Cytokinin Effects on Ribulose-1,5-Bisphosphate Carboxylase ...

Published November, 2004

Cytokinin Effects on Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Nitrogen Partitioning in Rice during Ripening Taiichiro Ookawa,* Yukiko Naruoka, Ayumi Sayama, and Tadashi Hirasawa

Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.

ABSTRACT

Graduate School of Agriculture, Tokyo Univ. of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan. Received 28 Dec. 2003. *Corresponding author ([email protected]).

kino et al., 1984). We demonstrated previously that Akenohoshi maintained larger amounts of nitrogen in leaves during ripening, which might be responsible for the difference in the maintenance of high leaf levels of Rubisco between cultivars (Ookawa et al., 2003). The high leaf nitrogen content in Akenohoshi is achieved by the greater accumulation of nitrogen in plants and, in addition, by the greater partitioning of nitrogen to leaves. It was also observed that larger amounts of cytokinins were transported to above-ground parts of the plant from the roots during the ripening stage in Akenohoshi than in Nipponbare (Soejima et al., 1992, 1995). Cytokinin can delay leaf senescence and recent reports have illustrated the importance of cytokinin in the control of senescence (Gan and Amasino, 1995; Buchanan-Wollaston et al., 2003). Cytokinins might also contribute to the maintenance of a high Rubisco content. However, it remains to be determined whether and how cytokinins might suppress a decline in Rubisco content. Several studies have reported that cytokinin can induce the expression of photosynthetic genes and promote protein synthesis (Lerbs et al., 1984; Sugiharto et al., 1992; Suzuki et al., 1994). Cytokinins affected the expression of genes for Rubisco in detached Cucurbita cotyledons (Lerbs et al., 1984) and for phosphoenolpyruvate carboxylase in detached leaves of maize (Zea mays L.) seedlings (Suzuki et al., 1994). It can be assumed as a possible mechanism that suppression of the decrease in the accumulation of transcripts of genes for Rubisco by cytokinin results in the maintenance of a high level of Rubisco during leaf senescence. In addition, cytokinins also affected nitrogen and biomass partitionings in wheat seedlings (Simpson et al., 1982) and a perennial herb (Wagner and Beck, 1993; Beck, 1996). They increased nitrogen levels in older leaves by promoting the accumulation of amino acids and other nitrogenous compounds in the leaves (Jordi et al., 2000). It can be also assumed as a possible mechanism that high nitrogen partitioning to leaves by cytokinin results in maintenance of high levels of Rubisco during leaf senescence. In the following experiments, our main aims were therefore (i) to compare the levels of Rubisco, nitrogen, and rbcL and rbcS transcripts between cultivars Nipponbare and Akenohoshi, (ii) to analyze the effects of exogenous cytokinin on the levels of Rubisco, nitrogen, and rbcL and rbcS transcripts in leaves of rice during the ripening stage, and (iii) in addition, to analyze the effects of exogenous cytokinin on the nitrogen content of leaves by determining nitrogen absorption and parti-

Published in Crop Sci. 44:2107–2115 (2004). © Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA

Abbreviations: BA, benzylaminopurine; NF, nitrogen fertilizer; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase.

High yield rice (Oryza sativa L.) cultivar Akenohoshi can realize a higher rate of photosynthesis during ripening by maintaining a higher level of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) than cv. Nipponbare. To clarify the causal factors for the difference in the levels of Rubisco in leaves between rice cultivars and the mechanisms of action of cytokinins on the maintenance of high levels of Rubisco in leaves, the level of rbcL and rbcS transcripts and the nitrogen content of leaves were investigated. The Rubisco content remained high not only in plants given additional nitrogen fertilizer (NF) but also in plants sprayed with 6-benzylaminopurine (BA). There was a close correlation between the Rubisco content and the level of rbcL and rbcS mRNAs. The maintenance of high leaf levels of Rubisco and of rbcL and rbcS mRNAs was related to the high nitrogen content in leaves, irrespective of the treatments and cultivars. The maintenance of high leaf nitrogen content was caused by an increase in nitrogen partitioning to leaves and not by an increase in nitrogen absorption by plants treated with BA. These results indicate that there are two probable effects of BA on the leaf level of Rubisco. One is the direct effect of BA inducing the synthesis of Rubisco. The other is the indirect effect of BA inducing the effective partitioning of nitrogen to leaves. Cytokinins could account for the difference in the reduction in leaf level of Rubisco during senescence between the rice cultivars.

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any factors contribute to the differences in dry matter production and grain yield among rice cultivars (Jiang et al., 1988a; Kuroda et al., 1989; Saitoh et al., 1990a, 1990b). One of these factors, the delay in leaf senescence, is considered very important. It was observed in many crops that the rate of leaf photosynthesis was kept high during ripening stage, and heavier dry matter and grain were produced in plants with slower leaf senescence (Hirasawa et al., 1994, 1998; Jiang et al., 1988b; Nakamura et al., 2003). Dry matter production and yield are higher in the rice cultivar Akenohoshi than in cv. Nipponbare. One of the important causes of this difference is the smaller decrease in the rate of photosynthesis during the ripening stage in Akenohoshi than in Nipponbare (Jiang et al., 1988b). The rate of photosynthesis during senescence is closely correlated with leaf levels of ribulose1,5-bisphosphate carboxylase/oxygenase (Rubisco) (Makino et al., 1985). Decreases in Rubisco content during leaf senescence are smaller in Akenohoshi than in Nipponbare during the ripening stage (Jiang et al., 1999). There is also a close correlation between nitrogen content and Rubisco content during leaf senescence (Ma-

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tioning of nitrogen to various organs in a whole plant, in an attempt to characterize the effects and significance of cytokinins in the maintenance of a high level of Rubisco as well as to clarify the cause of the difference in the rate of photosynthesis during senescence between Nipponbare and Akenohoshi. The effects of the application of cytokinin were also compared with those of additional NF. Experiments were performed in 2000 and 2001 and, since the same results were obtained in both years, only the experiments performed in 2001 are described in this report. MATERIALS AND METHODS Materials and Cultivation of Plants Rice seeds (Oryza sativa L., cvs. Nipponbare and Akenohoshi) were sown in nursery boxes on 27 May 2001, and seedlings at the 4th-leaf stage in both cultivars were transplanted to 15-L Wagner pots (Fujiwara Scientific Inc., Tokyo, Japan) filled with a mixture of Tama River alluvial soil and Kanto diluvial soil (1:1, v/v). The planting density was three plants per hill and four hills per pot. Compound fertilizer (7.1 g) that contained 14% each of N, P2O5, and K2O was applied to each pot as basal dressing. Compound fertilizer (6.3 g) that contained 16% each of N and K2O was applied to each pot as top dressing on 20 July and on 5 August. These fertilizers were compounded from ammonium nitrogen. Pots for each plot were placed randomly in the upland field of the Field Science Center, Tokyo University of Agriculture and Technology with a spacing of 80 by 50 cm. Plants were grown under submerged conditions throughout experiments. Heading occurred on 31 August both cultivars.

Application of Additional NF and Cytokinin In this study, the plants of the cultivar Nipponbare were treated with additional NF and cytokinin as follows. Ammonium sulfate was applied to some pots on 1 September at the early ripening stage at a rate of 10 g per pot as additional NF. For treatment with cytokinin, 30 mL of a 10⫺4 M solution of BA that contained 0.05% (v/v) Tween 20 [polyoxyethylene(20) sorbitan monolaurate], as surfactant, was sprayed on the entire aboveground parts of each hill in pots at 2-d intervals from 5 September onwards. Thirty milliliters of water containing 0.05% Tween 20 were sprayed on each hill in other pots that served as controls.

Determination of Levels of Rubisco and Nitrogen in Leaves Levels of Rubisco and nitrogen in the same leaves were determined as follows. The flag leaf and the 3rd leaf, counted from the flag leaf on the main culm, were collected and stored at ⫺80⬚C before analysis. The area and fresh weight of each leaf were measured and each leaf was separated into two equal parts for quantitation of Rubisco and nitrogen. The halves of leaves were homogenized separately with a mortar and pestle in a solution that contained 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10 mM MgCl2, 10 mM 2-mercaptoethanol, and 5% (w/w) insoluble polyvinylpyrolidone (Polyclar VT, Wako Chem. Industries, Tokyo, Japan). The homogenate was centrifuged at 10 000 ⫻ g for 10 min at 4⬚C. The supernatant was used for quantitation of Rubisco by the single radial immunodiffusion method (Sugiyama and Hirayama, 1983) with rabbit polyclonal antibodies raised against purified Rubisco from rice.

Nitrogen was quantified with a CN analyzer (MT-600, Yanaco Inc., Kyoto, Japan).

Analysis of Levels of rbcL and rbcS mRNAs Flag leaves on main culms were collected between 1000 and 1100 h on a clear day and immediately frozen in liquid N2 for quantitation of mRNAs. Total RNA was isolated by the cetyltrimethylammonium bromide (CTAB) method (Chang et al., 1993) and concentrations of total RNA were determined by spectrophotometry (U-3210, Hitachi Inc., Tokyo, Japan). Levels of rbcL and rbcS mRNAs were determined as follows. Extracted RNAs (10 ␮g) were dissolved in RNA sample buffer, heated at 65⬚C for 15 min, and then cooled on ice. Denatured RNAs were blotted onto a nylon membrane (Hybond-N⫹, Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA) with a dot blot apparatus (Milli Blot-N, Millipore, MA, USA). The membrane was soaked in 0.05 M NaOH for 5 min and washed in 2⫻ SSC (1⫻ SSC ⫽ 0.15 M NaCl and 15 mM sodium citrate) and then heated at 80⬚C for 2 h. 32P-Labeled DNA probes for rbcL and rbcS were prepared from a 565bp fragment of the rice rbcL gene (Hirai et al., 1985) and a 1.3-kbp fragment of the rice rbcS gene (Matsuoka et al., 1988), respectively. These probes were radiolabeled with a randomly primed DNA labeling system (RPN1607, Amersham Pharmacia Biotech Inc.). Hybridization with the 32P-labeled probes was performed in 50% (v/v) formamide, 0.1% (w/v) SDS, 50 mM sodium phosphate buffer (pH 6.5), 5⫻ SSC, 5⫻ Denhardt’s solution [0.1% (v/v) Ficoll, 0.1% (v/v) bovine serum albumin and 0.1% (w/v) polyvinylpyrrolidone], and 0.15 mg mL⫺1 denatured salmon sperm DNA. The membrane was incubated overnight at 42⬚C and then washed twice in 2⫻ SSC plus 0.1% SDS for 10 min. It was then incubated twice with 0.1⫻ SSC plus 0.1% SDS for 10 min at 65⬚C. Relative levels of mRNA were determined with a Bioimage analyzer (BAS1500, Fuji Film Inc., Tokyo, Japan).

Determination of the Nitrogen Content of Various Organs The levels of nitrogen in various organs were determined as follows. Three pots with plants having an average number of ears were selected from each treatment group, and all the plants in each plot separated into leaf blades, culms plus leaf sheaths, ears and roots. Pooled samples were dried in an oven at 90⬚C for 72h. After weighing, they were powdered with a ball mill (MB-1, Chuokako Inc., Aichi, Japan) for determinations of total nitrogen with the CN analyzer. Total nitrogen contents were calculated on a dry weight and nitrogen concentration basis.

RESULTS Levels of Rubisco in Leaves Comparison between Two Cultivars The time courses of changes in levels of Rubisco during ripening were compared between the untreated plants (control) of Nipponbare and the untreated plants of Akenohoshi (Fig. 1). In flag leaves, there were no differences between the two cultivars in terms of Rubisco content at the heading stage (31 August). After heading, the Rubisco content remained higher in Akenohoshi than in Nipponbare (Fig. 1A). In the 3rd leaf, the decline in Rubisco content with time after heading was larger in Nipponbare than in Akenohoshi (Fig. 1B).


OOKAWA ET AL.: CYTOKININ AND NITROGEN PARTITIONING IN RICE

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Fig. 1. Effects of 6-benzylaminopurine (BA) and additional nitrogen fertilizer (NF) applications on the Rubisco contents in the flag (A) and 3rd (B) leaves of Nipponbare (NI). The plants of Akenohoshi were grown in the same condition as the control for Nipponbare. Vertical bars represent standard deviations (n ⫽ 3). Symbols with different letters are significantly different at the 0.05 probability level (LSD).

Effects of Treatment with BA and NF The time courses of changes in levels of Rubisco during ripening were compared between the controls and the BA- and NF-treated plants of Nipponbare (Fig. 1). In controls of Nipponbare, the Rubisco content in flag leaves decreased with time after heading; however, the decrease was considerably smaller in the NF-treated plants than in controls (Fig. 1A). The effects of BA were similar to those of NF. The Rubisco content of the flag leaves of BA-treated plants remained significantly higher throughout the ripening stage than that of controls (Fig. 1A). In the 3rd leaf, the Rubisco content decreased much more markedly with time after heading than in flag leaves. The differences among 3rd leaves of plants subjected to the various treatments were identical to those observed in the case of the flag leaves (Fig. 1B). Rubisco content remained much higher in the plants treated with NF and BA than in the controls.

Nitrogen Contents of Leaves Comparison between Two Cultivars The time courses of changes in nitrogen content during ripening were compared between the untreated

plants (control) of Nipponbare and the untreated plants of Akenohoshi (Fig. 2). The nitrogen contents in flag leaves were higher in Nipponbare than in Akenohoshi at the heading stage. After heading, the nitrogen content decreased more slowly in Akenohoshi than in Nipponbare (Fig. 2A). In the 3rd leaf, nitrogen content decreased more markedly with time after heading in Nipponbare than in Akenohoshi, and nitrogen content remained significantly higher in Akenohoshi than in Nipponbare (Fig. 2B). Effects of Treatment with BA and NF The time courses of changes in nitrogen content during ripening were compared between the controls and the BA- and NF-treated plants of Nipponbare (Fig. 2). In the controls, the nitrogen content declined with time in both flag leaves (Fig. 2A) and 3rd leaves (Fig. 2B). By contrast, the nitrogen content of these leaves remained high in the NF-treated plants with almost no reduction during ripening. In the BA-treated plants, the nitrogen content decreased but remained relatively high during ripening compared with the controls.

Fig. 2. Effects of 6-benzylaminopurine (BA) and additional nitrogen fertilizer (NF) on the nitrogen content in flag (A) and 3rd (B) leaves of Nipponbare (NI). The plants of Akenohoshi were grown in the same condition as the control for Nipponbare. Vertical bars represent standard deviations (n ⫽ 3). Symbols with different letters are significantly different at the 0.05 probability level (LSD).


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Fig. 3. Effects of 6-benzylaminopurine (BA) and additional nitrogen fertilizer (NF) applications on the relative levels of rbcL mRNA (A) and rbcS mRNA (B) in flag leaves of Nipponbare (NI). The plants of Akenohoshi were grown in the same condition as the control for Nipponbare. The levels of mRNAs are given relative to levels of the mRNAs at the heading stage. Vertical bars represent standard deviations (n ⫽ 3). Symbols with different letters are significantly different at the 0.05 probability level (LSD).

Accumulation of rbcL and rbcS mRNAs Comparison between Two Cultivars The relative levels of rbcL and rbcS mRNAs in flag leaves were compared between the two cultivars (Fig. 3). There were no differences between the two cultivars in terms of the accumulation of rbcL and rbcS mRNAs at the heading stage (Fig. 3A, 3B). After heading, the level of rbcL mRNA decreased with time in Nipponbare; however, the level remained high in Akenohoshi through 20 September, the middle ripening stage (Fig. 3A). The decline in rbcS mRNA was larger compared with the level of rbcL mRNA in both cultivars. The level of rbcS mRNA remained higher in Akenohoshi than in Nipponbare at the middle ripening stage (Fig. 3B).

were investigated (Fig. 3). The levels of rbcL mRNA (Fig. 3A) and rbcS mRNA (Fig. 3B) decreased with time in the controls. However, the levels remained high in plants treated with NF and BA during ripening, except for rbcS mRNA at the end of September. Relationships between Rubisco Content, rbcL and rbcS mRNA Levels, and Nitrogen Content The relationship between the Rubisco content, nitrogen content and the levels of rbcL and rbcS mRNAs were compared among the untreated plants (control), the BA- and NF-treated plants of Nipponbare and the untreated plants of Akenohoshi.

Effects of Treatment with BA and NF

Relationships between Rubisco Content and Levels of rbcL and rbcS mRNAs

The effects of NF and BA on the relative levels of rbcL and rbcS mRNAs in flag leaves of Nipponbare

There were close relationships between Rubisco contents and levels of rbcL (Fig. 4A) and rbcS (Fig. 4B)

Fig. 4. Relationships between the relative levels of rbcL mRNA (A) and rbcS mRNA (B) and the Rubisco content of flag leaves. *** Significant at the 0.001 probability level.


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treated plants of Akenohoshi than in the NF-treated plants of Nipponbare.


Relationship between Levels of rbcL and rbcS mRNAs and Nitrogen Content There were also close correlations between the nitrogen content and the level of rbcL mRNA (Fig. 6A) and between the nitrogen content and the level of rbcS mRNA (Fig. 6B). At leaf nitrogen contents above 1.2 g m⫺2, however, the levels of both mRNAs tended to be higher in the BA-treated plants of Nipponbare and the untreated plants of Akenohoshi than in the NF-treated plants of Nipponbare.

Effects of BA and NF on the Accumulation and Partitioning of Nitrogen

Fig. 5. Relationship between the nitrogen content and the Rubisco content of flag leaves. *** Significant at the 0.001 probability level.

mRNAs irrespective of treatments and cultivars. It was suggested that the maintenance of high levels of rbcL and rbcS mRNAs contributed to the maintenance of high leaf levels of Rubisco during ripening in the BAand NF-treated plants in Nipponbare and also in the untreated plants of Akenohoshi. Relationships between Rubisco Content and Nitrogen Content There were close correlations between Rubisco content and nitrogen content over a wide range of nitrogen contents (Fig. 5). At leaf nitrogen contents above 1.2 g m⫺2, however, the Rubisco content tended to be greater in the BA-treated plants of Nipponbare and the un-

A high leaf nitrogen content was maintained not only in the NF-treated plants of Nipponbare, but also in the BA-treated plants of Nipponbare. Details of the accumulation and partitioning of nitrogen were compared between the controls and the BA- and NF-treated plants of Nipponbare. NF Treatment The amount of accumulated nitrogen in the entire plant at the late ripening stage was much larger after NF treatment than in the controls (Table 1). As a result, although the nitrogen contents of leaves decreased rapidly from the heading stage to the late ripening stage in the controls, the NF-treated plants retained larger amounts of nitrogen in their leaves, with no reduction from the heading stage in leaves, as well as in other organs (Table 2). There were large differences, in terms of the changes in nitrogen content of the organs between treatments. In leaves and culms plus leaf sheaths of

Fig. 6. Relationships between the nitrogen content and the relative levels of rbcL mRNA (A) and rbcS mRNA (B) in flag leaves. **, *** Significant at the 0.01 and 0.001 probability levels, respectively.

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Table 1. Effect of 6-benzylaminopurine (BA) and additional nitrogen fertilizer (NF) applications on the nitrogen content and the increase in nitrogen content of entire plants of Nipponbare. Nitrogen content


Treatment Control BA NF

Heading (31 August)

Late ripening (25 September)

Increase in nitrogen content

592.4 ⫾ 35.1† – –

mg hill⫺1 635.7 ⫾ 19.3a 653.9 ⫾ 15.8a 1024.6 ⫾ 6.2b

43.3 61.5 432.2

† Data represent means ⫾ standard deviations of results from three replicates. Means followed by different letters are significantly different at the 0.05 probability level (LSD).

controls, the nitrogen content decreased significantly, but it remained high in the NF-treated plants. The increase in the nitrogen content of panicles was significantly larger in the NF-treated plants than in controls. The partitioning of nitrogen to leaves increased significantly, but that to panicles and roots decreased as a result of NF treatment (Table 2). It appears, therefore, that the maintenance of high leaf nitrogen content in the NF-treated plants resulted not only from an increase in nitrogen accumulation by the entire plant but also from an increase in nitrogen partitioning to leaves. BA Treatment There was no difference, in terms of the increase in the nitrogen content of entire plants, between the BAtreated plants and controls from the heading stage to the late ripening stage, although the accumulation of nitrogen in the former tended to be slightly greater than in the latter (Table 1). The decrease in leaf nitrogen content after heading was smaller in the BA-treated plants than in the controls (Table 2). Moreover, increases in the nitrogen content of panicles were clearly smaller in the BA-treated plants than in the controls. The nitrogen content decreased in culms plus leaf sheaths and did not change in roots in the case of both BA-treated plants and controls. Differences in the ex-

tent of increase or decrease between treatments were small. The partitioning of nitrogen to leaves at the late ripening stage was 36.5% and was higher in the BAtreated plants than in the controls (22.6%) but less nitrogen was partitioned to panicles in the former than the latter. From these results, we can conclude that maintenance of a high leaf nitrogen content in the BA-treated plants was caused by an increase in the nitrogen partitioning to leaves and a decrease in the partitioning to panicles and not by an increase in accumulation of nitrogen by the entire plant.

DISCUSSION Causal Factors of the Maintenance of the Higher Level of Rubisco during Ripening in Akenohoshi than Nipponbare Akenohoshi can maintain higher levels of Rubisco in the leaves during ripening than Nipponbare (Fig. 1). This enables Akenohoshi to maintain a higher rate of leaf photosynthesis during ripening (Jiang et al., 1999). Akenohoshi maintained higher levels of leaf nitrogen and rbcL and rbcS mRNAs during ripening than did Nipponbare (Fig. 2, 3). Leaf Rubisco content correlated closely with nitrogen content and rbcL and rbcS mRNAs during ripening irrespective of treatments and cultivars (Fig. 4, 5). This means that a higher capacity of nitrogen accumulation in leaves and a higher capacity for the expression of Rubisco gene might be responsible for maintaining greater levels of Rubisco in Akenohoshi. It is known that the higher content of leaf nitrogen in Akenohoshi results from a higher capacity for nitrogen absorption and greater nitrogen partitioning to leaves (Ookawa et al., 2003). It was also reported that the amount of cytokinins transported from root to shoot was much greater in Akenohoshi than Nipponbare (Soejima et al., 1992, 1995). However, the effect of cytokinin on leaf levels of Rubisco and processes of cytokinin action were not examined in this paper.

Table 2. Effects of 6-benzylaminopurine (BA) and additional nitrogen fertilizer (NF) applications on the nitrogen contents, changes in nitrogen content and nitrogen partitioning to various organs in entire plants of Nipponbare. Nitrogen content Organ

Heading (31 Aug.)

Nitrogen partitioning

Late ripening (25 Sept.)

Changes in nitrogen content

Heading (31 Aug.)

mg hill⫺1 Leaves Control BA NF Culms ⫹ leaf sheaths Control BA NF Panicles Control BA NF Roots Control BA NF

Late ripening (25 Sept.) %

292.4 ⫾ 12.1† – –

142.9 ⫾ 4.9a 238.7 ⫾ 3.2b 307.2 ⫾ 3.5c

⫺149.5 ⫺53.7 14.8

49.4 ⫾ 1.0 – –

22.6 ⫾ 0.3a 36.5 ⫾ 1.3b 30.0 ⫾ 0.6c

202.0 ⫾ 18.2 – –

114.1 ⫾ 3.1a 115.2 ⫾ 0.3a 202.3 ⫾ 12.2b

⫺87.9 ⫺86.8 0.3

34.0 ⫾ 1.0 – –

17.9 ⫾ 0.5a 17.6 ⫾ 0.4a 19.7 ⫾ 1.0b

58.2 ⫾ 1.6 – –

328.7 ⫾ 8.9a 259.7 ⫾ 18.8b 459.4 ⫾ 3.1c

270.5 201.5 401.2

9.8 ⫾ 0.4 – –

51.5 ⫾ 0.7a 39.8 ⫾ 2.0b 44.8 ⫾ 0.5c

39.8 ⫾ 4.2 – –

50.1 ⫾ 7.4a 40.3 ⫾ 1.6b 55.7 ⫾ 0.6a

10.3 0.5 15.9

6.8 ⫾ 0.4 – –

8.0 ⫾ 1.0a 6.2 ⫾ 0.4b 5.5 ⫾ 0.0b

† Data represent means ⫾ standard deviations of results from three replicates. Nitrogen partitioning is expressed as the nitrogen content of the indicated organs as a percentage of the total nitrogen content of entire plant. Means followed by different letters are significantly different at the 0.05 probability level (LSD).



To clarify the effects of cytokinin on the maintenance of a high level of Rubisco in leaves during the ripening stage, as well as its effects on the leaf nitrogen content and the absorption and partitioning of nitrogen, plants of Nipponbare were treated with BA and additional NF. The Rubisco content and levels of rbcL mRNA and rbcS mRNA remained high during ripening in treated plants (Fig. 1, 3). There were close correlations between the Rubisco content and levels of rbcL mRNA and rbcS mRNA for all plants examined (Fig. 4). As another effect of application of BA, the nitrogen content of the flag and 3rd leaves remained high during ripening (Fig. 2). Maintenance of the high nitrogen content in leaves by the application of BA appeared to be caused by changes in the partitioning of nitrogen and not by an increase in nitrogen accumulation by the entire plant (Table 1). The partitioning of nitrogen to panicles and roots decreased in the BA-treated plants, with a resultant remarkably small decrease in leaf nitrogen content during senescence. From these results it could be concluded that the larger amount of cytokinins transported from roots to the shoot in Akenohoshi than in Nipponbare might cause the higher leaf level of Rubisco observed in Akenohoshi by retaining higher levels of rbcL and rbcS mRNAs and partitioning more nitrogen to leaves. It has been reported that cytokinin affects protein gene expression (Sugiharto et al., 1992; Suzuki et al., 1994) and nitrogen partitioning (Simpson et al., 1982; Jordi et al., 2000). Recent studies have shown that the regulation of genes does not proceed by a single signal pathway, but complex signaling networks are at work (Sakakibara, 2003). We propose that cytokinin regulates Rubisco content directly by affecting Rubisco gene expression at the leaf level and indirectly nitrogen partitioning at the whole plant level.

Direct Regulation of Rubisco Content by Cytokinin through Gene Expression The accumulation of rbcL and rbcS mRNAs decreased during leaf senescence, and there were close correlations between the decrease in Rubisco content and the levels of rbcL and rbcS mRNAs (Fig. 4). High levels of rbcL and rbcS mRNAs were maintained not only by the application of additional NF, but also by repeated application of BA during ripening (Fig. 3). In previous studies, cytokinins promoted the expression of genes for photosynthetic proteins by increasing transcription (Sugiharto et al., 1992; Suzuki et al., 1994). High levels of Rubisco were maintained in excised leaf segments floated on a solution that contains BA (Ookawa et al., 1997). Cytokinin levels in the xylem sap, transported from the roots to aboveground parts, were increased in plants supplied with additional NF in rice plants (Ookawa et al., 2003) and other plant species (Wagner and Beck, 1993; Takei et al., 2001). Therefore, it is also likely that cytokinins contributed to maintenance of high leaf levels of Rubisco in the NF- and BAtreated plants. It can be assumed that cytokinin might affect the turnover of Rubisco in leaves during senescence. In this

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study, the effects of exogenous BA and of additional NF on Rubisco content were investigated, focusing on the synthesis of Rubisco. However, many uncertainties remain with respect to the turnover of Rubisco. In rice, levels of rbcL and rbcS mRNAs were reported to decrease markedly before a decline in Rubisco content after heading, and these mRNAs remained at low levels during the ripening stage. The decrease in the Rubisco content of leaves was closely correlated with the degradation of Rubisco during senescence, rather than a decline in its synthesis (Suzuki et al., 2001). Levels of Rubisco decrease when the rate of degradation of Rubisco exceeds the rate of synthesis during senescence (Mae et al., 1983). In the cited study by Suzuki et al. (2001), the degradation of Rubisco was influenced significantly by specific treatments. When plants were grown in nutrient solution with a high concentration of nitrogen before heading and the concentration of nitrogen was gradually decreased after heading, the synthesis of Rubisco might have been limited by a decreased influx of nitrogen into leaves. Moreover, the rate of degradation of Rubisco might have increased, with a resultant reduction in Rubisco content after heading. It is unclear whether synthesis or the suppression of degradation is more important in the maintenance of a high level of Rubisco in rice leaves during senescence. There have been no studies, to our knowledge, that have compared the degradation and synthesis of Rubisco during senescence between leaves with different levels of Rubisco. The present research showed that the synthesis of Rubisco was significantly affected by growth conditions, and high levels of Rubisco and rbcL and rbcS mRNAs were maintained by BA and by the application of additional NF. The degradation of Rubisco might also be influenced by these treatments and should be the focus of future research. The accumulation of rbcL and rbcS mRNAs and the Rubisco content of leaves were related to nitrogen content of leaves in all plants, including those treated with BA. The Rubisco content and the levels of the two mRNAs at leaf nitrogen contents above 1.2 g m⫺2 tended to be higher in the BA-treated plants than in the NFtreated plants (Fig. 5, 6). This observation indicates that cytokinins contribute to the maintenance of a high Rubisco content, independently of nitrogen content. The efficiency of nitrogen use for photosynthesis also increased with the enhanced expression of genes for proteins involved in C4 photosynthesis that were induced specifically by cytokinins in the C4 plant maize (Suzuki et al., 1994; Takei et al., 2002). The accumulation of transcripts for Rubisco and the efficiency of nitrogen use for photosynthesis might also be increased specifically by cytokinins in rice, a C3 plant. Recent studies suggested that cytokinins affected the expression of the photosynthetic genes through a signal transduction pathway from roots to shoots (Takei et al., 2002; Sakakibara, 2003). The molecular mechanisms responsible for the control of Rubisco expression by cytokinins should be investigated in leaves during senescence.

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Indirect Regulation of Rubisco Content by Cytokinin by Whole-Plant Nitrogen Partitioning The nitrogen content of leaves remained high as a result of increases in the accumulation of nitrogen by the entire plant and also as a result of the partitioning of large amounts of nitrogen to leaves of the NF-treated plants. By contrast, in the BA-treated plants, the nitrogen content of leaves remained high as a result of the partitioning of more nitrogen to leaves, irrespective of the accumulation of nitrogen by the entire plant. In the BA- and NF-treated plants, the chlorophyll contents of leaves also remained high during senescence, and there was a close correlation between the nitrogen content and the chlorophyll content (data not shown). The maturity stages did not differ between the treatments. The greater partitioning of nitrogen to leaves in the BA-treated plants might have resulted from suppression of nitrogen translocation from leaves to other organs and/or an increase in the distribution of nitrogen to leaves. Increases in the nitrogen content of panicles were reduced in the BA-treated plants. The nitrogen content of panicles was approximately 40% of the total content in the entire plant at the late ripening stage and was lower than in the controls. The differences in nitrogen content of panicles and leaves between BAtreated plants and controls were far larger than the difference in the amount of nitrogen accumulated by entire plants during ripening. Therefore, maintenance of a high level of nitrogen in leaves might have resulted predominantly from a decrease in the efflux of nitrogen by translocation from leaves to the other organs in the cytokinin-treated plants. Nitrogen partitioning to aboveground parts of plants was enhanced and that to roots was reduced in wheat (Triticum aestivum L.) plants treated with cytokinins (Simpson et al., 1982). Jordi et al. (2000) used a 15N tracer to investigate patterns of nitrogen partitioning in transgenic tobacco (Nicotiana spp.) plants, in which the gene for a cytokinin-synthetic enzyme, isopentenyltransferase (ipt), was specifically expressed in senescent leaves. They found that high levels of Rubisco were maintained reflecting the partitioning of a large amount of 15N to leaves, with sink activities for nitrogen being enhanced in senescent leaves, where levels of cytokinins had been specifically increased. A large amount of nitrogen was partitioned to leaves in Akenohoshi with a high cytokinin activity in its xylem sap compared with those in Nipponbare (Ookawa et al., 2003). These results show that cytokinin might affect the nitrogen partitioning. These observations suggest that Rubisco synthesis is promoted through gene expression induced by cytokinin. However, it is known that cytokinin affects the transport of substances (Monthes and Engelbrecht, 1961). It has been suggested that cytokinin-mediated nitrogen signaling is mainly related to the control of nitrogen partitioning and development (Sakakibara, 2003). Recent studies have reported that cytokinin affected the activities of nitrogen assimilation and re-assimilation enzymes (Watanabe et al., 1994; Yu et al., 1998). The

activity and the gene expression of nitrate reductase increased with the application of cytokinin (Yu et al., 1998). It was also reported that an ammonium transporter, located in leaves, decreased during senescence (von Wiren et al., 2000). There are cytokinin-mediated nitrogen signaling pathways at the whole plant level (Takei et al., 2002; Sakakibara, 2003), and it can be assumed that cytokinin might affect the nitrogen transport, assimilation and re-assimilation in source and sink organs and therefore change the partitioning of nitrogen between source and sink organs. The biochemical and molecular mechanisms of the effects of cytokinins on nitrogen partitioning in whole plants remain to be clarified. This study and our previous study (Ookawa et al., 2003) show that the photosynthetic rate and the level of Rubisco in leaves during ripening were maintained by cytokinin. It is expected that dry matter production and yield might be increased by the application of BA. Akenohoshi achieves high dry matter production and yield by maintaining a high photosynthetic rate. Studies are underway on the effects of cytokinin on dry matter production and yield in rice plants. ACKNOWLEDGMENTS We thank Dr. A. Hirai of Tokyo University for providing the cDNA for rbcL and Dr. M. Matsuoka of Nagoya University for providing the cDNA for rbcS. We are also grateful to Prof. Kuni Ishihara for helpful discussion. This research was supported, in part, by a grant from the Ministry of Education, Science, Sports and Culture, Japan, and by a Grant-in-Aid (Bio Cosmos Program) from the Ministry of Agriculture, Forestry and Fisheries, Japan.

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