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Ultrastructural and biochemical changes in chloroplasts during Brassica napus senescence
Plant Physiol. Biochem. 39 (2001) 777−784 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0981942801012967/FLA
Ultrastructural and biochemical changes in chloroplasts during Brassica napus senescence Sibdas Ghosha*§, Sean R. Mahoneyb, Jon N. Pentermana, David Peirsonb, Erwin B. Dumbroffc a
Department of Biological Sciences, University of Wisconsin-Whitewater, Whitewater, Wisconsin 53190-1791, USA
b
Department of Biology, Wilfrid Laurier University, Waterloo, Ontario N2L 3C5, Canada
c
Kennedy-Leigh Centre for Horticultural Research, Faculty of Agriculture, Hebrew University of Jerusalem, P.O. Box 12, Rehovot, 76100, Israel
Received 14 August 2000; accepted 10 May 2001 Abstract – The ultrastructural and biochemical changes occurring in chloroplasts of canola (Brassica napus cv. Topas) cotyledons during senescence were characterized utilizing 1-, 2-, 3-, and 4-week-old plants. Chlorophyll a and b and carotenoid contents per unit cotyledon area were greatest 2 weeks after planting prior to the decline in total protein content. Photosystem II (PSII) activity per unit area of cotyledon was highest during the first week and declined through week 4. In contrast, photosystem I activity and CO2 assimilation remained relatively stable through week 2 and then declined. High levels of PSII activity were associated with large numbers of stacked grana; whereas loss of activity during week 2 was accompanied by reduced compactness of grana and the aggregation and enlargement of plastoglobuli. © 2001 Éditions scientifiques et médicales Elsevier SAS Brassica napus / chloroplast / cotyledon / phosphoenolpyruvate carboxylase / photosystem / plastoglobuli / Rubisco / senescence DMSO, dimethyl sulphoxide / PEPC, phosphoenolpyruvate carboxylase / PSI, photosystem I / PSII, photosystem II / RLSU, Rubisco large subunit / RSSU, Rubisco small subunit / Rubisco, ribulose-1,5-biphosphate carboxylase
1. INTRODUCTION Leaf senescence is the final stage of leaf development. Molecular studies have led to the discovery, identification and characterization of genes expressed during senescence in numerous plants [5, 6, 23]. Biochemical, physiological, morphological and ultrastructural analyses indicate that enhanced degradation of chloroplast components leading to a reduction in photosynthetic capacity is an integral part of the senescence process in green tissue [10, 12, 15, 16, 24, 28, 30]. Despite numerous studies, discrepancies exist in the literature regarding the sequence of events that lead to this loss of photosynthetic capability. For example, *Correspondence and reprints: fax +1 415 458 3755. E-mail address: [email protected] (S. Ghosh). § Present address: Dept of Natural Sciences and Mathematics, Dominican University of California, 50 Acacia Ave., San Rafael CA 94901-2298, USA.
some reports suggest that significant losses of photosystem II (PSII) activity precede those of photosystem I (PSI) during senescence [3, 17, 33], but others indicate that disablement of PSI occurs before PSII [28, 29]. Contradictions in the literature may be attributed to (a) chloroplasts in different plant species may undergo species-distinct changes during senescence, (b) chloroplasts from different tissues (e.g. leaves or cotyledons) could have dissimilar patterns of senescence, and (c) marked dissimilarities exist in the experimental approaches used by different research groups. For example, many researchers have examined chloroplast senescence in situ while others have studied senescence in vitro. Furthermore, changes in photosynthetic capacity were often monitored using different techniques, each having its own degree of sensitivity. Finally, no single comprehensive study has been performed that establishes direct correlations between the ultrastructural and biochemical changes in the chloroplast during senescence. Instead, many comparisons are based on ultrastructural information gath
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ered by a group using one plant species and biochemical data obtained by another group using a different species. The present study characterizes and correlates senescence-related ultrastructural and biochemical changes observed in the chloroplasts from cotyledons of Brassica napus. These cotyledons are epigeous and senesce completely in about 1 month, thus providing a convenient experimental system for a comprehensive study. Chloroplastic pigment concentrations were monitored and changes in photosynthetic capacity were characterized by measuring the activities of PSI, PSII, ribulose-1,5-bisphosphate carboxylase (Rubisco, EC 4.1.1.39) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in the cotyledons of 1-, 2-, 3-, and 4-week-old seedlings. Electron microscopy was used to determine ultrastructural changes of the chloroplasts.
Figure 2. Changes in the dry weight of cotyledons with age. Cotyledons were harvested from 1-, 2-, 3- and 4-week-old seedlings of B. napus, dried and their weights measured. Each data point represents the mean weight of twenty cotyledons. Vertical bars denote ± 1 SE when it exceeds the size of the symbol.
2.1. Morphological changes
protein are provided for comparison. The dry weights of the cotyledons increased between weeks 1 and 2, remained stable through week 3, and then declined through week 4 (figure 2). As noted in section 2.3, protein levels declined sharply through the last 3 weeks of the study.
Cotyledons of Brassica napus underwent marked expansion between 1 and 2 weeks after planting followed by a small and non-significant reduction in surface area apparently due to water loss (figures 1, 2). Surface area of the cotyledons from weeks 2 through 4 was, in fact, the most stable factor measured throughout this study. As such, expression of concentrations per unit of surface area provided a stable base for monitoring trends in other factors under test. Nevertheless, expression of changes in concentration and activity per unit dry weight of cotyledon and per unit
2.2. Pigment content The green color of the cotyledons began to fade after the second week. Total chlorophyll levels peaked at week 2 and then decreased steadily through harvest at the end of week 4 (figure 3). The ratio of chlorophyll a to chlorophyll b remained essentially constant. Carotenoid levels followed a trend similar to that of total chlorophyll with the highest levels per unit area observed in 2-week-old cotyledons followed by a decline through the fourth week (figure 4).
2. RESULTS AND DISCUSSION
2.3. PSI and PSII activity PSII activity per unit area of cotyledon (figure 5) decreased to less than one-half between weeks 1 and 2 and continued to drop to less than one-fifth of the first week’s readings when the cotyledons became highly senescent. In contrast, PSI activity (figure 6) remained stable through week 2 and then declined in parallel with the reduction in total protein content (figure 7). A decrease in PSII activity before a decline in PSI has also been observed in senescing monocot [3, 33] and dicot [17] leaves. However, in senescing wheat leaves [28] and pine needles [29], the decline in PSI activity preceded the loss in activity of PSII. Figure 1. Changes in surface area of cotyledons with age. Cotyledons were harvested from 1-, 2-, 3- and 4-week-old seedlings of B. napus and their areas measured. Each data point represents the mean area of twelve cotyledons. Vertical bars denote ± 1 SE when it exceeds the size of the symbol.
2.4. Carboxylating enzymes The specific activity of Rubisco increased over 30 % between the first and second week and thereafter
S. Ghosh et al. / Plant Physiol. Biochem. 39 (2001) 777–784
Figure 3. Changes in the content of (·) chlorophyll a, ([) chlorophyll b, and ( ) total chlorophyll with age. Two cotyledons from each of three seedlings were harvested from 1-, 2-, 3- and 4-week-old seedlings of B. napus and chlorophyll extracted in 5 mL DMSO at 60 °C for 1 h. Each data point represents the mean of three replicates. Vertical bars denote ± 1 SE when it exceeds the size of the symbol.
declined through week 4 (figure 8A). Protein and transcript levels of the chloroplastically synthesized large subunit of Rubisco (RLSU) (figures 9, 10) followed trends similar to that observed for Rubisco activity (figure 8A) with the highest levels observed in 2-week-old cotyledons. In contrast, protein levels of the nuclear encoded and cytoplasmically synthesized small subunit of Rubisco (RSSU) remained constant throughout the 4-week period (figure 9). These findings are consistent with observations of senescing primary leaves of soybean [1]. Jiang et al. [14] also observed a decrease of Rubisco in senescing soybean leaves, which correlated with reductions of RLSU and
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Figure 4. Changes in carotenoid content with age. Two cotyledons from each of three seedlings were harvested from 1-, 2-, 3- and 4-week-old seedlings of B. napus and carotenoids extracted in 5 mL DMSO at 60 °C for 1 h and absorbance measured at 480 nm. Each data point represents the mean of three replicates. Vertical bars denote ± 1 SE when it exceeds the size of the symbol.
RSSU mRNAs. In contrast, results from senescing flag leaves of barley were similar to our observations, i.e. decreased levels of RLSU concentrations correlated with losses in Rubisco activity while RSSU content remained stable [13]. Moreover, in the present study, losses in RLSU were closely associated with declining levels of the corresponding mRNA, therefore, suggesting transcriptional control of RLSU content or of the instability of RLSU mRNA [21]. PEPC did not appear to be a limiting factor in CO2 fixation during senescence since the specific activity of the enzyme increased between weeks 1 and 3 and showed only a moderate decline through week 4 (figure 8B). PEPC activity in the needles of black spruce
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Figure 5. Changes in PSII activity with age. Thylakoids isolated from cotyledons of 1-, 2-, 3- and 4-week-old seedlings of B. napus were used to measure the reduction of DCPIP during illumination with red light. Each data point represents the mean of three replicates. Vertical bars denote ± 1 SE when it exceeds the size of the symbol.
was also more stable than Rubisco during exposure to a combination of high light plus thermal stress [19]. The question of whether the greater stability of PEPC activity during stress and senescence compensates to a significant degree for the large reductions in Rubisco activity awaits further research.
2.5. Ultrastructural changes Ultrastructural changes in the chloroplasts observed in this study were generally consistent with those described by Woolhouse [31]. Over the 4-week period, there was also an increase in chloroplastic volume, due to the aggregation of the plastoglobuli (figure 11), as recently described in chloroplasts from other species
Figure 6. Changes in PSI activity with age. Thylakoids isolated from cotyledons of 1-, 2-, 3- and 4-week-old seedlings of B. napus were used to measure the oxidation of DCPIP during illumination with near far-red light. Each data point represents the mean of three replicates. Vertical bars denote ± 1 SE when it exceeds the size of the symbol.
[12, 15]. The outer envelope remained intact even in the highly senescent chloroplasts of 4-week-old cotyledons, providing further evidence that the outer membrane of the chloroplast is one of the last components to be dismantled during senescence [2]. During the second week of senescence, stroma and grana lamellae in the chloroplasts appeared swollen and granal stacking had become less compact (figure 11B). This decrease in granal stacking and the concurrent sharp decline in PSII activity suggest that an earlier loss of PSII relative to PSI activity was apparently related to the location of PSI in the unstacked stromal thylakoids as opposed to the location of PSII in the stacked intragranal regions. In 3-week-old cotyledons, reduc
S. Ghosh et al. / Plant Physiol. Biochem. 39 (2001) 777–784
Figure 7. Changes in total protein content of cotyledons with age. Protein was extracted from the cotyledons of 1-, 2-, 3- and 4-week-old seedlings of B. napus in 50 mM Hepes-KOH (pH 6.8), 2 % SDS (v/v), 1 mM PMSF, 1 mM benzamidine and 5 mM caproic acid. Each data point represents the mean of three replicates. Vertical bars denote ± 1 SE when it exceeds the size of the symbol.
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Figure 8. Specific activities of (A) Rubisco and (B) PEPC in 1-, 2-, 3- and 4-week-old cotyledons of B. napus. Each data point represents the mean of three replicates. Vertical bars denote ± 1 SE when it exceeds the size of the symbol.
tions in the abundance and size of the grana were also recorded (figure 11C), an observation reported earlier by Naito et al. [22] in senescing leaves of bean. By week 4, the integrity of the internal network of thylakoid membranes was almost completely lost (figure 11D). Moreover, loss of the integrity of both granal and stromal lamellae, measured in terms of aggregation of plastoglobuli and lumenal swelling, was accompanied by the nearly complete loss of the activities of both PSI and PSII.
3. CONCLUSION Chloroplast senescence in the cotyledons of Brassica napus progresses in an ordered and sequential manner that involves preferential dismantling of the photosynthetic apparatus. In 2-week-old cotyledons, a sharp decline in PSII activity accompanied by marked structural changes in the thylakoids provide the earliest manifestations of a loss in photosynthetic function. In contrast, chloroplast pigments are less sensitive
Figure 9. Immunological identification of the large (RLSU) and small (RSSU) subunits of Rubisco in western blots. After SDS-PAGE of protein extracts (2 µg) from 1-, 2-, 3- and 4-week-old cotyledons of B. napus, the samples were transferred to nitrocellulose membranes and incubated with antisera against RLSU and RSSU. The locations of the RLSU (52 kDa) and RSSU (15 kDa) are indicated by arrows.
indicators of senescence since chlorophyll and carotenoid levels rise through the second week and then decline. This observation is consistent with the view that a minimum level of thylakoid damage must
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in the dark for 10 min. The intact thylakoid membranes were separated from stroma by centrifugation at 4 °C for 2 min at 16 000 × g and resuspended in 1 mL 50 mM Epps NaOH (pH 8.0), 5 mM MgCl2 and 400 mM sorbital. The membranes were placed on ice in the dark and used immediately for PSI and PSII activity assays. Figure 10. Northern blot analysis of total RNA hybridized against RLSU DNA (pZMC461) from maize. RNA was prepared from 1-, 2-, 3- and 4-week-old cotyledons of B. napus. Total RNA (10 µg) was electrophoresed on formaldehyde-(1 %) agarose gels, transferred to a Biotrans nylon membrane and hybridized against a 5’-α-[32P] nicktranslated plasmid (pZMC461) carrying maize RLSU DNA. The putative transcript size (2.0 kb) was based on the relative migration of the components within the BRL RNA ladder.
precede chlorophyll loss during senescence [20, 31]. Changes in Rubisco activity and the amount of RLSU present in the cotyledons follow trends similar to those of the chloroplast pigments. Their initial gains and subsequent loss appear to be regulated by transcriptional modification and/or by the stability of RLSU mRNA. We should also note that a large rise in PEPC activity during senescence might compensate, to some degree, for the marked loss of Rubisco function.
4. METHODS 4.1. Plant materials and growth conditions Seeds of Brassica napus L. (cv. Topas) were germinated in flats of Pro-mix BX and placed in a growth chamber at 25 °C. Seedlings were watered daily and grown under a 16-h photoperiod with 140 µmol·m–2·s–1 mixed fluorescent and incandescent light. Cotyledons from 1-, 2-, 3-, and 4-week-old plants were excised, frozen in liquid nitrogen and stored at –70 °C for subsequent analysis.
4.2. Thylakoid membrane isolation Thylakoid membranes were prepared using a modification of the method of Paterson and Arntzen [25]. Briefly, six freshly cut cotyledons from 1-, 2-, 3-, and 4-week-old plants were ground in 2 mL ice-cold 50 mM Epps NaOH buffer (pH 8.0), 5 mM MgCl2 and 400 mM sorbital and the extracts filtered through miracloth to remove fibrous material. Chloroplasts were pelleted by centrifuging the filtrate at 16 000 × g for 2 min at 4 °C and resuspended in 1 mL hypotonic solution [50 mM Epps NaOH buffer (pH 8.0) and 5 mM MgCl2] at 4 °C
4.3. Biochemical and chemical analyses Photosynthetic pigments were extracted by incubating freshly cut cotyledons in 5 mL dimethyl sulphoxide (DMSO) for 1 h at 60 °C [11]. Chlorophyll and carotenoid concentrations were determined using the formulae described by Porra et al. [26] and Britton [4], respectively. PSI and PSII activities were measured using the method of Xiao et al. [32]. Total protein was extracted [8] and measured according to Ghosh et al. [7] using bovine serum album (fraction V, Sigma) as a standard. Proteins were fractionated by SDS-PAGE [18] and the RLSU and RSSU were visualized on western blots using monospecific polyclonal antibodies [8]. Total lithium chloride precipitable RNA was isolated, fractionated on a horizontal formaldehydeagarose (1 %) gel and then transferred onto a Biotrans nylon membrane for visualization of RLSU mRNA using northern blot analysis as described previously by Ghosh et al. [9]. Activities of Rubisco and PEPC were measured in extracts prepared from fresh-frozen cotyledons as described previously by Ghosh et al. [8].
4.4. Electron micrographs of chloroplasts Cotyledon tissue was prepared for electron microscopy using the method of Ronning et al. [27] as outlined in figure 10. One-mm2 sections of cotyledons from 1-, 2-, 3-, and 4-week old plants were fixed in 2.5 % glutaraldehyde at room temperature for 2 h. The tissue was then washed five times in 0.05 M phosphate (pH 6.8) followed by post-fixation in 2 % OsO4 in 0.05 M phosphate buffer (pH 6.8) at room temperature for 2 h. Samples were dehydrated in a concentration gradient of 20, 40, 60, 80, and 100 % acetone for 20 min each followed by two extra washes in 100 % acetone. Infiltration was done by incubating the dehydrated samples for 1 h each in 25, 50 and 75 % low-viscosity epoxy resin-acetone followed by four changes in 100 % resin. The infiltrated tissue was then embedded in 100 % resin and oven-cured at 60 °C for 12 h. Sectioning was done with an ultramicrotome and the tissue sections placed on copper grids for staining. Staining was done in 2 % uranyl acetate for 10 min followed by rinsing in deionized H2O and 5 min in
S. Ghosh et al. / Plant Physiol. Biochem. 39 (2001) 777–784
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Figure 11. Ultrastructural changes in thylakoid membranes of chloroplasts from (A) 1-, (B) 2-, (C) 3- and (D) 4-week-old seedlings of B. napus. GL, grana lamella; SL, stroma lamella; P, plastoglobulus; S, starch granule.
3 % lead citrate (pH 12). Potassium hydroxide pellets were placed in the staining chamber containing the lead citrate to minimize lead precipitation.
Acknowledgments. This study was supported, in part, by The University of Wisconsin-Whitewater Faculty Research Grant and The University of WisconsinWhitewater Undergraduate Research Grant. We are also grateful to Patricia Dumbroff for critical reading of this manuscript.
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[4] Britton G., General carotenoid methods, Methods Enzymol. 111 (1985) 113–144. [5] Buchanan-Wollaston V., The molecular biology of leaf senescence, J. Exp. Bot. 48 (1997) 818–899. [6] Gan S., Amasino R.M., Making sense of senescence, Plant Physiol. 113 (1997) 313–319. [7] Ghosh S., Gepstein S., Heikkila J., Dumbroff E.B., Use of a scanning densitometer or an ELISA plate reader for measurement of nanogram amounts of protein in crude extracts from biological tissues, Anal. Biochem. 169 (1988) 227–233. [8] Ghosh S., Gepstein S., Glick B.R., Heikkila J., Dumbroff E.B., Thermal regulation of phosphoenolpyruvate carboxylase and ribulose-1,5bisphosphate carboxylase in C3 and C4 plants native to hot and temperate climates, Plant Physiol. 90 (1989) 1298–1304. [9] Ghosh S., Glick B.R., Heikkila J., Dumbroff E.B., Gene expression under elevated temperature in C3 and C4 plants from hot and temperate climates, Plant Physiol. Biochem. 32 (1994) 45–54. [10] Ghosh S., Paliyath G., Peirson D., Fletcher R.A., Nitrogen mobilization during senescence, in: Srivastava H.S., Singh R.P. (Eds.), Nitrogen Nutrition in Higher Plants, Associated Publishing Co., New Delhi, India, 1995, pp. 337–365. [11] Hiscox J.D., Israelstam G.F., A method for the extraction of chlorophyll from leaf tissue without maceration, Can. J. Bot. 57 (1979) 1332–1334. [12] Hudak J., Photosynthetic apparatus, in: Pessaraki M. (Ed.), Handbook of Photosynthesis, Marcel Dekker, New York, 1997, pp. 27–48. [13] Humbeck K., Quast S., Krupinska K., Functional and molecular changes in the photosynthetic apparatus during senescence of flag leaves from field-grown barley plants, Plant Cell Environ. 19 (1996) 337–344. [14] Jiang C.Z., Rodermel S.R., Shibles R.M., Photosynthesis, rubisco activity and amount, and their regulation by transcription in senescing soybean leaves, Plant Physiol. 101 (1993) 105–112. [15] Kutik J., The development of chloroplast structure during leaf ontogeny, Photosynthetica 35 (1998) 481–505. [16] Kutik J., Kocova M., Hola D., Kornerova M., The development of chloroplast ultrastructure and hill reaction activity during leaf ontogeny in different maize (Zea mays L.) genotypes, Photosynthetica 36 (1999) 497–507. [17] La Rocca N., Barbato R., Casadoro G., Rascio N., Early degradation of photosynthetic membranes in carob and sunflower cotyledons, Physiol. Plant. 96 (1996) 513–518. [18] Laemmli U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [19] Mahoney S., Ghosh S., Peirson D., Dumbroff E.B., Paclobutrazol affects the resistance of black spruce to high light and thermal stress, Tree Physiol. 18 (1998) 121–127.
[20] Majumdar S., Ghosh S., Glick B., Dumbroff E.B., Activities of chlorophyllase, phosphoenolpyruvate carboxylase and ribulose-1,5-bisphosphate carboxylase in the primary leaves of soybean during senescence and drought, Physiol. Plant. 81 (1991) 473–480. [21] Mayak S., Tirosh T., Thompson J.E., Ghosh S., The fate of ribulose-1,5-bisphosphate carboxylase subunits during development of carnation petals, Plant Physiol. Biochem. 36 (1998) 835–841. [22] Naito K., Ueda K., Tsuji H., Differential effects of benzyladenine on the ultrastructure of chloroplasts in intact bean leaves according to their age, Protoplasma 105 (1981) 293–309. [23] Nam H.G., The molecular genetic analysis of leaf senescence, Curr. Opin. Biotech. 8 (1997) 200–207. [24] Nooden L.D., Guiamet J.J., John I., Senescence mechanisms, Physiol. Plant. 101 (1997) 746–753. [25] Paterson D.R., Arntzen C.J., Detection of altered inhibition of photosytem II reactions in herbicide resistant plants, in: Edelman M., Hallick R.B., Chua N.H. (Eds.), Methods in Chloroplast Molecular Biology, Elsevier Science Publications B.V, Amsterdam, 1981, pp. 985–1014. [26] Porra R.J., Thompson W.A., Kriedemann P.E., Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: Verification of the concentration of chlorophyll standards by atomic absorption spectroscopy, Biochim. Biophys. Acta 975 (1989) 348–394. [27] Ronning C.M., Bouwkamp J.C., Solomos T., Observations on the senescence of a mutant non-yellowing genotype of Phaseolus vulgaris L., J. Exp. Bot. 42 (1991) 235–241. [28] Sayeed S.A., Behera B.K., Mohanty P., Interaction of light quality and benzimidazole on pigment contents and on photochemical activity of chloroplasts isolated from detached senescing wheat (Triticum aestivum) leaves, Physiol. Plant. 64 (1985) 383–388. [29] Shinohara K., Murakami A., Changes in levels of thylakoid components in chloroplasts of pine needles of different ages, Plant Cell Physiol. 37 (1996) 1102–1107. [30] Watanabe M., Kawaski H., Itho Y., Watanabe Y., Senescence development of Brassica napus leaf protoplast during isolation and subsequent culture, J. Plant Physiol. 152 (1998) 487–493. [31] Woolhouse H.W., The biochemistry and regulation of senescence in chloroplasts, Can. J. Bot. 62 (1984) 2934–2942. [32] Xiao R., Ghosh S., Tanaka A.R., Greenberg B.M., Dumbroff E.B., A rapid spectrophotometric method for measuring photosystem I and photosystem II activities in a single sample, Plant Physiol. Biochem. 35 (1997) 411–417. [33] Xu Q., Paulsen A.Q., Guikema J.A., Paulsen G.M., Functional and ultrastructural injury to photosynthesis in wheat by high temperature during maturation, Environ. Exp. Bot. 35 (1995) 43–54.
Ultrastructural and biochemical changes in chloroplasts during Brassica napus senescence Sibdas Ghosha*§, Sean R. Mahoneyb, Jon N. Pentermana, David Peirsonb, Erwin B. Dumbroffc a
Department of Biological Sciences, University of Wisconsin-Whitewater, Whitewater, Wisconsin 53190-1791, USA
b
Department of Biology, Wilfrid Laurier University, Waterloo, Ontario N2L 3C5, Canada
c
Kennedy-Leigh Centre for Horticultural Research, Faculty of Agriculture, Hebrew University of Jerusalem, P.O. Box 12, Rehovot, 76100, Israel
Received 14 August 2000; accepted 10 May 2001 Abstract – The ultrastructural and biochemical changes occurring in chloroplasts of canola (Brassica napus cv. Topas) cotyledons during senescence were characterized utilizing 1-, 2-, 3-, and 4-week-old plants. Chlorophyll a and b and carotenoid contents per unit cotyledon area were greatest 2 weeks after planting prior to the decline in total protein content. Photosystem II (PSII) activity per unit area of cotyledon was highest during the first week and declined through week 4. In contrast, photosystem I activity and CO2 assimilation remained relatively stable through week 2 and then declined. High levels of PSII activity were associated with large numbers of stacked grana; whereas loss of activity during week 2 was accompanied by reduced compactness of grana and the aggregation and enlargement of plastoglobuli. © 2001 Éditions scientifiques et médicales Elsevier SAS Brassica napus / chloroplast / cotyledon / phosphoenolpyruvate carboxylase / photosystem / plastoglobuli / Rubisco / senescence DMSO, dimethyl sulphoxide / PEPC, phosphoenolpyruvate carboxylase / PSI, photosystem I / PSII, photosystem II / RLSU, Rubisco large subunit / RSSU, Rubisco small subunit / Rubisco, ribulose-1,5-biphosphate carboxylase
1. INTRODUCTION Leaf senescence is the final stage of leaf development. Molecular studies have led to the discovery, identification and characterization of genes expressed during senescence in numerous plants [5, 6, 23]. Biochemical, physiological, morphological and ultrastructural analyses indicate that enhanced degradation of chloroplast components leading to a reduction in photosynthetic capacity is an integral part of the senescence process in green tissue [10, 12, 15, 16, 24, 28, 30]. Despite numerous studies, discrepancies exist in the literature regarding the sequence of events that lead to this loss of photosynthetic capability. For example, *Correspondence and reprints: fax +1 415 458 3755. E-mail address: [email protected] (S. Ghosh). § Present address: Dept of Natural Sciences and Mathematics, Dominican University of California, 50 Acacia Ave., San Rafael CA 94901-2298, USA.
some reports suggest that significant losses of photosystem II (PSII) activity precede those of photosystem I (PSI) during senescence [3, 17, 33], but others indicate that disablement of PSI occurs before PSII [28, 29]. Contradictions in the literature may be attributed to (a) chloroplasts in different plant species may undergo species-distinct changes during senescence, (b) chloroplasts from different tissues (e.g. leaves or cotyledons) could have dissimilar patterns of senescence, and (c) marked dissimilarities exist in the experimental approaches used by different research groups. For example, many researchers have examined chloroplast senescence in situ while others have studied senescence in vitro. Furthermore, changes in photosynthetic capacity were often monitored using different techniques, each having its own degree of sensitivity. Finally, no single comprehensive study has been performed that establishes direct correlations between the ultrastructural and biochemical changes in the chloroplast during senescence. Instead, many comparisons are based on ultrastructural information gath
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S. Ghosh et al. / Plant Physiol. Biochem. 39 (2001) 777–784
ered by a group using one plant species and biochemical data obtained by another group using a different species. The present study characterizes and correlates senescence-related ultrastructural and biochemical changes observed in the chloroplasts from cotyledons of Brassica napus. These cotyledons are epigeous and senesce completely in about 1 month, thus providing a convenient experimental system for a comprehensive study. Chloroplastic pigment concentrations were monitored and changes in photosynthetic capacity were characterized by measuring the activities of PSI, PSII, ribulose-1,5-bisphosphate carboxylase (Rubisco, EC 4.1.1.39) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in the cotyledons of 1-, 2-, 3-, and 4-week-old seedlings. Electron microscopy was used to determine ultrastructural changes of the chloroplasts.
Figure 2. Changes in the dry weight of cotyledons with age. Cotyledons were harvested from 1-, 2-, 3- and 4-week-old seedlings of B. napus, dried and their weights measured. Each data point represents the mean weight of twenty cotyledons. Vertical bars denote ± 1 SE when it exceeds the size of the symbol.
2.1. Morphological changes
protein are provided for comparison. The dry weights of the cotyledons increased between weeks 1 and 2, remained stable through week 3, and then declined through week 4 (figure 2). As noted in section 2.3, protein levels declined sharply through the last 3 weeks of the study.
Cotyledons of Brassica napus underwent marked expansion between 1 and 2 weeks after planting followed by a small and non-significant reduction in surface area apparently due to water loss (figures 1, 2). Surface area of the cotyledons from weeks 2 through 4 was, in fact, the most stable factor measured throughout this study. As such, expression of concentrations per unit of surface area provided a stable base for monitoring trends in other factors under test. Nevertheless, expression of changes in concentration and activity per unit dry weight of cotyledon and per unit
2.2. Pigment content The green color of the cotyledons began to fade after the second week. Total chlorophyll levels peaked at week 2 and then decreased steadily through harvest at the end of week 4 (figure 3). The ratio of chlorophyll a to chlorophyll b remained essentially constant. Carotenoid levels followed a trend similar to that of total chlorophyll with the highest levels per unit area observed in 2-week-old cotyledons followed by a decline through the fourth week (figure 4).
2. RESULTS AND DISCUSSION
2.3. PSI and PSII activity PSII activity per unit area of cotyledon (figure 5) decreased to less than one-half between weeks 1 and 2 and continued to drop to less than one-fifth of the first week’s readings when the cotyledons became highly senescent. In contrast, PSI activity (figure 6) remained stable through week 2 and then declined in parallel with the reduction in total protein content (figure 7). A decrease in PSII activity before a decline in PSI has also been observed in senescing monocot [3, 33] and dicot [17] leaves. However, in senescing wheat leaves [28] and pine needles [29], the decline in PSI activity preceded the loss in activity of PSII. Figure 1. Changes in surface area of cotyledons with age. Cotyledons were harvested from 1-, 2-, 3- and 4-week-old seedlings of B. napus and their areas measured. Each data point represents the mean area of twelve cotyledons. Vertical bars denote ± 1 SE when it exceeds the size of the symbol.
2.4. Carboxylating enzymes The specific activity of Rubisco increased over 30 % between the first and second week and thereafter
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Figure 3. Changes in the content of (·) chlorophyll a, ([) chlorophyll b, and ( ) total chlorophyll with age. Two cotyledons from each of three seedlings were harvested from 1-, 2-, 3- and 4-week-old seedlings of B. napus and chlorophyll extracted in 5 mL DMSO at 60 °C for 1 h. Each data point represents the mean of three replicates. Vertical bars denote ± 1 SE when it exceeds the size of the symbol.
declined through week 4 (figure 8A). Protein and transcript levels of the chloroplastically synthesized large subunit of Rubisco (RLSU) (figures 9, 10) followed trends similar to that observed for Rubisco activity (figure 8A) with the highest levels observed in 2-week-old cotyledons. In contrast, protein levels of the nuclear encoded and cytoplasmically synthesized small subunit of Rubisco (RSSU) remained constant throughout the 4-week period (figure 9). These findings are consistent with observations of senescing primary leaves of soybean [1]. Jiang et al. [14] also observed a decrease of Rubisco in senescing soybean leaves, which correlated with reductions of RLSU and
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Figure 4. Changes in carotenoid content with age. Two cotyledons from each of three seedlings were harvested from 1-, 2-, 3- and 4-week-old seedlings of B. napus and carotenoids extracted in 5 mL DMSO at 60 °C for 1 h and absorbance measured at 480 nm. Each data point represents the mean of three replicates. Vertical bars denote ± 1 SE when it exceeds the size of the symbol.
RSSU mRNAs. In contrast, results from senescing flag leaves of barley were similar to our observations, i.e. decreased levels of RLSU concentrations correlated with losses in Rubisco activity while RSSU content remained stable [13]. Moreover, in the present study, losses in RLSU were closely associated with declining levels of the corresponding mRNA, therefore, suggesting transcriptional control of RLSU content or of the instability of RLSU mRNA [21]. PEPC did not appear to be a limiting factor in CO2 fixation during senescence since the specific activity of the enzyme increased between weeks 1 and 3 and showed only a moderate decline through week 4 (figure 8B). PEPC activity in the needles of black spruce
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Figure 5. Changes in PSII activity with age. Thylakoids isolated from cotyledons of 1-, 2-, 3- and 4-week-old seedlings of B. napus were used to measure the reduction of DCPIP during illumination with red light. Each data point represents the mean of three replicates. Vertical bars denote ± 1 SE when it exceeds the size of the symbol.
was also more stable than Rubisco during exposure to a combination of high light plus thermal stress [19]. The question of whether the greater stability of PEPC activity during stress and senescence compensates to a significant degree for the large reductions in Rubisco activity awaits further research.
2.5. Ultrastructural changes Ultrastructural changes in the chloroplasts observed in this study were generally consistent with those described by Woolhouse [31]. Over the 4-week period, there was also an increase in chloroplastic volume, due to the aggregation of the plastoglobuli (figure 11), as recently described in chloroplasts from other species
Figure 6. Changes in PSI activity with age. Thylakoids isolated from cotyledons of 1-, 2-, 3- and 4-week-old seedlings of B. napus were used to measure the oxidation of DCPIP during illumination with near far-red light. Each data point represents the mean of three replicates. Vertical bars denote ± 1 SE when it exceeds the size of the symbol.
[12, 15]. The outer envelope remained intact even in the highly senescent chloroplasts of 4-week-old cotyledons, providing further evidence that the outer membrane of the chloroplast is one of the last components to be dismantled during senescence [2]. During the second week of senescence, stroma and grana lamellae in the chloroplasts appeared swollen and granal stacking had become less compact (figure 11B). This decrease in granal stacking and the concurrent sharp decline in PSII activity suggest that an earlier loss of PSII relative to PSI activity was apparently related to the location of PSI in the unstacked stromal thylakoids as opposed to the location of PSII in the stacked intragranal regions. In 3-week-old cotyledons, reduc
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Figure 7. Changes in total protein content of cotyledons with age. Protein was extracted from the cotyledons of 1-, 2-, 3- and 4-week-old seedlings of B. napus in 50 mM Hepes-KOH (pH 6.8), 2 % SDS (v/v), 1 mM PMSF, 1 mM benzamidine and 5 mM caproic acid. Each data point represents the mean of three replicates. Vertical bars denote ± 1 SE when it exceeds the size of the symbol.
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Figure 8. Specific activities of (A) Rubisco and (B) PEPC in 1-, 2-, 3- and 4-week-old cotyledons of B. napus. Each data point represents the mean of three replicates. Vertical bars denote ± 1 SE when it exceeds the size of the symbol.
tions in the abundance and size of the grana were also recorded (figure 11C), an observation reported earlier by Naito et al. [22] in senescing leaves of bean. By week 4, the integrity of the internal network of thylakoid membranes was almost completely lost (figure 11D). Moreover, loss of the integrity of both granal and stromal lamellae, measured in terms of aggregation of plastoglobuli and lumenal swelling, was accompanied by the nearly complete loss of the activities of both PSI and PSII.
3. CONCLUSION Chloroplast senescence in the cotyledons of Brassica napus progresses in an ordered and sequential manner that involves preferential dismantling of the photosynthetic apparatus. In 2-week-old cotyledons, a sharp decline in PSII activity accompanied by marked structural changes in the thylakoids provide the earliest manifestations of a loss in photosynthetic function. In contrast, chloroplast pigments are less sensitive
Figure 9. Immunological identification of the large (RLSU) and small (RSSU) subunits of Rubisco in western blots. After SDS-PAGE of protein extracts (2 µg) from 1-, 2-, 3- and 4-week-old cotyledons of B. napus, the samples were transferred to nitrocellulose membranes and incubated with antisera against RLSU and RSSU. The locations of the RLSU (52 kDa) and RSSU (15 kDa) are indicated by arrows.
indicators of senescence since chlorophyll and carotenoid levels rise through the second week and then decline. This observation is consistent with the view that a minimum level of thylakoid damage must
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in the dark for 10 min. The intact thylakoid membranes were separated from stroma by centrifugation at 4 °C for 2 min at 16 000 × g and resuspended in 1 mL 50 mM Epps NaOH (pH 8.0), 5 mM MgCl2 and 400 mM sorbital. The membranes were placed on ice in the dark and used immediately for PSI and PSII activity assays. Figure 10. Northern blot analysis of total RNA hybridized against RLSU DNA (pZMC461) from maize. RNA was prepared from 1-, 2-, 3- and 4-week-old cotyledons of B. napus. Total RNA (10 µg) was electrophoresed on formaldehyde-(1 %) agarose gels, transferred to a Biotrans nylon membrane and hybridized against a 5’-α-[32P] nicktranslated plasmid (pZMC461) carrying maize RLSU DNA. The putative transcript size (2.0 kb) was based on the relative migration of the components within the BRL RNA ladder.
precede chlorophyll loss during senescence [20, 31]. Changes in Rubisco activity and the amount of RLSU present in the cotyledons follow trends similar to those of the chloroplast pigments. Their initial gains and subsequent loss appear to be regulated by transcriptional modification and/or by the stability of RLSU mRNA. We should also note that a large rise in PEPC activity during senescence might compensate, to some degree, for the marked loss of Rubisco function.
4. METHODS 4.1. Plant materials and growth conditions Seeds of Brassica napus L. (cv. Topas) were germinated in flats of Pro-mix BX and placed in a growth chamber at 25 °C. Seedlings were watered daily and grown under a 16-h photoperiod with 140 µmol·m–2·s–1 mixed fluorescent and incandescent light. Cotyledons from 1-, 2-, 3-, and 4-week-old plants were excised, frozen in liquid nitrogen and stored at –70 °C for subsequent analysis.
4.2. Thylakoid membrane isolation Thylakoid membranes were prepared using a modification of the method of Paterson and Arntzen [25]. Briefly, six freshly cut cotyledons from 1-, 2-, 3-, and 4-week-old plants were ground in 2 mL ice-cold 50 mM Epps NaOH buffer (pH 8.0), 5 mM MgCl2 and 400 mM sorbital and the extracts filtered through miracloth to remove fibrous material. Chloroplasts were pelleted by centrifuging the filtrate at 16 000 × g for 2 min at 4 °C and resuspended in 1 mL hypotonic solution [50 mM Epps NaOH buffer (pH 8.0) and 5 mM MgCl2] at 4 °C
4.3. Biochemical and chemical analyses Photosynthetic pigments were extracted by incubating freshly cut cotyledons in 5 mL dimethyl sulphoxide (DMSO) for 1 h at 60 °C [11]. Chlorophyll and carotenoid concentrations were determined using the formulae described by Porra et al. [26] and Britton [4], respectively. PSI and PSII activities were measured using the method of Xiao et al. [32]. Total protein was extracted [8] and measured according to Ghosh et al. [7] using bovine serum album (fraction V, Sigma) as a standard. Proteins were fractionated by SDS-PAGE [18] and the RLSU and RSSU were visualized on western blots using monospecific polyclonal antibodies [8]. Total lithium chloride precipitable RNA was isolated, fractionated on a horizontal formaldehydeagarose (1 %) gel and then transferred onto a Biotrans nylon membrane for visualization of RLSU mRNA using northern blot analysis as described previously by Ghosh et al. [9]. Activities of Rubisco and PEPC were measured in extracts prepared from fresh-frozen cotyledons as described previously by Ghosh et al. [8].
4.4. Electron micrographs of chloroplasts Cotyledon tissue was prepared for electron microscopy using the method of Ronning et al. [27] as outlined in figure 10. One-mm2 sections of cotyledons from 1-, 2-, 3-, and 4-week old plants were fixed in 2.5 % glutaraldehyde at room temperature for 2 h. The tissue was then washed five times in 0.05 M phosphate (pH 6.8) followed by post-fixation in 2 % OsO4 in 0.05 M phosphate buffer (pH 6.8) at room temperature for 2 h. Samples were dehydrated in a concentration gradient of 20, 40, 60, 80, and 100 % acetone for 20 min each followed by two extra washes in 100 % acetone. Infiltration was done by incubating the dehydrated samples for 1 h each in 25, 50 and 75 % low-viscosity epoxy resin-acetone followed by four changes in 100 % resin. The infiltrated tissue was then embedded in 100 % resin and oven-cured at 60 °C for 12 h. Sectioning was done with an ultramicrotome and the tissue sections placed on copper grids for staining. Staining was done in 2 % uranyl acetate for 10 min followed by rinsing in deionized H2O and 5 min in
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Figure 11. Ultrastructural changes in thylakoid membranes of chloroplasts from (A) 1-, (B) 2-, (C) 3- and (D) 4-week-old seedlings of B. napus. GL, grana lamella; SL, stroma lamella; P, plastoglobulus; S, starch granule.
3 % lead citrate (pH 12). Potassium hydroxide pellets were placed in the staining chamber containing the lead citrate to minimize lead precipitation.
Acknowledgments. This study was supported, in part, by The University of Wisconsin-Whitewater Faculty Research Grant and The University of WisconsinWhitewater Undergraduate Research Grant. We are also grateful to Patricia Dumbroff for critical reading of this manuscript.
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