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Increased protein content in transgenic Arabidopsis thaliana over-expressing nitrate reductase activity
Plant Science 130 (1997) 41 – 49
Increased protein content in transgenic Arabidopsis thaliana over-expressing nitrate reductase activity Ali Nejidat a, Genfa Zhang b, Michal Grinberg b, Yair M. Heimer b,* a
En6ironmental Microbiology Unit, The Jacob Blaustein Institute for Desert Research, Ben-Gurion Uni6ersity of The Nege6, Sede Boqer Campus 84993, Israel b Albert Katz Center for Desert Agrobiology, The Jacob Blaustein Institute for Desert Research, Ben-Gurion Uni6ersity of The Nege6, Sede Boqer Campus 84993, Israel Received 17 January 1997; received in revised form 21 July 1997; accepted 5 September 1997
Abstract Arabidopsis thaliana was engineered to over-express nitrate reductase (NR) by virtue of the light inducible chimeric gene Lhcb1*3::Nia1*2. The transgenic lines obtained displayed NR activity 2 – 4 times the level in the wild type, depending on the line. While not displaying advantage with respect to fresh or dry weight, 7-day-old transgenic seedlings did show up to 200% higher protein content than the wild type when grown on solid medium. Up to 30% increase in protein content was also obtained when grown in peat moss for at least 3 weeks. The increase in protein content was evident in several protein bands but was most apparent in that of the large subunit of RuBisCo. © 1997 Elsevier Science Ireland Ltd. Keywords: Arabidopsis thaliana; Chimeric gene; Nitrate; Nitrate reductase; Over-expression; Protein content
1. Introduction The ever-growing world population constantly imposes the need for increasing food supply. Thus, the potential of contributing of genetic engineering to crop improvement is of even greater significance.
* Corresponding author. Fax: +972 7 6596742; e-mail: [email protected]
Carbon and nitrogen are major constituents of plant biomass. Their availability in the environment and their incorporation into cellular components are major factors in plant growth and biomass production [1]. Nitrate is the most favorable nitrogen source for plants [2]. Other nitrogen fertilizers are readily converted to nitrate by soil microorganisms. Information is available pointing to interlocking between nitrogen and carbon assimilation in plants [3–5]. Because of to its key role in plant
0168-9452/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 1 6 8 - 9 4 5 2 ( 9 7 ) 0 0 2 0 3 - 3
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A. Nejidat et al. / Plant Science 130 (1997) 41–49
metabolism, one would expect plants to utilize any available nitrogen (nitrate) in their environment for the purpose of biomass production. However, providing plants with excess nitrogen fertilizers, a common agricultural practice, results in nitrate accumulation in the plant on the one hand and its leaching into ground water on the other, both having undesired environmental effects. Thus, there seems to be upper limits to resource utilization and, consequently, plant performance. The immediate question is, therefore, what are the factors which determine the upper limits of resource utilization. Genetic engineering techniques could make possible the exploration of those upper limits, which might provide an answer to that question. Modulation of the metabolic pathways of carbon fixation and nitrogen assimilation by means of genetic engineering has been attempted ([1,6] and references therein). The reduction of nitrate to nitrite carried out by the enzyme nitrate reductase (NR) has been claimed to be the rate-limiting step in the assimilation of nitrate nitrogen by plants [2,7]. However, there are puzzling exceptions, such as the mutants of barley, tobacco and Arabidopsis, which, while retaining only 10% of NR activity found in the wild type, can grow just as well on nitrate as the sole nitrogen source ([2] and references therein). NR, being the first reducing site in the nitrate assimilation pathway, might be the target of choice for genetic manipulation. Indeed, Quillere et al. [8] have reported that transgenic Nicotiana plumbaginifolia plants, constitutively over-expressing NR activity, display significant reduction in NO3− level and the implication of these findings to crop quality was suggested. These authors also reported an increase in the pools of free glutamine and malate in those transgenic lines. However, the N. plumbaginifolia system does not exhibit advantage with respect to total protein and biomass production when plants are grown under a variety of growth conditions [1,6,8]. At the time when the reports of Quillere et al. [8] were published we were addressing the same question using another plant system, that of Arabidopsis thaliana. Seven transgenic lines harboring the light inducible chimeric gene Lhcb1*3::Nia 1*2 [9] which over-express NR activity were exam-
ined. The results obtained indicate that A. thaliana responded differently than N. plumbaginifolia, particularly to over-expression of NR activity, by attaining higher protein contents.
2. Materials and methods
2.1. Plant material Wild type A. thaliana (L) Heyn. ecotype Wassilevskija (WS) and transgenic lines harboring the chimeric gene Lhcb1*3::Nia1*2 [9] were employed throughout this study. Five hundreds seeds from each line were surface sterilized as previously described [9], sown on Whatman No. 1 filter paper discs which were placed on top of Murashige and Skoog (MS) solid medium [10], and given a cold treatment (4°C) for 48 h in darkness. Seeds were also sown on peat moss in 4-inch pots watered with MS medium. Plants were grown under 16/8 h day/night cycles at 24°C. Photon flux density was 150 mmol m − 2 s − 1 (400–700 nm) emitted by fluorescent tubes (40 W/D/T12, day light, Sylvania Standard) supplemented with 60 W tungsten lamps. Plants were harvested, rinsed with distilled water to remove residual growth media, blotted to remove excess water, wrapped in tin foil, weighed and immediately placed in liquid nitrogen and stored at − 20°C. Precautions were taken to complete the entire harvest procedure rapidly to avoid undesirable loss of moisture from plant material.
2.2. Assay of nitrate reductase acti6ity Extraction and assay of NR were conducted as previously reported [9]. Frozen samples (see Section 2.1) were extracted in 100 mM K2PO4 buffer pH 7.5 (2.0 ml g − 1 fresh weight, FW), containing 1 mM EDTA, 5 mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride (PMSF) and 10 mM Leupeptin, using a pre-chilled mortar and pestle. After centrifugation in a microcentrifuge for 15 min at 4°C, 50 ml of the supernatant were used for each assay which was carried out in 50 mM K2PO4 buffer pH 7.5 containing 10 mM KNO3 and 0.1 mM NADH in a total volume of 500 ml at 30°C for 30 min. Assays were carried out in
A. Nejidat et al. / Plant Science 130 (1997) 41–49
triplicate. The reaction was stopped by the addition of 500 ml of 1% sulfanilamide in 3 N HCl followed by 500 ml of 0.02% N-naphthylethylenediamine dihydrochloride. A blank sample, in which sulfanilamide was added prior to the extract, was used for background reading. Absorbancy at 540 nm was measured. Enzyme activity was expressed as nmoles NO2− formed per hour on the basis of either milligrams of protein, unit fresh weight or 1000 seedlings.
2.3. Determination of nitrate content The extract used to determine NR activity was also used to determine nitrate content. The dissimilatory NR of Escherichia coli was employed [11] to reduce nitrate to nitrite, which was determined colorimetrically (see Section 2.2).
2.4. Protein extraction and analysis Total protein was extracted from frozen samples according to a published procedure [12,13], in a buffer (1.0 ml g − 1 FW) containing 125 mM Tris, 4 mM EDTA, 10 mM ascorbic acid, 10 mM KHSO3 (metabisulfite), 4 mM PMSF, 10 mM Leupeptin, 4.6% sodium dodecyl sulfate (SDS), 10% b-mercaptoethanol and 20% glycerol, using a pre-cooled mortar and pestle. The extract was incubated in boiling water for 5 min and subsequently centrifuged for 10 min in a microcentrifuge at maximum speed. Protein in the supernatant (100 ml aliquots) was precipitated by 10% trichloroacetic acid, washed twice with ethanol to remove low molecular weight compounds, dried and quantified by the method of Lowry et al. [14]. For analysis of the protein profile, aliquots were combined with sample buffer [13], boiled for 2 min and subjected to SDS-polyacrylamide gel electrophoresis (PAGE) in 12% acrylamide gels. The proteins were either stained with Coomasie Brilliant Blue or electrophoretically transferred onto nitrocellulose membrane for immuno-detection.
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3. Results and discussion
3.1. Nitrate reductase acti6ity in transgenic plants Seven independent transgenic lines, homozygous with respect to kanamycin resistance, a selectable marker which was introduced together with the Lhcb1*3::Nia 1*2 chimeric gene [9], were used in this study. In an earlier study the chimeric gene was shown to be expressed at the mRNA and the enzyme activity levels in line A4, when grown in the absence of nitrate and in the presence of L-glutamine as the sole nitrogen source, conditions which downregulate the expression of the endogenous NR genes [9]. In the present study we have analyzed all the transgenic lines obtained for the level of NR activity. As shown in Fig. 1, the seven transgenic lines displayed elevated NR activity as compared with the wild type when examined after 7 days of growth on solid MS medium containing 1 mM or 10 mM KNO3. Although the absolute values of NR activity in seedlings grown on 1 mM KNO3 were rather low (Fig. 1), the transgenic lines displayed up to 10 times the NR activity in the wild type. As expected, on 10 mM KNO3 the levels of NR activity were appreciably higher in the wild type and the transgenic lines than in those grown on 1 mM KNO3. The transgenic lines exhibited 117–300% of the NR activity of the wild type (Fig. 1). Although NR activity at 10 mM was higher than that at 1 mM, the extent of enhanced NR activity in the transgenic lines compared to the wild type was smaller at 10 mM nitrate in the growth medium than at 1 mM. One possible explanation for this is that there is an upper limit to the level of NR activity that the plant can accommodate. The growth of the wild type and the transgenic lines on 1 mM KNO3 was relatively poorer than that of those grown on 10 mM KNO3 as judged by the fresh and dry weights (Fig. 2). Apparently, the enhanced NR activity in the transgenic lines was not accompanied by an increase in biomass production (Fig. 2). These results correspond with those reported by Quillere et al. [8]. The level of NR activity in the wild type and transgenic lines exhibited light–dark fluctuations, being highest at the mid-light period and lowest just before the
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Fig. 1. Nitrate reductase activity in wild type and transgenic lines as a function of nitrate concentration in the growth medium. Five hundreds seeds of each of the lines were sown on filter paper and grown for 7 days (as described in Section 2) on solid medium containing 1 or 10 mM KNO3. Seedlings were removed from the filter paper, placed in liquid nitrogen and kept frozen at −20°C until used. The results are the average 9S.D. of five experiments. WT, wild type; A1 and A3 – A8, transgenic lines.
end of the dark period (Fig. 3). This was expected since, on one hand, the expression of the endogenous NR gene was shown to be light dependent [15] while, on the other hand, the chimeric gene used was driven by a light inducible promoter [9]. Elevated levels (up to 3-fold) of NR activity were also observed in seedlings grown in peat moss for 3 weeks (data not shown). In the present study we used seeds from T4 – T6 generations. We did not observe decrease in NR activity in six consecutive generations (data not shown).
3.2. Protein and biomass production Quillere et al. [8] were able to show a 32 – 47% decrease in the nitrate level of leaves of N. plumbaginifolia plants which constitutively over-expressed NR. However, in the transgenic Arabidopsis lines the levels of endogenous nitrate were similar to those of the wild type or even somewhat higher (Table 1). This indicates that the nitrate uptake machinery is not the rate-limiting step in nitrate assimilation in Arabidopsis under our experimental conditions.
Since the reduction of nitrate to nitrite and subsequently to ammonia is a prerequisite for the synthesis of amino acids and proteins, total protein content in the wild type and transgenic lines was determined. Seedlings of the transgenic lines exhibited higher contents of total protein when grown for 7 days on solid medium (Fig. 4) containing 1, 10 or 20 mM nitrate. The elevated level of total protein (up to 200%) was more significant at 10 or 20 mM nitrate. Apparently, the advantage of over-expression of NR in the transgenic lines was manifested when the ambient nitrate concentration exceeded that which could be utilized by the wild type. The increase in protein content was not correlated with the extent of over-expression of NR activity. This may be explained by a possible different position effect of the introduced chimeric gene in each of the independent transgenic lines. The available methodologies do not yet offer a control over the site of the introduction of transgenes. A significant increase (up to 30%) in total protein content was also observed in seedlings grown on peat moss for
A. Nejidat et al. / Plant Science 130 (1997) 41–49
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Fig. 2. Fresh and dry weights of seedlings of wild type and transgenic lines as a function of nitrate concentration in the growth medium. Experimental details as in Fig. 1.
at least 3 weeks (data not shown). SDS-PAGE analysis of total protein extracted from 7-day-old seedlings grown on peat moss indicated that the increase in protein content was evident in many protein bands (Fig. 5) and was most apparent in that of the large subunit of RuBisCo. The promoter used for constructing the chimeric gene was that of the Lhcb1*3 gene. It was thus of interest to determine whether it affected the level of the Lhcb protein. Total protein extracts of the various lines (from a different
experiment than those reported in Fig. 5) were subjected to a Western blot analysis using rabbit anti-Lhcb antibodies in order to determine the level of the Lhcb protein in the extracts (Fig. 6). The results clearly indicate that the introduction of the chimeric gene did not inhibit the accumulation of the Lhcb protein. On the contrary, the level of this protein was significantly higher in most of the transgenic lines than in the wild type, which is in agreement with the results of the SDS-PAGE analysis (Fig. 5). Apparently, if there
A. Nejidat et al. / Plant Science 130 (1997) 141–149
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Fig. 3. Level of extractable NR activity during light and dark periods in wild type and transgenic Arabidopsis lines. Growth conditions as in Fig. 1. Seedlings were harvested either at the mid-light period, the mid-dark period or at the end of the dark period. The extractable activity of wild type plants at the middle of the light period was taken as 100%. WT, wild type; A1, A3, A4 and A8, transgenic lines.
was an interaction between the promoter of the chimeric gene and that of the host Lhcb genes it did not cause cosuppression as previously reported [16]. One is tempted to compare the results obtained in the present study with those of Quillere et al. [8]. It appears that transgenic A. thaliana and transgenic N. plumbaginifolia responded differently to elevated NR activity. N. plumbaginifolia plants which constitutively over-express NR activ-
ity by virtue of the 35S cauliflower mosaic virus (CaMV) promoter and display up to 5-fold increased NR activity as compared with the wild type, show a significant decrease in nitrate content and significant increase in the free pool of several amino acids, mainly L-glutamine. However, they do not display an increase in total protein content. On the other hand, transgenic Arabidopsis which over-express NR by virtue of a light inducible promoter, do not display decreased nitrate
Table 1 Nitrate content in A. thaliana wild type and transgenic lines over-expressing NR Line WT −1 mmol NO− 61.2 (2.2) 3 g FW % of WT 100
A1 63.4 (2.5) 103.5
A3 62.6 (6.1) 102.2
A4 72.4 (6.7) 118.3
A5 75.1 (5.9) 122.7
A6 74.6 (6.8) 121.8
A7 64.8 (3.5) 105.8
A8 71.1 (4.2) 116.1
Seedlings were grown for 7 days on MS medium containing 20 mM KNO3. the results are the average of three experiments (standard deviation in parentheses). FW, fresh weight; WT, wild type.
A. Nejidat et al. / Plant Science 130 (1997) 141–149
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Fig. 4. Total protein content in wild type and transgenic lines as a function of nitrate concentration in the growth medium. Five hundred seeds of each of the lines were sowed on filter paper and grown for 7 days (as described in Section 2) on solid medium containing 1, 10 or 20 mM KNO3. Seedlings were removed from the filter paper, placed in liquid nitrogen and kept frozen at − 20°C until used. Total protein were extracted and determined as described in Section 2. The results are the average 9S.D. of five experiments. WT, wild type; A1 and A3–A8, transgenic lines.
content but do show a significant increase in total protein content. Several explanations could account for this different response. The simplest is that different plants species respond to genetic manipulation of nitrogen metabolism in a different manner, reflecting the uniqueness of their genetic make-up. This could have also been affected by the fact that N. plumbaginifolia is a cultivated species selected for specific performance, whereas A. thaliana is a wild species. Another explanation is that the different responses reflect the nature of the chimeric genes used to transform the two plant species. The 35S CaMV promoter used to drive the expression of the NR cDNA in N. plumbaginifolia and the Lhcb1*3 promoter used to drive the expression of the NR gene in Arabidopsis are known to have different tissue specificities [17,18]. Furthermore, the Lhcb1*3 promoter is light regulated. It should be noted that NR can be found in the cytosol of cells of Arabidopsis shoots and roots [2]. Indeed, both
wild type and transgenic Arabidopsis lines displayed NR activity in the roots being higher in the latter (data not shown). Several arguments support the causal relationship between elevated protein contents and overexpression of NR: (1) the over-expression of NR on the one hand, and the concomitant elevation of protein content on the other, occurred in seven independent transgenic lines in which the sites of insertion of the chimeric gene are likely to be different; (2) somaclonal variation, a rather rare event, could not account for that either, since all the transformed lines tested displayed similar results; (3) the over-expression of NR and the elevated protein content occurred in every experiment in which the plants grew either on solid medium or on peat moss, under growth-room or greenhouse conditions; (4) the elevated protein content becomes significantly apparent only at high ambient nitrate, the utilization of which is limited by the level of NR in the wild type.
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mining crop quality and thus points to a possible approach for crop improvement. On the other hand, the different results obtained with N. plumbaginifolia should be taken as a note of caution, indicating that success is not certain. Each crop plant may require particular attention especially with respect to the construction of the appropriate chimeric gene to be introduced to fit its specific genetic make-up.
Acknowledgements Ali Nejidat was supported by a Deichman Fellowship. Genfa Zhang was a postdoctorate fellow of The Jacob Blaustein International Center. The authors thank Prof. Rachel Nechushtai, for supplying antibodies against Lhcb protein, and Dr. Elliott Birnbaum for editing the manuscript. This research was supported in part by a grant from The Moriah Foundation.
References
Fig. 5. Increased abundance of various proteins in transgenic plants. Five microliters of total protein extracts of 1-week-old wild type and transgenic lines grown on peat moss were subjected to SDS-PAGE. Protein bands were visualized by Coomasie Brilliant Blue. WT, wild type; A1, A3, A4 and A8, transgenic lines; M, molecular weight markers.
The consequence of over-expression of NR in A. thaliana was the increase in protein content mainly at high ambient nitrate concentrations. This feature is of immense importance in deter-
Fig. 6. Immunoblot analysis of the abundance of Lhcb protein in wild type and transgenic lines. Transferred proteins (see Section 2) were reacted with rabbit antibodies against Lhcb protein followed with alkaline phosphatase-conjugated goat anti-rabbit antibodies. WT, wild type; A1 and A3–A8, transgenic lines.
[1] C.H. Foyer, S.F. Ferrario, Modulation of carbon and nitrogen metabolism in transgenic plants with a view to improve biomass production, Biochem. Soc. Trans. 22 (1994) 909 – 915. [2] N.M. Crawford, Nitrate, phosphate, and iron assimilation in Arabidopsis, in: E.M. Meyerowitz, C.R. Somerville (Eds.), Arabidopsis, Laboratory Press, Cold Spring Harbor, 1994, pp. 1119 – 1146. [3] C.-L. Cheng, G.N. Acedo, M. Cristinsin, M.A. Conkling, Sucrose mimics the light induction of Arabidopsis nitrate reductase gene transcription, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 1861 – 1864. [4] I. Suzuki, C. Cretin, T. Omata, T. Sugiyama, Transcriptional and post-transcriptional regulation of nitrogen-responding expression of phosphoenolpyruvate carboxylase gene in maize, Plant Physiol. 105 (1994) 1223 – 1229. [5] M. Vincentz, T. Moureaux, M.T. Leydecker, H. Vaucheret, M. Caboche, Regulation of nitrate and nitrite reductase expression in Nicotiana plumbaginifolia leaves by nitrogen and carbon metabolites, Plant J. 3 (1993) 315 – 324. [6] C.H. Foyer, M. Chaumont, E. Murchie, N. Galtier, S. Ferrario, End-product modulation of carbon partitioning with a view to improve biomass production, in: M.A. Madore, W.J. Lucas (Eds.), Carbon Partitioning and Source – Sink Interactions in Plants, American Society of Plant Physiologists, 1995, pp. 45 – 55.
A. Nejidat et al. / Plant Science 130 (1997) 41–49 [7] L. Beevers, R.H. Hageman, Nitrate reduction in higher plants, Annu. Rev. Plant Physiol. 20 (1969) 495–522. [8] I. Quillere, C.H. Dufosse, Y. Roux, C.H. Foyer, M. Caboche, J.F. Morot-Gaudry, The effects of deregulation of NR gene expression on growth and nitrogen metabolism of Nicotiana plumbaginifolia plants, J. Exp. Bot. 45 (1994) 1205 –1211. [9] Y.M. Heimer, J.A. Brusslan, D. Kenigsbuch, E.M. Tobin, A chimeric Lhcb::Nia gene: an inducible counter selection system for mutants in the phytochrome signal transduction pathway, Plant Mol. Biol. 27 (1995) 129–136. [10] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassay with tobacco tissue culture, Physiol. Plant. 15 (1962) 473–497. [11] A.L. McNamara, G.B. Meeker, P.D. Shaw, R.H. Hageman, Use of dissimilatory nitrate reductase from Escherichia coli and formate as a reducing system for nitrate assays, J. Agric. Food Chem. 19 (1971) 229–231. [12] W.J. Hurkman, C.K. Tanaka, Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis, Plant Physiol. 81 (1986) 802–806.
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[13] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680 – 685. [14] O.H. Lowry, N.J. Rosenbrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265 – 275. [15] V.K. Rajasekhar, G. Gowri, W.H. Campbell, Phytochrome-mediated light regulation of nitrate reductase expression in squash cotyledons, Plant Physiol. 88 (1988) 242 – 244. [16] H. Vaucheret, M. Caboche, Induction of nitrate reductase host gene expression has a negative effect on the expression of transgenes driven by the nitrate reductase promoter, Plant Sci. 107 (1995) 95 – 104. [17] P.N. Benfy, N.-H. Chua, Regulated genes in transgenic plants, Science 244 (1989) 174 – 182. [18] P.N. Benfy, L. Ren, N.-H. Chua, The CaMV 35S enhancer contains at least two domains which can confer deferent developmental and tissue specific expression pattern, EMBO J. 8 (1989) 2195 – 2202.
Increased protein content in transgenic Arabidopsis thaliana over-expressing nitrate reductase activity Ali Nejidat a, Genfa Zhang b, Michal Grinberg b, Yair M. Heimer b,* a
En6ironmental Microbiology Unit, The Jacob Blaustein Institute for Desert Research, Ben-Gurion Uni6ersity of The Nege6, Sede Boqer Campus 84993, Israel b Albert Katz Center for Desert Agrobiology, The Jacob Blaustein Institute for Desert Research, Ben-Gurion Uni6ersity of The Nege6, Sede Boqer Campus 84993, Israel Received 17 January 1997; received in revised form 21 July 1997; accepted 5 September 1997
Abstract Arabidopsis thaliana was engineered to over-express nitrate reductase (NR) by virtue of the light inducible chimeric gene Lhcb1*3::Nia1*2. The transgenic lines obtained displayed NR activity 2 – 4 times the level in the wild type, depending on the line. While not displaying advantage with respect to fresh or dry weight, 7-day-old transgenic seedlings did show up to 200% higher protein content than the wild type when grown on solid medium. Up to 30% increase in protein content was also obtained when grown in peat moss for at least 3 weeks. The increase in protein content was evident in several protein bands but was most apparent in that of the large subunit of RuBisCo. © 1997 Elsevier Science Ireland Ltd. Keywords: Arabidopsis thaliana; Chimeric gene; Nitrate; Nitrate reductase; Over-expression; Protein content
1. Introduction The ever-growing world population constantly imposes the need for increasing food supply. Thus, the potential of contributing of genetic engineering to crop improvement is of even greater significance.
* Corresponding author. Fax: +972 7 6596742; e-mail: [email protected]
Carbon and nitrogen are major constituents of plant biomass. Their availability in the environment and their incorporation into cellular components are major factors in plant growth and biomass production [1]. Nitrate is the most favorable nitrogen source for plants [2]. Other nitrogen fertilizers are readily converted to nitrate by soil microorganisms. Information is available pointing to interlocking between nitrogen and carbon assimilation in plants [3–5]. Because of to its key role in plant
0168-9452/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 1 6 8 - 9 4 5 2 ( 9 7 ) 0 0 2 0 3 - 3
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A. Nejidat et al. / Plant Science 130 (1997) 41–49
metabolism, one would expect plants to utilize any available nitrogen (nitrate) in their environment for the purpose of biomass production. However, providing plants with excess nitrogen fertilizers, a common agricultural practice, results in nitrate accumulation in the plant on the one hand and its leaching into ground water on the other, both having undesired environmental effects. Thus, there seems to be upper limits to resource utilization and, consequently, plant performance. The immediate question is, therefore, what are the factors which determine the upper limits of resource utilization. Genetic engineering techniques could make possible the exploration of those upper limits, which might provide an answer to that question. Modulation of the metabolic pathways of carbon fixation and nitrogen assimilation by means of genetic engineering has been attempted ([1,6] and references therein). The reduction of nitrate to nitrite carried out by the enzyme nitrate reductase (NR) has been claimed to be the rate-limiting step in the assimilation of nitrate nitrogen by plants [2,7]. However, there are puzzling exceptions, such as the mutants of barley, tobacco and Arabidopsis, which, while retaining only 10% of NR activity found in the wild type, can grow just as well on nitrate as the sole nitrogen source ([2] and references therein). NR, being the first reducing site in the nitrate assimilation pathway, might be the target of choice for genetic manipulation. Indeed, Quillere et al. [8] have reported that transgenic Nicotiana plumbaginifolia plants, constitutively over-expressing NR activity, display significant reduction in NO3− level and the implication of these findings to crop quality was suggested. These authors also reported an increase in the pools of free glutamine and malate in those transgenic lines. However, the N. plumbaginifolia system does not exhibit advantage with respect to total protein and biomass production when plants are grown under a variety of growth conditions [1,6,8]. At the time when the reports of Quillere et al. [8] were published we were addressing the same question using another plant system, that of Arabidopsis thaliana. Seven transgenic lines harboring the light inducible chimeric gene Lhcb1*3::Nia 1*2 [9] which over-express NR activity were exam-
ined. The results obtained indicate that A. thaliana responded differently than N. plumbaginifolia, particularly to over-expression of NR activity, by attaining higher protein contents.
2. Materials and methods
2.1. Plant material Wild type A. thaliana (L) Heyn. ecotype Wassilevskija (WS) and transgenic lines harboring the chimeric gene Lhcb1*3::Nia1*2 [9] were employed throughout this study. Five hundreds seeds from each line were surface sterilized as previously described [9], sown on Whatman No. 1 filter paper discs which were placed on top of Murashige and Skoog (MS) solid medium [10], and given a cold treatment (4°C) for 48 h in darkness. Seeds were also sown on peat moss in 4-inch pots watered with MS medium. Plants were grown under 16/8 h day/night cycles at 24°C. Photon flux density was 150 mmol m − 2 s − 1 (400–700 nm) emitted by fluorescent tubes (40 W/D/T12, day light, Sylvania Standard) supplemented with 60 W tungsten lamps. Plants were harvested, rinsed with distilled water to remove residual growth media, blotted to remove excess water, wrapped in tin foil, weighed and immediately placed in liquid nitrogen and stored at − 20°C. Precautions were taken to complete the entire harvest procedure rapidly to avoid undesirable loss of moisture from plant material.
2.2. Assay of nitrate reductase acti6ity Extraction and assay of NR were conducted as previously reported [9]. Frozen samples (see Section 2.1) were extracted in 100 mM K2PO4 buffer pH 7.5 (2.0 ml g − 1 fresh weight, FW), containing 1 mM EDTA, 5 mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride (PMSF) and 10 mM Leupeptin, using a pre-chilled mortar and pestle. After centrifugation in a microcentrifuge for 15 min at 4°C, 50 ml of the supernatant were used for each assay which was carried out in 50 mM K2PO4 buffer pH 7.5 containing 10 mM KNO3 and 0.1 mM NADH in a total volume of 500 ml at 30°C for 30 min. Assays were carried out in
A. Nejidat et al. / Plant Science 130 (1997) 41–49
triplicate. The reaction was stopped by the addition of 500 ml of 1% sulfanilamide in 3 N HCl followed by 500 ml of 0.02% N-naphthylethylenediamine dihydrochloride. A blank sample, in which sulfanilamide was added prior to the extract, was used for background reading. Absorbancy at 540 nm was measured. Enzyme activity was expressed as nmoles NO2− formed per hour on the basis of either milligrams of protein, unit fresh weight or 1000 seedlings.
2.3. Determination of nitrate content The extract used to determine NR activity was also used to determine nitrate content. The dissimilatory NR of Escherichia coli was employed [11] to reduce nitrate to nitrite, which was determined colorimetrically (see Section 2.2).
2.4. Protein extraction and analysis Total protein was extracted from frozen samples according to a published procedure [12,13], in a buffer (1.0 ml g − 1 FW) containing 125 mM Tris, 4 mM EDTA, 10 mM ascorbic acid, 10 mM KHSO3 (metabisulfite), 4 mM PMSF, 10 mM Leupeptin, 4.6% sodium dodecyl sulfate (SDS), 10% b-mercaptoethanol and 20% glycerol, using a pre-cooled mortar and pestle. The extract was incubated in boiling water for 5 min and subsequently centrifuged for 10 min in a microcentrifuge at maximum speed. Protein in the supernatant (100 ml aliquots) was precipitated by 10% trichloroacetic acid, washed twice with ethanol to remove low molecular weight compounds, dried and quantified by the method of Lowry et al. [14]. For analysis of the protein profile, aliquots were combined with sample buffer [13], boiled for 2 min and subjected to SDS-polyacrylamide gel electrophoresis (PAGE) in 12% acrylamide gels. The proteins were either stained with Coomasie Brilliant Blue or electrophoretically transferred onto nitrocellulose membrane for immuno-detection.
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3. Results and discussion
3.1. Nitrate reductase acti6ity in transgenic plants Seven independent transgenic lines, homozygous with respect to kanamycin resistance, a selectable marker which was introduced together with the Lhcb1*3::Nia 1*2 chimeric gene [9], were used in this study. In an earlier study the chimeric gene was shown to be expressed at the mRNA and the enzyme activity levels in line A4, when grown in the absence of nitrate and in the presence of L-glutamine as the sole nitrogen source, conditions which downregulate the expression of the endogenous NR genes [9]. In the present study we have analyzed all the transgenic lines obtained for the level of NR activity. As shown in Fig. 1, the seven transgenic lines displayed elevated NR activity as compared with the wild type when examined after 7 days of growth on solid MS medium containing 1 mM or 10 mM KNO3. Although the absolute values of NR activity in seedlings grown on 1 mM KNO3 were rather low (Fig. 1), the transgenic lines displayed up to 10 times the NR activity in the wild type. As expected, on 10 mM KNO3 the levels of NR activity were appreciably higher in the wild type and the transgenic lines than in those grown on 1 mM KNO3. The transgenic lines exhibited 117–300% of the NR activity of the wild type (Fig. 1). Although NR activity at 10 mM was higher than that at 1 mM, the extent of enhanced NR activity in the transgenic lines compared to the wild type was smaller at 10 mM nitrate in the growth medium than at 1 mM. One possible explanation for this is that there is an upper limit to the level of NR activity that the plant can accommodate. The growth of the wild type and the transgenic lines on 1 mM KNO3 was relatively poorer than that of those grown on 10 mM KNO3 as judged by the fresh and dry weights (Fig. 2). Apparently, the enhanced NR activity in the transgenic lines was not accompanied by an increase in biomass production (Fig. 2). These results correspond with those reported by Quillere et al. [8]. The level of NR activity in the wild type and transgenic lines exhibited light–dark fluctuations, being highest at the mid-light period and lowest just before the
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Fig. 1. Nitrate reductase activity in wild type and transgenic lines as a function of nitrate concentration in the growth medium. Five hundreds seeds of each of the lines were sown on filter paper and grown for 7 days (as described in Section 2) on solid medium containing 1 or 10 mM KNO3. Seedlings were removed from the filter paper, placed in liquid nitrogen and kept frozen at −20°C until used. The results are the average 9S.D. of five experiments. WT, wild type; A1 and A3 – A8, transgenic lines.
end of the dark period (Fig. 3). This was expected since, on one hand, the expression of the endogenous NR gene was shown to be light dependent [15] while, on the other hand, the chimeric gene used was driven by a light inducible promoter [9]. Elevated levels (up to 3-fold) of NR activity were also observed in seedlings grown in peat moss for 3 weeks (data not shown). In the present study we used seeds from T4 – T6 generations. We did not observe decrease in NR activity in six consecutive generations (data not shown).
3.2. Protein and biomass production Quillere et al. [8] were able to show a 32 – 47% decrease in the nitrate level of leaves of N. plumbaginifolia plants which constitutively over-expressed NR. However, in the transgenic Arabidopsis lines the levels of endogenous nitrate were similar to those of the wild type or even somewhat higher (Table 1). This indicates that the nitrate uptake machinery is not the rate-limiting step in nitrate assimilation in Arabidopsis under our experimental conditions.
Since the reduction of nitrate to nitrite and subsequently to ammonia is a prerequisite for the synthesis of amino acids and proteins, total protein content in the wild type and transgenic lines was determined. Seedlings of the transgenic lines exhibited higher contents of total protein when grown for 7 days on solid medium (Fig. 4) containing 1, 10 or 20 mM nitrate. The elevated level of total protein (up to 200%) was more significant at 10 or 20 mM nitrate. Apparently, the advantage of over-expression of NR in the transgenic lines was manifested when the ambient nitrate concentration exceeded that which could be utilized by the wild type. The increase in protein content was not correlated with the extent of over-expression of NR activity. This may be explained by a possible different position effect of the introduced chimeric gene in each of the independent transgenic lines. The available methodologies do not yet offer a control over the site of the introduction of transgenes. A significant increase (up to 30%) in total protein content was also observed in seedlings grown on peat moss for
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Fig. 2. Fresh and dry weights of seedlings of wild type and transgenic lines as a function of nitrate concentration in the growth medium. Experimental details as in Fig. 1.
at least 3 weeks (data not shown). SDS-PAGE analysis of total protein extracted from 7-day-old seedlings grown on peat moss indicated that the increase in protein content was evident in many protein bands (Fig. 5) and was most apparent in that of the large subunit of RuBisCo. The promoter used for constructing the chimeric gene was that of the Lhcb1*3 gene. It was thus of interest to determine whether it affected the level of the Lhcb protein. Total protein extracts of the various lines (from a different
experiment than those reported in Fig. 5) were subjected to a Western blot analysis using rabbit anti-Lhcb antibodies in order to determine the level of the Lhcb protein in the extracts (Fig. 6). The results clearly indicate that the introduction of the chimeric gene did not inhibit the accumulation of the Lhcb protein. On the contrary, the level of this protein was significantly higher in most of the transgenic lines than in the wild type, which is in agreement with the results of the SDS-PAGE analysis (Fig. 5). Apparently, if there
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Fig. 3. Level of extractable NR activity during light and dark periods in wild type and transgenic Arabidopsis lines. Growth conditions as in Fig. 1. Seedlings were harvested either at the mid-light period, the mid-dark period or at the end of the dark period. The extractable activity of wild type plants at the middle of the light period was taken as 100%. WT, wild type; A1, A3, A4 and A8, transgenic lines.
was an interaction between the promoter of the chimeric gene and that of the host Lhcb genes it did not cause cosuppression as previously reported [16]. One is tempted to compare the results obtained in the present study with those of Quillere et al. [8]. It appears that transgenic A. thaliana and transgenic N. plumbaginifolia responded differently to elevated NR activity. N. plumbaginifolia plants which constitutively over-express NR activ-
ity by virtue of the 35S cauliflower mosaic virus (CaMV) promoter and display up to 5-fold increased NR activity as compared with the wild type, show a significant decrease in nitrate content and significant increase in the free pool of several amino acids, mainly L-glutamine. However, they do not display an increase in total protein content. On the other hand, transgenic Arabidopsis which over-express NR by virtue of a light inducible promoter, do not display decreased nitrate
Table 1 Nitrate content in A. thaliana wild type and transgenic lines over-expressing NR Line WT −1 mmol NO− 61.2 (2.2) 3 g FW % of WT 100
A1 63.4 (2.5) 103.5
A3 62.6 (6.1) 102.2
A4 72.4 (6.7) 118.3
A5 75.1 (5.9) 122.7
A6 74.6 (6.8) 121.8
A7 64.8 (3.5) 105.8
A8 71.1 (4.2) 116.1
Seedlings were grown for 7 days on MS medium containing 20 mM KNO3. the results are the average of three experiments (standard deviation in parentheses). FW, fresh weight; WT, wild type.
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Fig. 4. Total protein content in wild type and transgenic lines as a function of nitrate concentration in the growth medium. Five hundred seeds of each of the lines were sowed on filter paper and grown for 7 days (as described in Section 2) on solid medium containing 1, 10 or 20 mM KNO3. Seedlings were removed from the filter paper, placed in liquid nitrogen and kept frozen at − 20°C until used. Total protein were extracted and determined as described in Section 2. The results are the average 9S.D. of five experiments. WT, wild type; A1 and A3–A8, transgenic lines.
content but do show a significant increase in total protein content. Several explanations could account for this different response. The simplest is that different plants species respond to genetic manipulation of nitrogen metabolism in a different manner, reflecting the uniqueness of their genetic make-up. This could have also been affected by the fact that N. plumbaginifolia is a cultivated species selected for specific performance, whereas A. thaliana is a wild species. Another explanation is that the different responses reflect the nature of the chimeric genes used to transform the two plant species. The 35S CaMV promoter used to drive the expression of the NR cDNA in N. plumbaginifolia and the Lhcb1*3 promoter used to drive the expression of the NR gene in Arabidopsis are known to have different tissue specificities [17,18]. Furthermore, the Lhcb1*3 promoter is light regulated. It should be noted that NR can be found in the cytosol of cells of Arabidopsis shoots and roots [2]. Indeed, both
wild type and transgenic Arabidopsis lines displayed NR activity in the roots being higher in the latter (data not shown). Several arguments support the causal relationship between elevated protein contents and overexpression of NR: (1) the over-expression of NR on the one hand, and the concomitant elevation of protein content on the other, occurred in seven independent transgenic lines in which the sites of insertion of the chimeric gene are likely to be different; (2) somaclonal variation, a rather rare event, could not account for that either, since all the transformed lines tested displayed similar results; (3) the over-expression of NR and the elevated protein content occurred in every experiment in which the plants grew either on solid medium or on peat moss, under growth-room or greenhouse conditions; (4) the elevated protein content becomes significantly apparent only at high ambient nitrate, the utilization of which is limited by the level of NR in the wild type.
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mining crop quality and thus points to a possible approach for crop improvement. On the other hand, the different results obtained with N. plumbaginifolia should be taken as a note of caution, indicating that success is not certain. Each crop plant may require particular attention especially with respect to the construction of the appropriate chimeric gene to be introduced to fit its specific genetic make-up.
Acknowledgements Ali Nejidat was supported by a Deichman Fellowship. Genfa Zhang was a postdoctorate fellow of The Jacob Blaustein International Center. The authors thank Prof. Rachel Nechushtai, for supplying antibodies against Lhcb protein, and Dr. Elliott Birnbaum for editing the manuscript. This research was supported in part by a grant from The Moriah Foundation.
References
Fig. 5. Increased abundance of various proteins in transgenic plants. Five microliters of total protein extracts of 1-week-old wild type and transgenic lines grown on peat moss were subjected to SDS-PAGE. Protein bands were visualized by Coomasie Brilliant Blue. WT, wild type; A1, A3, A4 and A8, transgenic lines; M, molecular weight markers.
The consequence of over-expression of NR in A. thaliana was the increase in protein content mainly at high ambient nitrate concentrations. This feature is of immense importance in deter-
Fig. 6. Immunoblot analysis of the abundance of Lhcb protein in wild type and transgenic lines. Transferred proteins (see Section 2) were reacted with rabbit antibodies against Lhcb protein followed with alkaline phosphatase-conjugated goat anti-rabbit antibodies. WT, wild type; A1 and A3–A8, transgenic lines.
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