicle)Fertility (%)One spikelet weight (mg)
  1. All lines were grown in soil until full maturity. The wild-type Nipponbare, heterozygote, and no insertion null lines were harvested at 112 days after planting, whereas the homozygote was allowed to grow for 140 days. Fertility was determined using spikelets on a main stem and spikelet weight was measured using the superior spikelet. Values are mean ± SD.

Wild type (n = 4)62 ± 1092 ± 1024.4 ± 0.5
Homozygote (n = 8)26 ± 112 ± 417.3 ± 0.5
Heterozygote (n = 4)67 ± 586 ± 1024.0 ± 0.3
null (n = 3)68 ± 1290 ± 1023.7 ± 0.7

The ND8037 mutant transcribed two abnormal mRNAs (Figure 2a). Sequencing of these mRNAs revealed that an RNA larger than normal OsGS1;1 mRNA resulted from insertion of Tos17 at 1553 bp of the corresponding GS1;1 cDNA, whereas a smaller band was a 27-bp-truncated RNA from 640 to 666 bp in the normal OsGS1;1 mRNA. The other line, NC2373, also transcribed two abnormal mRNAs: one was larger, as in ND8037, and the other was smaller (data not shown). The latter resulted from truncation of 29 bp between 759 and 787 bp in the OsGS1;1 mRNA with further addition of 18 bp nucleotides (5′-GTTGCAAGTAAGTTAAGA-3′), originated from Tos17, that formed a new stop codon. The GS1 protein (Figure 2b) and its activity (Figure 2c) in the leaf blades were hardly detected in these mutants. A faint band for the GS1 protein was detected in the leaf sheath of the mutants (Figure 2b). This was probably the OsGS1;2 protein, as our GS1 antibody was unable to distinguish between OsGS1;1 and OsGS1;2, and OsGS1;2 was expressed not only in roots but also in the leaf sheath. In line NC2327 also, the GS1 protein was not detected in the leaf blade (data not shown).

image

Figure 2. Analyses of gene products from OsGS1;1-knockout mutants. (a) RT-PCR detection of OsGS1;1 mRNA from roots (lanes 1 and 3) and leaf blades (lanes 2 and 4) in 26-day-old seedlings of the wild type (lanes 1 and 2) and homozygote lines (lanes 3 and 4). (b) Immunodetection of GS1 protein in extract from mature leaf blades (lanes 1 and 2) and leaf sheaths (lanes 3 and 4) of 26-day-old seedlings. We separated 5 μg of soluble protein in crude extract prepared from the wild type (lanes 1 and 3) and homozygote lines (lane 2 and 4). Affinity-purified anti-GS1-peptide IgG was used for immunoblotting. (c) Elution profiles of GS1-semisynthetic activity of extracts from the leaf blades of 26-day-old seedlings. Total activities loaded on the anion-exchange column were 191 and 123 nkat for wild type (open circle) and homozygote lines (filled circle), respectively.

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Complementation of knockout mutants by introducing OsGS1;1 cDNA

Wild-type rice contains two copies of endogenous Tos17 (Hirochika et al., 1996). A genomic DNA blot showed that these three mutant lines possessed four to six additional copies of Tos17 (results not shown). We backcrossed the homozygote with the wild type to reduce the copy number (two to four) and sequenced both the 3′- and 5′-flanking regions of Tos17 insertions in the genome. Although the location of one copy of Tos17 in the rice genome remains unclear, additional copies of Tos17 were not inserted into any other putative open reading frames (results not shown), except into OsGS1;1. Because the heterozygote and null lines showed normal phenotypes and because the homozygote of three allelic knockout lines showed exactly the same phenotypes (Figure 1), we conclude that the severely retarded growth and productivity are attributable to the lack of functional OsGS1;1.

To confirm this conclusion, a full-length cDNA for OsGS1;1 was introduced into the heterozygote plants under the control of its own promoter, using Agrobacterium-mediated transformation (Kojima et al., 2000b). We obtained 148 seeds of generation T1 by self-fertilization. These plants were grown for 26 days after germination and we analyzed their genotypes and phenotype (Figure 3). Diagrams for genotypes and the representative phenotype are shown in Figure 3(a,b), respectively. Genotypes for Tos17 inserted into endogenous OsGS1;1 and the presence of transgene, OsGS1;1-promoter::OsGS1;1 cDNA, were confirmed using PCR (Figure 3c). Table 2 summarizes the phenotypes for all lines at the seedling stage. Of 148 transformant plants, 28 possessed homozygous Tos17 in OsGS1;1 (Table 2). Of these 28 plants, 22 contained the transgene of OsGS1;1-promoter::OsGS1;1 cDNA; these plants showed little or no visible difference in phenotype compared with the wild type. In the remaining six progeny, the OsGS1;1 transgene was segregated out during self-fertilization, engendering severe growth retardation caused by the knockout of OsGS1;1 (Figure 3b). Other genotypes, i.e., heterozygote and no insertion, showed no apparent differences in phenotype between the lines in the presence and absence of the transgene. Therefore, re-introduction of OsGS1;1 cDNA into the knockout mutants successfully complemented the loss of functioning OsGS1;1. Expression of the transgene in the transformants was confirmed (Figure 3d).

image

Figure 3. Complementation of knockout mutants by introduction of OsGS1;1 cDNA. (a) Diagrammatic representation of various genotypes. Terms of homozygote, heterozygote or no insertion are used for the Tos17 inserted into endogenous OsGS1;1. Transgene (+) indicates the presence of OsGS1;1-promoter::OsGS1;1 cDNA in progeny. Arabic numbers correspond to those in Figure 3b,c. (b) Comparison of phenotype of self-pollinated progeny after transformation. OsGS1;1 cDNA under the control of its own promoter was introduced into heterozygote lines. Progeny with [transgene (+)] or without [transgene (−)] were compared in their phenotype with wild type (WT) after 26-day growth. (c) Confirmation of the presence or absence of Tos17-containing OsGS1;1 structural gene (upper panel), that of native OsGS1;1 structural gene, and the transgene, OsGS1;1-promoter::OsGS1;1 cDNA (lower panel) in progeny. Lane 1, homozygote on Tos17 without OsGS1;1-cDNA transgene; lane 2, homozygote with OsGS1;1 cDNA; lane 3, heterozygote without OsGS1;1 cDNA; lane 4, heterozygote with OsGS1;1 cDNA; lane 5, no insertion without OsGS1;1 cDNA; lane 6, no insertion with OsGS1;1 cDNA; lane 7, wild type. (d) Confirmation of expression of transgene in homozygote (upper panel) with RT-PCR. Expression of Tos17-inserted GS1;1 was also confirmed in homozygote (middle panel). Actin mRNA was determined as a positive control (lower panel). Lane 1, homozygote on Tos17 without OsGS1;1 cDNA-transgene; lane 2, homozygote with OsGS1;1 cDNA; lane 3, wild type. PCR was carried out using a suitable set of primers, as described in Experimental procedures. The red and white areas of the scale bar in (b) are 10 cm.

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Table 2.  Complementation of OsGS1;1-knockout mutants by introducing OsGS1;1 cDNA under the control of the OsGS1;1-promoter
LinePlant height (cm)Tiller numberLeaf age
  1. T1 seeds obtained from the transformant were grown for 26 days as described in Figure 3. All lines were tested for the occurrence of Tos17 in endogenous OsGS1;1 as well as for the presence of transgene, OsGS1;1-promoter::OsGS1;1 cDNA, to determine the genotype by PCR, as described in Figure 3(b).

Wild type (n = 6)37.0 ± 1.74 ± 06.6 ± 0.1
Homozygote without OsGS1;1 cDNA (n = 6)9.1 ± 4.53 ± 14.0 ± 1.2
Homozygote with OsGS1;1 cDNA (n = 22)32.7 ± 3.23 ± 15.5 ± 0.4
Heterozygote without OsGS1;1 cDNA (n = 13)31.5 ± 5.03 ± 15.4 ± 0.6
Heterozygote with OsGS1;1 cDNA (n = 45)32.7 ± 5.33 ± 15.5 ± 0.7
null without OsGS1;1 cDNA (n = 25)32.5 ± 5.03 ± 15.5 ± 0.7
null with OsGS1;1 cDNA (n = 37)30.6 ± 5.73 ± 15.6 ± 0.7

Expression profiles of the OsGS1 gene family in rice

Full-length cDNA for GS1;3 was isolated from total RNA of a rice spikelet using RT-PCR and submitted to DDBJ, EMBL, GenBank under accession number AB180689. OsGS1;1 was located on chromosome 2 (Obara et al., 2001). Information for two other genes was obtained from the Rice Genome Database (http://www.dna.affrc.go.jp/misc/bank/index.html) using these cDNA sequences: OsGS1;2 and OsGS1;3 were both detected in BAC clones, AC105364 located in the 106346–111134 minus strand, 30.5 cM, and AC082645 located in the 121625–125570 minus strand, 127.1 cM, on chromosome 3.

Real-time PCR indicated that OsGS1;1 was expressed in all tested organs, i.e., root, leaf blade, and leaf sheath of wild-type rice at seedling or vegetative growth stage, in addition to spikelet at regeneration stage of the plants, under normal growth conditions in the presence of nitrogen supply (Figure 4a). In wild-type rice plants, OsGS1;3 was mainly expressed in spikelets (Figure 4a). As expected, expression of OsGS1;1 in these organs was undetectable in the knockout mutants at both growth stages (Figure 4b). Expression of OsGS1;2 in leaf blades and sheath was identical between the mutants and wild-type plants. As normal spikelets were rarely obtained from the mutants (Table 1), the expression profile of the OsGS1-gene family in this organ could not be shown. The OsGS1;2 transcript was slightly abundant in the roots of the knockout mutants than those in wild-type seedlings (Figure 4a,b). We conclude that OsGS1;2 could provide a certain level of glutamine for growth of the mutants.

image

Figure 4. Accumulation of OsGS1;1, OsGS1;2, and OsGS1;3 mRNAs in wild type and OsGS1;1-knockout rice plants. Quantitative real-time PCR was performed using gene-specific primers and values were normalized relative to the values of actin mRNA in each organ, as described in Experimental procedures. (a) Accumulation of OsGS1;1 (black bars), OsGS1;2 (hatched bars), and OsGS1;3 (open bars) mRNAs in leaf blades (LB), leaf sheaths (LS), and roots (R) of 24-day-old seedlings (seedling) or in those at the eighth position of the ninth leaf stage of rice (mature) grown hydroponically, and spikelet (SP) at 5 days after flowering. (b) Accumulation of OsGS1;1 (black bars), OsGS1;2 (hatched bars), and OsGS1;3 (open bars) mRNAs in the OsGS1;1-knockout. Samples were of the same age as in (a).

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Comparison of amino acid pools in various organs between wild type and the mutants

To analyze the physiological effect of the knockout mutation, the composition of free amino acids in various organs was compared between the mutant and wild-type seedlings (Table 3). The seedlings were the same ones used for the analysis of mRNAs as in Figure 4. Total free amino acids were identical between the roots, i.e., 16.7 and 15.8 μmol g−1 fresh weight of roots for wild type and the mutants, respectively, at 48 h after the supply of 1 mm NH4Cl. Glutamine, asparagine, alanine, and proline were major free amino acids in rice roots. The glutamine content at 48 h was slightly lower in roots of OsGS1;1 knockout mutants than the wild type, whereas glutamate and serine content was significantly higher in the mutant than in the wild type. In the leaf blade of the knockout mutants, glutamine content at 48 h was 8.13 ± 1.22 μmol g−1 fresh weight, which was approximately 60% lower than that of the wild type. Total free amino acids in the leaf blade of wild type and the mutants were 56.6 and 44.2 μmol g−1 fresh weight, respectively. Thus, the lack of OsGS1;1 in the leaf blade caused a significant reduction in total amino acids, which was mainly caused by the reduction in glutamine content in the mutants. Slightly less glutamine was also observed in the leaf sheath of the mutants. Some fluctuations in contents of other amino acids were also seen in the leaf blade and leaf sheath between the mutants and wild type, but their contributions to the total amino acid pool were relatively small. As senescing rice leaf blades contain only a very small amount of glutamine (Kamachi et al., 1991), it is unlikely that the function of OsGS1;1 in young leaves will be the same as in the senescing leaves as reported previously (Kamachi et al., 1992; Sakurai et al., 1996).

Table 3.  Comparison of free amino acids in roots and leaves of wild type with those of OsGS1;1 knockout mutants
Amino acidsRootLeaf bladeLeaf sheath
Wild typeHomozygoteWild typeHomozygoteWild typeHomozygote
  1. Amino acid contents are μmol g−1 FW with SD. Triplicate independent samples were used for the extraction of free amino acids. Wild-type rice and the OsGS1;1 knockout mutants were grown in water for 17 days after germination and in nutrient solution without nitrogen for another 5 days. Then, 1.0 mm NH4Cl was supplied to the nutrient solution and the seedlings were grown for another 48 h.

Gln3.99 ± 0.832.54 ± 0.1021.7 ± 6.678.13 ± 1.2220.2 ± 7.9117.7 ± 3.04
Glu0.19 ± 0.060.39 ± 0.252.24 ± 0.412.58 ± 0.110.38 ± 0.180.72 ± 0.02
Asn6.94 ± 0.785.80 ± 0.2911.8 ± 2.148.62 ± 0.089.81 ± 3.3614.6 ± 4.36
Asp0.52 ± 0.060.58 ± 0.072.11 ± 0.141.56 ± 0.151.20 ± 0.341.95 ± 0.05
Ser0.55 ± 0.070.90 ± 0.091.19 ± 0.131.45 ± 0.021.88 ± 0.624.01 ± 0.11
Gly0.13 ± 0.050.07 ± 0.033.05 ± 0.643.32 ± 0.240.92 ± 0.391.99 ± 0.41
Ala0.75 ± 0.150.81 ± 0.044.75 ± 0.639.55 ± 2.061.52 ± 0.592.89 ± 1.46
Tyr0.04 ± 0.010.05 ± 0.010.16 ± 0.010.15 ± 0.010.09 ± 0.040.15 ± 0.03
Phe0.05 ± 0.010.05 ± 0.030.10 ± 0.010.07 ± 0.020.05 ± 0.020.12 ± 0.03
Lys0.12 ± 0.020.07 ± 0.050.63 ± 0.090.44 ± 0.060.17 ± 0.060.31 ± 0.03
Thr0.12 ± 0.020.13 ± 0.031.18 ± 0.151.04 ± 0.080.59 ± 0.201.07 ± 0.02
Met0.03 ± 0.010.03 ± 0.020.13 ± 0.020.12 ± 0.010.05 ± 0.020.10 ± 0.01
Val0.12 ± 0.020.13 ± 0.040.30 ± 0.030.36 ± 0.030.21 ± 0.070.44 ± 0.11
Leu0.07 ± 0.010.07 ± 0.052.34 ± 0.332.18 ± 0.000.32 ± 0.120.23 ± 0.04
Ileu0.07 ± 0.010.06 ± 0.020.16 ± 0.020.10 ± 0.010.10 ± 0.020.10 ± 0.01
Pro1.25 ± 0.161.51 ± 0.363.34 ± 0.213.77 ± 0.261.33 ± 0.372.54 ± 0.32
Arg0.14 ± 0.020.12 ± 0.081.22 ± 0.110.65 ± 0.041.09 ± 0.391.60 ± 0.24
Cys0.01 ± 0.000.02 ± 0.000.09 ± 0.010.08 ± 0.040.01 ± 0.000.03 ± 0.00
His0.03 ± 0.010.04 ± 0.010.13 ± 0.030.08 ± 0.010.12 ± 0.050.20 ± 0.01
NHinline image1.64 ± 0.062.42 ± 0.040.78 ± 0.110.94 ± 0.010.66 ± 0.131.61 ± 0.22

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Using reverse genetics, we have extended the fundamental understanding of physiological function of GS1;1 in rice. Three independent lines, mutated by the random insertion of an endogenous retrotransposon Tos17 into the OsGS1;1 structural gene, were screened from the Mutant Panel of the Project for Rice Genome Research, the Ministry of Agriculture, Forestry and Fisheries of Japan. These mutants are genetically stable and homozygously inserted progeny showed exactly the same phenotype, i.e., severe reduction in growth rate and grain filling. This severe phenotype was complemented successfully by transformation of OsGS1;1 cDNA under the control of its own promoter. Recent progress in transgenic techniques, enabling either decreasing or over-expressing the GS1-gene, should help in elucidating its role in metabolism. However, transgenic approaches to investigating metabolism pose a severe problem: it is often not repeatable in another laboratory or even the same laboratory because there is no method available to control the insertion site and copy number of a transgene. Hence, genetic approaches are important for comprehensive understanding of ammonium assimilation. Failure to isolate any GS1 knockout mutants in other species could be caused by (i) a lethal mutation or (ii) complementation of GS1 function by another member of the GS1-gene family. Fortunately in rice, it was not a lethal mutation. Nevertheless, meticulous care was required to select these mutants at the seedling stage because of severe growth retardation in the presence of normal nitrogen nutrition.

The OsGS1;1 gene was expressed in all organs tested in wild-type rice plants (Figure 4). In contrast, OsGS1;2 was expressed in roots and in the leaf sheath. In roots, expression of OsGS1;2 was upregulated following the supply of 1 mm NH4Cl into the nutrient solution and became the major transcript for the OsGS1 gene family (Ishiyama et al., 2004b). OsGS1;3 was expressed mainly in spikelets. Similar results were obtained in maize in which one of the five GS1 genes is also preferentially expressed in kernel pedicels (Rastogi et al., 1998). The lack of GS1;1 in the mutants severely reduced growth rate and biomass production (Figure 1). Although the OsGS1;1 knockout mutants showed normal expression profiles for OsGS1;2 and OsGS1;3 (Figure 4b) in all organs tested, neither OsGS1;2 nor OsGS1;3 was able to complement the function of OsGS1;1. Vmax values for all substrates (glutamate, NHinline image, and ATP) of recombinant OsGS1;1 were twice as high as those of recombinant OsGS1;2 (Ishiyama et al., 2004b). This means that OsGS1;1 is more efficient in glutamine synthesis than OsGS1;2. Even at the very young stage of rice seedlings, lack of OsGS1;1 caused significant reduction in the glutamine pool in roots and especially in leaf blades (Table 3). In the leaf blade of the mutants, the large reduction in the glutamine pool was the major factor for the reduction of total free amino acids under the conditions tested. In contrast to glutamine, there were no major changes in NHinline image ions and glutamate pools in the leaf blade between the mutants and the wild-type plants. This result suggests that alternative pathways, such as glutamate dehydrogenase (Tercé-Laforgue et al., 2004), may be involved in the assimilation of NHinline image ions when OsGS1;1 is missing. Fluctuation of amino acid pools were relatively small in roots and leaf sheath where OsGS1;2 was expressed, suggesting that OsGS1;2 could provide a certain level of glutamine in the mutants. Occurrence of OsGS1;2 in roots and sheaths and OsGS1;3 in spikelets probably prevented the mutation from being lethal. In leaf blades, OsGS1;1 gene product was located in companion cells and parenchyma cells of vascular tissues (Sakurai et al., 1996). As suggested previously (Kamachi et al., 1992; Sakurai et al., 1996), OsGS1;1 should be responsible for the generation of glutamine in the remobilization of nitrogen via phloem. Because of the severe reduction in biomass production at the vegetative stage, there should be a shortage of nitrogen and carbon assimilates in senescing source organs, which caused the limitation of remobilization into sink organs. Thus, severe reduction in grain filling could occur in the knockout mutants.

Co-localization of quantitative trait loci for OsGS1;1 protein content and those for agronomic elements for productivity in our earlier study (Obara et al., 2001) suggests that the function of OsGS1;1 is closely related to panicle weight, tiller number, and total spikelet number. Genetic variability of nitrogen use efficiency has also been studied in maize (Gallais and Hirel, 2004), barley (Mickelson et al., 2003), and Arabidopsis thaliana (Loudet et al., 2003). In rice, the effect of quantitative trait loci was further confirmed recently by using chromosome-substituted lines in chromosome 2 (Obara et al., 2004). This inference agrees well with the severe phenotype found in knockout mutants, and strongly suggests that OsGS1;1 is unequivocally the major component of the cytosolic glutamine synthetic pathway in rice leaf blades. As suggested by Ishiyama et al. (2004b) in tissue-specific expression studies, OsGS1;2 is probably important in the primary assimilation of NHinline image taken up by roots. The OsGS1;3 gene was expressed specifically in spikelets, but relative abundance of mRNA for this gene was lower than that of mRNA for OsGS1;1 and OsGS1;2 in this organ. Thus, the three isoenzymes of GS1 in rice plants apparently have distinct functions. Use of knockout mutants or knockdown plants by an RNA interference technique for OsGS1;2 and OsGS1;3 may provide additional evidence for the specialized functions of these GS1 genes in rice plants.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Plant materials

The Project for Rice Genome Research, the Ministry of Agriculture, Forestry and Fisheries of Japan (mutant panel: http://tos.nias.affrc.go.jp/miyao/pub/tos17/) kindly provided 20 seeds each from five lines (ND8037, ND8901, NC2373, NF1130, and NC0866) of rice (O. sativa L. cv. Nipponbare) in which retrotransposon Tos17 could potentially have been inserted into theOsGS1;1 gene. For identification of these knockout mutants, border sequences of Tos17 were surveyed against a sequence of OsGS1;1 from a Sasanishiki cultivar (Kojima et al., 2000a) using the blast program (Altschul et al., 1990). A Nipponbare wild type was also used. Seeds were germinated in distilled water at 30°C for 48 h in the dark. Each germinated seed was planted on a synthetic culture soil in a small container. Twenty days later, one seedling each of these mutants and wild type was transplanted into a 1.3-l plastic pot with 1.0 g of slow-release fertilizer and grown in a greenhouse with irrigation, as described by Obara et al. (2001). For DNA gel blot analysis, 2 g fresh weight of leaf blades on tillers was harvested when the tenth leaf was fully expanded on a main stem, and immediately frozen in liquid nitrogen. A fully expanded leaf blade at the fifth position from the bottom on a main stem was also harvested from 26-day-old plants, weighed, and frozen in liquid nitrogen for preparation of total RNA and soluble protein for immunoblotting and activity measurement. For quantitative real-time PCR analysis, two different growth stages of rice plants were used. The plants were first grown in water for 17 days and then a quarter-strength of nutrient solution (Yamaya et al., 1995) without nitrogen for another 5 days. At 23 days after germination, 1 mm NH4Cl was supplied to the nutrient solution and the seedlings were grown for 48 h. For the analysis with vegetative mature leaves, a fully expanded leaf blade at the eighth position and roots were also harvested. Five-day-old spikelets after flowering were also used for the real-time PCR. Total RNA was prepared from the roots, the fully expanded leaf blade and sheath at 24 h after the supply of NH4Cl. Free amino acids were also determined using the same seedlings, but grown for 48 h in the presence of 1 mm NH4Cl. Total RNA was also prepared from the spikelets of wild-type plants at 5 days after flowering. These samples were stored at −80°C until required.

Screening for the GS1;1 mutant by PCR amplification

We screened 39 mutant panels (NC0–NC8, ND0–ND9, NE0–NE9, and NF0–NF9) for isolation of GS1;1 mutants. NC–NF were three-dimensional (12 × 8 × 10) panels. Two Tos17-specific primers (T17F-1, 5′-ACCACTTCAGAGATTGTGTGGTGC-3′ and T17R-1, 5′-CAGCAACGATGTAGATGGTCAAGC-3′) and five GS1;1-specific primers (GS1;1cDNAF6-25, 5′-TTCTCTCACCGATCTCGTCA-3′; GS1;1cDNAF93-112, 5′-GGATCTCAGGAGCAAGGCTA-3′; GS1;1cDNAR831-812, 5′-GTGCCTGAGCTTGAGCTTCT-3′; GS1;1cDNAR1073-1054, 5′-CTTCAGGGCTTCCAGATGAT-3′; and GS1;1cDNAR1266-1247, 5′-TGGTAGCATGCAACCAAATC-3′) were used in all possible combinations for PCR amplification of 50 ng of genomic DNA from each pooled DNA sample. We used additional Tos17-specific primers (T17F-2, 5′-GACAACACCGGAGCTATACAAATCG-3′ and T17R-2, 5′-AGGAGGTTGCTTAGCAGTGAAACG-3′) for nested PCR to eliminate non-specific amplifications. The amplified DNA fragments were cloned and sequenced to confirm the insertion of Tos17.

Re-introduction of OsGS1;1 cDNA into the mutants

His6-tag codons and a stop codon was added at the C-terminus of a full-length cDNA for OsGS1;1 as described by Ishiyama et al. (2004a)). The entire 2464 kbp 5′-upstream region to the translation start codon of OsGS1;1 (Kojima et al., 2000a) was fused with this construct in pBI101Hm (Kojima et al., 2000b). Agrobacterium-mediated transformation was carried out as described by Kojima et al. (2000b). As host plants, we used calli generated from 68 lines of the mutants harboring Tos17 in OsGS1;1 in a heterozygous manner. We regenerated 17 calli that were confirmed for the presence of transgene; 15 transgenic plants were grown further until maturity. For the complementation test, we used 148 self-pollinated seeds obtained from the transformants. They were grown for 26 days in soil, and phenotypic characteristics as well as genotypic properties, i.e., the presence of Tos17 in OsGS1;1 and of OsGS1;1::OsGS1;1 cDNA, were analyzed. A set of primers (Tos17, 5′-ATTGTTAGGTTGCAAGTTAGTTAAGA-3′; GS1;1cDNAR831-812, 5′-GTGCCTGAGCTTGAGCTTCT-3′) was used for detection of Tos17 in OsGS1;1, whereas another set (GS1;1cDNAF397-416, 5′-ACCCTCCTCCAGAAGGACAT-3′; GS1;1cDNAR831-812, 5′-GTGCCTGAGCTTGAGCTTCT-3′) was adapted for detection of transgene by PCR. Expression of the transgene in the complemented lines was confirmed using RT-PCR. A set of primers (GS1;1cDNAF791-810 5′-AGATCATCAAGTCCGCCATT-3′; GS1;1cDNAR1064-1068-HisTag 5′-ATGGTCAATGATGATGATGATGATGGGGCT-3′) was used for detection of mRNA from the transgene, whereas a set of those described above was adapted for detection of Tos17 in OsGS1;1.

Cloning of OsGS1;3 cDNA

Molecular biological experiments were performed according to standard protocols (Sambrook et al., 1989). A predicted coding sequence (BAC accession number AC082645, location 121625–125570 bp; chromosome 3) for OsGS1;3 was picked from the database of the rice genomic sequence, using the deduced amino acid sequence for OsGS1;1 (cDNA accession number AB037595; Kojima et al., 2000a). A cDNA for OsGS1;3 was isolated from rice-spikelet total RNA using RT-PCR. Total RNA was extracted using CTAB methods with a Sepasol-RNAI solution (Nacalai Tesque Inc., Kyoto, Japan) according to the instruction manual. SuperScriptTM First-Strand Synthesis System for RT-PCR (Invitrogen Corp., Tokyo, Japan) was used to prepare the first-strand cDNA. Two cDNA fragments were amplified using the following primers: GS1;3cDNAF-167–141, 5′-CCTCTGCCTATATAGGCAGGAGCAAAG-3′; GS1;3cDNAR522-498 5′-AACGATGTCGCGCCCGTACGATTTA-3′, and GS1;3cDNAF278-302 5′-TGTGTGACTGTTATGCGCCGAATGG-3′; GS1;3cDNAR1375-1339 5′-ATTGTCATTATTGAGGAGAGTAGATGGTATCTGGAGG-3′. The amplified fragments were cloned into the Hinc II site of pUC118 and sequenced. Fragments of RT-PCR products were ligated using an internal NcoI site to create the full-length cDNA of OsGS1;3 (accession no. AB180689). Alignment of protein sequences was performed using the GENETYX software system ver. 7 (Software Development Co. Ltd, Tokyo, Japan).

DNA gel blotting

DNA gel blot analysis was carried out as described previously (Suenaga et al., 2003). To exclude PCR errors, all PCR products were produced in duplicates and cloned; PCR clones were fully sequenced. A genomic clone of OsGS1;1 (AB037664) was used as a template; DIG-labeled probes were obtained using A PCR DIG Probe Synthesis Kit (Roche Diagnostics Corp., Tokyo, Japan). The following primers were used for OsGS1;1: as forward primer, 5′-ACCTCCTCCAGAAGGACAT-3′; and as the reverse primer, 5′-GTGCCTGAGCTTGAGCTTCT-3′. For DNA gel blotting of Tos17 in the mutants, PCR was performed against a gag-region (+1514 to +2411) of Tos17 (Hirochika et al., 1996) as a template. The following primers were used: as the forward primer, 5′-TGAAGCATCGGTCTCAGCTA-3; and as the reverse primer, 5′-GTAGGTTGGGAGGGTTGTGA-3′.

RT-PCR

Extraction of RNA and synthesis of corresponding cDNA were performed as described above. RT-PCR analysis (Figure 2a) was carried out using OsGS1;1 mRNA-specific primers as follows: GS1;1cDNAF-30–6 5′-GCCTCTTGCTTCCTCCTCCTCATCG-3′ and GS1;1cDNAR831-812 5′-GTGCCTGAGCTTGAGCTTCT-3′. The set of primers for detection of actin mRNA was used as described previously (Sonoda et al., 2003). PCR amplification was performed using TaKaRa Ex-TaqTM polymerase. Amplified fragments were obtained after 35 cycles of amplification. They were electrophoresed on 1.5% (w/v) agarose gel and visualized by ethidium bromide staining (Suenaga et al., 2003). Amplified fragments were all sequenced to confirm their reliability.

Quantitative real-time PCR

Extraction of RNA was performed as described above for cDNA cloning. SuperScriptTM First-Strand Synthesis System for RT-PCR (Invitrogen Corp.) was used to prepare the first-strand cDNA during quantitative real-time PCR. Quantitative real-time PCR analysis was varied using gene-specific primers as follows: GS1;1cDNAF492-519 5′-CAAGTCTTTTGGGCGTGATATTGTTGAC-3′ and GS1;1cDNAR666-650 5′- CTCAAGAATGTAGCGAG-3′ for OsGS1;1 mRNA; GS1;2cDNAF492-519 5′- AAAGGCGTTCGGCCGCGACATCGTGGAC-3′; and GS1;2cDNAR642-615 5′- CACTTGGTCAGCAGCGGCGATGCCAACT-3′ for OsGS1;2 mRNA; GS1;3cDNAF498-525 5′-TAAATCGTACGGGCGCGACATCGTTGAT-3′; and GS1;3cDNAR648-621 5′- GACATGATCCCCTGCGGAGACGCCAA-3′ for OsGS1;3 mRNA. Constitutive expression of Actin mRNA was determined to confirm the equality of mRNA (Sonoda et al., 2003). The values in Figure 4 of three GS1 mRNAs were normalized relative to the values of Actin mRNA. The PCR products were detected as SYBR green fluorescence (Roche Diagnostics Corp.) using LightCycler II (Roche Diagnostics Corp.). The mRNA content was determined quantitatively using a purified cDNA clone as a standard.

Immunoblotting and activity measurement of GS1 protein

Approximately 0.1 g of frozen leaf blade was used for immunoblotting of GS1 subunit protein. Extraction of soluble protein and immunoblotting procedures were essentially the same as those described by Sakurai et al. (1996). Affinity-purified IgG raised against synthetic 17-mer peptide of OsGS1;1 was used for detection of the GS1 subunit protein, as described previously (Yamaya et al., 1992). Activity of GS1 was assayed after separation from GS2 on an ion-exchange column RESOURCETMQ (Amersham Pharmacia Biotech, Inc., Tokyo, Japan) as described previously (Sakurai et al., 1996).

Determination of free amino acids

Triplicate independent seedlings were used for the analysis of free amino acids. Frozen leaf blade (8.2–14.3 mg), leaf sheath (8.4–14.9 mg), or root samples (48.6–92.4 mg) were first powdered in liquid nitrogen and then homogenized in 10 volumes (10 μl for 1 mg sample) of 10 mm HCl. The homogenate was centrifuged and the supernatant fraction was filtered though Ultrafree-MC filter (Millipore, Bedford, MA, USA) and the resulting filtrate was used for analysis of amino acids. Amino acids were determined using Alliance HPLC System (Waters 2475 Multi-λ Fluorescence Detector and Waters 2695 Separation Module with Pico Tag column; Waters Corp. Tokyo, Japan), according to the instruction manual.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank Dr H. Hirochika and Dr A. Miyao, The Project for Rice Genome Research, the Ministry of Agriculture, Forestry and Fisheries of Japan, Tsukuba, Japan, for providing seeds of Tos17-inserted rice mutants. We are grateful to Dr Alyson Tobin, University of St Andrews, UK, for helpful comments and critical reading of the manuscript. We also thank Dr T. Hayakawa and Dr M. Obara for valuable discussions and Mr Y. Hongo and Miss T. Umetsu for technical assistance. This work was supported in part by a Grant-in-aid for Scientific Research (no. 14360035) and that on Priority Area (16085201) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by Core Research for the Evolutional Science and Technology (CREST) of Japan Science and Technology, and in part by The Project for Rice Genome Research (IP1016) from the Ministry of Agriculture, Forestry and Fisheries of Japan.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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Accession numbers: AB037664 for the OsGS1;1 gene, AB037595, AB180688, and AB180689 for cDNAs of OsGS1;1, OsGS1;2, and OsGS1;3.