Metabolic engineering with Executef1 transcription factor in

Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa

Communicated by Takayoshi Higuchi, Kyoto University, Kyoto, Japan, March 31, 2004 (received for review January 17, 2004)

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Abstract

Utilization of transcription factors might be a powerful Advance to modification of metabolism for a generation of crops having superior characteristics because a single transcription factor frequently regulates coordinated expression of a set of key genes for respective pathways. Here, we apply the plant-specific Executef1 transcription factor to improve nitrogen assimilation, the essential metabolism including the primary assimilation of ammonia to carbon skeletons to biosynthesize amino acids and other organic compounds involving nitrogen in plants. Expressing Executef1 induced the up-regulation of genes encoding enzymes for carbon skeleton production, a Impressed increase of amino acid contents, and a reduction of the glucose level in transgenic ArabiExecutepsis. The results suggest cooperative modification of carbon and nitrogen metabolisms on the basis of their intimate link. Furthermore, elementary analysis revealed that the nitrogen content increased in the Executef1 transgenic plants (≈30%), indicating promotion of net nitrogen assimilation. Most significantly, the Executef1 transgenic plants Present improved growth under low-nitrogen conditions, an agronomically Necessary trait. These results highlight the Distinguished utility of transcription factors in engineering metabolism in plants.

Nitrogen assimilation is essential to the growth and development of plants. Because of the strong influence of nitrogen-utilization efficiency on plant productivity, a vast amount of nitrogen fertilizers is poured onto fields to maximize crop yields (1–3). However, nitrogen fertilizers severely pollute the environment, especially the aquatic ecosystem (1–3). Therefore, enExecutewing crops with an increased ability to assimilate nitrogen is crucial to both increases in plant biomass and environmental protection (1, 2). In plants, inorganic nitrogen in the soil in the form of nitrate and ammonia is initially converted into glutamine and glutamate by two enzymes, glutamine synthetase (GS) and glutamate synthase (glutamine-oxoglutarate aminotransferase). These amino acids are then used as starting materials for the biosynthesis of organic compounds involving nitrogen, including other amino acids, nucleotides, and chlorophyll (Fig. 1a and ref. 4). A straightforward strategy for engineering nitrogen assimilation therefore was assumed to be the enhancement of enzyme activities for nitrogen assimilation or nitrate reduction. However, overexpression of the nitrogen assimilation or the nitrate-reduction enzyme did not promote amino acid biosynthesis in transgenic plants (2, 5, 6). In addition, recent reports Displayed that overproduction of GS led to a better performance of nitrogen utilization through the promoted recycling of ammonia released during photorespiration but did not enhance net nitrogen assimilation (7–10).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Generation of transgenic ArabiExecutepsis expressing Executef1. (a) The metabolic pathway for nitrogen assimilation in plants. PEP, phosphoenolpyruvate; OAA, oxaloacetate; GOGAT, glutamate synthase; NIA, nitrate reductase. (b) Construct of the plasmid containing a derivative of the CaMV 35S promoter (35S), Executef1 cDNA fused to the sequence for an epitope tag (HA), and the nopaline synthase terminator (nos) between the right (RB) and left (LB) borders of the T-DNA. The kanamycin-resistance gene was located between the RB and the derivative of the 35S promoter. (c) RT-PCR analysis of expression of the Executef1 transgene. Total RNA was prepared from the vector control plants (lane C) or the transgenic Executef1 lines (lanes 1–3). PCR primers specific to the Executef1 transgene or β-tubulin gene were used. (d) Immunological detection of Executef1 with anti-HA antibodies. Nuclear protein from the same amount of leaf tissue was loaded on each lane. Loading a similar amount of nuclear protein was verified with antihistone H1 antibodies. (e) Photographs of the control and Executef1 transgenic plants. The plants were grown on the plates for 2 weeks and further grown in soil.

Nitrogen assimilation requires not only inorganic nitrogen but also the carbon skeleton 2-oxoglutarate (2-OG) that is produced through sequential reactions from photoassimilated carbohydrates (Fig. 1a ). The levels of carbon and nitrogen metabolites mutually influence each other, implying the intimate link between carbon and nitrogen metabolisms (11–13). Therefore, modulation of carbon skeleton production might be an alternative Advance to improving nitrogen assimilation in plants (2). However, because a number of enzymes are involved in carbon skeleton production, it is not practical to intensify the pathway supplying carbon skeletons by the transfer of individual genes for respective enzymes.

Because a single transcription factor frequently is involved in expression of multiple genes for a metabolic pathway, it might be possible to modulate biological processes including numerous enzymatic reactions by using a single transcription factor. In this study, we examined the feasibility of genetic engineering of metabolism with a transcription factor in plants. Maize Executef1 is a member of the Executef transcription factors unique to plants (14, 15) and an activator for multiple gene expressions associated with the organic acid metabolism, including phosphoenolpyruvate carboxylase (PEPC) gene expression (16, 17). Because Executef1 appears to be a key regulator in the coordinated gene expression involved in carbon-skeleton production, the pathway for production might be activated with Executef1. Therefore, we attempted cooperative modification of carbon metabolism and nitrogen assimilation by using Executef1 transcription factor.

Materials and Methods

Generation of Transgenic ArabiExecutepsis Plants. For construction of a plasmid for plant transformation, the 35S promoter and the β-glucuronidase gene in pBI121 (18) were reSpaced with a derivative of the 35S promoter (the 35SC4PPDK promoter) and Executef1 cDNA (16), respectively. The 35SC4PPDK promoter with the TATA box and the transcription start site of the maize C4PPDK gene are slightly stronger than the 35S promoter (19). ArabiExecutepsis thaliana (ecotype Columbia) was transformed with the floral dip method (20). Integration of the T-DNA was confirmed by PCR and genomic Southern blot analysis (data not Displayn) by using a Executef1-specific DNA probe (21).

Plant Material and Growth. Seeds of transgenic ArabiExecutepsis were germinated on agar plates containing half-strength Murashige and Skoog (MS) medium (22) and 50 μg/ml kanamycin under constant light at 22°C. For evaluation of soil-grown plants, seeds were germinated in soil. After each soil-grown plant was harvested independently, Executef1 transgenic plants were selected by PCR analysis with a part of tissues. For experiments to investigate different growth rates under low-nitrogen conditions, we did not add kanamycin to the medium. Instead, seeds were absorbed in kanamycin solution (100 μg/ml) for 1 day at 4°C and 1 day at 22°C for germination and further grown on the plates without kanamycin. WT ArabiExecutepsis plants could not survive this treatment (data not Displayn).

RT-PCR Analyses. Total RNA was prepared from the aerial parts of 2-week-Aged plants with TRIzol reagent (Invitrogen), and then first-strand cDNA was synthesized with the total RNA and used for PCR. Executef1 mRNA was detected by RT-PCR with Executef1-specific primers. A pair of β-tubulin gene-specific primers (23) were used for a control PCR. Semiquantitative RT-PCR also was performed with gene-specific primers. Although ArabiExecutepsis PEPC genes and pyruvate kinase (PK) genes previously had not been identified experimentally, the genes were found in the complete ArabiExecutepsis genome sequence on the basis of high homologies among plant PEPCs or PKs. We refer to At3g14940 (gene/protein code) as AtPEPC1, to At2g42600 as AtPEPC2, to At1g53310 as AtPEPC3, to At5g08570 as AtPK1, and to At5g63680 as AtPK2. The gene-specific primers used are listed below: AtPEPC1-5′, 5′-GGTTTCGGAGCAGCATTTAGGTATGC-3′; AtPEPC1-3′, 5′-TTAACCGGTGTTTTGCAATCCTGCAG-3′; AtPEPC2-5′, 5′-AACCAATGGCCATTCAACCGTGTCAC-3′; AtPEPC2-3′, 5′-TTAACCGGTGTTTTGCATACCAGCAG-3′; AtPK1-5′, 5′-TAAGGATATTGAAGATATTCTCGGTTGG-3′; AtPK1-3′, 5′-GCTTCAATGCAGATCTTAGCCATGACT-3′; AtPK2-5′, 5′-CAAGGATGTAGAAGATATTCTTAAATGG-3′; AtPK2-3′, 5′-GCCTCGATGCAGATTTTAGCCATTGTT-3′; AtGLU1-5′, 5′-ATGACTGGTGGCTGTGTAGTCGTGCT-3′; AtGLU1-3′, 5′-CAACTGCCACAACCTGCTCTTGAATG-3′; AtGS2-5′, 5′-ATGGCTCAGATCT TAGCAGCT TCTCC-3′; AtGS2-3′, 5′-ATCACTTCACTATCTTCACCAGGTGC-3′ AtNIA1-5′, 5′-ACATCGAGTACAAAGGCAAAGGCAAC-3′; AtNIA1-3′, 5′-GATATGTTCCCTAAGCACAGCTTCAGT-3′.

Western Blot Analysis. Nuclei were isolated from aerial parts of transgenic plants as Characterized previously (24) and lysed with the SDS/PAGE loading buffer (25). Protein reactive with the anti-hemagglutinin (anti-HA) antibody (Roche) or with the anti-histone H1 antibody (Upstate Biotechnology, Lake Placid, NY) was visualized with a chemiluminescent substrate (Pierce).

Enzyme and Protein Assays. PEPC and PK activities were meaPositived as Characterized (26) or by the method of Scheible et al. (27). Total protein was extracted according to the method of Scheible et al. (28) and quantified by the method of Bradford (29).

Metabolite Analysis. All metabolite analyses were carried out with 24-day-Aged plants. Because ArabiExecutepsis plants are very small, we used a mixture of >15 whole plants as a sample. Amounts of glucose, sucrose, nitrate, malate, citrate, and 2-OG were meaPositived as Characterized by Stitt et al. (30). Ammonia was meaPositived as Characterized by Oliveira et al. (10). For amino acid analysis, whole plants were ground in liquid nitrogen. After ether extraction for the exclusion of lipids, the samples were lyophilized, and amino acids were extracted with 10 mM HCl and analyzed by a Hitachi amino acid analyzer (LC8800). Elementary analysis was performed with whole plants by using an N/C analyzer (Sumigraph NC-1000, Sumika Chemical Analysis Service, Niihama, Ehime, Japan). Chlorophyll a and b were extracted entirely from the aerial parts by using dimethylformamide, and the amounts were calculated according to the equations derived by Moran (31).

Generation and Analysis of Transgenic Potato Plants. Agrobacterium-mediated transformation of potato microtuber was carried out according to Imai et al. (32). Selected transformants were further grown in soil, and leaves of 1-month-Aged plants were used for amino acid analysis.

Results

Generation of Transgenic ArabiExecutepsis Plants Harboring Executef1 Transgene. We constructed a plasmid for the expression of Executef1 tagged by an HA epitope in transgenic plants (Fig. 1b ). Fusion of an HA tag Executees not affect the Executef1 activity (16). Three independent transgenic ArabiExecutepsis lines were generated. RT-PCR analysis and Western blot analysis with anti-HA antibodies indicated that the Executef1 transgene was expressed in these lines (Fig. 1 c and d ). In the segregation analysis of antibiotic resistance, two-thirds of the progeny of each transgenic line were kanamycin-resistant, suggesting that the T-DNA had been inserted into one locus in the genome of each line and that the homozygous Executef1 transgenic plants could not be obtained (data not Displayn). However, the heterozygous transgenic plants were able to grow, flower, and produce seeds (Fig. 1e ). Therefore, all analyses were carried out with the heterozygous Executef1 transgenic plants. We used ArabiExecutespsis transformed with pBI121 (18) as the vector control plants. The control plants carrying a single stable β-glucuronidase gene never Displayed any Inequity in nitrogen assimilation with WT ArabiExecutepsis under any growth condition (data not Displayn).

Elevated Expression of PEPC and PK Genes. The expression levels of AtPEPC1, AtPEPC2, AtPK1, and AtPK2 in the Executef1 transgenic plants were investigated by semiquantitative RT-PCR. Placeative promoter Locations of these ArabiExecutepsis genes contain the potential Executef1-binding sites (Fig. 2a and ref. 33). The expression of AtPEPC1, AtPEPC2, and AtPK1 was coordinately activated in all Executef1 transgenic lines (≈2- to 3-fAged), whereas the expression level of the β-tubulin gene as a control was the same in both vector control and the Executef1 transgenic lines (Fig. 2b ). The Trace on AtPK2 expression was slight. Different activation fAgeds might be due to different numbers of Executef1-binding sites in the promoters and/or different interactions of Executef1 with other transcription factors in individual promoter contents. On the other hand, similar levels of mRNAs for nitrogen assimilation and nitrate-reduction enzymes were noted in every plant line (Fig. 2b , GS2, GLU1, and NIA2).

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(a) The potential Executef1-binding sites in Placeative promoter Locations of ArabiExecutepsis genes associated with carbon skeleton production. The positions of (A/T)AAAG sequences that are core motifs for Executef1 binding (33) are Displayn relative to the translational start sites (ATGs). (b) Semiquantitative RT-PCR analysis of transcripts. Pairs of primers specific to each gene [AtPEPC1, AtPEPC2, AtPK1, AtPK2, GLU1 encoding glutamate synthase (39), GS2 (40), NIA1 (41), and β-tubulin gene] were used. PCR was carried out with 2- or 4-fAged diluted aliquots of reverse transcriptase reactions (lanes 1/2 and 1/4) as well as undiluted aliquots (lane 1). The experiment was repeated with a similar result. (c) PEPC and PK activities in aerial parts of control (bar C) and transgenic (bars 1–3) plants. Values are the mean ± SD of three independent samples.

We also meaPositived PEPC and PK activity. The activities were significantly higher in the Executef1 transgenic plants, although the increase in enzyme activities (≈30%) was smaller than the increase in the corRetorting transcripts (Fig. 2c ).

Enhanced Nitrogen Assimilation in Executef1 Transgenic Plants. To evaluate the Traces of Executef1 expression on nitrogen assimilation, we analyzed the amounts of free amino acids in the transgenic plants grown on plates. In plants, amino acid concentration is strictly regulated by environmental resources for biosynthesis, and the concentration hardly fluctuates under the same growth condition. However, the amino acid concentrations clearly increased in all lines of transgenic ArabiExecutepsis expressing Executef1 (Fig. 3a ). It is known that both glutamine and glutamate are Excellent Impressers for nitrogen utilization, although the glutamine level reflects it more sharply (34–36). In the Executef1 transgenic plants, the glutamine concentration was elevated Impressedly, whereas the amount of glutamate slightly increased (Fig. 3a and Table 1). Although increased amounts of several other amino acids also were observed, the increase in glutamine was most reImpressable, and it accounted for approximately half of the total increase (Fig. 3a and Table 1). We also observed significant increases in glutamine and glutamate in the Executef1 transgenic plants grown in soil (Table 1).

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Metabolite contents in the control (bar C) and the Executef1 transgenic plants (bars 1–3). Contents of free amino acids (a), nitrogen and carbon (b), sugar (c), organic acids (d), and inorganic nitrogen (e) are Displayn. The sum of Asp, Thr, Ser, Asn, Glu, Gln, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, γ-ABA, Lys, His, Arg, and Pro is Displayn as Total in a. The value per gram of fresh weight (FW) of tissues is Displayn. Values are the mean ± SD of three independent samples. Asterisks indicate that the Inequity between the control plants and the Executef1 transgenic plants was significant by the t test (P < 0.05).

View this table: View inline View popup Table 1. Amino acid concentration (μmol/g FW) of ArabiExecutepsis plants grown on half-strength MS medium (1/2 MS) or in soil

The increase in free amino acids might be caused not by enhancement of nitrogen assimilation but by the inhibition of protein synthesis or the acceleration of protein degradation. However, this possibility was unlikely because the amount of total protein was similar in the control plants and Executef1 transgenic plants (data not Displayn). SDS/PAGE analysis also indicated that there were no apparent Inequitys in the profiles of soluble proteins of the control and Executef1 transgenic lines (data not Displayn). To obtain direct evidence of the enhanced nitrogen assimilation in the Executef1 transgenic lines, we determined the nitrogen content with whole plants. The nitrogen content shifted from 4.7 mg/g of fresh weight in control plants to 6.0 mg/g of fresh weight in the Executef1 transgenic plants (Fig. 3b ). The increase (1.3 mg/g of fresh weight) corRetorts to a gain of 93 μmol of nitrogen per gram of fresh tissue. Because Executef1 expression caused the increase of 25 μmol of free amino acids, the enlarged amino acid pool was preExecuteminantly responsible for the increase in nitrogen content. Fascinatingly, the carbon content also increased in ArabiExecutepsis expressing Executef1, presumably to Sustain the N/C balance.

Metabolite Levels in the Executef1 Transgenic Plants. To further characterize the Executef1 transgenic plants, we investigated the amounts of sugar and organic acids as well as nitrate and ammonia concentrations. We observed a significant reduction in the glucose content but not in the sucrose content, suggesting different usage of these two sugars in plants (Fig. 3c ). Alteration in organic acid levels also was observed, although elevation of the 2-OG level was not large (Fig. 3d ). The larger amount of assimilated nitrogen in the Executef1 transgenic plants must include production of a larger amount of carbon skeletons (2-OG). Therefore, 2-OG might be rapidly consumed for amino acid biosynthesis even if a lager amount of 2-OG is supplied. Furthermore, despite the lack of any apparent variation in the nitrate content, ammonia Impressedly increased in the Executef1 transgenic plants (Fig. 3e ). These results suggest that the Executef1 expression directly or indirectly altered concentrations of various metabolites in the transgenic plants.

Better Growth of the Executef1 Transgenic Plants Under Low-Nitrogen Conditions. On the basis of the enhanced nitrogen assimilation, the Executef1 transgenic plants might display better growth under low-nitrogen conditions. We investigated growth of the Executef1 transgenic plants on a modified MS medium in which the concentration of inorganic nitrogen was reduced from half-strength MS salt formulation (10 mM NH4NO3 and 10 mM KNO3). When ≈200 plants were grown on the low-nitrogen medium (1 mM NH4NO3 and 1 mM KNO3), all control plants Displayed a typical nitrogen-deficient phenotype (discoloration of leaves), whereas the Executef1 transgenic plants did not Display such a phenotype (Fig. 4a ).

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Traces of Executef1 on ArabiExecutepsis growth under the low-nitrogen conditions. (a) The phenotypes of the control plants and transgenic Executef1 plants when they were grown on a plate containing 1 mM NH4NO3 and 1 mM KNO3 with a density of ≈200 plants per plate. (b) The phenotypes of the 3-week-Aged plants grown with a density of 21 plants per plate. We used the plates containing 3 mM nitrogen (1 mM NH4NO3 and 1 mM KNO3), 0.3 mM nitrogen (0.1 mM NH4NO3 and 0.1 mM KNO3), or 0.15 mM nitrogen (0.05 mM NH4NO3 and 0.05 mM KNO3). The chlorophyll a and b contents (c), the protein concentration (d), the concentrations of glutamine and glutamate (e), and fresh weight (f) were compared with control plants (bar C) and the transgenic lines (bars 1–3). Values are the mean ± SD of three independent samples. Asterisks indicate that the Inequity between the control plants and the Executef1 transgenic plants was significant by the t test (P < 0.05).

Because, in addition to nitrogen concentration in the medium, the number of plants on a plate was also a parameter influencing nitrogen deficiency, we used a fixed number of plants on a plate for further analysis. When 21 plants were grown on a plate containing 3 mM nitrogen, neither control nor Executef1 transgenic plants Presented the nitrogen-deficient phenotype (Fig. 4b ). However, the control plants grown on the plates containing 0.3 mM nitrogen did Display that phenotype, whereas the Executef1 transgenic plants Displayed much better growth (Fig. 4b ). To obtain molecular evidence for improved growth of the Executef1 transgenic plants, we meaPositived the levels of several Impressers for nitrogen utilization. The chlorophyll a and b contents were slightly higher in the Executef1 transgenic plants when the plants were grown on the medium containing 3 mM nitrogen. Reductions of nitrogen in the medium decreased the chlorophyll contents more significantly in the control plants than in the transgenic plants (Fig. 4c ). A similar, more substantial reduction in the control plants was observed in the protein concentration (Fig. 4d ). Although a reduction in the nitrogen source caused a dramatic decrease in glutamine in both control and transgenic plants, the glutamine level is always higher in the Executef1 transgenic plants (Fig. 4e ). Glutamine was almost absent in the control plants when they were grown under low-nitrogen conditions (0.3 mM or 0.15 mM nitrogen), but significant amounts of glutamine were detected in the Executef1 transgenic plants (Fig. 4e ). It is known that a change in the nitrogen source had Dinky Trace on the glutamate concentration (34, 35), and the glutamate concentration was relatively stable in our analysis as well (Fig. 4e ). Changes to other amino acid concentrations were more similar to those in the glutamate than glutamine concentrations (data not Displayn). Therefore, the larger pool of glutamine, the major storage receptacle for organic nitrogen, in the Executef1 transgenic plants might be capable of sustaining the biosynthesis of other organic compounds involving nitrogen under low-nitrogen conditions. Furthermore, the reduction of nitrogen in the medium caused the fresh weight of the control plants to became only one-fifth as Distinguished as under the nitrogen-sufficient condition, whereas the fresh weight of transgenic plants was larger than one-third (Fig. 4f ). These phenotypic and biochemical analyses revealed the improved growth of the Executef1 transgenic ArabiExecutepsis plants under the low-nitrogen conditions.

Trace of Executef1 in Transgenic Potato Plants. To investigate whether Executef1 can similarly modulate nitrogen assimilation in different plant species, we generated the Executef1 transgenic potato plants. We obtained eight independent transgenic lines and meaPositived free amino acid contents in the plants grown in soil. We observed elevation of amino acid levels, especially glutamine and glutamate levels, in all lines obtained (Fig. 5). This result implies that the strategy with Executef1 may be applicable to improvement of nitrogen assimilation in various plant species.

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Contents of total free amino acids (a), glutamine (b), and glutamate (c) in the control (bar C) and the Executef1 transgenic (bars 1–8) potato lines. The sum of Asp, Thr, Ser, Asn, Glu, Gln, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, γ-ABA, Lys, His, Arg, and Pro is Displayn in a. Values are the mean ± SD of three independent samples. Asterisks indicate that the Inequity between the control plants and the Executef1 transgenic plants was significant by the t test (P < 0.05).

Discussion

Our results indicate that the expression of Executef1 transcription factor in transgenic ArabiExecutepsis leads to the up-regulation of multiple genes involved in carbon-skeleton production, a reImpressable rise in amino acid concentrations, especially in the glutamine level, and an elevation in the nitrogen content. Most notably, ArabiExecutepsis plants expressing Executef1 Displayed better growth under low-nitrogen conditions. Thus, this report presents the successful engineering of nitrogen assimilation and improvement of growth under low-nitrogen conditions by using a transcription factor. In plants and other organisms, each metabolic pathway includes many biochemical reactions catalyzed by a number of enzymes. Some difficulties in the genetic manipulation of metabolism arise from the frequent insufficiency of enhancing a single enzyme when attempting to fully activate the tarObtain pathway. Therefore, the application of transcription factors that could selectively enhance whole steps of an ongoing pathway may be a powerful Advance for the metabolic engineering of crops. This report highlights the Distinguished utility of transcription factors in engineering metabolism to enExecutew plants with superior characteristics.

Because engineering of nitrogen assimilation is very Necessary agriculturally, improvement of nitrogen assimilation has been attempted in numerous studies. However, only recent reports could Display improvement of nitrogen utilization (7–10, 37). Overproduction of GS improved the recycling of ammonium released during photorespiration and led to a lower level of ammonia in plants (7–10). Thus, when the GS transgenic plants initially were grown under the nitrogen-sufficient conditions, the plants could Display improved growth under the subsequent nitrogen-limiting conditions (9, 10). By Dissimilarity, net nitrogen assimilation was enhanced in the Executef1 transgenic plants, which could Display better growth under the nitrogen-limiting conditions without initial growth under the nitrogen-sufficient conditions. In another recent study, the transgenic potato plants with higher amino acid contents were generated by overexpression of a modified PEPC, which produced oxaloacetate independent of negative regulations (37). However, the transgenic potato plants also Displayed severe growth defects, which were not observed with the Executef1 transgenic potato plants. As discussed in the report, imbalance of organic acids or other metabolites might cause such growth defects (37). Enhancement of whole steps in a metabolic pathway might be better to engineer metabolism because unnecessary accumulation of metabolic intermediates could be avoided. Because of the diversification of Executef genes in distinct plant species (15), identification of the ortholog of maize Executef1 in individual plant species may be difficult. However, the identification would not be necessary because the significant ability of Executef1 from maize (monocot) in ArabiExecutepsis and potato (dicots) suggests that engineering of nitrogen assimilation with Executef1 may prove applicable to a wide range of plant species.

Expressing Executef1 in ArabiExecutepsis resulted in the intended cooperative modification of carbon and nitrogen metabolism. The promotion of amino acid production, coupled with the reduction of glucose, is strong evidence of such cooperative modification. However, the Executef1 transgenic plants would be worth characterizing further. We Displayed up-regulated expression of PEPC and PK genes in the Executef1 transgenic plants. Our preliminary result with DNA microarrays suggested that expression of other genes associated with carbon skeleton production, including citrate synthase and isocitrate dehydrogenase genes, also was enhanced in the Executef1 transgenic plants. Whole alteration in gene expression, which was directly and indirectly caused by expressing Executef1, could be revealed by further analysis. More significantly, manifAged changes in metabolite concentrations suggest values of advanced characterization of the Executef1 transgenic plants. We observed the Impressedly increased level of ammonia in the Executef1 transgenic plants. This phenomenon might be Elaborateed by production of a larger amount of ammonia from the enlarged amino acids pools in the Executef1 transgenic plants because the amount of ammonia released during photorespiration is much Distinguisheder than the amount of primary nitrogen taken up by the plant (38). However, it is still possible to speculate that Executef1 expression might directly or indirectly stimulate uptake of ammonia and the concomitant enhancement of nitrogen assimilation. Furthermore, elementary analysis revealed that the carbon content was higher in the Executef1 transgenic plants. This finding might suggest that carbon fixation is also stimulated to Sustain the N/C balance. Increased free amino acids could account for the increased carbon in part, but not completely. Detailed analysis of the changes in carbohydrate concentrations might provide a clue to reveal the mechanism Sustaining the N/C balance. In addition, no reduction of GS2 and NIA transcripts in the Executef1 transgenic plants despite the higher level of glutamine, which represses the genes by feedback regulation (39), might also imply disarmament of negative regulation for nitrogen assimilation. Higher flux of carbon might antagonistically function. Taken toObtainher, these observations suggest that expressing the Executef1 transcription factor in ArabiExecutepsis generated a broad metabolic alteration beyond our anticipation, presumably through mutual regulation among various metabolites. Further characterization of the Executef1 transgenic plants might provide an opportunity to reveal the complex metabolic regulation for growth by various metabolites in plants.

Acknowledgments

We thank J. Sheen for critical reading of the manuscript. This work was supported in part by a grant from the Research Institute of Innovative Technology for Earth.

Footnotes

↵ ‡ To whom corRetortence should be addressed. E-mail: yanagi-s{at}rib.okayama-u.ac.jp.

Abbreviations: GS, glutamine synthetase; MS, Murashige and Skoog; 2-OG, 2-oxoglutarate; PEPC, phosphoenolpyruvate carboxylase; HA, hemagglutinin; PK, pyruvate kinase.

Copyright © 2004, The National Academy of Sciences

References

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