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Keywords:

  • Oryza sativa L.;
  • chloroplast DNA;
  • 15N;
  • protein degradation;
  • protein synthesis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Changes in the amount of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4·1·1·39) synthesized and degraded and the levels of rbcL and rbcS mRNAs were examined in the eighth leaf blades of rice from emergence to senescence. Synthesis of Rubisco was very active during leaf expansion, became quite low at the time of full expansion and then declined further during senescence. The changes in the levels of rbcL and rbcS mRNAs co-ordinated approximately with those in the amount of Rubisco synthesized. Thus, it is suggested that the amount of Rubisco synthesized is determined primarily by the levels of rbcL and rbcS mRNAs during the life span of the leaves. Degradation of Rubisco started just before the time of full expansion and became far more active than its synthesis during senescence. Since the synthesis of Rubisco during senescence scarcely contributed to its amount, it can be concluded that the degradation of Rubisco is the major determinant for the amount of Rubisco in senescent leaves. The decline in the level of rbcL mRNA occurred much earlier in the developmental stage and proceeded at a much faster rate than that of rbcL DNA, indicating that the level of rbcL DNA is not a major determinant for the level of rbcL mRNA in senescent leaves of rice.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

As a key enzyme for carbon fixation, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4·1·1·39) plays a central role in photosynthesis. The rate of CO2 assimilation at ambient CO2 partial pressure under high irradiance correlates well with the total amount of Rubisco throughout a leaf’s life (Makino, Mae & Ojima 1985; Evans 1986). Rubisco is the most abundant protein in the mature leaves of C3 plants, and it accounts for 15–30% of total nitrogen (N) (Evans 1989; Makino et al. 1992). Rubisco content increases rapidly during leaf expansion, reaches its maximum around maturation, and then declines gradually during senescence. Rubisco is degraded during senescence, and its degradation products are re-utilized as a source of N for developing tissues (Mae, Makino & Ohira 1983; Makino, Mae & Ohira 1984). Changes in the amount of Rubisco in leaves are thus directly related to carbon and N economy in plants.

The amount of Rubisco in a leaf is the result of a balance between its synthesis and degradation. It has been shown previously that in rice leaves, the amount of Rubisco synthesized is very high during leaf expansion but becomes quite low at the time of full expansion, and that the amount of Rubisco degraded is greater than the amount synthesized during senescence by using 15N as a tracer (Mae et al. 1983; Makino et al. 1984). Changes in the levels of rbcL and rbcS mRNAs and their relationship to the synthesis of Rubisco have been studied in amaranth leaves at various points of time from expansion to senescence (Nikolau & Klessig 1987) and in bean (Phaseolus vulgaris L.) leaves during senescence (Bate, Rothstein & Thompson 1991). These observations suggest that the rate of synthesis of the large and small subunits of Rubisco is controlled not only at the transcriptional level, but also at the translational level (at least in part) in senescent leaves. Although these studies provide some molecular basis for the changes in Rubisco synthesis during leaf senescence, changes in the absolute amount of Rubisco synthesized and degraded have not been examined simultaneously with changes in the levels of both mRNAs from leaf emergence to senescence. Therefore, it remains unclear how changes in the balance between these factors determine the amount of Rubisco during senescence.

The aim of this study was to elucidate the major determinants of the amount of Rubisco throughout the life span of rice leaves, particularly during senescence. The amounts of Rubisco synthesized and degraded were determined as in previous reports (Mae et al. 1983; Makino et al. 1984), and these were further compared with the levels of rbcL and rbcS mRNAs. In addition, the level of rbcL DNA was analysed because it has been found that the amount of chloroplast DNA declines in the early developmental stages of coleoptiles and leaves of rice (Sodmergen et al. 1991; Inada et al. 1998a, 1998b, 1999); it has also been suggested that such a decline could limit the level of rbcL mRNA during leaf senescence in soybeans (Jiang, Rodermel & Shibles 1993) and tobacco (Jiang & Rodermel 1995; Miller et al. 2000).

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Plant materials and 15N-labelling

Rice (Oryza sativa L. cv. Notohikari) plants were grown hydroponically in an environmentally controlled growth chamber (Makino, Nakano & Mae 1994). The chamber was operated with a 14 h photoperiod, 26/20 °C day/night temperature, 60% relative humidity and a photosynthetic photon flux density of 1000 µmol quanta m−2 s−1 at plant level during the daytime. One hundred 2-week-old seedlings were transplanted into a 20 L plastic vat containing a nutrient solution. The basal nutrient solution was as previously described by Mae & Ohira (1981). The nutrient solution was renewed once a week and its pH was adjusted to 5·5 with 1 m HCl. The strength of the nutrient solution was varied depending on the age of the plants (days after germination): 1/8 strength, 14–20 d; 1/6 strength, 21–27 d; 1/4 strength, 28–34 d; 3/8 strength, 35–41 d. The eighth leaf blades emerged 42 d after germination. 15N-labelling of the plant and collection of the samples were then carried out in two ways. In the first experiment (Figs 1–4), the nutrient solution was replaced for 4 d (commencing on the 42nd day) by a nutrient solution of 1/5 strength containing 0·2 mm (15NH4)2SO4 (30·4 atom% excess) instead of 0·2 mm NH4NO3. Thereafter, the plants were cultured again in a non-labelled nutrient solution of 1/5 strength. The whole eighth leaf blades were collected as samples every 4 d. In the second experiment (Figs 5–7), the plants were grown with a nutrient solution of 1/5 strength for 4 d from 42 to 45 d after germination, and a nutrient solution of 1/10 strength for the next 2 d. Then, the nutrient solution was replaced for 2 d by a 1/10 strength nutrient solution containing 0·1 mm (15NH4)2SO4 (30·3 atom% excess) instead of 0·1 mm NH4NO3. The strength of the nutrient solution was adjusted to supply an equal amount of nutrients per day. After 15N-labelling, the eighth, seventh and fifth leaf blades were collected as samples. All collections were performed between 1100 and 1300 h. The collected leaves were weighed and frozen in liquid nitrogen immediately, then stored at −80 °C until analysis.

image

Figure 1. Changes in length (a) and FW (b) of the eighth leaf blades of rice, and changes in the amounts of nitrogen (c) and chlorophyll (d) from emergence through to senescence. Data represent the means for three leaf blades. Vertical bars indicate standard error; arrows indicate the time of full expansion.

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image

Figure 2. Changes in the amount of Rubisco (a), its 15N abundance (b) and its synthesis (c) and degradation (d) in the eighth leaf blades of rice from emergence to senescence. Data for the amount of Rubisco and its 15N abundance represent the means for three leaf blades. Vertical bars indicate standard error. Data for the Rubisco synthesized and degraded were estimated from the mean amount of Rubisco and its 15N abundance for three leaf blades. The arrow indicates the time of the full expansion.

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image

Figure 3. Changes in the amount of total RNA (a) and total DNA (b) in the eighth leaf blades of rice from emergence through to senescence. Data represent the means for three leaf blades. Vertical bars indicate standard error; arrows indicate the time of the full expansion.

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image

Figure 4. Changes in the relative levels of rbcL and rbcS mRNAs and rbcL and rbcS DNAs in the eighth leaf blades of rice from emergence to senescence. (a) Gel blot analysis of rbcL and rbcS mRNAs and rbcL and rbcS DNAs. Total RNA (1·5 µg) and total DNA digested with EcoRI (2·0 µg) were separated by agarose gel electrophoresis. The SYBR green stained rRNA bands (Molecular Probes, Eugene, OR, USA) are shown as loading controls for RNA gel blot analysis. (b) Changes in the relative levels of rbcL and rbcS mRNAs and rbcL and rbcS DNAs. Total RNA (0·5 µg) and total DNA digested with EcoRI (0·3 µg) were slot-blotted onto a positively charged nylon membrane. Data represent the means for three leaf blades. Vertical bars indicate standard error; arrows indicate the time of the full expansion.

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image

Figure 5. The amounts of chlorophyll (a), nitrogen (b), total RNA (c) and total DNA (d) on the basis of unit leaf FW in the eighth, seventh and fifth leaf blades 9 d after the emergence of the eighth leaf blades. Data represent the means for three leaf blades. Vertical bars indicate standard error. Within each panel, bars with different letters are significantly different (Student’s t-test, P < 0·05).

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image

Figure 6. The amount of Rubisco (a) and its synthesis (b) on the basis of unit leaf FW in the eighth, seventh and fifth leaf blades 9 d after the emergence of the eighth leaf blades. Data represent the means for three leaf blades. Vertical bars indicate standard error. Within each panel, bars with different letters are significantly different (Student’s t-test, P < 0·05).

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image

Figure 7. The relative levels of rbcL and rbcS mRNAs and rbcL and rbcS DNAs in the eighth, seventh and fifth leaf blades 9 d after the emergence of the eighth leaf blades. (a) Gel blot analysis of rbcL and rbcS mRNAs and rbcL and rbcS DNAs. (b) Changes in the relative levels of rbcL and rbcS mRNAs and rbcL and rbcS DNAs. Analyses were carried out as in Fig. 4. Data represent the means for three leaf blades. Vertical bars indicate standard error. Within each panel, bars with different letters are significantly different (Student’s t-test, P < 0·05).

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Determination of Rubisco, chlorophyll and total N and 15N analysis of isolated Rubisco

Rubisco, chlorophyll and total N were determined as described in Makino et al. (1992), except that 50 mm sodium-phosphate buffer, pH 7·5, containing 0·8% (v/v) 2-mercaptoethanol, 2 mm iodoacetic acid and 5% glycerol was used as the homogenization buffer.

Isolation of Rubisco, its 15N analysis and estimation of the amounts of Rubisco synthesized and degraded were carried out as described in Mae et al. (1983), with some modifications. The leaf was homogenized in the homogenization buffer for chlorophyll, Rubisco and total N determination [containing 0·2% (v/v) Triton X-100] at a ratio of leaf to buffer of 1 : 2–3 (g : ml) in a chilled mortar and pestle with acid-washed quartz sand. An aliquot of the homogenate was centrifuged at 12 000 × g at 4 °C for 30 min, and the supernatant was further centrifuged at 12 000 × g at 4 °C for 10 min. An aliquot of the supernatant mixed with an equal volume of glycerol was applied to a polyacrylamide minislab gel without sodium dodecyl sulfate (SDS) (3·5% stacking gel, 6·25% running gel). The section of the gel corresponding to Rubisco, which could be detected by light refraction because of its quite high concentration, was cut out immediately, macerated in 50 mm sodium phosphate buffer, pH 7·5, containing 0·05% (w/v) sodium azide and 0·2% (v/v) Triton X-100, and then shaken vigorously at 37 °C for 12–16 h. After centrifugation at 12 000 × g at 4 °C for 10 min, protein in an aliquot of the supernatant was precipitated by the addition of 45% (w/v) trichloroacetic acid to final concentration of 7·5% (w/v). The precipitate collected by centrifugation at 12 000 × g at 4 °C for 15 min was washed twice with 5% (w/v) trichloroacetic acid and once with 99·5% (v/v) ethanol. The precipitate thus obtained was essentially pure Rubisco, according to SDS-PAGE. The precipitate was dissolved in 0·1 m NaOH. 15N abundance in this solution was determined by emission spectrography (Yoneyama, Arima & Kumazawa 1975) with a 15N-analyser (N-151; JASCO, Tokyo, Japan). The amounts of labelled Rubisco-N were calculated using the following equation:

  • image(1)

where 15NR and 15NF are 15N atom% excess of purified Rubisco and (15NH4)2SO4 fed to the plant, respectively. The amount of Rubisco synthesized or degraded for each 4 d period during the life span of the leaves was calculated using the following equations (here, the degradation of Rubisco was assumed to be zero while its labelled N content was increasing):

  • image(2)
  • image(3)

where t is the first day of each 4 d period, t′ is the fourth day after t, Rubisco degradedt-t′ and Rubisco synthesizedt-t′ are the amounts of Rubisco degraded and synthesized in the leaves between t and t′, respectively, 15NRt and 15NRt′ are the amounts of labelled Rubisco in the leaf at t and t′, respectively, and NRt and NRt′ are the amounts of Rubisco-N in the leaf at t and t′, respectively.

RNA extraction and Northern blot analysis

A new method for ribonucleic acid (RNA) preparation (Suzuki, Makino & Mae 2001), which enables us to determine the absolute amount of total RNA with its high extraction efficiency, was employed. Our previous experiments showed that 81–95% of RNA in the leaf blades of rice could be extracted, irrespective of leaf age, with the extraction method employed here. Part of the total RNA was subjected to formaldehyde-agarose gel electrophoresis according to the method of Sambrook, Fritsch & Maniatis (1989). RNA in the gel was then blotted onto membrane as described in Yamaya et al. (1995), except for the use of a positively charged nylon membrane (Hybond-N+ Amersham Pharmacia Biotech, Piscataway, NJ, USA) in our experiment. Part of the total RNA was slot-blotted onto positively charged nylon membrane with slot-blot apparatus (Bio-Dot; Bio-Rad, Hercules, CA, USA) according to the method of Maniatis et al. (1982). Hybridization and washing of the membranes were carried out according to the digoxlgenin (DIG) system user’s guide (Roche Diagnostics, Indianapolis, IN, USA). The DIG-labelled DNA probes for rbcL and rbcS were prepared from fragments of the rice rbcL gene (Hirai et al. 1985) and rbcS gene (Matsuoka et al. 1988), respectively, with the PCR DIG probe synthesis kit (Roche Diagnostics). Signals was detected with a DIG luminescent detection kit (Roche Diagnostics). In the case of slot-blot analysis, the signal intensities were determined with a densitometer (GS-700; Bio-Rad). A calibration curve for slot-blot analysis was made with sequentially diluted total RNA extracted from expanding rice leaves.

DNA extraction and Southern blot analysis

DNA was extracted with an improved method based on the reports of Zhu, Qu & Zhu (1993) and Jhingan (1992). Frozen leaves were briefly ground to a powder with a mortar and pestle in the presence of liquid nitrogen in a cold room. The resulting leaf powder was weighed and placed in denaturing solution [100 mm Tris-HCl buffer and 40 mm ethylenediaminetetraacetic acid (EDTA), pH 8·5, containing 6·94 mm potassium O-ethyl dithiocarbonate, 2% (w/v) sodium dodecyl sulfate (SDS), 5% (v/v) 2-mercaptoethanol] at a ratio of denaturing solution to leaves of 10 : 15 mL g−1 fresh weight (FW). The homogenate was further macerated with a small amount of quartz sand with a mortar and pestle. An aliquot (2·0 mL) of the homogenate was transferred to a 10 mL Teflon tube and 2·0 mL of benzyl chloride was added. The mixture was shaken vigorously at 60 °C for 30 min. Thereafter, 1·0 mL of 3 m sodium acetate, pH 5·2, was added. The tube was then kept on ice for 15 min, followed by centrifugation at 12 000 × g at 4 °C for 15 min. The aqueous phase after the removal of debris was transferred to a new 10 mL Teflon tube. The extraction mixture was re-extracted and phase-separated, then the aqueous phase was combined with the aqueous phase prepared previously. DNA was isopropanol-precipitated from the combined aqueous phase and dissolved in Tris-EDTA (TE) buffer containing 1 µg mL−1 of RNaseA. The amount of DNA in the solution was determined by fluorescence with Hoechst 33258 with a spectrofluorophotometer (RF-1500; Shimadzu, Kyoto, Japan). A calibration curve was made with sequentially diluted lambda DNA. The ratio of the amount of DNA collected by re-extraction to that collected by the first extraction was constant, irrespective of leaf age (data not shown). The amount of DNA in the whole aqueous phase was calculated and defined as the amount of total DNA. Our preliminary experiments showed that about 80% of DNA in the leaf blades of rice could be extracted with the extraction method employed here. The loss of DNA during extraction was about 15%. These results suggest that the method of DNA extraction employed here can be used for the determination of total DNA. The extracted DNA was digested with EcoRI, phenol-extracted, then ethanol-precipitated and dissolved in TE buffer. Part of the total DNA fraction was subjected to agarose gel electrophoresis and blotting onto a positively charged nylon membrane according to the methods of Goto et al. (1998). Part of the total DNA fraction was slot-blotted onto a positively charged nylon membrane with slot-blot apparatus (Bio-Dot; Bio-Rad) according to the methods of Sambrook, Fritsch & Maniatis (1989). The methods for the hybridization, detection and determination of signal intensities were the same as those described for the Northern analysis, except the calibration curve for slot-blot analysis was made with sequentially diluted DNA extracted from expanding rice leaves and digested with EcoRI as described above.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Changes in the indexes of leaf age in the eighth leaf blades

The tip of the eighth leaf blade emerged from the seventh leaf sheath 42 d after germination (defined as the first day after leaf emergence) (Fig. 1). Leaf length and FW of the leaf blade increased rapidly after its emergence and reached its maximum the 13th day after emergence (full expansion). Thereafter, both values remained constant during senescence. The amounts of chlorophyll and total N increased rapidly during expansion, reached their maxima on the 13th day after emergence, and then declined slowly.

Changes in the amounts of Rubisco synthesized and degraded

The amount of Rubisco increased rapidly after leaf emergence, reached its maximum around full expansion, and then declined gradually (Fig. 2). 15N abundance of purified Rubisco was the highest at the end of 15N-labelling, and then declined gradually with leaf age. The amount of Rubisco synthesized or degraded during each 4 d period was estimated from changes in the amount of Rubisco and its 15N abundance. Rubisco was actively synthesized during expansion, but the amount of Rubisco synthesized declined rapidly just before full expansion. It had already declined to about one fifth of the maximal amount at the time of full expansion, and declined further to about one tenth of the maximal amount during senescence. In contrast, the degradation of Rubisco started just before full expansion, and became far more active than its synthesis during senescence.

Changes in the amounts of total RNA and total DNA

The amount of total RNA increased rapidly soon after leaf emergence, reached its maximum on the fifth day after emergence, and then declined gradually (Fig. 3). On the other hand, the amount of total DNA increased slightly during leaf expansion, and then declined slightly during senescence.

Changes in the levels of rbcL mRNA, rbcS mRNA, rbcL DNA and rbcS DNA

Figure 4a shows the results of electrophoresis of total RNA, gel blot analysis of rbcL and rbcS mRNAs and rbcL and rbcS DNAs. The respective single bands were observed for rbcL and rbcS mRNAs and rbcL and rbcS DNAs. The levels of these mRNAs and DNAs were quantified by slot-blot analysis and were expressed on a leaf-blade basis by calculation based on the amount of total RNA and total DNA shown in Fig. 3 (Fig. 4b). The levels of rbcL and rbcS mRNAs increased rapidly just after leaf emergence and reached their maxima on the fifth day after emergence. Thereafter, the levels of rbcL and rbcS mRNAs declined quickly to about one fifth and one third of their maxima, respectively, at the time of full expansion, and declined further to less than one tenth during senescence. These changes in the levels of both mRNAs were approximately co-ordinated with the changes in the amount of Rubisco synthesized, and their maxima obviously preceded that of the amount of Rubisco (see Fig. 2). The level of rbcL DNA increased gradually after leaf emergence, reached its maximum just before full expansion, and then declined to about half of its maximum during senescence. The level of rbcS DNA also increased after leaf emergence and remained almost constant thereafter, whereas the level of rbcL DNA declined significantly during senescence. However, this decline in the level of rbcL DNA started later and was much slower than that of rbcL mRNA.

Differences among respective leaf positions

Similar analyses were carried out on leaf blades at different positions on the main culm. Samplings were carried out 9 d after the emergence of the eighth leaf blades. The eighth, seventh and fifth leaf blades were chosen as being just before maturation, when mature and when slightly senescent, respectively. Figure 5 shows the amounts of chlorophyll, total N, total RNA and total DNA on a FW basis for each leaf blade. The amounts of chlorophyll and total N tended to be highest in the seventh leaf blades and lowest in the fifth. The amount of total RNA was highest in the eighth leaf blades and lowest in the fifth. The amount of total DNA was slightly lower in the seventh and fifth leaf blades than in the eighth.

The amount of Rubisco was almost the same in the eighth and seventh leaf blades, but in the fifth, it was 50% lower (Fig. 6). Estimated synthesis of Rubisco was highest in the eighth leaf blades and considerably lower in the seventh and fifth leaf blades – about one third and one fifth, respectively, of that in the eighth leaf blades.

Figure 7a shows the results of electrophoresis of total RNA, gel blot analysis of rbcL and rbcS mRNAs and of rbcL and rbcS DNAs. The levels of these mRNAs and DNAs were quantified with slot-blot analysis and were expressed on a FW basis for each leaf blade by calculations based on the amount of total RNA and total DNA shown in Fig. 5 (Fig. 7b). The levels of rbcL and rbcS mRNAs were highest in the eighth leaf blades and were quite low in the seventh and fifth leaf blades. In the seventh and fifth leaf blades, the levels of rbcL mRNA were about one fifth and one fourth, respectively, of that in the eighth leaf blades, and the levels of rbcS mRNA were much lower – about one tenth of that in the eighth leaf blades. The changes in both mRNAs were approximately co-ordinated with those in the amount of Rubisco synthesized but were largely different from the absolute amount of Rubisco protein (Fig. 6). The level of rbcL DNA tended to be slightly less in the fifth leaf blades. The level of rbcS DNA was not different between the eighth and seventh leaf blades, but was slightly lower in the fifth. The decline in the level of rbcL DNA was thus much slower than that of rbcL mRNA.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The changes in the levels of rbcL and rbcS mRNAs were approximately co-ordinated with those in the amount of Rubisco synthesized (Figs 2, 4, 6 & 7). Therefore, it is suggested that the amount of Rubisco synthesized is primarily determined by the levels of rbcL and rbcS mRNAs during the life span of the leaves. These tendencies are fundamentally similar to those found in the processes of leaf expansion and greening. For example, changes in the rate of Rubisco synthesis are well correlated with those in the levels of rbcL and rbcS mRNAs in the greening process of etiolated pea leaves (Sasaki et al. 1985; Sasaki, Nakamura & Matsuno 1987).

The data in Figs 2 and 4 are likely to show that the levels of rbcL and rbcS mRNAs begin to decrease early in development, before decreases occur in the rates of Rubisco synthesis, and indicate that post-transcriptional controls might have a role in regulating Rubisco synthesis at the early stage of leaf development to some extent. However, the levels of rbcL and rbcS mRNAs were measured 1, 5, 9 and 13 d after the leaf emergence and the rate of Rubisco synthesis was estimated as an average value of a 4 d interval between these days. If average values of the mRNAs between 1 and 5, 5 and 9 and 9 and 13 d after leaf emergence are taken, changing patterns for both mRNAs become close to those of Rubisco synthesis. It is difficult to discuss definitively the role of the post-transcriptional control in regulating the Rubisco synthesis from the presented data only.

The amount of Rubisco degraded in the senescent leaves was much greater than that synthesized (Fig. 2). This clearly indicates that degradation of Rubisco is the major determinant of changes in the amount of Rubisco in the senescent leaves of rice. Our findings on the large differences in Rubisco synthesis and the levels of rbcL and rbcS mRNAs between the early stage of leaf expansion and the various stages of leaf senescence indicate that the contribution of Rubisco synthesis controlled by the levels of rbcL and rbcS mRNAs to changes in the amount of Rubisco is much smaller in senescent leaves than in expanding leaves (Figs 2 & 4).

The results in Figs 4 and 7 show that the decline in the level of rbcL mRNA occurred much earlier in the developmental stage and proceeded at a much faster rate than that of rbcL DNA. For example, 80% of the maximal level of rbcL DNA remained in the eighth leaf blades just after full expansion, while only 20% of the maximal level of rbcL mRNA remained. These observations indicate that the level of rbcL DNA is not a major determinant of the level of rbcL mRNA in senescent leaves of rice.

It has been suggested that the decline in the level of rbcL DNA can limit the level of rbcL mRNA, at least in part, during leaf senescence. For example, the level of rbcL mRNA declined a little faster during senescence than that of rbcL DNA in soybean (Jiang et al. 1993), and declined almost in parallel with the level of rbcL DNA in tobacco (Jiang & Rodermel 1995; Miller et al. 2000). The reason for the discrepancy between these reports and ours is not known. Although this difference could be attributed to a species-dependent difference, it is possible that it is due to differences in the manners in which these parameters are expressed. In our study, the level of rbcL DNA was compared directly with that of rbcL mRNA on a leaf-blade or FW basis. In the other reports, these levels were expressed on a total DNA, 18 S rDNA or total RNA basis and compared with each other. However, the amounts of total DNA – especially chloroplast DNA – and total RNA change with leaf age and these changes were not parallel with each other during the life span of the leaves (see Fig. 3). In addition, it is possible that the extraction efficiency of DNA or RNA using a conventional method often changes depending on the leaf age of samples. We used an improved method for DNA and RNA extraction with high efficiency (Suzuki et al. 2001; see also Materials and methods).

It has been observed that the amount of chloroplast DNA declined rapidly in the leaf or the coleoptile of rice before their soluble protein content reached a maximum (Sodmergen et al. 1991; Inada et al. 1998a, 1998b, 1999). The reason for the difference between these reports and ours is not known. One possible reason is a difference in the experimental methods. In the above-mentioned reports, chloroplast nucleoids were counted with fluorescence microscopy or electron microscopy. In our study, rbcL DNA was measured using Southern blot analysis.

It has also been proposed that a decline in the amount of chloroplast DNA occurs in the maturation process of barley leaves and it would probably reflect a decline in the demand for protein synthesis (Baumgartner, Rapp & Mullet 1989). This means that a decline in the level of mRNAs occurs at the same time. However, there have been some reports that the level of psbA mRNA was still significant, even in senescent leaves (Bate et al. 1991; Thomas & de Villiers 1996). The levels of mRNAs are generally considered to be regulated by both transcriptional activity and stability (Shirsat, Gatehouse & Robinson 1999). Although the balance between these factors remains unclear in senescent leaves, it is possible that some mRNAs are still transcribed and that chloroplast DNA is preserved to some extent and serves as templates for these mRNAs. Therefore, the retention observed in the level of rbcL DNA may be reasonable.

In conclusion, the amount of Rubisco was determined mainly by the degradation of Rubisco in senescent leaves of rice, whereas the synthesis of Rubisco controlled by the levels of rbcL and rbcS mRNAs contributed to a lesser extent. The level of rbcL DNA was not considered as a major determinant for the level of rbcL mRNA in the senescent leaves. It is interesting, and remains to be studied, how environmental factors such as nutrition level and light intensity affect the balance between the factors involved in the synthesis and degradation of Rubisco.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We thank Drs Ko Iba and Kensuke Kusumi (Kyushu University) for the kind gift of cDNA clones of rice and their advice on the experiment, and Drs Tomoyuki Yamaya and Toshihiko Hayakawa and members of their laboratory for their instruction and fruitful discussion and for the use of their equipment. We also thank Dr Atsuko Niimi (Nagoya University) for her kind help with the slot-blot analysis. This work was supported by: Grant-in-Aid for Science Research 12460028 and Grant-in-Aid for Science Research in Priority Areas 12025202 from the Ministry of Education, Science and Culture of Japan; by Grant-JSPS-RFTF 96L00604 for Research for the Future from the Japan Society for the Promotion of Science, and by the Bio Design Program (BDP-01-I-1-X) of the Ministry of Agriculture, Forestry and Fisheries, Japan.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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