- Email: [email protected]
Transgenic rice with low sucrose-phosphate synthase activities retain more soluble protein and chlorophyll during flag leaf senescence
Plant Physiol. Biochem., 1999, 37 (12), 949−953 / © 1999 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0981942899001060/FLA
Short Paper Transgenic rice with low sucrose-phosphate synthase activities retain more soluble protein and chlorophyll during flag leaf senescence Kiyomi Ono*, Ken Ishimaru, Naohiro Aoki, Ryu Ohsugi§ Laboratory of Carbon Metabolism, Department of Plant Physiology, National Institute of Agrobiological Resources, Kannondai 2-1-2, Tsukuba 305-8602, Japan
* Author to whom correspondence should be addressed (fax +81 298 38 8347; e-mail [email protected]) (Received July 12, 1999; accepted October 15, 1999) Abstract — We investigated whether changes in sucrose-phosphate synthase (EC 2.4.1.14, SPS) activity could alter N remobilization during leaf senescence. Transgenic rice (Oryza sativa L. cv. Nipponbare) with low SPS activities and wild-type rice plants were grown with basal N (1.0 mM NH4NO3) until the late vegetative stage. Subsequently, half of the plants were transferred to a low N (0.1 mM NH4NO3) condition to accelerate leaf senescence, and the others were continuously grown with basal N. With low N supply, the amounts of chlorophyll and soluble protein in flag leaf blades decreased after anthesis in both the low SPS plants and wild-type plants, although the decrease was less in the low SPS plants. Panicle weights were significantly lower in the low SPS plant than in the wild-type plant. These results suggest that the remobilization of N from flag leaves was diminished by suppressing the development of reproductive sinks in the low SPS plant. © 1999 Éditions scientifiques et médicales Elsevier SAS Nitrogen / Oryza sativa / senescence / sucrose-phosphate synthase / transgenic plant Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase / SPS, sucrose-phosphate synthase
1. INTRODUCTION Sucrose is the most transportable photoassimilate in plants and is synthesized from UDP-glucose and fructose-6-phosphate in a sequence of two reactions catalyzed by sucrose-phosphate synthase (EC 2.4.1.14, SPS) and sucrose-phosphate phosphatase (EC 3.1.3.24). It is generally thought that SPS is the major limiting enzyme for sucrose synthesis [5, 16]. In addition, a strong correlation was found between the SPS activity in source leaves and the elongation rate of expanding leaves [14]. In recent years, with the development of transgenic techniques, a maize SPS gene has been overexpressed in tomato [2, 3, 11, 20], Arabidopsis [15] and potato [18]. Some studies [11, 18] reported that overexpression of the maize SPS §
Present address: Agriculture, Forestry & Fisheries, Research Council Secretariat, MAFF, Kasumigaseki 1-2-1, Chiyoda-ku, Tokyo 100-9850, Japan.
gene increased fruit and tuber dry weight. Therefore, it is probable that changing the SPS activity alters the growth and development of reproductive sinks. To develop new parts of a plant, both C and N are necessary. During the grain filling stage, N is absorbed at a low rate. The stored N in the leaves and the culm is the major source of N for grain filling and is translocated to the developing panicles. In many studies investigating the interaction between the sourcesink relationship and leaf senescence, the source-sink balance has been altered by growing plants under different nutrient and/or light conditions [1, 13] or by removing sink organs (flowers, pods, ears) [4, 7]. It has been suggested that the source-sink relationship influences the whole plant N economy and the remobilization of resources during leaf senescence. It is probable that N remobilization changes in plants whose sink development is altered by manipulating the SPS activity. We tested this hypothesis by using transgenic rice (Oryza sativa L. cv. Nipponbare) plants that have lower SPS activity than the wild-type [12].
Plant Physiol. Biochem., 0981-9428/99/12/© 1999 E´ditions scientifiques et médicales Elsevier SAS. All rights reserved
950
K. Ono et al.
2. RESULTS AND DISCUSSION The apparent Vmax activities of SPS in flag leaf blades were calculated by a Lineweaver-Burk plot (table I). The SPS activities in transgenic rice plants with small amounts of SPS protein (#139-4, low SPS plants) were half of those in wild-type rice plants under both N conditions. In our previous work [12], a western blot analysis was carried out with an anti-rice SPS antibody which showed that transgenic rice #139-4 had lower amounts of rice SPS protein than wild-type rice plants. In the present study, the maximal SPS activity slightly increased under the low N condition in both wild-type and low SPS plants (table I). Using detached spinach leaves, Huber et al. [6] showed that N supply to plants affects the activation state of SPS. In our study, we measured the maximal SPS activity, which is independent of the activation state and reflects the amount of SPS protein. Makino et al. [10] showed that N allocation to SPS protein decreased with increasing N supply to plants and that N allocation to chlorophyll was independent of N supply. Because we expressed the SPS activity on a chlorophyll basis in this study, the difference in the activity between the N treatments is probably caused by the changes in N partitioning. Anthesis began between September 1st and 8th in almost all panicles. The flowering time was not significantly different between the low SPS plants and the wild-type plants. Therefore, the number of days after anthesis was counted from September 1st in both groups. CO2 assimilation rates of the flag leaf blades under saturated light and ambient CO2 conditions did not differ significantly between the low SPS plants and wild-type plants in the early ripening stage (12–13 d after anthesis) (table II). The amounts of chlorophyll, soluble protein and Rubisco in the flag leaf blades were measured from before heading until the end of the ripening period (figure 1). With low N supply, chlorophyll, soluble protein and Rubisco decreased after anthesis Table I. Maximal SPS activities of flag leaf blades sampled between 10.00 and 12.00 hours at 8 d after anthesis. Maximal SPS activities were calculated by Lineweaver-Burk plots. Values show mean ± standard error for three plants. SPS activity (µmol⋅mg–1 chl⋅h–1)
Wild-type Low SPS
Plant Physiol. Biochem.
Low N
Basal N
82.6 ± 18.7 41.4 ± 2.8
61.1 ± 8.2 28.0 ± 4.3
Table II. CO2 assimilation rates of flag leaf blades at 12–13 d after anthesis. CO2 assimilation rates were measured between 9.00 and 12.00 hours, under saturated light (1 000 µmol⋅m–2⋅s–1) and ambient CO2 (350 µL⋅L–1). Values show mean ± standard error for three plants. CO2 assimilation rates (µmol CO2⋅m–2⋅s–1)
Wild-type Low SPS
Low N
Basal N
12.8 ± 2.8 12.3 ± 1.9
13.1 ± 1.4 14.5 ± 0.5
(figure 1 A–C), while they did not decrease with basal N supply during this period (figure 1 D–F). With basal N supply, the leaves maintained higher levels of soluble protein, suggesting that the N required for panicle development was translocated from the lower and older leaves rather than from the flag leaves. At 32 d after anthesis, the low SPS plants retained more chlorophyll, soluble protein and Rubisco than did the wild-type plants. The observed differences were highly significant (P < 0.05 for chlorophyll and Rubisco, and P < 0.005 for soluble protein), suggesting that N mobilization and reallocation had been altered in SPS transgenic rice. Under both N conditions, the final dry weights of shoots except for panicles and roots did not differ significantly between the wild-type plants and the low SPS plants (table III). Panicle weights of the low SPS plants were significantly lower than those of the wild-type plants with low N supply (P < 0.01) and with basal N supply (P < 0.05). The panicle weights of the low SPS plants were 75.7 % (under low N condition) and 80.6 % (under basal N condition) of those of wild-type plants. In rice plants, because the number of panicles and spikelets is highly dependent on the amount of N uptake and partitioning [17], we were unable to correlate the panicle weight with SPS activity of the plants grown under different N conditions. The subsequent panicles growth is mainly determined by the availability of carbohydrates [17]. This may be why panicle weights were significantly lower in the low SPS plants than in wild-type plants (table III). The carbohydrate supply to panicles may be limited by low translocation efficiency of photoassimilates and/or by low photosynthetic activities. The photosynthetic rates of flag leaves did not differ significantly between low SPS plants and wild-type plants when they were measured between 9.00 and 12.00 hours at 12–13 d after anthesis (table II), and low SPS plants retained more chlorophyll, protein and Rubisco for a longer period (figure 1 A–C). However, we cannot exclude the possibility that photosynthesis
Leaf senescence of transgenic plants with low SPS activity
951
The present results (figure 1 A–C, table III) indicate that a decrease in SPS activities limited the reproductive sink development and suppressed N allocation to the developing grain. However, no significant difference was observed in the dry weights of vegetative parts (shoots except for panicles, or roots) between wild-type plants and the low SPS plants (table III). It is well known that carbon requirement during the vegetative stage is much less than during the reproductive stage. Therefore, the major carbon limitations occur during the grain filling stage and the reduction in yield became obvious in the low SPS rice. Seneweera et al. [14] reported that the sink strength and the SPS activity change during the vegetative stage of rice plants. At the early vegetative stage, growing leaves are the major sinks for carbon. As plants reach the mid-tillering stage, tillers become the major sinks. At this stage both the Vmax and Vlimiting of SPS activity were higher than in the early vegetative stage [14]. Therefore, it is probable that the impact of the reduced SPS activity on the plant growth depends on growth stages. Young caryopses of the panicle is the strongest sink for carbon compared to leaf sheaths, culms and roots, suggesting that grain filling is mostly affected by SPS activity. Our results suggest that the strength of the reproductive sink is stronger than that of vegetative sinks and is affected more by the reduced SPS activity. Figure 1. Amounts of chlorophyll (A, D), soluble protein (B, E) and Rubisco (C, F) in flag leaves of transgenic rice plants with low SPS activities (•) and wild-type rice plants (●). Leaves were sampled on August 27th (5 d before anthesis), September 9th (8 d after anthesis), September 21st (20 d after anthesis), October 3rd (32 d after anthesis, low N supply) and October 11th (40 d after anthesis, basal N supply). A–C, Values in plants grown with low N supply; D–F, values in plants grown with basal N supply. The values are means of three different plants. Bars indicate standard errors. * and *** indicate statistically significant differences at P < 0.05 and P < 0.005, respectively.
declines during the afternoon due to Pi limitation. Therefore, it is necessary to further investigate the translocation rates of photoassimilates to the panicles in our transgenic rice.
3. CONCLUSION Under low N condition, chlorophyll, soluble protein and Rubisco decreased after anthesis, although the decreases in these components were suppressed in the low SPS plants. In addition, panicle weights were significantly lower in the low SPS plants than in the wild-type plants. It seems reasonable to conclude that a decrease in SPS activity reduced reproductive sink development and suppressed N mobilization from flag leaf blades.
Table III. Final dry weights and panicle weights in wild-type rice plants and low SPS plants. Values show mean ± standard error for three plants. * and ** indicate statistically significant differences at P < 0.05 and P < 0.01, respectively. Dry weight
Low N
–1
Shoot (g⋅plant ) Root (g⋅plant–1) Panicle weight (g⋅panicle–1)
Basal N
Wild-type
Low SPS
Wild-type
Low SPS
24.8 ± 3.0 4.2 ± 0.87 1.03 ± 0.12
27.8 ± 4.8 4.0 ± 1.0 0.78 ± 0.04**
62.9 ± 14.3 20.1 ± 1.5 1.29 ± 0.21
63.3 ± 13.0 19.7 ± 0.42 1.04 ± 0.17*
vol. 37 (12) 1999
952
K. Ono et al.
4. METHODS 4.1. Plant material Transgenic rice (Oryza sativa L. cv. Nipponbare) plants containing the maize sucrose-phosphate synthase (EC 2.4.1.14, SPS) cDNA [12] and wild-type rice plants (control) were used. Among the T2 plants, we selected one of the transgenic plants which had very small amounts of SPS protein and low SPS activities (#139-4). Seeds of transgenic and wild-type rice plants were soaked in tap water at 28 °C for 4 d, and the seedlings (T3 plants) were grown in a growth chamber (Koitotron, Koito, Japan) for 18 d on a plastic net floating on tap water adjusted to pH 5.5. The first and the second leaves were sampled on June 18th, when the 5th leaf was developing. Using these samples, transgenic rice plants were selected by a western blot analysis against anti-maize SPS antibody and anti-rice SPS antibody. Transgenic plants and wild-type plants were grown hydroponically in a glass room under natural sunlight. The temperature of the room was maintained at 25 °C. Seedlings were transplanted to 100-L containers containing 75 L nutrient solution. Fifteen seedlings were grown in each container. The basal nutrient solution is described in Makino et al. [9]. The solution was renewed every 10 d. Plants were supplied with basal N (1.0 mM NH4NO3) until July 24th (late stage of vegetative growth). Half of the plants were continuously grown with basal N and the remaining were transferred to a low N (0.1 mM NH4NO3) condition. The youngest fully expanded leaves were sampled on July 21st, and again the presence of maize SPS protein or the reduction of rice SPS protein was checked. Flag leaf blades were sampled prior to the heading stage (August 27th), just after anthesis (September 9th), during ripening (September 21st) and after ripening (October 3rd with low N supply and October 11th with basal N supply). Flag leaf blades were sampled between 10.00 and 12.00 hours. For measurements of SPS activity and protein level, leaves were immediately frozen with liquid nitrogen and stored at –80 °C.
4.2. Gas exchange measurements CO2 assimilation rates were measured using a portable gas-exchange system (LI-6400; Li-COR Inc., Lincoln NE, USA). Measurements were made using intact flag leaf blades between 9.00 and 12.00 hours on September 13th and 14th. Light was provided by a LED source (red/blue, 6400-02 LED source; LiCOR Inc., Lincoln NE, USA). The photon flux density was Plant Physiol. Biochem.
1 000 µmol photons⋅m–2⋅s–1, leaf temperature was 25 °C, and the reference CO2 concentration was 350 µL⋅L–1.
4.3. Measurements of proteins, chlorophyll and SPS activity Soluble protein was extracted as described previously [12]. For determination of ribulose-1,5bisphosphate carboxylase/oxygenase (EC 4.1.1.39, Rubisco) content, 1 µg protein was loaded onto a 12.5 % (w/v) polyacrylamide gel containing 0.1 % (w/v) sodium dodecylsulfate. The amounts of Rubisco protein were quantified using an image analyzer (UMAX PowerLook 2000, UMAX Data System, Inc., Taipei, ROC, ZERO Dscan, MA, USA). Bovine serum albumin was used as a standard to quantify the relative amount of Rubisco. For chlorophyll measurements, one or two fresh leaf disks (0.28 cm2⋅disk–1) were taken from the leaves and soaked in 1 mL 96 % ethanol until the disks became colorless. Chlorophyll concentration was measured by the method of Wintermans and De Mots [19]. For measurements of SPS activity, flag leaves were sampled on September 9th and measurements were made according the method of Lunn and Hatch [8] with a slight modification [12].
4.4. Measurements of dry weights Plants were carefully separated into shoots and roots, and all the fractions were dried at 80 °C for more than 48 h before determining dry mass. Panicles were weighed after drying at room temperature for a few days.
Acknowledgments We thank Drs S.P. Seneweera and O. Ueno for critical readings of this manuscript. This work was supported in part by a Grant-in-Aid-(Bio Cosmos Program) No. BDP-99-I-1-6 from the Ministry of Agriculture, Forestry and Fisheries, Japan and CREST (Core Research for Evolution Science and Technology) of Japan Science and Technology Corporation (JST).
Leaf senescence of transgenic plants with low SPS activity
REFERENCES [1] Anten N.P.R., Werger M.J.A., Canopy structure and nitrogen distribution in dominant and subordinate plants in a dense stand of Amaranthus dubius L. with a size hierarchy of individuals, Oecologia 105 (1996) 30–37. [2] Galtier N., Foyer C.H., Huber J., Voelker T.A., Huber S.C., Effects of elevated sucrose-phosphate synthase activity on photosynthesis, assimilate partitioning, and growth in tomato (Lycopersicon esculentum var UC82B), Plant Physiol. 101 (1993) 535–543. [3] Galtier N., Foyer C.H., Murchie E., Alred R., Quick P., Voelker T.A., Thepenier C., Lasceve G., Betsche T., Effects of light and atmospheric carbon dioxide enrichment on photosynthesis and carbon partitioning in the leaves of tomato (Lycopersicon esculentum L.) plants over-expressing sucrose phosphate synthase, J. Exp. Bot. 46 (1995) 1335–1344. [4] Guitmann M.R., Arnozis A.A., Barneix A.J., Effects of source-sink relations and nitrogen nutrition on senescence and N mobilization in the flag leaf of wheat, Physiol. Plant. 82 (1991) 278–284. [5] Huber S.C., Role of sucrose-phosphate synthase in partitioning of carbon in leaves, Plant Physiol. 71 (1983) 818–821. [6] Huber S.C., Bachmann M., McMichael Jr R.W., Huber J.L., Regulation of sucrose-phosphate synthase by reversible protein phosphorylation: Manipulation of activation and inactivation in vivo, in: Points H.G., Salerno G.L., Echeverria E.J. (Eds.), International Symposium on Source Metabolism, American Society of Plant Physiologists, 1995, pp. 6–13. [7] Khanna-Chopra R., Reddy P.V., Regulation of leaf senescence by reproductive sink intensity in cowpea (Vigra unguiculata L. Walp), Ann. Bot. 61 (1988) 655–658. [8] Lunn J.E., Hatch M.D., The role of sucrose-phosphate synthase in the control of photosynthate partitioning in Zea mays leaves, Aust. J. Plant Physiol. 24 (1997) 1–8. [9] Makino A., Mae T., Ohira K., Differences between wheat and rice in the enzyme properties of ribulose1,5-bisphosphate carboxylase/oxygenase and the relationship to photosynthetic gas exchange, Planta 174 (1988) 30–38. [10] Makino A., Nakano H., Mae T., Responses of ribulose1,5-bisphosphate carboxylase, cytochrome f, and sucrose synthesis enzymes in rice leaves to leaf nitrogen and their relationships to photosynthesis, Plant Physiol. 105 (1994) 173–179.
953
[11] Micallef B.J., Haskins K.A., Vanderveer P.J., Poh K.S., Shewmaker C.K., Sharkey T.D., Altered photosynthesis, flowering, and fruiting in transgenic tomato plants that have an increased capacity for sucrose synthesis, Planta 196 (1995) 327–334. [12] Ono K., Ishimaru K., Aoki N., Takahashi S., Ozawa K., Ohkawa Y., Ohsugi R., Characterization of a maize sucrose-phosphate synthase protein and its effect on carbon partitioning in transgenic rice plants, Plant Prod. Sci. 2 (1999) 172–177. [13] Ono K., Terashima I., Watanabe A., Interaction between nitrogen deficit of a plant and nitrogen content in the old leaves, Plant Cell Physiol. 37 (1996) 1083–1089. [14] Seneweera S.P., Basra A.S., Barlow E.W., Conroy J.P., Diurnal regulation of leaf blade elongation in rice by CO2, Plant Physiol. 108 (1995) 1471–1477. [15] Signora L., Galtier N., Skot L., Lucas H., Foyer C.H., Overexpression of sucrose phosphate synthase in Arabidopsis thaliana results in increased foliar carbohydrate accumulation in plants after prolonged growth with CO2 enrichment, J. Exp. Bot. 49 (1998) 669–680. [16] Stitt M., Huber S., Kerr P., Control of photosynthetic sucrose formation, in: Hatch M.D., Boardmann N.K. (Eds.), The Biochemistry of Plants: Photosynthesis, Academic Press, New York, 1987, pp. 327–409. [17] Takeoka Y., Shimizu M., Wada T., Panicles, in: Matsuo T., Hoshikawa K. (Eds.), Science of the Rice Plant, vol. 1, Food and Agriculture Policy Research Center, Tokyo, 1993, pp. 295–338. [18] Tobias D.J., Hirose T., Ishimaru K., Ishige T., Ohkawa Y., Kano-Murakami Y., Matsuoka M., Ohsugi R., Elevated sucrose-phosphate synthase activity in source leaves of potato plants transformed with the maize SPS gene, Plant Prod. Sci. 2 (1999) 92–99. [19] Wintermans J.F.G.M., De Mots A., Spectrophotometric characteristics of chlorophylls and their pheophytins in ethanol, Biochim. Biophys. Acta 109 (1965) 448–453. [20] Worrell A.C., Bruneau J.M., Summerfelt K., Boersig M., Voelker T.A., Expression of a maize sucrose phosphate synthase in tomato alters leaf carbohydrate partitioning, Plant Cell 3 (1991) 1121–1130.
vol. 37 (12) 1999
Short Paper Transgenic rice with low sucrose-phosphate synthase activities retain more soluble protein and chlorophyll during flag leaf senescence Kiyomi Ono*, Ken Ishimaru, Naohiro Aoki, Ryu Ohsugi§ Laboratory of Carbon Metabolism, Department of Plant Physiology, National Institute of Agrobiological Resources, Kannondai 2-1-2, Tsukuba 305-8602, Japan
* Author to whom correspondence should be addressed (fax +81 298 38 8347; e-mail [email protected]) (Received July 12, 1999; accepted October 15, 1999) Abstract — We investigated whether changes in sucrose-phosphate synthase (EC 2.4.1.14, SPS) activity could alter N remobilization during leaf senescence. Transgenic rice (Oryza sativa L. cv. Nipponbare) with low SPS activities and wild-type rice plants were grown with basal N (1.0 mM NH4NO3) until the late vegetative stage. Subsequently, half of the plants were transferred to a low N (0.1 mM NH4NO3) condition to accelerate leaf senescence, and the others were continuously grown with basal N. With low N supply, the amounts of chlorophyll and soluble protein in flag leaf blades decreased after anthesis in both the low SPS plants and wild-type plants, although the decrease was less in the low SPS plants. Panicle weights were significantly lower in the low SPS plant than in the wild-type plant. These results suggest that the remobilization of N from flag leaves was diminished by suppressing the development of reproductive sinks in the low SPS plant. © 1999 Éditions scientifiques et médicales Elsevier SAS Nitrogen / Oryza sativa / senescence / sucrose-phosphate synthase / transgenic plant Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase / SPS, sucrose-phosphate synthase
1. INTRODUCTION Sucrose is the most transportable photoassimilate in plants and is synthesized from UDP-glucose and fructose-6-phosphate in a sequence of two reactions catalyzed by sucrose-phosphate synthase (EC 2.4.1.14, SPS) and sucrose-phosphate phosphatase (EC 3.1.3.24). It is generally thought that SPS is the major limiting enzyme for sucrose synthesis [5, 16]. In addition, a strong correlation was found between the SPS activity in source leaves and the elongation rate of expanding leaves [14]. In recent years, with the development of transgenic techniques, a maize SPS gene has been overexpressed in tomato [2, 3, 11, 20], Arabidopsis [15] and potato [18]. Some studies [11, 18] reported that overexpression of the maize SPS §
Present address: Agriculture, Forestry & Fisheries, Research Council Secretariat, MAFF, Kasumigaseki 1-2-1, Chiyoda-ku, Tokyo 100-9850, Japan.
gene increased fruit and tuber dry weight. Therefore, it is probable that changing the SPS activity alters the growth and development of reproductive sinks. To develop new parts of a plant, both C and N are necessary. During the grain filling stage, N is absorbed at a low rate. The stored N in the leaves and the culm is the major source of N for grain filling and is translocated to the developing panicles. In many studies investigating the interaction between the sourcesink relationship and leaf senescence, the source-sink balance has been altered by growing plants under different nutrient and/or light conditions [1, 13] or by removing sink organs (flowers, pods, ears) [4, 7]. It has been suggested that the source-sink relationship influences the whole plant N economy and the remobilization of resources during leaf senescence. It is probable that N remobilization changes in plants whose sink development is altered by manipulating the SPS activity. We tested this hypothesis by using transgenic rice (Oryza sativa L. cv. Nipponbare) plants that have lower SPS activity than the wild-type [12].
Plant Physiol. Biochem., 0981-9428/99/12/© 1999 E´ditions scientifiques et médicales Elsevier SAS. All rights reserved
950
K. Ono et al.
2. RESULTS AND DISCUSSION The apparent Vmax activities of SPS in flag leaf blades were calculated by a Lineweaver-Burk plot (table I). The SPS activities in transgenic rice plants with small amounts of SPS protein (#139-4, low SPS plants) were half of those in wild-type rice plants under both N conditions. In our previous work [12], a western blot analysis was carried out with an anti-rice SPS antibody which showed that transgenic rice #139-4 had lower amounts of rice SPS protein than wild-type rice plants. In the present study, the maximal SPS activity slightly increased under the low N condition in both wild-type and low SPS plants (table I). Using detached spinach leaves, Huber et al. [6] showed that N supply to plants affects the activation state of SPS. In our study, we measured the maximal SPS activity, which is independent of the activation state and reflects the amount of SPS protein. Makino et al. [10] showed that N allocation to SPS protein decreased with increasing N supply to plants and that N allocation to chlorophyll was independent of N supply. Because we expressed the SPS activity on a chlorophyll basis in this study, the difference in the activity between the N treatments is probably caused by the changes in N partitioning. Anthesis began between September 1st and 8th in almost all panicles. The flowering time was not significantly different between the low SPS plants and the wild-type plants. Therefore, the number of days after anthesis was counted from September 1st in both groups. CO2 assimilation rates of the flag leaf blades under saturated light and ambient CO2 conditions did not differ significantly between the low SPS plants and wild-type plants in the early ripening stage (12–13 d after anthesis) (table II). The amounts of chlorophyll, soluble protein and Rubisco in the flag leaf blades were measured from before heading until the end of the ripening period (figure 1). With low N supply, chlorophyll, soluble protein and Rubisco decreased after anthesis Table I. Maximal SPS activities of flag leaf blades sampled between 10.00 and 12.00 hours at 8 d after anthesis. Maximal SPS activities were calculated by Lineweaver-Burk plots. Values show mean ± standard error for three plants. SPS activity (µmol⋅mg–1 chl⋅h–1)
Wild-type Low SPS
Plant Physiol. Biochem.
Low N
Basal N
82.6 ± 18.7 41.4 ± 2.8
61.1 ± 8.2 28.0 ± 4.3
Table II. CO2 assimilation rates of flag leaf blades at 12–13 d after anthesis. CO2 assimilation rates were measured between 9.00 and 12.00 hours, under saturated light (1 000 µmol⋅m–2⋅s–1) and ambient CO2 (350 µL⋅L–1). Values show mean ± standard error for three plants. CO2 assimilation rates (µmol CO2⋅m–2⋅s–1)
Wild-type Low SPS
Low N
Basal N
12.8 ± 2.8 12.3 ± 1.9
13.1 ± 1.4 14.5 ± 0.5
(figure 1 A–C), while they did not decrease with basal N supply during this period (figure 1 D–F). With basal N supply, the leaves maintained higher levels of soluble protein, suggesting that the N required for panicle development was translocated from the lower and older leaves rather than from the flag leaves. At 32 d after anthesis, the low SPS plants retained more chlorophyll, soluble protein and Rubisco than did the wild-type plants. The observed differences were highly significant (P < 0.05 for chlorophyll and Rubisco, and P < 0.005 for soluble protein), suggesting that N mobilization and reallocation had been altered in SPS transgenic rice. Under both N conditions, the final dry weights of shoots except for panicles and roots did not differ significantly between the wild-type plants and the low SPS plants (table III). Panicle weights of the low SPS plants were significantly lower than those of the wild-type plants with low N supply (P < 0.01) and with basal N supply (P < 0.05). The panicle weights of the low SPS plants were 75.7 % (under low N condition) and 80.6 % (under basal N condition) of those of wild-type plants. In rice plants, because the number of panicles and spikelets is highly dependent on the amount of N uptake and partitioning [17], we were unable to correlate the panicle weight with SPS activity of the plants grown under different N conditions. The subsequent panicles growth is mainly determined by the availability of carbohydrates [17]. This may be why panicle weights were significantly lower in the low SPS plants than in wild-type plants (table III). The carbohydrate supply to panicles may be limited by low translocation efficiency of photoassimilates and/or by low photosynthetic activities. The photosynthetic rates of flag leaves did not differ significantly between low SPS plants and wild-type plants when they were measured between 9.00 and 12.00 hours at 12–13 d after anthesis (table II), and low SPS plants retained more chlorophyll, protein and Rubisco for a longer period (figure 1 A–C). However, we cannot exclude the possibility that photosynthesis
Leaf senescence of transgenic plants with low SPS activity
951
The present results (figure 1 A–C, table III) indicate that a decrease in SPS activities limited the reproductive sink development and suppressed N allocation to the developing grain. However, no significant difference was observed in the dry weights of vegetative parts (shoots except for panicles, or roots) between wild-type plants and the low SPS plants (table III). It is well known that carbon requirement during the vegetative stage is much less than during the reproductive stage. Therefore, the major carbon limitations occur during the grain filling stage and the reduction in yield became obvious in the low SPS rice. Seneweera et al. [14] reported that the sink strength and the SPS activity change during the vegetative stage of rice plants. At the early vegetative stage, growing leaves are the major sinks for carbon. As plants reach the mid-tillering stage, tillers become the major sinks. At this stage both the Vmax and Vlimiting of SPS activity were higher than in the early vegetative stage [14]. Therefore, it is probable that the impact of the reduced SPS activity on the plant growth depends on growth stages. Young caryopses of the panicle is the strongest sink for carbon compared to leaf sheaths, culms and roots, suggesting that grain filling is mostly affected by SPS activity. Our results suggest that the strength of the reproductive sink is stronger than that of vegetative sinks and is affected more by the reduced SPS activity. Figure 1. Amounts of chlorophyll (A, D), soluble protein (B, E) and Rubisco (C, F) in flag leaves of transgenic rice plants with low SPS activities (•) and wild-type rice plants (●). Leaves were sampled on August 27th (5 d before anthesis), September 9th (8 d after anthesis), September 21st (20 d after anthesis), October 3rd (32 d after anthesis, low N supply) and October 11th (40 d after anthesis, basal N supply). A–C, Values in plants grown with low N supply; D–F, values in plants grown with basal N supply. The values are means of three different plants. Bars indicate standard errors. * and *** indicate statistically significant differences at P < 0.05 and P < 0.005, respectively.
declines during the afternoon due to Pi limitation. Therefore, it is necessary to further investigate the translocation rates of photoassimilates to the panicles in our transgenic rice.
3. CONCLUSION Under low N condition, chlorophyll, soluble protein and Rubisco decreased after anthesis, although the decreases in these components were suppressed in the low SPS plants. In addition, panicle weights were significantly lower in the low SPS plants than in the wild-type plants. It seems reasonable to conclude that a decrease in SPS activity reduced reproductive sink development and suppressed N mobilization from flag leaf blades.
Table III. Final dry weights and panicle weights in wild-type rice plants and low SPS plants. Values show mean ± standard error for three plants. * and ** indicate statistically significant differences at P < 0.05 and P < 0.01, respectively. Dry weight
Low N
–1
Shoot (g⋅plant ) Root (g⋅plant–1) Panicle weight (g⋅panicle–1)
Basal N
Wild-type
Low SPS
Wild-type
Low SPS
24.8 ± 3.0 4.2 ± 0.87 1.03 ± 0.12
27.8 ± 4.8 4.0 ± 1.0 0.78 ± 0.04**
62.9 ± 14.3 20.1 ± 1.5 1.29 ± 0.21
63.3 ± 13.0 19.7 ± 0.42 1.04 ± 0.17*
vol. 37 (12) 1999
952
K. Ono et al.
4. METHODS 4.1. Plant material Transgenic rice (Oryza sativa L. cv. Nipponbare) plants containing the maize sucrose-phosphate synthase (EC 2.4.1.14, SPS) cDNA [12] and wild-type rice plants (control) were used. Among the T2 plants, we selected one of the transgenic plants which had very small amounts of SPS protein and low SPS activities (#139-4). Seeds of transgenic and wild-type rice plants were soaked in tap water at 28 °C for 4 d, and the seedlings (T3 plants) were grown in a growth chamber (Koitotron, Koito, Japan) for 18 d on a plastic net floating on tap water adjusted to pH 5.5. The first and the second leaves were sampled on June 18th, when the 5th leaf was developing. Using these samples, transgenic rice plants were selected by a western blot analysis against anti-maize SPS antibody and anti-rice SPS antibody. Transgenic plants and wild-type plants were grown hydroponically in a glass room under natural sunlight. The temperature of the room was maintained at 25 °C. Seedlings were transplanted to 100-L containers containing 75 L nutrient solution. Fifteen seedlings were grown in each container. The basal nutrient solution is described in Makino et al. [9]. The solution was renewed every 10 d. Plants were supplied with basal N (1.0 mM NH4NO3) until July 24th (late stage of vegetative growth). Half of the plants were continuously grown with basal N and the remaining were transferred to a low N (0.1 mM NH4NO3) condition. The youngest fully expanded leaves were sampled on July 21st, and again the presence of maize SPS protein or the reduction of rice SPS protein was checked. Flag leaf blades were sampled prior to the heading stage (August 27th), just after anthesis (September 9th), during ripening (September 21st) and after ripening (October 3rd with low N supply and October 11th with basal N supply). Flag leaf blades were sampled between 10.00 and 12.00 hours. For measurements of SPS activity and protein level, leaves were immediately frozen with liquid nitrogen and stored at –80 °C.
4.2. Gas exchange measurements CO2 assimilation rates were measured using a portable gas-exchange system (LI-6400; Li-COR Inc., Lincoln NE, USA). Measurements were made using intact flag leaf blades between 9.00 and 12.00 hours on September 13th and 14th. Light was provided by a LED source (red/blue, 6400-02 LED source; LiCOR Inc., Lincoln NE, USA). The photon flux density was Plant Physiol. Biochem.
1 000 µmol photons⋅m–2⋅s–1, leaf temperature was 25 °C, and the reference CO2 concentration was 350 µL⋅L–1.
4.3. Measurements of proteins, chlorophyll and SPS activity Soluble protein was extracted as described previously [12]. For determination of ribulose-1,5bisphosphate carboxylase/oxygenase (EC 4.1.1.39, Rubisco) content, 1 µg protein was loaded onto a 12.5 % (w/v) polyacrylamide gel containing 0.1 % (w/v) sodium dodecylsulfate. The amounts of Rubisco protein were quantified using an image analyzer (UMAX PowerLook 2000, UMAX Data System, Inc., Taipei, ROC, ZERO Dscan, MA, USA). Bovine serum albumin was used as a standard to quantify the relative amount of Rubisco. For chlorophyll measurements, one or two fresh leaf disks (0.28 cm2⋅disk–1) were taken from the leaves and soaked in 1 mL 96 % ethanol until the disks became colorless. Chlorophyll concentration was measured by the method of Wintermans and De Mots [19]. For measurements of SPS activity, flag leaves were sampled on September 9th and measurements were made according the method of Lunn and Hatch [8] with a slight modification [12].
4.4. Measurements of dry weights Plants were carefully separated into shoots and roots, and all the fractions were dried at 80 °C for more than 48 h before determining dry mass. Panicles were weighed after drying at room temperature for a few days.
Acknowledgments We thank Drs S.P. Seneweera and O. Ueno for critical readings of this manuscript. This work was supported in part by a Grant-in-Aid-(Bio Cosmos Program) No. BDP-99-I-1-6 from the Ministry of Agriculture, Forestry and Fisheries, Japan and CREST (Core Research for Evolution Science and Technology) of Japan Science and Technology Corporation (JST).
Leaf senescence of transgenic plants with low SPS activity
REFERENCES [1] Anten N.P.R., Werger M.J.A., Canopy structure and nitrogen distribution in dominant and subordinate plants in a dense stand of Amaranthus dubius L. with a size hierarchy of individuals, Oecologia 105 (1996) 30–37. [2] Galtier N., Foyer C.H., Huber J., Voelker T.A., Huber S.C., Effects of elevated sucrose-phosphate synthase activity on photosynthesis, assimilate partitioning, and growth in tomato (Lycopersicon esculentum var UC82B), Plant Physiol. 101 (1993) 535–543. [3] Galtier N., Foyer C.H., Murchie E., Alred R., Quick P., Voelker T.A., Thepenier C., Lasceve G., Betsche T., Effects of light and atmospheric carbon dioxide enrichment on photosynthesis and carbon partitioning in the leaves of tomato (Lycopersicon esculentum L.) plants over-expressing sucrose phosphate synthase, J. Exp. Bot. 46 (1995) 1335–1344. [4] Guitmann M.R., Arnozis A.A., Barneix A.J., Effects of source-sink relations and nitrogen nutrition on senescence and N mobilization in the flag leaf of wheat, Physiol. Plant. 82 (1991) 278–284. [5] Huber S.C., Role of sucrose-phosphate synthase in partitioning of carbon in leaves, Plant Physiol. 71 (1983) 818–821. [6] Huber S.C., Bachmann M., McMichael Jr R.W., Huber J.L., Regulation of sucrose-phosphate synthase by reversible protein phosphorylation: Manipulation of activation and inactivation in vivo, in: Points H.G., Salerno G.L., Echeverria E.J. (Eds.), International Symposium on Source Metabolism, American Society of Plant Physiologists, 1995, pp. 6–13. [7] Khanna-Chopra R., Reddy P.V., Regulation of leaf senescence by reproductive sink intensity in cowpea (Vigra unguiculata L. Walp), Ann. Bot. 61 (1988) 655–658. [8] Lunn J.E., Hatch M.D., The role of sucrose-phosphate synthase in the control of photosynthate partitioning in Zea mays leaves, Aust. J. Plant Physiol. 24 (1997) 1–8. [9] Makino A., Mae T., Ohira K., Differences between wheat and rice in the enzyme properties of ribulose1,5-bisphosphate carboxylase/oxygenase and the relationship to photosynthetic gas exchange, Planta 174 (1988) 30–38. [10] Makino A., Nakano H., Mae T., Responses of ribulose1,5-bisphosphate carboxylase, cytochrome f, and sucrose synthesis enzymes in rice leaves to leaf nitrogen and their relationships to photosynthesis, Plant Physiol. 105 (1994) 173–179.
953
[11] Micallef B.J., Haskins K.A., Vanderveer P.J., Poh K.S., Shewmaker C.K., Sharkey T.D., Altered photosynthesis, flowering, and fruiting in transgenic tomato plants that have an increased capacity for sucrose synthesis, Planta 196 (1995) 327–334. [12] Ono K., Ishimaru K., Aoki N., Takahashi S., Ozawa K., Ohkawa Y., Ohsugi R., Characterization of a maize sucrose-phosphate synthase protein and its effect on carbon partitioning in transgenic rice plants, Plant Prod. Sci. 2 (1999) 172–177. [13] Ono K., Terashima I., Watanabe A., Interaction between nitrogen deficit of a plant and nitrogen content in the old leaves, Plant Cell Physiol. 37 (1996) 1083–1089. [14] Seneweera S.P., Basra A.S., Barlow E.W., Conroy J.P., Diurnal regulation of leaf blade elongation in rice by CO2, Plant Physiol. 108 (1995) 1471–1477. [15] Signora L., Galtier N., Skot L., Lucas H., Foyer C.H., Overexpression of sucrose phosphate synthase in Arabidopsis thaliana results in increased foliar carbohydrate accumulation in plants after prolonged growth with CO2 enrichment, J. Exp. Bot. 49 (1998) 669–680. [16] Stitt M., Huber S., Kerr P., Control of photosynthetic sucrose formation, in: Hatch M.D., Boardmann N.K. (Eds.), The Biochemistry of Plants: Photosynthesis, Academic Press, New York, 1987, pp. 327–409. [17] Takeoka Y., Shimizu M., Wada T., Panicles, in: Matsuo T., Hoshikawa K. (Eds.), Science of the Rice Plant, vol. 1, Food and Agriculture Policy Research Center, Tokyo, 1993, pp. 295–338. [18] Tobias D.J., Hirose T., Ishimaru K., Ishige T., Ohkawa Y., Kano-Murakami Y., Matsuoka M., Ohsugi R., Elevated sucrose-phosphate synthase activity in source leaves of potato plants transformed with the maize SPS gene, Plant Prod. Sci. 2 (1999) 92–99. [19] Wintermans J.F.G.M., De Mots A., Spectrophotometric characteristics of chlorophylls and their pheophytins in ethanol, Biochim. Biophys. Acta 109 (1965) 448–453. [20] Worrell A.C., Bruneau J.M., Summerfelt K., Boersig M., Voelker T.A., Expression of a maize sucrose phosphate synthase in tomato alters leaf carbohydrate partitioning, Plant Cell 3 (1991) 1121–1130.
vol. 37 (12) 1999