Journal of Experimental Botany, Vol. 52, No. 362, pp. 1805-1810,
September 1, 2001
© 2001 Oxford University Press
Original Papers |
Characterization of photosynthetic pigment composition, photosystem II photochemistry and thermal energy dissipation during leaf senescence of wheat plants grown in the field
1 Photosynthesis Research Centre, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, PR China
2 Department of Biology, Hong Kong Baptist University, Kowloon, Hong Kong, PR China
Received 7 March 2001; Accepted 5 June 2001
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Abstract |
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Photosynthetic pigment composition and photosystem II (PSII) photochemistry were characterized during the flag leaf senescence of wheat plants grown in the field. During leaf senescence, neoxanthin and ß-carotene decreased concomitantly with chlorophyll, whereas lutein and xanthophyll cycle pigments were less affected, leading to increases in lutein/chlorophyll and xanthophyll cycle pigments/chlorophyll ratios. The chlorophyll a/b ratio also increased. With the progression of senescence, the maximal efficiency of PSII photochemistry decreased only slightly in the early morning (low light conditions), but substantially at midday (high light conditions). Actual PSII efficiency, photochemical quenching and the efficiency of excitation capture by open PSII centres decreased significantly both early in the morning and at midday and such decreases were much greater at midday than in the early morning. At the same time, non-photochemical quenching, zeaxanthin and antheraxanthin contents at the expense of violaxanthin increased both early in the morning and at midday, with a greater increase at midday. The results in the present study suggest that a down-regulation of PSII occurred in senescent leaves and that the xanthophyll cycle plays a role in the protection of PSII from photoinhibitory damage in senescent leaves by dissipating excess excitation energy, particularly when exposed to high light.
Key words: Chlorophyll fluorescence, pigment composition, photosystem II photochemistry, xanthophyll cycle, wheat (Triticum aestivum L.).
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Introduction |
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Leaf senescence is the sequential degradation process that leads to a massive mobilization and export of nitrogen and minerals and eventually to leaf death (Buchanan-Wolleston, 1997
). The most remarkable event in leaf senescence is the disassembly of the photosynthetic apparatus within chloroplasts and thus the concomitant decrease in photosynthetic activity (Woolhouse, 1984; Grover and Mohanty, 1992). A key event leading to the decrease in photosynthetic activity during leaf senescence is associated with the loss of RuBP carboxylase/ oxygenase (Crafts-Brandner et al., 1990; Grover, 1993). The loss of major thylakoid proteins is usually delayed to the later senescence phases relative to stromal enzymes and is less directly correlated to the decrease in photosynthetic activity (Okada et al., 1992; Mae et al., 1993). Thus, the decrease in photosynthetic CO2 fixation during leaf senescence normally occurs earlier than that in the maximal efficiency of photosystem II (PSII) photochemistry (Humbeck et al., 1996). Previous studies in this laboratory have shown that PSII activity seemed to be affected only slightly whereas a substantial decrease in photosynthetic capacity occurred during leaf senescence (Lu and Zhang, 1998a, b).
A substantial decrease in photosynthetic capacity accompanied by only a slight decrease in PSII photochemistry in senescent leaves can potentially expose the senescent leaves to excess excitation energy, which, if not safely dissipated, may result in photodamage to PSII because of an overreduction of reaction centres (Demmig-Adams and Adams, 1992). Excess excitation energy can be harmlessly dissipated in the antennae complexes of PSII as heat through a process which involves the xanthophyll cycle and a low thylakoid pH. The xanthophyll cycle pigments zeaxanthin (Z) and antherxanthin (A) are formed from violaxanthin (V) under conditions of excess excitation energy and are both thought to be involved in the photoprotective dissipation process (Demmig-Adams and Adams, 1992; Gilmore, 1997).
The role of the xanthophyll cycle under conditions of cold-temperature stress (Verhoeven et al., 1999), water stress (Munné-Bosch and Alegre, 2000), UV-B elevation (Levall and Bornman, 2000), nitrogen deficiency (Verhoeven et al., 1997), and iron deficiency (Morales et al., 2000), has been widely investigated. However, the changes in the xanthophyll cycle pigments and other photosynthetic pigments during leaf senescence have not been fully characterized. Moreover, it is not yet clear whether the xanthophyll cycle plays any role in the dissipation of excess light energy in senescent leaves with decreased photosynthetic capacity.
Senescence is usually studied in some model systems, in which leaf senescence is induced by either incubating detached leaves or whole plants in the dark. Although these systems provide many practical advantages and valuable information on the mechanisms of leaf senescence phenomena, effects observed in these model systems are complicated by the wounding of the tissues and/or of light/dark transition. Even experiments with intact plants grown under a diurnal light/dark cycle in a controlled growth chamber may not represent all aspects of senescence occurring in a natural habitat, where simultaneous changes in environmental factors affect the senescent process (Smart, 1994). Until now, only few reports on the senescence of field-grown plants have been published (Adams et al., 1990; Humbeck et al., 1996; Murchie et al., 2000). Moreover, the characterization of PSII photochemistry, thermal energy dissipation and the xanthophyll cycle in either senescent leaves induced by above model systems or in naturally senescent leaves of plants grown under natural habitat has received little attention. It is of interest to perform a detailed characterization of photosynthetic pigment composition, in particular these pigments related to the xanthophyll cycle, and PSII photochemistry during leaf senescence occurring in natural habit.
In the present study, flag leaves were chosen to investigate the changes in the photosynthetic characteristics during leaf senescence of wheat plants grown in the field since the photosynthesis of flag leaves is most important for grain-filling. The objectives of this study were (1) to characterize fully photosynthetic pigment composition, (2) to examine if and how down-regulation of PSII happens in senescent leaves when exposed to excess light energy, and (3) to determine if the xanthophyll cycle plays a role in dissipating excess light energy during leaf senescence. To these ends, the changes in the xanthophyll cycle pigments and their de-epoxidation status have been investigated as well as other photosynthetic pigment composition and fluorescence quenching parameters during leaf senescence in response to low (early morning) and high light conditions (at midday).
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Materials and methods |
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Plant material
Winter wheat (Triticum aestivum L. cv. Beijing 3348) was grown in 1999–2000 in a field situated at Beijing. Seeds were sown on 2 October, 1999 and a clay soil was used. Nutrients and water were supplied sufficiently throughout and thus potential nutrients and drought stress were avoided.
Flag leaves were used for all analyses beginning on 12 May at the stage of flowering and ending on 10 June. During this period, the weather showed the typical Beijing spring weather with a mean daily air temperature between 19–23 °C and an average PPFD at the leaf level around 1400 µmol m-2 s-1 at midday.
Chl fluorescence
Modulated Chl fluorescence measurements were made in attached leaves in the field with a PAM-2000 portable fluorometer (Walz, Effeltrich, Germany) connected to a notebook computer with data acquisition software (DA-2000, Heinz, Walz). Essentially, the experimental protocol of Demmig-Adams et al. was followed (Demmig-Adams et al., 1996). The minimal fluorescence level (Fo) in dark-adapted state was measured by the measuring modulated light which was sufficiently low (<0.1 µmol m-2 s-1) not to induce any significant variable fluorescence. To determine the minimal fluorescence level during illumination (F'o), a black cloth was rapidly placed around the leaf and the leaf-clip holder in the presence of far-red light (7 µmol m-2 s-1) in order to oxidize the PSII centres fully. Upon darkening of the leaf, fluorescence dropped to the F'o level and immediately rose again within several seconds. The maximal fluorescence level in the dark-adapted state (Fm) and the maximal fluorescence level during natural illumination (F'm) were measured by a 0.8 s saturating pulse at 8000 µmol m-2 s-1. Fm was measured after 30 min of dark adaptation. F'm was measured when morning and midday PPFDs were approximately 200 and 1400 µmol m-2 s-1, respectively. The steady-state fluorescence level during exposure to natural illumination (Fs) was also measured when morning and midday PPFDs were approximately 200 and 1400 µmol m-2 s-1, respectively. All measurements of Fo and F'o were performed with the measuring beam set to a frequency of 600 Hz, whereas all measurements of Fm and F'm were performed with the measuring beam automatically switching to 20 kHz during the saturating flash.
The actual PSII efficiency (
PSII) and the efficiency of excitation capture by open PSII centres were calculated as (F'm - Fs)/F'm and F'v/F'm, respectively (Genty et al., 1989). Photochemical quenching (qP) was calculated as (F'm - Fs)/(F'm - F'o) (van Kooten and Snel, 1990). Non-photochemical quenching (NPQ) was calculated as (Fm/F'm) - 1 (Bilger and Björkman, 1990). It should be noted that the Fm values at predawn which were fully recovered after the 30 min dark adaptation were used for the calculation of NPQ.
Pigment analyses
Leaf samples were taken and immediately frozen in liquid nitrogen. Leaf samples were extracted in ice-cold 100% acetone and the pigment extracts were filtered through a 0.45 µm membrane filter. Pigments were separated and quantified by HPLC essentially as described earlier (Thayer and Björkman, 1990).
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Results |
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Pigment composition
Total chlorophyll (Chl) content and the ratio of Chl a/b did not change considerably until 20 d after flowering. From the 20th to the 28th day, the Chl content decreased remarkably from 524 to 110 µmol m-2 and the ratio Chl a/b increased from 2.4 to 3.3. No difference was observed in the total Chl content and the ratio Chl a/b between samples taken early in the morning and at midday (Fig. 1
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). Morning and midday PPFDs were approximately 200 and 1400 µmol photons m-2 s-1. Data are means±SE of 3–5 independent measurements.Figure 2
shows the changes in carotenoid contents expressed on a total chlorophyll basis during the senescence of flag leaves. Neoxanthin and ß-carotene contents were largely unchanged after flowering, both early in the morning and at midday (Fig. 2A, B). Lutein content remained unchanged until the 20th day after flowering and then increased significantly from 70 to 150 mmol mol-1 Chl on the 28th day. There was no significant difference in the lutein content between the samples taken early in the morning and at midday (Fig. 2C). V, A and Z contents also remained unchanged until 20 d after flowering and then increased significantly starting from 20 d after flowering. However, the V content was lower and the A and Z contents were higher in samples taken at midday than in samples taken early in the morning (Fig. 2E, F, G).
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Chl fluorescence
Figure 3 shows the changes in several fluorescence parameters during the senescence of flag leaves. When measured early in the morning, the maximal efficiency of PSII photochemistry (Fv/Fm) was virtually kept at a high value of around 0.82 until 26 d after flowering and decreased slightly only on the 28th day with a value of 0.72. When measured at midday, Fv/Fm maintained a value of around 0.82 until 20 d after flowering; thereafter it decreased significantly with senescence progressing to a value of 0.45 on the 28th day (Fig. 3A). The actual PSII efficiency (PSII), the efficiency of excitation capture by open PSII centres (F'v/F'm), and the photochemical quenching coefficient (qP) decreased significantly only from 20 d after flowering and their decreases were much greater when measured at midday than early in the morning (Fig. 3B, C, D). Non-photochemical quenching (NPQ) was largely unchanged until around 20 d after flowering both in the morning and at midday and increased significantly thereafter. The NPQ values were much higher at midday than early in the morning (Fig. 3E).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Characterization of pigment composition
In the present study, changes in the photosynthetic pigment composition were investigated during the senescence of flag leaves of wheat plants grown in the field. The changes in pigment contents demonstrate that senescence of flag leaves was not induced until around 20 d after flowering. The leaves in this period are defined here as ‘the control leaves’. Senescence of flag leaves started 20 d after flowering. With senescence progressing, absolute photosynthetic pigment contents expressed on a leaf area basis decreased dramatically. However, they degraded to different extents. Neoxanthin and ß-carotene decreased equally with chlorophyll, whereas lutein and the carotenoids within the xanthophyll cycle were much less affected. Thus, a relative enrichment in lutein and the xanthophyll cycle pigments was observed (Fig. 2
). In addition, an increase in the Chl a/b ratio in senescent leaves suggests that Chl b was more affected than Chl a (Fig. 1). These results indicate that there was a change in the photosynthetic pigment stoichiometry during leaf senescence.
The carotenoids within the xanthophyll cycle underwent epoxidations and de-epoxidations to different extents in control and senescent leaves in response to different light conditions. During the night, most of the xanthophyll cycle pool was in the epoxidated form V in both control and senescent leaves with (Z+A)/(V+Z+A) ratio around 0.05 and 0.12, respectively. Either in the early morning or at midday, the de-epoxidated forms A and Z increased significantly with senescence progressing at the expense of V. Such an increase in the de-epoxidated forms A and Z was much greater at midday than in the morning (Fig. 2E, F, G).
Down-regulation of PSII efficiency
The maximal efficiency of PSII photochemistry (Fv/Fm) decreased only slightly when measured in the early morning, but decreased substantially in severely senescent leaves at midday. Such a midday decrease in Fv/Fm became larger with senescence progressing and could recover to the values that were similar to those in the morning (Fig. 3A). Similarly, compared to the control leaves, the actual PSII efficiency (PSII) decreased markedly in senescent leaves both early in the morning and at midday. Such a decrease was more evident at midday than early in the morning and could also recover to the values that were similar to those in the morning (Fig. 3B). These reversible changes in Fv/Fm and PSII can be ascribed as a down-regulation of PSII that may reflect the protective or regulatory mechanisms to avoid photodamage to the photosynthetic apparatus (Krause, 1988; Demmig-Adams, 1990; Demmig-Adams and Adams, 1992). Such a down-regulation of PSII in senescent leaves can be explained by the increase in the proportion of closed PSII centres (estimated from decreased qP) and the decrease in the efficiency of excitation energy capture (F'v/F'm) (Fig. 3C, D).
A considerable decrease in F'v/F'm in senescent leaves both in the morning and at midday, but only a slight decrease in Fv/Fm measured in the morning, suggests that the decreased F'v/F'm in senescent leaves could be associated with an increase in energy dissipation in the PSII antennae (Demmig-Adams et al., 1995, 1996; Demmig-Adams and Adams, 1996). The data from this study show that under low light conditions (early in the morning), an increase in non-photochemical quenching (NPQ) with the progression of senescence was accompanied by an increase in A and Z contents, when compared to the control leaves (Figs 2F, G; 3E), suggesting that the xanthophyll cycle-related thermal dissipation was significantly enhanced already at low light in senescent leaves (Horton et al., 1996; Gilmore, 1997). Under high light conditions (at midday), senescent leaves had a greater increase in NPQ, which was again associated with a greater increase in A and Z contents (Figs 2F, G; 3E), indicating that a higher level of thermal dissipation involved in the xanthophyll cycle occurred in senescent leaves when exposed to high light. The results in the present study suggest that xanthophyll cycle plays a role in dissipating excess light energy during leaf senescence, particularly when exposed to high light.
In summary, the photosynthetic pigment composition and PSII photochemistry during flag leaf senescence of wheat plants grown in the field has been investigated. These results indicate that there was a change in the photosynthetic pigment stoichiometry during leaf senescence and that a down-regulation of PSII occurred in senescent leaves particularly when exposed to high light. The present results demonstrate that the xanthophyll cycle-related thermal dissipation in the PSII antennae was enhanced significantly in senescent leaves, which may protect the photosynthetic apparatus from photoinhibitory damage in senescent leaves when exposed to high light.
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Acknowledgments |
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This work was supported by the Program of 100 Distinguished Young Scientists of the Chinese Academy of Sciences, the State Key Basic Research and Development Plan of China (No. G1998010100), the Innovative Foundation of the Laboratory of Photosynthesis Research, Institute of Botany, Chinese Academy of Sciences (CL), as well as the Hong Kong Baptist University (FRG grant) (CL, JZ).
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Notes |
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3 To whom correspondence should be addressed. Fax: +86 10 62590833. E-mail: congminglu{at}yahoo.com
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Abbreviations |
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A, antheraxanthin; Chl, chlorophyll; Fm, F'm, maximal fluorescence level in the dark or during illumination; Fo, F'o, minimal fluorescence in the dark or during illumination; Fv, F'v, maximal variable fluorescence level in the dark or during illumination; Fs, steady-state fluorescence yield during illumination; Fv/Fm, maximal efficiency of PSII photochemistry;
PSII, actual PSII efficiency; F'v/F'm, efficiency of excitation energy capture by open PSII centres; NPQ, non-photochemical fluorescence quenching; PPFD, photosynthetic photon flux density; PSII, photosystem II; qP, photochemical quenching coefficient; V, violaxanthin; Z, zeaxanthin. |
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