ARTICLE IN PRESS Journal of Plant Physiology 165 (2008) 1954—1963
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Preservation of photosynthetic electron transport from senescence-induced inactivation in primary leaves after decapitation and defoliation of bean plants Ivan Yordanova, Vasilij Goltsevb,, Detelin Stefanova, Petko Chernevb, ¨rg Strasserc Ivelina Zaharievab, Maria Kirovab, Velichka Gechevaa, Reto Jo a
Institute of Plant Physiology, Bulgarian Academy of Sciences, Sofia, Bulgaria Department of Biophysics and Radiobiology, Faculty of Biology, St. Kliment Ohridski University of Sofia, 8, Dragan Tzankov Blvd., 1164 Sofia, Bulgaria c Bioenergetics Laboratory, University of Geneva, Jussy-Geneva, Switzerland b
Received 28 January 2008; received in revised form 12 May 2008; accepted 14 May 2008
KEYWORDS Decapitated plants; Defoliation; Delayed fluorescence; JIP-test; Variable chlorophyll a fluorescence
Summary The comparative effects of decapitation and defoliation on the senescence-induced inactivation of photosynthetic activity in primary leaves of bean plants were investigated. Decapitation was performed during different phases of bean plant ontogenesis, immediately after the appearance of the 1st, 2nd, 3rd and 4th composite leaf. In addition, we examined a variant with primary leaves and stem with an apical bud, but without composite leaves, i.e. defoliated plants. Analyses of chlorophyll fluorescence, millisecond delayed fluorescence and absorption at 830 nm in primary leaves were undertaken to investigate the alterations in photosystems II and I electron transport during the decapitation-induced delayed senescence in the non-detached leaves. Analysis of the OKJIP transients using the JIP-test (see [Strasser R, Srivastava A, Tsimilli-Michael M. Analysis of the chlorophyll a fluorescence transient. In: Papageorgiou G, Govindjee, editors. Chlorophyll a fluorescence: a signature of photosynthesis. The Netherlands: Kluwer Academic Publishers, 2004; pp. 321–362]) showed an increase in several biophysical parameters of photosystem II in decapitated plants, specifically, the density of
Abbreviation: D, decapitated bean plants; F0 Fm and Ft, chlorophyll fluorescence intensities – initial maximal and at any moment t after start of leaf illumination by actinic light respectively; FR, – far red; ND, non-decapitated plants; OIDP, rapid fluorescence transient at moderate actinic light intensities; OKJIP, rapid fluorescence transient at high actinic light intensities; PFD, photon flux density; PI, performance index on a chlorophyll basis; PS I and PS II, photosystems I and II; QA and QB, the first and second stable quinone acceptors in photosystem II, respectively; TEM, transmission electron microscopy. Corresponding author. Tel.: +359 2 8167 370; fax: +359 2 8656 641. E-mail address:
[email protected]fia.bg (V. Goltsev). 0176-1617/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2008.05.003
ARTICLE IN PRESS PS II electron transport after decapitation and defoliation of bean plants
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active reaction centers on a chlorophyll basis, the yields of trapping and electron transport, and the performance index. We also observed a decrease in the absorbed light energy per reaction center. Such a decrease in light absorption could be a result of the photosystem II down regulation that appeared as an increase in QB-nonreducing photosystem II centers. The effect was identical when all leaves except the primary leaves were removed. The variant with a preserved apical bud, the defoliated plant, showed values similar to those of decapitated plants with primary leaves only. The changes in the induction curves of the delayed fluorescence also indicated an acceleration of electron transport beyond photosystem II in the decapitated and in defoliated plants. In these plants, the photosystem I-driven electron transport was accelerated, and the size of the plastoquinone pool was enhanced. It was established that decapitation can retard the senescence of primary leaves, can expand leaf life span and can cause activation of both photosystems I and II electron transport. The decapitation procedure shows similarities to the process of defoliation. The overcompensation effect that is developed after defoliation could initially be manifested as an acceleration of the linear photosynthetic electron flow in the rest of the leaves. & 2008 Elsevier GmbH. All rights reserved.
Introduction During leaf ontogenesis, including leaf senescence, the structure and activity of the photosynthetic apparatus are altered. Changes in decapitated bean plants (D) have been studied extensively in our laboratory (Yordanov, 1984). The results have revealed that decapitation of bean plants above the primary leaves or cotyledons considerably delays their senescence (Yordanov, 1984; Wilhelmova et al., 1997; Kutik et al., 1998). Studies on leaf senescence after decapitation can be considered as informative, because decapitation can be used as a model system for investigation of defoliation, which is often observed in nature, and induced by various pests as insects, herbivores, hail, etc. The (over)compensation for the defoliation effect via delayed leaf senescence is also frequently observed (Li et al., 2005). The appearance of delayed senescence phenomena after defoliation or during artificial decapitation has shown similarities in plant responses during their development (Yordanov et al., 2008). The role of apical dominance phenomena should be of great importance for defoliated plants in relation to their further development. Apical dominance is the control exerted by the shoot apex over lateral bud outgrowth (Cline, 1997). One set of studies examined whether the influence of the apical dominance on the photosynthesis of the primary leaves would be lower in plants having only primary leaves and a stem with an intact apical bud than in intact (ND) plants. Young leaves behave as sink leaves, while mature and old senescing leaves behave as source leaves. The source–sink relation-
ship, which was influenced by both genotype and environmental factors, contributed to the variation in photosynthesis and photosynthate partitioning in plants. Increased leaf net photosynthetic rate as a result of source reduction after partial defoliation and decreased net photosynthesis after sink reduction were also observed (Buchanan-Wollaston, 1997; Nooden et al., 1997). Information is limited regarding the influence of plant decapitation at different stages of ontogenesis. The aim of this paper was to compare the photosynthetic behavior of primary leaves of bean plants of the same ontogenetic age, but at different physiological states, created by decapitation. Using fluorescence methods, we investigated the alterations in photosystem (PS) II function during plant senescence in decapitated/defoliated bean plants. As a criterion, we used the photosynthetic electron transport that depends on the modification of the sink–source interaction in an experimentally created system.
Materials and methods Material and treatments Phaseolus vulgaris, cv. ‘‘Black Starozagorski’’ was grown in a growing chamber at controlled temperature (22–23 1C), light (120–130 mmol m2 s1), humidity (60–65%) and photoperiod 12/12 h, as a water (hydroponics) culture on Knopp nutrient solution medium. After the appearance of the primary leaves the bean plants were divided into 6 groups (Figure 1): (1) control plants that were non-decapitated (ND) (i.e. intact); (2) plants decapitated after the formation of primary leaves
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Figure 1. Scheme of experimental plants: in variants D0–D3 the apical bud was removed after appearance of 1st, 2nd, 3rd and 4th trifoliate leaf, respectively, in 10–21 d-old plants. In the defoliated variant–Df, all newly formed young trifoliate leaves were removed. and appearance of the first trifoliate leaf, D0; (3) plants decapitated after formation of the first and appearance of the second trifoliate leaf, D1; (4) plants decapitated after formation of second and appearance of the third trifoliate leaf, D2; (5) plants decapitated after emergence of fourth trifoliate leaf, D3; and (6) plants that contained only primary leaves and a stem with an apical bud, Df. Decapitation was performed by removing the stem, buds, apex and the newly appearing leaves just above the already formed leaves. As a result, the plants in groups D0–D3 were left with 0–3 trifoliate leaves, respectively. For all groups consisting of decapitated plants, the emerging buds and the apex of the central stem were removed over the following days. Transmission electron microscopy (TEM) Samples for TEM were prepared as was described in Yordanov (1984). Briefly, thin leaf slices were taken from the middle part of the lamina. The pieces were vacuum infiltrated with 2% water solution of KMnO4 for additional fixation procedures. The KMnO4 was removed from the samples by rinsing 10 times with water. Series of ethanol solutions were used for dehydration. The material was embedded in EPON (Fluka, Germany), followed by cutting in ultra-thin slices by ultramicrotome Tesla (Prague, Czech Republic). The electron micrographs were taken using a JEM 100 B (Japan) electron microscope. Chlorophyll a fluorescence measurements The photosynthetic activity was measured in primary leaves of 30-d-old bean plants. The induction transients of prompt chlorophyll fluorescence were registered for 1 s with a fluorometer Handy PEA (Plant Efficiency Analyser, Hansatech Instruments Ltd, King’s Lynn, Nor-
I. Yordanov et al. folk, UK) at an actinic light intensity of 3000 mmol m2 s1 photosynthetic photon flux density (PFD). The fluorescence transients were analyzed according to the JIP-test equations (Strasser et al., 1995; Strasser et al., 2004). JIP-test parameters present the energy fluxes and their bifurcations, as well as the efficiencies/ yields, which are defined as ratios of the energy fluxes. The maximum quantum yield of primary photochemistry (jPo) was calculated from the equation: jPo ¼ 1F0/FM, the quantum yield of electron transport from Q A to the intersystem electron acceptors (jEo) was calculated by equation jEo ¼ 1FJ/FM, and the quantum yield of electron transport from Q A to the PS I end electron acceptors (jRo) – by formulae jRo ¼ 1FI/FM (TsimilliMichael and Strasser, 2008; Smith et al., 2008). The photosynthetic performance index (PIABS), which is a combined measure of the amount of photosynthetic reaction centers (RC/ABS), the maximal energy flux which reaches the PS II reaction center and the electron transport at the onset of illumination, can be calculated as PIABS ¼ gRC =ð1 gRC ÞjPo =ð1 jPo ÞcEo =ð1 cEo Þ where gRC is the fraction of chlorophylls acting as a RC of PS II, jPo is the fraction of excitons trapped per photon absorbed and cEo is the probability that an electron moves further than Q A . This PI can be expressed in the following experimentally available fluorescence intensities: PIABS ¼
1 ðF 0 =F M Þ ðF M F 0 Þ ðF M F J Þ 4ðF 300 ms F 0 Þ=ðF J F 0 Þ F0 ðF J F 0 Þ
In vivo modulated Chl fluorescence was measured at room temperature using a PAM fluorometer (H. Walz, Effeltrich, Germany, model PAM 101-103). The F0 level was sensitized at an instrument frequency of 1.6 kHz with the measuring beam set at the largest possible amplification without causing photosynthetic induction (0.050 mmol m2 s1 PFD). Red actinic light illumination 32 mmol m2 s1 PFD was provided by PAM 102 unit connected to red light-emitting diode PAM 102L for registration of rapid phase of fluorescence induction curve, OIDP. QB-non-reducing PS II centers were calculated by the ratio (OI)/(OP) following the procedure proposed by Laza ´r (1999). All fluorescence measurements were carried out after 30 min dark adaptation. Data analysis (Tyystjarvi and Karunen, 1990) was carried out by FIP software (QA-Data, Turku, Finland). Delayed fluorescence measurement Delayed fluorescence induction curves were registered by measuring system FL-2006, Test, Russia, described in detail in Zaharieva and Goltsev (2003). P700 oxidation measurement The redox state of P700 was investigated in vivo with a dual wavelength (810/860 nm) unit (Walz ED 700DW-E) attached to a PAM101E main control unit (Klughammer
ARTICLE IN PRESS PS II electron transport after decapitation and defoliation of bean plants and Schreiber, 1998) in the reflectance mode (Schreiber et al., 1988). P700 was oxidized by irradiation with far red, FR, light (13.4 W m2) provided by a photodiode (FR102, Walz, Effeltrich, Germany) that was controlled by the PAM 102 unit. Most of the data were the mean values of four independent experiments with at least six repetitions.
Results Decapitated plants display characteristics of delayed senescence. As we have shown previously, D0 plants preserved high pigment amounts during the period of the investigation (Yordanov, 1984). The leaves of decapitated plants were dark green, thicker and had larger leaf areas (Figure 1). In parallel, ND plants showed the development of primary leaves with senescence-related yellowing of the leaves, the appearance of necrotic spots during late stages of leaf senescence, and lower O2 evolution and CO2 assimilation rate (Yordanov, 1984). Chloroplast structure determines photosynthetic capacity of leaf cells. The chloroplast ultrastructure in mesophyll cells of the bean primary leaves is shown in Figure 2. The changes in chloroplast structure in 10-d-old-grown (young), 18-d-old-
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grown (mature) and 30-d-old-grown (old) primary leaves of ND and D bean plants were examined. A comparison of ND and D chloroplast ultrastructure showed no difference in 10-d-old plants as a result of the decapitation procedure. They were enriched in grana and lamellar thylakoids. Clearly expressed differences in thylakoid architecture in chloroplasts of 18-d and 30-d primary leaves of ND and D bean plants were observed. While chloroplast structure in mature (18 d) primary leaves displayed well expressed and arranged grana and stroma thylakoids, the chloroplast ultrastructure in 30-d-old ND bean plants was highly modified. In contrast to ND, chloroplast granal ultrastructure in D bean plants was well preserved (Figure 2). The changes in anatomical properties of the primary leaves and changes in their chloroplast ultrastructure in ND and D bean plants (Figure 2) suggest altered metabolism in leaves, especially in the photosynthetic apparatus. We investigated PS II and PS I activities using chlorophyll a fluorescence induction (Schreiber et al., 1986; Strasser et al., 2004) and using FR-induced changes in leaf absorbance at the near-infrared region, about 830 nm (Schreiber et al., 1988), respectively. A PI parameter (PIABS or simply PI) introduced by a JIP-test (see Strasser et al., 2004) was used to characterize PS II structural and functional
Figure 2. Electron micrographs of chloroplasts in mesophyll cells of primary leaf of the decapitated (D) and nondecapitated (ND) bean plants at different plant ages (left column of numbers). The numbers on the right-hand side are days after decapitation (d.a.d.) in variant D0. The bars show a distance of 1 mm.
ARTICLE IN PRESS 1958 alterations in control and decapitated bean plants, with a goal to investigate changes in leaf senescence modulated by different decapitation procedures. The changes in PI are presented in Figure 3. The PI was examined on the primary bean leaves in all investigated variants at different stages of leaf ontogenesis, beginning on the 6th day after the emergence of the first leaf (mature leaves) to the 22nd day when the primary leaves in ND plants had almost totally lost their functional activity. PI values decreased with the aging of the primary leaves. The decapitation procedure applied after the emergence of the primary leaves and of the first trifoliate leaves (D0 n D1 variants) caused a visible increase in PI (Figure 3). PI remained relatively unchanged in D2 during the entire period of investigation. D3 plants were similar to the control plants, but the inactivation of their photosynthesis was less pronounced. Defoliation that was carried out after the 8th day and after emergence of the subsequent leaves did not significantly change PI (Figure 3). The changes in the OKJIP fluorescence transient are presented in Figure 4A as a difference of each of the (Ft–F0)/(Fm–F0) kinetics from the curve of the D0 samples, which is characterized by maximal functional activity, i.e. lower extent of leaf senescence. Two main phases were observed
Figure 3. Influence of decapitation/defoliation on the dynamics of aging-induced changes in PS II activity in primary bean leaves during 10–30 d of plant development. After the emergence of the first (D0), second (D1), third (D2) and fourth (D3) trifoliate leaves the apical bud together with newly emerging leaves were removed. In the variant ‘‘defoliated plants’’ (Df) the newly appearing leaves were immediately detached. Chlorophyll fluorescence transients were registered on primary leaves in intact plants (in situ) by a Handy PEA (Hansatech, Norfolk) fluorometer at photosynthetic PFD of 3000 mmol m2 s1 after 3 min dark adaptation. The values of performance index (PI) were calculated according to Strasser et al. (2004).
I. Yordanov et al.
Figure 4. Changes in double normalized chlorophyll fluorescence transients measured in primary leaves of 30-d-old bean plants modified and marked according to the experimental scheme (see Figure 1). (A) Thin lines show double normalized chlorophyll fluorescence transients (right axis), and thick lines – the differences between (temporal) moment values of relative variable fluorescence for each variant and the corresponding value for D0 (left axis). (B) Changes in JIP-test parameters, calculated according to Strasser et al. (2004); Tsimilli-Michael and Strasser (2008). Gray empty symbols show values of parameters in defoliated plants (Df). Chlorophyll fluorescence was measured as in Figure 3.
(Figure 4A). The first phase, which coincides with the OKJ transient, showed that, in ND plants, the electron flow beyond QA was suppressed (i.e. increased J). We also observed a decreased rate of electron donation by the oxygen-evolving complex, i.e. increased K values (Strasser, 1997). The second phase displayed complex behavior and likely reflects changes in electron withdrawal from PS II, depending on the activity of PS I (Schansker et al., 2005). This phase (Figure 4A) shows decreased PS I activity in ND plants. All changes reported in Figure 4A are explicable in terms of delayed leaf senescence in D plants compared to ND plants. The delay of the leaf senescence was reduced in the following order: D04Df4D14D24D34ND. The changes in some JIP-test parameters are presented in Figure 4B. We observed no significant changes in the structural stability of PS II, which remained active during leaf senescence as evaluated by the Fv/Fm ratio. Maximal values of RC/ABS
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were reached in D0 plants. The Df plants had values of RC/ABS similar to those of D0. The changes in PI(ABS), Sm and j(Eo) were similar to the changes of RC/ABS after different decapitation procedures. The new parameter j(Ro) introduced by Tsimilli-Michael and Strasser (2008) reflects the reduction capacity of the PS I end electron acceptors shows a rising inactivation of PS I in the sequence DfED14D24D34ND as compared to D0 plants (Figure 4B). The rise kinetics of fluorescence during the Kautsky effect, OIDP, was measured under moderate intensities of red light (32 mmol m2 s1 PFD). Under such conditions, equilibrium between the quinone electron acceptors of PS II, QA and QB is achieved, and the plastoquinone pool is not fully reduced in the P peak. The ratio (OI)/(OP) reflects the proportion of QB-non-reducing PS II centers (Figure 5). The proportion of inactive QB-non-reducing PS II centers changed in a manner similar to the delay in the leaf senescence, evaluated by the OJIP transient (Figure 4A). QB-non-reducing PS II centers decrease in the following order: D04D14Df4D24D34ND. Photosystem I activity can be studied by the changes in A830 leaf absorbance induced by FR light (Figure 5). The level of FR-induced P700 oxidation decreases as follows: D04D14Df4D2 4D34ND. Delayed chlorophyll fluorescence is an informative test of the physiological state of plants. It is highly correlated with photosynthetic activity of whole leaves and isolated chloroplasts during plant senescence (Zhang et al., 2007). The induction
curve of millisecond delayed fluorescence during the first several minutes can be described as the combined result of at least six superimposed peaks, denoted in the order of their occurrence on the timescale as I1–I6, according to nomenclature previously proposed by Goltsev and co-workers (Goltsev et al., 2003; Zaharieva and Goltsev, 2003). The I1–I2–D2 transition in the rapid phase reflects the appearance and disappearance of ‘‘light-emitting’’ states of PS II reaction centers and correlates with the rate of electron transport reactions in the PS II acceptor side. The delayed fluorescence decrease after I2 is a result of lightinduced closure of reaction centers and disappearance of the ‘‘light-emitting’’ states. The decrease of the I2/D2 ratio indicates an acceleration of the intersystem electron flow (Zaharieva et al., 2001). The delayed fluorescence drop after I2 was diminished in decapitated plants and lacked in variant D0 (Figure 6), which supposes an acceleration of the electron transport beyond PS II in decapitated and defoliated plants. The enhancement of the electron transport is a result of delayed senescence in the primary leaves of the decapitated bean plants, and could be explained by changes in the sink– source interactions.
Figure 5. Changes in relative absorption at 830 nm (DA/ A – left axis) and share of QB non-reducing PS II reaction centers (estimated using parameter (OI)/(OP) of chlorophyll fluorescence transient – right axis) measured in primary leaves of 30-d-old bean plants modified and marked according to the experimental scheme (see Figure 1). Ten seconds OIDP fluorescence transient at low actinic light intensities of 32 mmol m2 s1 PFD registered after 30 min dark adaptation with PAM fluorometer (Walz, Germany) in leaf disks.
Figure 6. Changes in delayed chlorophyll fluorescence transients measured in primary leaves of 30-d-old bean plants modified and marked according to the experimental scheme (see Figure 1). Leaf disks were dark adapted for 3 min and delayed fluorescence induction curves were recorded for 3 min using the fluorometer FL2006 (‘‘TEST’’, Krasnojarsk, Russia) at actinic light intensity 1200 mmol m2 s1 PFD according to Zaharieva and Goltsev (2003). The delayed fluorescence values are normalized to their maximal level.
Discussion Decapitation of the plants (Figure 1) modified the leaf senescence program, as do other internal and
ARTICLE IN PRESS 1960 external stimuli such as light, temperature, drought, elevated CO2, age, reproduction, plant growth regulators, defoliation, etc. (Yordanov and Popov, 1967; Wilhelmova et al., 1997; Jordi et al., 2000). These factors can interact with each other in triggering, delaying or even reversing of leaf senescence. Usually, delayed senescence is promoted by plant growth regulators such as cytokinins, gibbrellins and auxins (Richmond and Lang, 1957; Gan, 2004). Some external factors such as dark treatment (Weaver and Amasino, 2001) or pathogen acting (Panstruga, 2003) could also induce delayed senescence. Modulated life spans in the leaves, involving delaying or reversing (rejuvenation) of senescence as a result of decapitation, has also been investigated (Yordanov and Popov, 1967; Wilhelmova et al., 1997; Kutik et al., 1998). Modulation of the leaf life span of the primary leaves has been revealed by changes in leaf senescence of Phaseolus vulgaris L. as evaluated by changes in chloroplast ultrastructure (Figure 2) and by altered PS I and PS II activity (Figures 3–6). Earlier, its characteristics were also modulated by epicotyl decapitation (Wilhelmova et al., 1997; Kutik et al., 1998). We investigated the changes in some photosynthetic processes in decapitated (D0, D1, D2 and D3) and in artificially defoliated plants (Df) as compared to control ND plants that expressed typical senescence symptoms during their development (Figure 1). When the leaf is no longer required by the plant, the senescence process is induced and recycling of all mobilizable nutrients occurs (Buchanan-Wollaston et al., 2003) in the life of both annual and perennial plants. During its life span, a leaf undergoes at least three phases of development. Initially, the young leaf expands rapidly, importing carbon and nitrogen, and undergoes rapid protein synthesis until its full capacity for photosynthesis is reached. Then the mature leaf becomes an asset to the plant, contributing to the supply of carbon, during which time, protein turnover is at a consistently low level. This continues until internal or external conditions initiate the onset of senescence. Leaf senescence is a period of massive mobilization of nitrogen, carbon and minerals from the mature leaf to other parts of the plant and is a highly regulated, ordered series of events involving cessation of photosynthesis, disintegration of chloroplasts, breakdown of leaf proteins, loss of chlorophyll and removal of amino acids. The final stage of this process is leaf death, but this is actively delayed until all nutrients have been removed through the processes of developmental senescence. The chloroplasts/photosynthetic ma-
I. Yordanov et al. chineries are expressed as the most long-lived cell organelles/processes. Chloroplast breakdown is not a primary cause of cell death. It is important in that a large proportion of the plant’s resources, e.g. nitrogen, are tied up in the leaves, mostly in the chloroplasts. These resources are generally redistributed for reuse elsewhere, e.g., new leaf formation, developing seeds (Lawlor, 1993). When the sink–source relationships were altered by decapitation, a process of re-greening and lack of leaf fall was observed in the rest of the leaves (Figure 1). Even in the yellow primary leaves on their final stage of senescence the decapitation above them could induce the re-greening effect (data not shown). Our results showed that decapitation of bean plants above the primary leaves reversed (D0, D1 and Df, variants on Figure 3) or considerably delayed (D2 and D3) their senescence. The process of re-greening of the primary leaves was observed (Figure 1). Also, preserved chloroplast ultrastructure in D bean plants was observed (Figure 2). The pigment content and the rate of photosynthesis increased (Popov et al., 1969; Yordanov et al., 1977). The primary leaves of decapitated bean plants are also characterized by high levels of proteins and RNA (Yordanov and Nikolova, 1971). Decapitated plants were more resistant to high temperature (Yordanov and Weis, 1984) and some antibiotics (Chichev and Yordanov, 1971). It has also been suggested that the degenerative changes were postponed or slowed down in decapitated plants under the influence of root-produced cytokinins that are induced by plant decapitation (Dei, 1978; Wilhelmova et al., 1997). When ageing was retarded in decapitated plants, the photosynthesis rate, determined by CO2 assimilation or O2 evolution, was high (data not shown). It is evident that the sink/source mechanism plays a very important role in enhanced photosynthetic rate. This has clearly been illustrated by experiments showing that senescence is reversible. A leaf that is completely yellow and has mobilized the great majority of its nutrient content can be induced to re-green through various treatments (Zavaleta-Mancera et al., 1999a, b; Thomas and Donnison, 2000). The most peculiar phenomena that are part of the reversion of senescence are recovering of structural features of the chloroplasts (Figure 2), synthesis of chloroplast proteins that occurs after decapitation (Yordanov, 1984) and restarting of photosynthesis (Figure 3). The reversibility of leaf senescence in the direction of the rejuvenation depends on the decapitation procedure carried out at different plant ages. For younger leaves, reversed senescence occurred
ARTICLE IN PRESS PS II electron transport after decapitation and defoliation of bean plants more easily than in older ones (Figure 3). Regreening phenomena were more evident when decapitation was carried out in old plants (data not shown). In this case rejuvenation, i.e. reversing of senescence, was observed. The decapitation effect expressed as a preservation of chloroplast structure was more pronounced in old bean plants (30-d old in Figure 2). It can also be regarded as a stress. Similar to other stresses, decapitation can induce rejuvenation in old bean plants (Nyitrai et al., 2004). Nyitrai et al. demonstrated that low doses of stressors such as Cd, Pb, Ni, Ti salts and DCMU delayed the senescence of chloroplasts and stimulated chlorophyll synthesis and photosynthetic activity. Defoliation (leaf loss), which has often been described as an aspect of herbivory effects on plant canopies, was carried out artificially in our experiments (Figure 1). Defoliation is a frequent phenomenon in herbaceous plants and in tropical forests. Delayed senescence in response to leaf loss and regreening of yellow senescent leaves was reported 40 years ago (Woolhouse, 1967; Wareing et al., 1968). Generally, herbivory has negative effects on plant performance, but consumption of vital plant organs sometimes appears to increase plant fitness (Prins and Verkaar, 1992). The main physiological responses to defoliation include stimulation of photosynthetic rate, changed hormonal balance, delayed plant senescence and redistribution of carbohydrates and mineral nutrients as a result of changed sink–source relationships. Defoliation induced artificially in Nicotiana tabacum (KolodnyHirsch et al., 1986) is accompanied by increased efficiency in assimilating dry matter. Richards et al. (1988) found that there are differences between decapitation (apical bud removal) and defoliation in relation to changed apical dominance. Our experiments revealed that Df plants expressed characteristics of delayed senescence but without rise of the PI above the value in young control ND plants as the one observed in decapitated plants D0 and D1 (Figure 3). We suggest that the lack of activation of photosynthesis in primary leaves in Df plants is an expression of a suppression effect of apical dominance. For a more detailed study on the altered photosynthesis after decapitation and defoliation, we investigated PS II and PS I functioning by the changes in the JIP-test parameters (Figures 4B and 5), OIDP fluorescence transient (Figure 4A), A830 leaf absorbance (Figure 5) and delayed fluorescence induction (Figure 6). At the level of PS II the delay of the leaf senescence (D0, D1 and Df) was observed as a higher intactness both in the PS II donor side (which
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was expressed as a decrease of the first maximum denoted as K peak of the chlorophyll fluorescence transients and presented as difference kinetics of the relative variable fluorescence V ¼ (Ft–F0)/ (Fm–F0) on Figure 4A) and acceptor side (shown by altered electron transport rate–j(Eo) in Figure 4 B). In decapitated and defoliated plants (D0, D1 and Df), the PS I-driven electron transport for the reduction of the end acceptors (RE) was accelerated (lowered second maximum on Figure 4A, higher level of j(Ro) ¼ RE/ABS on Figure 4B, and increased P700 oxidation – Figure 5) and the size of the plastoquinone pool was increased (high levels of Sm, standing for the total number of electrons transported per RC of PS II until the Fm is reached in Figure 4B). The rapid phase of the induction curve of the delayed chlorophyll fluorescence (ratio I1/I2 in Figure 6) is evidence of the increase in electron transport activity beyond QA in leaves of D0, D1 and Df plants. Similarly, Zhang et al. (2007) found that altered delayed chlorophyll fluorescence parameters correlated with photosynthetic activity changes induced by plant decapitation during the senescence of the primary leaves. We suggest, therefore, an increase in linear electron transport, which is caused by higher activity of the Calvin– Benson–Basham cycle reactions (Yordanov and Popov, 1967) and photosynthate partitioning (Zavaleta-Mancera et al., 1999a, b; Thomas and Donnison, 2000) as a result of changed sink–source relationships in decapitated/defoliated plants. The effects on linear electron transport observed in bean may be connected to hormonal changes evoked by decapitation or defoliation. Our earlier investigations showed that decapitation-induced activation of photosynthetic activity in primary leaves of D bean plants was accompanied by increased cytokinin content (Yordanov and Iliev, 1983). On the other hand, there were no observable differences in P700+ re-reduction kinetics after cessation of FR illumination in any of the studied samples (data not shown). Such P700+ re-reduction kinetics reflect changes in the dynamics in the PS I cyclic electron transport. In contrast to increased linear photosynthetic electron transport, the lack of observable alterations in the PS I cyclic electron flow between ND and decapitated bean plants is proposed. In normal senesced primary leaves, the relative number of active PS II reaction centers per chlorophyll concentration decreased (RC/ABS parameter in Figure 4B), and the antenna size was higher (ABS/RC) than that in the decapitated plants. This means that the degradation of the antenna pigment protein complexes occurs after
ARTICLE IN PRESS 1962 the inactivation of the PS II reaction centers during senescence. In spite of the increased relative part of the active PS II reaction centers in primary leaves in D0, D1 and Df plants, the PS II activity is down regulated effectively by an increased share of QB non-reducing centers (Figure 4B). This may be the other reason for the enhanced FR-induced P700 oxidation in addition to the higher activity of PS I acceptor side. Our results suggest that the decapitation procedure which prevents the growth of the new leaves demonstrates similarities with the process of defoliation with regard to senescence of the primary leaves as estimated by changes in PS I and II electron transport. The strength of these effects depends on the duration of decapitation/ defoliation action, i.e. the effect is decreased when decapitation is performed late (or when fewer leaves are removed). The overcompensation effect is expressed as an acceleration of the linear photosynthetic electron flow in the remaining leaves after defoliation. In conclusion, decapitation can reverse the senescence of the primary leaves, expand leaf life span, and can cause activation of both PS I and II electron transport. We suggest that modulated source–sink relationships in decapitated plants affect the studied photosynthetic parameters.
Acknowledgments This paper is dedicated to the 100th birthday of the Bulgarian plant physiologist Prof. DSci Kiril Popov. We thank Dr. Elena Chakalova for the preparation of the electron micrographs.
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