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Photosynthetic light-harvesting during leaf senescence in Panicum miliaceum
plan cience ELSEVIER SCIENTIFIC Pt.IB[ ISHERS IRELAND
Plant Science 95 (1994) 141-152
Photosynthetic light-harvesting during leaf senescence in Panicum miliaceum J. Lorene Embry't, Eugene A. Nothnagel* Department of Botany and Plant Sciences, University of California, Riverside, California 92521-0124, USA
(Received 8 July 1993; accepted 13 September 1993)
Abstract
During plant senescence, cai'otenoid pigments are lost from the leaves slower than chlorophyll pigments. To determine if the decreasing chlorophyll to carotenoid content ratio leads to a relative increase in photosynthetic lightharvesting by carotenoids, the excitation spectrum of chlorophyll fluorescence at 25°C in thylakoid preparations from proso millet (Pa,icum miliaceum L var Minco) was measured. During the time-course of either drought-accelerated senescence of intact plants or induced senescence of excised leaves, the observed changes in the shape of the excitation spectrum were usually small ( < 15%) and mostly reflective of changes in the chlorophyll a to chlorophyll h ratio. A small increase in excitation around 535 nm occurred during drought-accelerated senescence. The near constancy of the shape of the excitation spectrum shows that the relative contribution by carotenoid pigments to photosynthetic lightharvesting does not increase significantly during natural senescence and suggests that loss of light-harvesting capacity occurs in a coordinated manner. Key words. Panicum miliaceum: Leaf senescence; Chlorophyll fluorescence; Carotenoids: Photosynthesis: Water stress
I. Introduction
Loss of photosynthetic pigments is the most visible change associated with the onset o f leaf senescence, and quantitation of chlorophyll loss is commonly used as a measure of the progress of senescence [I,2]. In most plants, the chlorophyll a and chlorophyll b levels fall in near synchrony until late in senescence when a decrease in the chlorophyll a to chlorophyll h ratio occurs [2-6].
* Corresponding author. tDeceased.
Although less frequently studied, total carotenoid pigment content generally persists longer than chlorophyll content, thus leading to a decreasing chlorophyll to carotenoid ratio during senescence [3,5] and to the familiar yellowing of leaves. Because appreciable proportions o f the total carotenoids in senescent leaves exist as carotenoid esters in plastoglobuli [7,8], the level of thylakoidassociated carotenoids is not as persistent as the level of total carotenoids during senescence [4,5]. What are the functional consequences of these changes in photosynthetic pigment levels during leaf senescence? Changes in CO~ fixation rate or other indicators of total photosynthesis often
0168-9452/94/$06.00 © 1994 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0168-9452(93)03743-F
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roughly parallel the loss of chlorophyll during leaf senescence [1,2,9]. Although many aspects of this overall loss of photosynthesis during senescence have been investigated, other details of photosynthetic function remain to be elucidated. Carotenoid pigments have several functions that include protecting chlorophyll from photooxidation [10,11]; harvesting light energy for transfer to photosynthetic reaction centers [10,11]; and, as studies of the xanthophyll cycle have shown [12], dissipating excess excitation energy during periods of high illumination [13]. Our interest in carotenoid pigments in this work arises from their function as light-harvesting pigments that help supply the energy to drive photosynthesis. The light-harvesting function of carotenoid pigments has been reviewed by Siefermann-Harms [10,11]. The efficiency of energy transfer from carotenoid pigments to chlorophyll a has been shown to be 100% in light-harvesting complex II of Lactuca sativa [10,11], although transfer efficiencies considerably less than 100% are observed in most photosynthetic systems and in model systems [14]. While development of the ability to transfer excitation energy from carotenoids to chlorophyll a has been investigated during the greening of etiolated bean [15,16] and barley [17] leaves, the maintenance of energy transfer from carotenoids to chlorophyll a during leaf senescence is a phenomenon worthy of further investigation. Does the decreasing chlorophyll to carotenoid ratio during senescence imply that carotenoid pigments play a relatively increasing role in harvesting light energy for photosynthesis during leaf senescence? To address this and other questions about lightharvesting during leaf senescence, we are using proso millet (Panicum mileaceum L. var Minco) as a model system. Proso millet is a cereal plant with a short seed-to-seed cycle, and as shown in our previous work [5,18], this plant provides a convenient model system for the investigation of both drought-accelerated senescence in intact plants and induced senescence in excised leaves. Our principal approach is to investigate light harvesting in photosystem II by examining the chlorophyll fluorescence excitation spectrum at room temperature [10,11,15,16,19-22], i.e., the ac-
J.L. Embrv, E.A. Nothnagel/ Plant Sci. 95 (1994) 141-152
tion spectrum for the generation of chlorophyll fluorescence, in thylakoid membranes prepared from proso millet leaves in various stages of senescence. 2. Methods
2.1. Plant material and growth Proso millet (Panicum miliaceum L. var Minco) seeds were germinated and grown to seedlings in a controlled environment chamber or to mature plants in a glasshouse under soil, temperature, and light conditions reported previously [5]. In experiments on induced senescence, seedlings were grown for 11 days in the controlled environment chamber, and then the first leaf was excised, trimmed at the tip to 3 cm, and floated on 0.2% (v/v) Tween 80 in Petri dishes in the dark for various incubation times. In each experiment, replicate Petri dishes, each containing five or ten excised leaves, were used at each time point. After incubation, the leaves were blotted dry and extracted for total chlorophyll determination or processed to yield broken thylakoid membranes which were used for measurements of chlorophyll fluorescence excitation spectra and for determinations of chlorophyll and carotenoid pigment ratios. In experiments on drought-accelerated senescence, plants were grown in the glasshouse, one plant per 4-1 pot, and were watered daily with nutrient solution until 25 days after planting [5], about the time of anthesis. After that date, all water and nutrients were withheld. During the subsequent slow drying of the soil in the pots, the new seeds continued to develop and became viable as soon as 32 days after planting [5]. Leaf pigments and chlorophyll fluorescence excitation were measured for leaf three (numbered from the bottom of the plant), which was harvested at 5-day intervals as water stress and senescence increased from 25 days onward. Relative water content was determined for leaf four on each harvest date as described [5]. As necessitated by the destructive nature of the sampling, separate lots of 5-20 plants each were used at each harvest date in each experiment. A total of 16 experiments on drought-
J.L. Embry, E.A. Nothnagel/Plant Sci. 95 (1994) 141-152
accelerated senescence were conducted over a 4year period. Because the rate of soil drying was not controlled after water was withheld starting at 25 day, the time course of change in leaf relative water content was not precisely matched between the 16 separate experiments and sometimes differed considerably, such as between summer and winter experiments. Thus while the changes observed with senescence in the 16 experiments were generally similar, the time periods during which these changes occurred were variable. Each set of results presented here are averages from three experiments which had similar time courses of change in leaf relative water content.
2.2. Thylakoid preparation Processing of harvested leaves to yield broken thylakoid membranes was by the method of Percival and Baker [23], modified as described [5]. The composition of the resuspension buffer used with the thylakoid membrane fractions during the present work was 100 mM sorbitol, 5 mM NaCI, 5 mM MgC12, and 10 mM [N-2-hydroxyethyl piperazine-N'-2 ethane sulfonic acid (Hepes) titrated with NaOH to pH 7.0. In a typical experiment, thylakoid membranes were prepared from 0.3 g of fresh leaf tissue and then stored frozen at liquid nitrogen temperature in resuspension buffer plus 15% (w/v) glycerol. Prior to measurements of chlorophyll fluorescence excitation spectra, pigment content ratios, and/or relative 2,6dichlorophenolindophenol reduction rates, the frozen thylakoid membranes were rapidly thawed and washed by centrifugation in resuspension buffer. As shown previously [5], the total chlorophyll to total carotenoid ratio is higher for these broken thylakoid membranes than for whole millet leaves because use of homogenization conditions that rupture whole chloroplasts enables at least partial separation of the thylakoid membranes from the carotenoid ester-containing plastoglobuli [7,8]. 2.3. Pigment content determination Pigment extraction from leaves or from thylakoid membranes was done with acetone as described [5]. Quantitation of chlorophyll a, chlorophyll b, and total carotenoids in the
143
resulting 80% (v/v) acetone solutions was done spectrophotometrically by measuring the absorbances at 460, 645, and 660 nm and then using the equations described [5].
2.4. Fluorescence spectroscopy Fluorescence excitation and emission spectra were measured at 25°C with a Fluorolog 2 model 112A spectrofluorometer (Spex Industries, Edison, N J). Slit widths of 2.50 mm were used and resulted in 9.2 nm bandpass resolution in the single excitation spectrometer and 4.5 nm bandpass resolution in the double emission spectrometer. Emission spectra were corrected by reference to the output of a standard lamp which was traceable to the National Bureau of Standards. Excitation spectra were corrected for variations in excitation energy and differences in optical paths by use of 1,1 ', 3,3,3 ',3' -hexamethylindot ricarbocyanine perchlorate as a quantum counter dye [24]. A KG-3 colored-glass filter (Fish-Schurman Corp., New Rochelle, NY) was used in the excitation path to attenuate stray light from the very strong near infrared emission lines of the Xe arc lamp [24]. Even the double emission spectrometer could not completely eliminate scattered light, which was especially intense with thylakoid preparations from highly senescent leaves. To further suppress stray and scattered light while recording excitation spectra, a 700 nm short-wave pass interference filter (700FL07-50 from Andover Corp., Salem, NH) was used in the excitation path, and a 730 nm longwave pass colored-glass filter (RG-9 from Schott Optical Glass, Inc., Duryea, PA) was used in the emission path. The thylakoid suspensions were held in 1 cm x 1 cm × 4.5 cm polystyrene cuvettes during the fluorescence measurements and were magnetically stirred to prevent settling. When the 2.50 mm excitation slits were used, the excitation spectrometer illuminated a 3.3 mm × 8.0 mm area on the front face of the cuvette. As the excitation spectrometer was scanned from 400 to 700 nm, the incident light intensity in this illuminated area varied between 60 and 230/~mol.m -z • s -l, with the average being 160 ~tmol.m -2 • s -l. Front-face collection of emitted light, i.e., collection at an
144
angle of 22.5 ° relative to the incident beam, was used routinely. Chlorophyll fluorescence emission from thylakoid suspensions was measured at 734 nm in the band arising from vibrational sublevels in the main chlorophyll transition [20]. Prior to fluorescence measurements, suspensions of thylakoid membranes were diluted to 2/~g chlorophyll/ml in a final volume of 3 ml of resuspension buffer. Except when noted otherwise, 3-(3,4-dichlorophenyl)-l,l-dimethylurea was added from a 10 mM stock solution in 95% (v/v) ethanol to give a final concentration of 20 #M. The thylakoid suspensions were held in the dark and then illuminated in the spectrofluorometer for 30 s prior to initiating the excitation scan. Control experiments involving fluorescence measurements at fixed excitation and emission wavelengths showed that specimens prepared under these 3-(3,4dichlorophenyl)-l,l-dimethylurea and preillumination conditions exhibited no time-dependent fluorescence induction kinetics over a time period equal to that required to complete an excitation scan. Excitation spectra to be averaged were first normalized to unit area intensity integrated between 400 and 700 nm before summing for an average. In the calculation of excitation difference spectra for a senescence sequence, the averaged spectrum for the earliest time point in the sequence was taken as the reference. This reference spectrum was scaled to relative intensity of 100 at the long wavelength peak which occurred in the 660-680 nm range. Then prior to subtracting the reference spectrum, the averaged spectrum at each later time point was scaled so that its area intensity integrated between 660 and 680 nm matched that of the reference spectrum.
2.5. Electron transJer determination Relative electron transfer rates at two excitation wavelengths were estimated by using 2,6dichlorophenolindophenol as an electron acceptor from photosystem II [22]. A suspension of thylakoid membranes was prepared, and its chlorophyll fluorescence excitation spectrum was recorded as described in the previous section, except that no 3-(3,4-dichlorophenyl)-l,l-dimethylurea was added and the preillumination period
J.L. Embry. E.A. Nothnagel/Plant Sci. 95 (1994) 141-152
was extended to 2 min. After the excitation spectrum was recorded, the cuvette containing the thylakoid suspension was transported to a spectrophotometer, and absorbances at 535, 605, and 670 nm were recorded. An aliquot from a stock solution of 20 mM 2,6-dichlorophenolindophenol in dimethyl sulfoxide was then added and mixed to achieve a final 2,6-dichlorophenolindophenol concentration of 20 /,M. The thylakoid suspension was again measured for absorbance at 605 nm and then transported in the dark back to the spectrofluorometer. The widths of the excitation slits were set to 5.0 mm (19 nm bandpass), and the thylakoid suspension was irradiated for 2 min with 535 nm light. The specimen was then transported in the dark to the spectrophotometer where absorbance at 605 nm was again quickly recorded. The sequence continued with irradiation for 2 min at 670 nm and measurement of absorbance at 605 nm. The cycle of irradiation at 535 and 670 nm was repeated until the total irradiation time was 30 min, or until the rate of 2,6-dichlorophenolindophenol reduction became too low for reliable measurement. With the 5.0 mm excitation slits in the spectrofluorometer, the irradiated area at the front face of the cuvette was 4.7 mm × 8.0 mm. The total light power incident into this area, as measured with a power meter (model 815, Newport Corp., Fountain Valley, CA), was 0.017 /~mol/s at 535 nm or 0.012 ~mol/s at 670 rim. Analysis of 2,6-dichlorophenolindophenol reduction, observed as decreased absorbance at 605 nm, involved several calculations that were incorporated into a single computer program. One calculation corrected for the unequal incident light powers at the two wavelengths of irradiation. Another calculation involved application of the Beer-Lambert law [22] to correct for the decrease in available incident light power due to absorption of some of the light by 2,6-dichlorophenolindophenol at 535 nm and 670 rim. No correction was required for loss of incident light power to reduced 2,6-dichlorophenolindophenol, since reduced 2,6dichlorophenolindophenol does not absorb appreciably at these two wavelengths. To compensate for the slow overall decline in the rate of 2,6-dichlorophenolindophenol reduction that oc-
J.L Embry, E.A. Nothnagel/ Plant Sci. 95 (1994) 141-152
curred during the irradiation period, short periods of irradiation with each of the wavelengths were applied cyclically as described. Each determination of 2,6-dichlorophenolindophenol reduction with 535 nm irradiation was then calculated as the average of a pair of rates expressed relative to the rate for the intervening period of 670 nm irradiation.
2.6. Putative chlorophyll catabolite extraction Thylakoid membrane fractions from senescent, mature millet leaves were used as the starting material for the partial purification of a pigment fraction which had characteristics similar to the reported 'pink pigment' catabolites of chlorophyll [25-28]. The procedures described by Matile et al. [25,26] were used with minor modifications. Thylakoid membranes were precipitated from suspension by centrifugation for 90 s at 2900 x g. The resulting membrane pellet was resuspended in just enough 5% (v/v) formic acid in methanol to completely wet the sample, and then the pigments were extracted by the addition of chloroform. The pigment extract was filtered through cotton wool and then loaded onto a 2.6 cm x 0.5 cm diameter column of silica gel. The column was washed with chloroform and then eluted with methanol as described [25]. The methanol eluate containing the 'pink pigments', was further analyzed by thin layer chromatography as described [25], by colorimetric assay for tetrapyrrole catabolites [25], and by absorption and fluorescence spectroscopies. The colorimetric assay for tetrapyrrole catabolites was a slight modification of the method of Matile et al. [25]. The diazotized reagent was prepared by adding 40 ml of 0.2 N HCI to 0.05 g (0.297 mmol) of 2-methoxy-4-nitroaniline (Aldrich Chemical Co., Milwaukee, WI), stirring the solution on an ice bath while slowly (dropwise) adding 10 ml of 0.0297 M NaNO2, and then continuing to stir on the ice bath for 1 h. A control reagent was prepared in a parallel manner except that no NaNO2 was added. Duplicate sets of test tubes were prepared to contain 2 ml of I'Y,, (w/v) Triton X-100 in 0.1 M Bicine, pH 10.0, and either unknowns from the methanol eluate of the silica gel column or various amounts of a standard bilirubin stock solution (0.5 mg/ml in 20 mM
145
NaOH). Diazotized reagent (0.1 ml) was added to each of the test tubes in one set, while control reagent (0.1 ml) was added to each of the test tubes in the duplicate set. All of the test tubes were vortexed and held initially for 15 min on an ice bath and then finally for 20 min at room temperature. Absorbance at 520 nm was measured against a blank of 1% (w/v) Triton X-100 in 0.1 M Bicine, pH 10.0. Absorbance differences between corresponding specimens prepared with diazotized reagent or with control reagnt were used to construct a standard curve and to determine unknowns. 3. Results
3.1. Avoidance of self-absorption artifacts In principle, chlorophyll fluorescence excitation spectra can be recorded directly from whole leaves or leaf segments. In practice, however, excitation spectra recorded directly from leaves are distorted due to self-absorption effects wherein some of the emitted fluorescence is absorbed by other chlorophyll molecules before it can escape the leaf. Artifacts arising from this self-absorption effect have been discussed in detail by Percival and Baker [23] and are very significant in intact leaves where the chlorophyll concentration is very high. These artifacts can be diminished in experiments on thylakoid membrane suspensions by diluting the preparations to low chlorophyll concentrations. Results obtained in the present project (Fig. 1) demonstrated that self-absorption effects were less severe with front face collection of fluorescence and became negligible at chlorophyll concentrations less than 5 ~g/ml. Front face collection of emitted light and chlorophyll concentrations of 2 /zg/ml were used in all subsequent chlorophyll fluorescence measurements in this project. 3.2. Drought-accelerated senescence of intact plants Changes in leaf constituents, including soluble protein, c~-amino nitrogen and total chlorophyll, have been previously reported [5] for P. miliaceum plants undergoing the program of droughtaccelerated senescence described in Methods. In the present work, relative total chlorophyll content per leaf was used as an indicator of the timecourse of senescence (Table 1).
J.L. Embry, E.A. Nothnagel/Phmt Sci. 95 (1994) 141-152
146
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Fig. I. Assessment of self-absorption effect in suspensions of thylakoid membranes from the first leaf of l 1-day-old P. miliaeeum seedlings. Suspensions of various chlorophyll concentrations were illuminated with 480 nm excitation light, and the emission spectra were recorded. The ratio of apparent emission intensities at the main peak of chlorophyll a fluorescence
(681 nm) and in the weaker band (734 nm) due to vibrational levels [20] was determined at each chlorophyll concentration. Since light absorption by chlorophyll is substantial at 681 nm, but negligible at 734 nm, the 681 nm to 734 nm fluorescence ratio provides a relative measure of self-absorption [23]. The fluorescence light was collected at an angle of either 90° (right angle mode, O) or 22.5° (front face mode, O) relative to the excitation light path.
Table 1 Pigments in leaf three of P. miliaceum plants in various stages of drought-accelerated senescence Plant age (days)
RWC (%) Total Chl (%)
Chl a/Chl h (w/w)
Chl/carotenoid (w/w)
25 30 35 40
99 92 61 38
3.68 4.15 2.87 2.13
6.02 4.91 4.60 4.70
± + + +
2 10 46 38
100 74 + 12 30 ± 34 11 ± 6
+ ± + +
0.43 0.78 1.66 0.99
+ + ± 4-
1.40 0.26 1.21 1.27
Relative water content (RWC) was measured in leaf four. Final watering of the plants was on day 25. Total chlorophyll (Chl) content per leaf segment is expressed relative to the value at day 25. Ratios of chlorophylls and carotenoids were measured in thylakoid preparations to enable closest comparison with fluorescence excitation spectra (Fig. 2). Data are expressed as means ± S.D. from three experiments conducted over a 2-year period.
Analysis of pigment ratios in thylakoid membrane fractions from leaf three of P. mil&ceum showed that the chlorophyll a to chlorophyll h ratio declined from about 35 days onward as senescence and water stress increased (Table 1). A decline in the total chlorophyll to total carotenoid ratio was also evident but occurred earlier than the decline in chlorophyll a to chlorophyll h ratio (Table 1). A typical chlorophyll fluorescence excitation spectrum measured from P. miliaceum thylakoids is shown in Fig. 2A. As expected, the spectrum showed strong excitation bands in the violet to blue wavelengths of 400-500 nm and in the red wavelengths of 600-700 nm. The bands in the 400-500 nm region are due to light-harvesting by the Soret bands of chlorophyll a and h and by carotenoid pigments [10,11]. The band in the 600-700 nm region is due solely to the first excited electronic states of chlorophyll a and b, however, with no contribution from carotenoid pigments. Since the primary interest in this work was detection of changes in relative light harvesting by carotenoid pigments, chlorophyll excitation spectra recorded at different time points in the senescence sequence were scaled to match at the red band and then subtracted to facilitate detection of changes in the violet to blue bands. The difference spectra thus obtained (Figs. 2 B - D ) showed changes that amounted to no more than 16%, as referenced to the height of the red band observed with thylakoids from 25-day-old plants. The most prominent of these changes were centered in the red region around 670 rim, instead of in the violet to blue region of carotenoid absorbance. In addition, a smaller band developed around 535 nm in the difference spectra. This 535 nm band was further investigated by using reduction of 2,6-dichlorophenolindophenol as an independent measure of photosynthetic light-harvesting (Table 2). Although the ratios of activities measured at 535 and 670 nm for 2,6dichlorophenolindophenol reduction were slightly larger than the ratios measured for chlorophyll fluorescence excitation, both techniques indicated a small increase in the 535-670 nm ratio o f activities as senescence progressed. In a few experiments, a large 530 nm band ap-
J.L. Embry, E.A. Nothnagel/Plant Sci. 95 (1994) 141-152
147 Table 2 Comparison of relative chlorophyll (Chl) fluorescence excitation and relative 2,6-dichlorophenolindophenol (DCIP) reduction for 535 and 670 nm incident light
100 80 60 40 20 i
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25 30 35 40
0.120 0.128 0.151 0.181
0.137 0.184 0.227 0.259
4± ± +
0.003 0.001 0.041 0.061
± ± ± ±
0.084 0.067 0.080 0.115
Fluorescence excitation ratios were calculated from the excitation spectra used to generate the difference spectra shown in Fig. 2. Relative DCIP reduction rates were measured and analyzed as described in Methods. Results are expressed as the parameter measured with 535 nm incident light relative to the same parameter measured with 670 nm incident light. Data represent means ± S.D. from three (fluorescence), or six to eight (DCIP) measurements made in two or three experiments conducted over a 2-year period.
-8
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Fig. 2. Excitation of chlorophyll fluorescence in thylakoid membranes from P. miliaceum plants in various stages of drought-accelerated senescence. Corresponding pigment and relative water content data are presented in Table 1. (A) Excitation spectrum for thylakoids from 25-day-old plants: (B) excitation difference spectrum, 30-day-old plants minus 25-day-old plants; (C) excitation difference spectrum, 35-day-old plants minus 25-day-old plants; (D) excitation difference spectrum, 40-day-old plants minus 25-day-old plants. Spectra are averages from three experiments conducted over a 2-year period.
peared in the excitation difference spectra at times very late in the program of drought-accelerated senescence (Fig. 3). Since comparably large increases in 2,6-dichlorophenolindophenol reduction were not observed (not shown), this large 530 nm excitation band seemed to be unrelated to pho-
tosynthetic light-harvesting. Thus, it was necessary to investigate other mechanisms that might account for the observed increase in 530 nm excitation of 734 nm emission. Several articles [25-28] have reported that 'pink pigments' accumulate in senescent leaf tissues and seem to be breakdown products of chlorophyll. When the extraction and partial purification techniques of Matile et al. [25,26] were applied to thylakoid preparations from highly senescent leaves of drought-stressed P. miliaceum, a pigment fraction eluted from the silica gel column with methanol, as expected for the putative chlorophyll catabolites [25,26]. The presence of tetrapyrrole catabolites in this methanol fraction was confirmed by a color±metric assay which detected 16/~g of bilirubin equivalents/g fresh weight of senescent leaf tissue. This amount of tetrapyrrole catabolites corresponded to about 0.9% (w/w) of the chlorophyll content of mature, non-senescent P. miliaceum leaves. The spectral properties of the methanol fraction from the silica gel column were analyzed (Fig. 4), and a general similarity between its fluorescence excitation spectrum and the difference spectrum in Fig. 3C was observed. The relative sizes of the
148
J.L. Emhry, E.A. Nothnagel/Plant Sci. 95 (1994) 141-152
i i 0.8
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Fig. 3 . Example of large 530 nm band that sometimes developed in the excitation spectrum of 734 nm fluorescence in thylakoid preparations during drought-accelerated senescence in P. miliaceum. Corresponding pigment and relative water content data were generally similar to those shown in Table 1, except in these experiments the pigment changes progressed farther with the chlorophyll a to chlorophyll b ratio reaching 1.19 4- 0.40 and the total chlorophyll to carotenoid ratio reaching 3.18 4- 0.29 in the 40-day-old plants. (A) Excitation difference spectrum, 30-day-old plants minus 25-day-old plants; (B) excitation difference spectrum, 35-day-old plants minus 25-day-old plants: (C) excitation difference spectrum, 40-day-old plants minus 25-day-old plants. Spectra are averages from three experiments. Note the differences in the ordinate scales between these graphs and those of Fig. 2.
peaks in the spectra of Fig. 4 varied somewhat between different preparations (data not shown). This observation suggested that the methanol fraction contained a mixture of pigments, a conclusion confirmed by thin layer chromatography of the fraction (not shown). Further separation and chemical characterization of this fraction were not attempted. 3.3. Induced senescence of excised leaf segments Changes in leaf constituents, including soluble protein, o~-amino nitrogen, and total chlorophyll,
Fig. 4. Absorption and fluorescence spectra of putative chlorophyll catabolites extracted from the thylakoid membrane fraction obtained from mature, highly senescent P. miliaceum leaves. Fluorescence emission was measured at 734 nm when recording the excitation spectrum, and excitation at 530 nm was used when recording the fluorescence emission spectrum. To avoid spectral distortions due to self-absorption effects, a diluted aliquot of the pigments was used when recording these fluorescence spectra. Methanol was used as the solvent for all three spectra.
Table 3 Pigments in excised P. miliaceum leaves in various stages of dark-induced senescence Incubation time (h)
Total Chl (%)
Chl a/Chl h (w/w)
Chl/carotenoid (w/w)
0 6 16 48 72 120
100 99 492 472 439 49 4-
3.92 4.16 4.33 4.41 4.63 0.99
5.80 5.75 5.00 4.89 3.61 3.50
17 17 12 14 4
+ 44+ 44-
0.89 0.56 0.28 1.28 0.54 0.12
444+ 44-
1.23 1.06 0.12 1.31 0.17 0.60
The first leaves were excised from 11-day-old seedlings at time 0 h and thereafter incubated floating on 0.2% (v/v) Tween 80 in the dark at 26°C until extraction of chlorophyll (Chl) or preparation of thylakoids. Total chlorophyll content per leaf is expressed relative to the value at 0 h. Ratios of chlorophylls and carotenoids were measured in thylakoid preparations to enable closest comparison with fluorescence excitation spectra (Fig. 5). Data are expressed as means 4- S.D. from four determinations made in two separate experiments.
J.L. Embry, E.A. Nothnagel/ Plant Sci. 95 (1994) 141-152
have been previously reported [5] for induced senescence of excised first leaves of P. miliaceum. In the present work, relative total chlorophyll content per leaf was used as an indicator of the timecourse of senescence (Table 3). The chlorophyll a to chlorophyll b ratio in thylakoid membrane fractions from excised P. miliaceum leaves exhibited a slight upward drift until a substantial drop occurred very late in senescence (Table 3). The total chlorophyll to total carotenoid ratio gradually decreased throughout indu~:ed senscence. Changes in the chlorophyll fluorescence excitation spectrum during the time-course of induced senescence (Fig. 5) were generally 10% or less until very late in senescence (Fig. 5E) when a significant
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500 600 W A V E L E N G T H (nm)
/ /
700
Fig. 5. Excitation of chlorophyll fluorescence in thylakoid membranes from excised P. miliaceum leaves in various stages of dark-induced senescence. Corresponding pigment ratios are presented in Table 3. Spectra are excitation difference spectra with the spectrum of thylakoids from freshly excised leaves subtracted from spectra of thylakoids from leaf segments excised and incubated for: (A) 6 h; (B) 16 h; (C) 48 h: (D) 72 h; (El 120 h. Spectra are averages from two experiments.
149
reduction of excitation occurred in the 430-500 nm region. A change at the red wavelengths, similar to that observed during droughtaccelerated senescence (Fig. 2), was also evident at this late stage of senescence (Fig. 5E). 4. Discussion Panicum miliaceum plants undergoing droughtaccelerated senescence exhibit the decreasing chlorophyll a to chlorophyll b and total chlorophyll to total carotenoid ratios (Table l) that are typical of other senescing plants [2-6]. One method to assess the effects of these pigment changes on photosynthetic light-harvesting is to examine the excitation, or action, spectrum for the generation of chlorophli fluorescence from broken thylakoids in the presence of 3-(3,4-dichlorophenyl)-l,l-dimethylurea. At 25°C most of this fluorescence arises from chlorophyll a in the photosystem II reaction center, although a small contribution from photosystem I cannot be ruled out when fluorescence emission is measured at 734 nm as in the present work [20,22,23]. The shape of the fluorescence excitation spectrum reflects the combined effectiveness of all of the pigments in the antenna system that absorb incident light and transfer excitation energy to the photosynthetic reaction center [10,11,15-17,19,21]. The data of Fig. 2 show that the excitation spectrum changes during the course of droughtaccelerated senescence. The most important aspect of these observations is that the changes in fluorescence excitation are remarkably small (less than 16%) compared to the changes in pigment ratios (up to nearly two-fold, Table 1). In particular, no remarkable increase in excitation occurs in the 400-500 nm wavelength range that is characteristic of carotenoid absorption [13]. No appreciable excitation enhancement is evident even at 487 nm, which is a wavelength at which the carotenoid contribution to light-harvesting is relatively prominent [10,11]. This observation shows that the relative importance of lightharvesting by carotenoids does not increase during senescence, in spite of a decrease in the chlorophyll to carotenoid content ratio. Within the 600-700 nm range of red light,
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chlorophyll b absorbance in thylakoid membranes is strongest in a band peaking around 650 nm, while chlorophyll a absorbance is most prominent in a band peaking between 670 and 680 nm [10,11,29]. Thus, the change centered at 670 nm that develops in the excitation difference spectrum during senescence (Fig. 2) indicates that fluorescence excitation through chlorophyll a is decreasing relative to fluorescence excitation through chlorophyll b. This change in fluorescence excitation is consistent with the decreasing chlorophyll a to chlorophyll b content ratio that was observed in this (Table 1) and many previous [2-6,30] studies of leaf senescence. The mechanism of these changes in relative pigment content and fluorescence excitation likely involves the relative stability of the various pigment-protein complexes of the thylakoid membrane. Within the thylakoid membrane, chlorophyll b is predominantly associated with light-harvesting complex II [30]. Work by KuraHotta et al. [31] has shown that the decrease in the chlorophyll a to chlorophyll b ratio with leaf age is paralleled by a decrease in the reaction center to light-harvesting complex II ratio. This and other work has led to the conclusion that the decrease in the chlorophyll a to chlorophyll b ratio is due to a more rapid loss of reaction center proteins, especially those associated with photosystem I, than light-harvesting complex proteins during senescence [2]. Studies of the turnover of the various polypeptide chains of the pigment-protein complexes during senescence have indicated that the relatively prolonged abundance of some complexes can be due either to slow degradation, as in the case of light-harvesting complex I1, or to continued synthesis, as in the case of the D-1 polypeptide of photosystem II [32,33]. The changes in fluorescence excitation around 670 nm in Fig. 2 might then be due to prolonged abundance of light-harvesting complex II. This interpretation is especially appealing since, under low and moderate light levels, light-harvesting complex 1I is predominantly associated with photosystem II [30], the source of most chlorophyll fluorescence at 25°C. It is also important to consider that uncoupling of excitation energy transfer might occur during senescence,
ZL. Embrv, E.4. Nothnagel/Plant SoL 95 (1994) 141-152
however, in which case the proportion of chlorophyll fluorescence emitted directly from the antenna system would be increased. Since fluorescence emission from isolated light-harvesting complex I1 extends only very weakly to 734 nm [34], the measurements at this wavelength in the present work would have likely missed most of the fluorescence emitted directly from the antenna system. It remains possible, however, that some of the observed change in fluorescence excitation could be due to uncoupling of energy transfer. Although the small relative increase in fluorescence excitation around 535 nm that was observed during drought-accelerated senescence (Fig. 2) seemed to be confirmed by measurements of electron transport activity (Table 2), the mechanism of this increase remains obscure. Also unclear is why a far larger increase in excitation in this wavelength region occurred in a few experiments (Fig. 3) but not in others. It remains possible that the putative chlorophyll catabolites (Fig. 4) that seem to be responsible for the large band in Fig. 3C are also responsible for this small increase at 535 nm in Fig. 2, This hypothesis would seem to require that a small portion of the light energy absorbed by the chlorophyll catabolites is able to drive electron transport, a rather surprising conclusion, if correct. On the other hand, Thomas et al. [28] have shown that at least the initial catabolites of chlorophyll a remain associated with the light-harvesting complex II. Although the mechanism of this 535 nm increase in excitation remains uncertain, the results presented in Fig. 4 point to the need for caution when using fluorescence excitation alone to assess photosynthetic light-harvesting. In the case of induced senescence of excised leaf segments, a small increase in excitation in the 450-520 nm range is evident in the first 48 h after excision (Figs. 5A-C). This increase in excitation occurs before the decline in total chlorophyll to total carotenoid content ratio is very far advanced, however, and is then reversed by a much larger decrease in 430-500 nm excitation that occurs by late senescence (Fig. 5E). This large negative band may be due to light loss to carotenoids or other pigments that do not effectively transfer excitation energy to the reaction centers.
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The changes in the 660-680 nm region of the excitation spectra for induced senescence (Fig. 5) are generally consistent with the observed changes in the chlorophyll a to chlorophyll b content ratio (Table 3). A very slight indication of enhanced light-harvesting by chlorophyll a is evident until late in senescence (Fig. 5E) when a significant reversal in the shape of the difference spectrum occurs and signals enhanced light-harvesting by chlorophyll b. The central result of this work is the demonstration that only small (usually < 15%) changes occur in the shape of the chlorophyll excitation spectrum while as much as 90% of the total chlorophyll is lost during senescence. No evidence of a significant increase in relative light-harvesting by carotenoid pigments is observed, even when the chlorophyll to carotenoid content ratio is significantly decreased. These observations tend to confirm the notion [2] that disassembly of the photosynthetic system during senescence is a coordinated process wherein loss of function occurs by units rather than by degrees. More specifically, loss of chlorophyll from a pigment/protein complex seems to signal total loss of function for that complex, i.e., loss of chlorophyll is not compensated by increased light-harvesting by carotenoid pigments.
5
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5. Acknowledgements 14
This study was supported by an Intramural Research Grant (5-547570-19900) from the University of California, Riverside.
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6. References 16 I
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3
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K.V. Thimann, The senescence of leaves, in: K.V. Thimann (Ed.), Senescence in Plants, CRC Press, Boca Raton, Florida, 1980, pp. 85-115. S. Gepstein, Photosynthesis, in: L.D. Nooden and A.C. Leopold (Eds.), Senescence and Aging in Plants, Academic Press, San Diego, 1988, pp. 85-109. A. Grover, S.C. Sabat and P.R. Mohanty, Relative sensitivity of various spectral forms of photosynthetic pigments to leaf senescence in wheat (Triticum aestivum L.). Photosynth. Res., 10 (1986) 223-229. H. Gut, C. Rutz, P. Matile and H . L Thomas, Leaf senescence in a non-yellowing mutant of Festuca pratensis, degradation of carotenoids. Physiol. Plant., 70 (1987) 659-663.
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18
19
J.L. Embry and E.A. Nothnagel, Leaf development and senescence in Panicum miliaceum L., a cereal with a short seed-to-seed cycle. Plant Sci., 55 t1988) 129-136. J.J. Guiamet, E. Schwartz, E. Pichersky and L,D. Nooden, Characterization of cytoplasmic and nuclear mutations affecting chlorophyll and chlorophyll-binding proteins during senescence in soybean. Plant Physiol., 96 (1991) 227-231. D. Steinmuller and M. Tevini, Composition and function of plastoglobuli, 1. Isolation and purification from chloroplasts and chromoplasts. Planta, 163 (1985) 201-207. M. Tevini and D. Steinmuller, Composition and function of plastoglobuli, 11. Lipid composition of leaves and plastoglobuli during beech leaf senescence. Planta, 163 (1985) 91-96. W.W. Adams, III, K. Winter, U. Schreiber and P. Schramel, Photosynthesis and chlorophyll fluorescence characteristics in relationship to changes in pigment and element composition of leaves of Platanus occidentalis L. during autumnal leaf senescence. Plant Physiol., 93 (1990) 1184-1190. D. Siefermann-Harms. Carotenoids in photosynthesis., I. Location in photosynthetic membranes and lightharvesting function. Biochim. Biophys. Acta, 811 (1985) 325-355. D. Siefermann-Harms, The light-harvesting and protective functions of carotenoids in photosynthetic membranes. Physiol. Plant., 69 (1987) 561-568. B. Demmig, K. Winter, A. Kruger and F.-C. Czygan. Photoinhibition and zeaxanthin formation in intact leaves, a possible role of the xanthophyll cycle in the dissipation of excess light energy. Plant Physiol.. 84 (1987) 218-224. H.K. Lichtenthaler, Chlorophylls and carotenoids. pigments of photosynthetic biomembranes. Methods Enzymol., 148 (1987) 350-382. V.A. Sineshchekov, F.F. Litvin and M. Das, Chlorophyll a and carotenoid aggregates and energy migration in monolayers and thin films. Photochem. Photobiol.. 15 (1972) 187-197. W.L. Butler, Chloroplast development, energy transfer and structure. Arch. Biochem. Biophys., 92 (1961) 287-295. M. Koizumi, H. Kano, N. Katsura and K. Inada, The effect of chlorophyll content on changes of photochemical reactions in intact kidney bean leaves. Plant Cell Environ., 13 (1990) 65-71. L.I. Fradkin, A.G. Samoilenko and A.A. Shlyk, Development of energy transfer from carotenoids to chlorophyll a during the Shibata shift. Doklady Biophys., 281 (1985) 100-103. Q. Li and E.A. Nothnagel, Fluorescence and delayed light emission from mesophyll and bundle sheath cells in leaves of normal and salt-treated Panicum mi/iaceum. Plant Physiol., 89 (1989) 211-219. J.C. Goedheer, Fluorescence action spectra of algae and bean leaves at room and at liquid nitrogen temperatures. Biochim. Biophys. Acta, 102 (1965) 73-89.
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22
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26
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J.L. Emho', E.A. Nothnagel/Phmt Sci. 95 (1994) 141-152 J.C. Goedheer, Fluorescence in relation to photosynthesis. Annu. Rev. Plant Physiol., 23 (1972) 87-I 12. C.W. Vertucci, J.L. Ellenson and A.C. Leopold, Chlorophyll fluorescence characteristics associated with hydration level in pea cotyledons. Plant Physiol., 79 (19851 248-252. M.F. Hipkins and N.R. Baker. Spectroscopy. in: M.F. Hipkins and N.R. Baker (Eds.), Photosynthesis and Energy Transduction, A Practical Approach, IRL Press, Oxford, 1986, pp. 51-101. M.P. Percival and N.R. Baker, Comparison of chlorophyll fluorescence emission characteristics of wheat leaf tissue and isolated thylakoids as a function of excitation wavelength. Plant Cell Environ., 8 (1985) 41-48. E.A. Nothnagel, Quantum counter for correcting fluorescence excitation spectra at 320 to 800 nm wavelengths. Anal. Biochem., 163 (1987) 224-237. P. Matile, S. Ginsburg, M. Schellenberg and H. Thomas, Catabolites of chlorophyll in senescent leaves. J. Plant Physiol., 129 (1987) 219-228. P. Matile, S. Ginsburg, M. Schellenberg and H. Thomas, Catabolites of chlorophyll in senescing barley leaves are localized in the vacuoles of mesophyll cells. Proc. Natl. Acad. Sci., USA, 85 (1988) 9529-9532. P. Matile, T. Duggelin, M. Schellenberg, D. Rentsch, K. Bortlik, C. Peisker and H. Thomas, How and why is chlorophyll broken down in senescent leaves. Plant Physiol. Biochem., 27 (1989) 595-604.
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H. Thomas, K. Bortlik, D. Rentsch, M. Schellenberg and P. Matile, Catabolism of chlorophyll in vivo, significance of polar chlorophyll catabolites:in a non-yellowing senescence mutant of Festuca pratensis Huds. New Phytol., 111 (1989) 3-8. K. Sauer and M. Calvin, Absorption spectra of spinach quantasomes and bleaching of the pigments. Biochim. Biophys. Acta, 64 (1962) 324-339. K. Gounaris, J. Barber and J.L. Harwood, The thylakoid membranes of higher plant chloroplasts. Biochem. J., 237 (1986) 313-326. M. Kura-Hotta, K. Satoh and S. Katoh, Relationship between photosynthesis and chlorophyll content during leaf senescence of rice seedlings. Plant Cell Physiol., 28 (1987) 1321-1329. D.R. Roberts, J.E. Thompson, E.B. Dumbroff, S. Gepstein and A.K. Mattoo, Differential changes in the synthesis and steady-state levels of thylakoid proteins during bean leaf senescence. Plant Mol. Biol., 9 (19871 343-353. N.J. Bate, N.A. Straus and J.E. Thompson, Expression of chloroplast photosynthesis genes during leaf scnesccncc. Physiol. Plant., 80 (1990) 217-225. D. Siefermann-Harms, Fluorescence properties of isolated chlorophyll protein complexes, in: H.K. Lichtenthaler (Ed.), Applications of Chlorophyll Fluorescence, Kluwer Academic Publishers, Boston, 1988, pp. 45-54.
Plant Science 95 (1994) 141-152
Photosynthetic light-harvesting during leaf senescence in Panicum miliaceum J. Lorene Embry't, Eugene A. Nothnagel* Department of Botany and Plant Sciences, University of California, Riverside, California 92521-0124, USA
(Received 8 July 1993; accepted 13 September 1993)
Abstract
During plant senescence, cai'otenoid pigments are lost from the leaves slower than chlorophyll pigments. To determine if the decreasing chlorophyll to carotenoid content ratio leads to a relative increase in photosynthetic lightharvesting by carotenoids, the excitation spectrum of chlorophyll fluorescence at 25°C in thylakoid preparations from proso millet (Pa,icum miliaceum L var Minco) was measured. During the time-course of either drought-accelerated senescence of intact plants or induced senescence of excised leaves, the observed changes in the shape of the excitation spectrum were usually small ( < 15%) and mostly reflective of changes in the chlorophyll a to chlorophyll h ratio. A small increase in excitation around 535 nm occurred during drought-accelerated senescence. The near constancy of the shape of the excitation spectrum shows that the relative contribution by carotenoid pigments to photosynthetic lightharvesting does not increase significantly during natural senescence and suggests that loss of light-harvesting capacity occurs in a coordinated manner. Key words. Panicum miliaceum: Leaf senescence; Chlorophyll fluorescence; Carotenoids: Photosynthesis: Water stress
I. Introduction
Loss of photosynthetic pigments is the most visible change associated with the onset o f leaf senescence, and quantitation of chlorophyll loss is commonly used as a measure of the progress of senescence [I,2]. In most plants, the chlorophyll a and chlorophyll b levels fall in near synchrony until late in senescence when a decrease in the chlorophyll a to chlorophyll h ratio occurs [2-6].
* Corresponding author. tDeceased.
Although less frequently studied, total carotenoid pigment content generally persists longer than chlorophyll content, thus leading to a decreasing chlorophyll to carotenoid ratio during senescence [3,5] and to the familiar yellowing of leaves. Because appreciable proportions o f the total carotenoids in senescent leaves exist as carotenoid esters in plastoglobuli [7,8], the level of thylakoidassociated carotenoids is not as persistent as the level of total carotenoids during senescence [4,5]. What are the functional consequences of these changes in photosynthetic pigment levels during leaf senescence? Changes in CO~ fixation rate or other indicators of total photosynthesis often
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roughly parallel the loss of chlorophyll during leaf senescence [1,2,9]. Although many aspects of this overall loss of photosynthesis during senescence have been investigated, other details of photosynthetic function remain to be elucidated. Carotenoid pigments have several functions that include protecting chlorophyll from photooxidation [10,11]; harvesting light energy for transfer to photosynthetic reaction centers [10,11]; and, as studies of the xanthophyll cycle have shown [12], dissipating excess excitation energy during periods of high illumination [13]. Our interest in carotenoid pigments in this work arises from their function as light-harvesting pigments that help supply the energy to drive photosynthesis. The light-harvesting function of carotenoid pigments has been reviewed by Siefermann-Harms [10,11]. The efficiency of energy transfer from carotenoid pigments to chlorophyll a has been shown to be 100% in light-harvesting complex II of Lactuca sativa [10,11], although transfer efficiencies considerably less than 100% are observed in most photosynthetic systems and in model systems [14]. While development of the ability to transfer excitation energy from carotenoids to chlorophyll a has been investigated during the greening of etiolated bean [15,16] and barley [17] leaves, the maintenance of energy transfer from carotenoids to chlorophyll a during leaf senescence is a phenomenon worthy of further investigation. Does the decreasing chlorophyll to carotenoid ratio during senescence imply that carotenoid pigments play a relatively increasing role in harvesting light energy for photosynthesis during leaf senescence? To address this and other questions about lightharvesting during leaf senescence, we are using proso millet (Panicum mileaceum L. var Minco) as a model system. Proso millet is a cereal plant with a short seed-to-seed cycle, and as shown in our previous work [5,18], this plant provides a convenient model system for the investigation of both drought-accelerated senescence in intact plants and induced senescence in excised leaves. Our principal approach is to investigate light harvesting in photosystem II by examining the chlorophyll fluorescence excitation spectrum at room temperature [10,11,15,16,19-22], i.e., the ac-
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tion spectrum for the generation of chlorophyll fluorescence, in thylakoid membranes prepared from proso millet leaves in various stages of senescence. 2. Methods
2.1. Plant material and growth Proso millet (Panicum miliaceum L. var Minco) seeds were germinated and grown to seedlings in a controlled environment chamber or to mature plants in a glasshouse under soil, temperature, and light conditions reported previously [5]. In experiments on induced senescence, seedlings were grown for 11 days in the controlled environment chamber, and then the first leaf was excised, trimmed at the tip to 3 cm, and floated on 0.2% (v/v) Tween 80 in Petri dishes in the dark for various incubation times. In each experiment, replicate Petri dishes, each containing five or ten excised leaves, were used at each time point. After incubation, the leaves were blotted dry and extracted for total chlorophyll determination or processed to yield broken thylakoid membranes which were used for measurements of chlorophyll fluorescence excitation spectra and for determinations of chlorophyll and carotenoid pigment ratios. In experiments on drought-accelerated senescence, plants were grown in the glasshouse, one plant per 4-1 pot, and were watered daily with nutrient solution until 25 days after planting [5], about the time of anthesis. After that date, all water and nutrients were withheld. During the subsequent slow drying of the soil in the pots, the new seeds continued to develop and became viable as soon as 32 days after planting [5]. Leaf pigments and chlorophyll fluorescence excitation were measured for leaf three (numbered from the bottom of the plant), which was harvested at 5-day intervals as water stress and senescence increased from 25 days onward. Relative water content was determined for leaf four on each harvest date as described [5]. As necessitated by the destructive nature of the sampling, separate lots of 5-20 plants each were used at each harvest date in each experiment. A total of 16 experiments on drought-
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accelerated senescence were conducted over a 4year period. Because the rate of soil drying was not controlled after water was withheld starting at 25 day, the time course of change in leaf relative water content was not precisely matched between the 16 separate experiments and sometimes differed considerably, such as between summer and winter experiments. Thus while the changes observed with senescence in the 16 experiments were generally similar, the time periods during which these changes occurred were variable. Each set of results presented here are averages from three experiments which had similar time courses of change in leaf relative water content.
2.2. Thylakoid preparation Processing of harvested leaves to yield broken thylakoid membranes was by the method of Percival and Baker [23], modified as described [5]. The composition of the resuspension buffer used with the thylakoid membrane fractions during the present work was 100 mM sorbitol, 5 mM NaCI, 5 mM MgC12, and 10 mM [N-2-hydroxyethyl piperazine-N'-2 ethane sulfonic acid (Hepes) titrated with NaOH to pH 7.0. In a typical experiment, thylakoid membranes were prepared from 0.3 g of fresh leaf tissue and then stored frozen at liquid nitrogen temperature in resuspension buffer plus 15% (w/v) glycerol. Prior to measurements of chlorophyll fluorescence excitation spectra, pigment content ratios, and/or relative 2,6dichlorophenolindophenol reduction rates, the frozen thylakoid membranes were rapidly thawed and washed by centrifugation in resuspension buffer. As shown previously [5], the total chlorophyll to total carotenoid ratio is higher for these broken thylakoid membranes than for whole millet leaves because use of homogenization conditions that rupture whole chloroplasts enables at least partial separation of the thylakoid membranes from the carotenoid ester-containing plastoglobuli [7,8]. 2.3. Pigment content determination Pigment extraction from leaves or from thylakoid membranes was done with acetone as described [5]. Quantitation of chlorophyll a, chlorophyll b, and total carotenoids in the
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resulting 80% (v/v) acetone solutions was done spectrophotometrically by measuring the absorbances at 460, 645, and 660 nm and then using the equations described [5].
2.4. Fluorescence spectroscopy Fluorescence excitation and emission spectra were measured at 25°C with a Fluorolog 2 model 112A spectrofluorometer (Spex Industries, Edison, N J). Slit widths of 2.50 mm were used and resulted in 9.2 nm bandpass resolution in the single excitation spectrometer and 4.5 nm bandpass resolution in the double emission spectrometer. Emission spectra were corrected by reference to the output of a standard lamp which was traceable to the National Bureau of Standards. Excitation spectra were corrected for variations in excitation energy and differences in optical paths by use of 1,1 ', 3,3,3 ',3' -hexamethylindot ricarbocyanine perchlorate as a quantum counter dye [24]. A KG-3 colored-glass filter (Fish-Schurman Corp., New Rochelle, NY) was used in the excitation path to attenuate stray light from the very strong near infrared emission lines of the Xe arc lamp [24]. Even the double emission spectrometer could not completely eliminate scattered light, which was especially intense with thylakoid preparations from highly senescent leaves. To further suppress stray and scattered light while recording excitation spectra, a 700 nm short-wave pass interference filter (700FL07-50 from Andover Corp., Salem, NH) was used in the excitation path, and a 730 nm longwave pass colored-glass filter (RG-9 from Schott Optical Glass, Inc., Duryea, PA) was used in the emission path. The thylakoid suspensions were held in 1 cm x 1 cm × 4.5 cm polystyrene cuvettes during the fluorescence measurements and were magnetically stirred to prevent settling. When the 2.50 mm excitation slits were used, the excitation spectrometer illuminated a 3.3 mm × 8.0 mm area on the front face of the cuvette. As the excitation spectrometer was scanned from 400 to 700 nm, the incident light intensity in this illuminated area varied between 60 and 230/~mol.m -z • s -l, with the average being 160 ~tmol.m -2 • s -l. Front-face collection of emitted light, i.e., collection at an
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angle of 22.5 ° relative to the incident beam, was used routinely. Chlorophyll fluorescence emission from thylakoid suspensions was measured at 734 nm in the band arising from vibrational sublevels in the main chlorophyll transition [20]. Prior to fluorescence measurements, suspensions of thylakoid membranes were diluted to 2/~g chlorophyll/ml in a final volume of 3 ml of resuspension buffer. Except when noted otherwise, 3-(3,4-dichlorophenyl)-l,l-dimethylurea was added from a 10 mM stock solution in 95% (v/v) ethanol to give a final concentration of 20 #M. The thylakoid suspensions were held in the dark and then illuminated in the spectrofluorometer for 30 s prior to initiating the excitation scan. Control experiments involving fluorescence measurements at fixed excitation and emission wavelengths showed that specimens prepared under these 3-(3,4dichlorophenyl)-l,l-dimethylurea and preillumination conditions exhibited no time-dependent fluorescence induction kinetics over a time period equal to that required to complete an excitation scan. Excitation spectra to be averaged were first normalized to unit area intensity integrated between 400 and 700 nm before summing for an average. In the calculation of excitation difference spectra for a senescence sequence, the averaged spectrum for the earliest time point in the sequence was taken as the reference. This reference spectrum was scaled to relative intensity of 100 at the long wavelength peak which occurred in the 660-680 nm range. Then prior to subtracting the reference spectrum, the averaged spectrum at each later time point was scaled so that its area intensity integrated between 660 and 680 nm matched that of the reference spectrum.
2.5. Electron transJer determination Relative electron transfer rates at two excitation wavelengths were estimated by using 2,6dichlorophenolindophenol as an electron acceptor from photosystem II [22]. A suspension of thylakoid membranes was prepared, and its chlorophyll fluorescence excitation spectrum was recorded as described in the previous section, except that no 3-(3,4-dichlorophenyl)-l,l-dimethylurea was added and the preillumination period
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was extended to 2 min. After the excitation spectrum was recorded, the cuvette containing the thylakoid suspension was transported to a spectrophotometer, and absorbances at 535, 605, and 670 nm were recorded. An aliquot from a stock solution of 20 mM 2,6-dichlorophenolindophenol in dimethyl sulfoxide was then added and mixed to achieve a final 2,6-dichlorophenolindophenol concentration of 20 /,M. The thylakoid suspension was again measured for absorbance at 605 nm and then transported in the dark back to the spectrofluorometer. The widths of the excitation slits were set to 5.0 mm (19 nm bandpass), and the thylakoid suspension was irradiated for 2 min with 535 nm light. The specimen was then transported in the dark to the spectrophotometer where absorbance at 605 nm was again quickly recorded. The sequence continued with irradiation for 2 min at 670 nm and measurement of absorbance at 605 nm. The cycle of irradiation at 535 and 670 nm was repeated until the total irradiation time was 30 min, or until the rate of 2,6-dichlorophenolindophenol reduction became too low for reliable measurement. With the 5.0 mm excitation slits in the spectrofluorometer, the irradiated area at the front face of the cuvette was 4.7 mm × 8.0 mm. The total light power incident into this area, as measured with a power meter (model 815, Newport Corp., Fountain Valley, CA), was 0.017 /~mol/s at 535 nm or 0.012 ~mol/s at 670 rim. Analysis of 2,6-dichlorophenolindophenol reduction, observed as decreased absorbance at 605 nm, involved several calculations that were incorporated into a single computer program. One calculation corrected for the unequal incident light powers at the two wavelengths of irradiation. Another calculation involved application of the Beer-Lambert law [22] to correct for the decrease in available incident light power due to absorption of some of the light by 2,6-dichlorophenolindophenol at 535 nm and 670 rim. No correction was required for loss of incident light power to reduced 2,6-dichlorophenolindophenol, since reduced 2,6dichlorophenolindophenol does not absorb appreciably at these two wavelengths. To compensate for the slow overall decline in the rate of 2,6-dichlorophenolindophenol reduction that oc-
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curred during the irradiation period, short periods of irradiation with each of the wavelengths were applied cyclically as described. Each determination of 2,6-dichlorophenolindophenol reduction with 535 nm irradiation was then calculated as the average of a pair of rates expressed relative to the rate for the intervening period of 670 nm irradiation.
2.6. Putative chlorophyll catabolite extraction Thylakoid membrane fractions from senescent, mature millet leaves were used as the starting material for the partial purification of a pigment fraction which had characteristics similar to the reported 'pink pigment' catabolites of chlorophyll [25-28]. The procedures described by Matile et al. [25,26] were used with minor modifications. Thylakoid membranes were precipitated from suspension by centrifugation for 90 s at 2900 x g. The resulting membrane pellet was resuspended in just enough 5% (v/v) formic acid in methanol to completely wet the sample, and then the pigments were extracted by the addition of chloroform. The pigment extract was filtered through cotton wool and then loaded onto a 2.6 cm x 0.5 cm diameter column of silica gel. The column was washed with chloroform and then eluted with methanol as described [25]. The methanol eluate containing the 'pink pigments', was further analyzed by thin layer chromatography as described [25], by colorimetric assay for tetrapyrrole catabolites [25], and by absorption and fluorescence spectroscopies. The colorimetric assay for tetrapyrrole catabolites was a slight modification of the method of Matile et al. [25]. The diazotized reagent was prepared by adding 40 ml of 0.2 N HCI to 0.05 g (0.297 mmol) of 2-methoxy-4-nitroaniline (Aldrich Chemical Co., Milwaukee, WI), stirring the solution on an ice bath while slowly (dropwise) adding 10 ml of 0.0297 M NaNO2, and then continuing to stir on the ice bath for 1 h. A control reagent was prepared in a parallel manner except that no NaNO2 was added. Duplicate sets of test tubes were prepared to contain 2 ml of I'Y,, (w/v) Triton X-100 in 0.1 M Bicine, pH 10.0, and either unknowns from the methanol eluate of the silica gel column or various amounts of a standard bilirubin stock solution (0.5 mg/ml in 20 mM
145
NaOH). Diazotized reagent (0.1 ml) was added to each of the test tubes in one set, while control reagent (0.1 ml) was added to each of the test tubes in the duplicate set. All of the test tubes were vortexed and held initially for 15 min on an ice bath and then finally for 20 min at room temperature. Absorbance at 520 nm was measured against a blank of 1% (w/v) Triton X-100 in 0.1 M Bicine, pH 10.0. Absorbance differences between corresponding specimens prepared with diazotized reagent or with control reagnt were used to construct a standard curve and to determine unknowns. 3. Results
3.1. Avoidance of self-absorption artifacts In principle, chlorophyll fluorescence excitation spectra can be recorded directly from whole leaves or leaf segments. In practice, however, excitation spectra recorded directly from leaves are distorted due to self-absorption effects wherein some of the emitted fluorescence is absorbed by other chlorophyll molecules before it can escape the leaf. Artifacts arising from this self-absorption effect have been discussed in detail by Percival and Baker [23] and are very significant in intact leaves where the chlorophyll concentration is very high. These artifacts can be diminished in experiments on thylakoid membrane suspensions by diluting the preparations to low chlorophyll concentrations. Results obtained in the present project (Fig. 1) demonstrated that self-absorption effects were less severe with front face collection of fluorescence and became negligible at chlorophyll concentrations less than 5 ~g/ml. Front face collection of emitted light and chlorophyll concentrations of 2 /zg/ml were used in all subsequent chlorophyll fluorescence measurements in this project. 3.2. Drought-accelerated senescence of intact plants Changes in leaf constituents, including soluble protein, c~-amino nitrogen and total chlorophyll, have been previously reported [5] for P. miliaceum plants undergoing the program of droughtaccelerated senescence described in Methods. In the present work, relative total chlorophyll content per leaf was used as an indicator of the timecourse of senescence (Table 1).
J.L. Embry, E.A. Nothnagel/Phmt Sci. 95 (1994) 141-152
146
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Fig. I. Assessment of self-absorption effect in suspensions of thylakoid membranes from the first leaf of l 1-day-old P. miliaeeum seedlings. Suspensions of various chlorophyll concentrations were illuminated with 480 nm excitation light, and the emission spectra were recorded. The ratio of apparent emission intensities at the main peak of chlorophyll a fluorescence
(681 nm) and in the weaker band (734 nm) due to vibrational levels [20] was determined at each chlorophyll concentration. Since light absorption by chlorophyll is substantial at 681 nm, but negligible at 734 nm, the 681 nm to 734 nm fluorescence ratio provides a relative measure of self-absorption [23]. The fluorescence light was collected at an angle of either 90° (right angle mode, O) or 22.5° (front face mode, O) relative to the excitation light path.
Table 1 Pigments in leaf three of P. miliaceum plants in various stages of drought-accelerated senescence Plant age (days)
RWC (%) Total Chl (%)
Chl a/Chl h (w/w)
Chl/carotenoid (w/w)
25 30 35 40
99 92 61 38
3.68 4.15 2.87 2.13
6.02 4.91 4.60 4.70
± + + +
2 10 46 38
100 74 + 12 30 ± 34 11 ± 6
+ ± + +
0.43 0.78 1.66 0.99
+ + ± 4-
1.40 0.26 1.21 1.27
Relative water content (RWC) was measured in leaf four. Final watering of the plants was on day 25. Total chlorophyll (Chl) content per leaf segment is expressed relative to the value at day 25. Ratios of chlorophylls and carotenoids were measured in thylakoid preparations to enable closest comparison with fluorescence excitation spectra (Fig. 2). Data are expressed as means ± S.D. from three experiments conducted over a 2-year period.
Analysis of pigment ratios in thylakoid membrane fractions from leaf three of P. mil&ceum showed that the chlorophyll a to chlorophyll h ratio declined from about 35 days onward as senescence and water stress increased (Table 1). A decline in the total chlorophyll to total carotenoid ratio was also evident but occurred earlier than the decline in chlorophyll a to chlorophyll h ratio (Table 1). A typical chlorophyll fluorescence excitation spectrum measured from P. miliaceum thylakoids is shown in Fig. 2A. As expected, the spectrum showed strong excitation bands in the violet to blue wavelengths of 400-500 nm and in the red wavelengths of 600-700 nm. The bands in the 400-500 nm region are due to light-harvesting by the Soret bands of chlorophyll a and h and by carotenoid pigments [10,11]. The band in the 600-700 nm region is due solely to the first excited electronic states of chlorophyll a and b, however, with no contribution from carotenoid pigments. Since the primary interest in this work was detection of changes in relative light harvesting by carotenoid pigments, chlorophyll excitation spectra recorded at different time points in the senescence sequence were scaled to match at the red band and then subtracted to facilitate detection of changes in the violet to blue bands. The difference spectra thus obtained (Figs. 2 B - D ) showed changes that amounted to no more than 16%, as referenced to the height of the red band observed with thylakoids from 25-day-old plants. The most prominent of these changes were centered in the red region around 670 rim, instead of in the violet to blue region of carotenoid absorbance. In addition, a smaller band developed around 535 nm in the difference spectra. This 535 nm band was further investigated by using reduction of 2,6-dichlorophenolindophenol as an independent measure of photosynthetic light-harvesting (Table 2). Although the ratios of activities measured at 535 and 670 nm for 2,6dichlorophenolindophenol reduction were slightly larger than the ratios measured for chlorophyll fluorescence excitation, both techniques indicated a small increase in the 535-670 nm ratio o f activities as senescence progressed. In a few experiments, a large 530 nm band ap-
J.L. Embry, E.A. Nothnagel/Plant Sci. 95 (1994) 141-152
147 Table 2 Comparison of relative chlorophyll (Chl) fluorescence excitation and relative 2,6-dichlorophenolindophenol (DCIP) reduction for 535 and 670 nm incident light
100 80 60 40 20 i
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25 30 35 40
0.120 0.128 0.151 0.181
0.137 0.184 0.227 0.259
4± ± +
0.003 0.001 0.041 0.061
± ± ± ±
0.084 0.067 0.080 0.115
Fluorescence excitation ratios were calculated from the excitation spectra used to generate the difference spectra shown in Fig. 2. Relative DCIP reduction rates were measured and analyzed as described in Methods. Results are expressed as the parameter measured with 535 nm incident light relative to the same parameter measured with 670 nm incident light. Data represent means ± S.D. from three (fluorescence), or six to eight (DCIP) measurements made in two or three experiments conducted over a 2-year period.
-8
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Fig. 2. Excitation of chlorophyll fluorescence in thylakoid membranes from P. miliaceum plants in various stages of drought-accelerated senescence. Corresponding pigment and relative water content data are presented in Table 1. (A) Excitation spectrum for thylakoids from 25-day-old plants: (B) excitation difference spectrum, 30-day-old plants minus 25-day-old plants; (C) excitation difference spectrum, 35-day-old plants minus 25-day-old plants; (D) excitation difference spectrum, 40-day-old plants minus 25-day-old plants. Spectra are averages from three experiments conducted over a 2-year period.
peared in the excitation difference spectra at times very late in the program of drought-accelerated senescence (Fig. 3). Since comparably large increases in 2,6-dichlorophenolindophenol reduction were not observed (not shown), this large 530 nm excitation band seemed to be unrelated to pho-
tosynthetic light-harvesting. Thus, it was necessary to investigate other mechanisms that might account for the observed increase in 530 nm excitation of 734 nm emission. Several articles [25-28] have reported that 'pink pigments' accumulate in senescent leaf tissues and seem to be breakdown products of chlorophyll. When the extraction and partial purification techniques of Matile et al. [25,26] were applied to thylakoid preparations from highly senescent leaves of drought-stressed P. miliaceum, a pigment fraction eluted from the silica gel column with methanol, as expected for the putative chlorophyll catabolites [25,26]. The presence of tetrapyrrole catabolites in this methanol fraction was confirmed by a color±metric assay which detected 16/~g of bilirubin equivalents/g fresh weight of senescent leaf tissue. This amount of tetrapyrrole catabolites corresponded to about 0.9% (w/w) of the chlorophyll content of mature, non-senescent P. miliaceum leaves. The spectral properties of the methanol fraction from the silica gel column were analyzed (Fig. 4), and a general similarity between its fluorescence excitation spectrum and the difference spectrum in Fig. 3C was observed. The relative sizes of the
148
J.L. Emhry, E.A. Nothnagel/Plant Sci. 95 (1994) 141-152
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-160 400
500 600 WAVELENGTH (nm)
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Fig. 3 . Example of large 530 nm band that sometimes developed in the excitation spectrum of 734 nm fluorescence in thylakoid preparations during drought-accelerated senescence in P. miliaceum. Corresponding pigment and relative water content data were generally similar to those shown in Table 1, except in these experiments the pigment changes progressed farther with the chlorophyll a to chlorophyll b ratio reaching 1.19 4- 0.40 and the total chlorophyll to carotenoid ratio reaching 3.18 4- 0.29 in the 40-day-old plants. (A) Excitation difference spectrum, 30-day-old plants minus 25-day-old plants; (B) excitation difference spectrum, 35-day-old plants minus 25-day-old plants: (C) excitation difference spectrum, 40-day-old plants minus 25-day-old plants. Spectra are averages from three experiments. Note the differences in the ordinate scales between these graphs and those of Fig. 2.
peaks in the spectra of Fig. 4 varied somewhat between different preparations (data not shown). This observation suggested that the methanol fraction contained a mixture of pigments, a conclusion confirmed by thin layer chromatography of the fraction (not shown). Further separation and chemical characterization of this fraction were not attempted. 3.3. Induced senescence of excised leaf segments Changes in leaf constituents, including soluble protein, o~-amino nitrogen, and total chlorophyll,
Fig. 4. Absorption and fluorescence spectra of putative chlorophyll catabolites extracted from the thylakoid membrane fraction obtained from mature, highly senescent P. miliaceum leaves. Fluorescence emission was measured at 734 nm when recording the excitation spectrum, and excitation at 530 nm was used when recording the fluorescence emission spectrum. To avoid spectral distortions due to self-absorption effects, a diluted aliquot of the pigments was used when recording these fluorescence spectra. Methanol was used as the solvent for all three spectra.
Table 3 Pigments in excised P. miliaceum leaves in various stages of dark-induced senescence Incubation time (h)
Total Chl (%)
Chl a/Chl h (w/w)
Chl/carotenoid (w/w)
0 6 16 48 72 120
100 99 492 472 439 49 4-
3.92 4.16 4.33 4.41 4.63 0.99
5.80 5.75 5.00 4.89 3.61 3.50
17 17 12 14 4
+ 44+ 44-
0.89 0.56 0.28 1.28 0.54 0.12
444+ 44-
1.23 1.06 0.12 1.31 0.17 0.60
The first leaves were excised from 11-day-old seedlings at time 0 h and thereafter incubated floating on 0.2% (v/v) Tween 80 in the dark at 26°C until extraction of chlorophyll (Chl) or preparation of thylakoids. Total chlorophyll content per leaf is expressed relative to the value at 0 h. Ratios of chlorophylls and carotenoids were measured in thylakoid preparations to enable closest comparison with fluorescence excitation spectra (Fig. 5). Data are expressed as means 4- S.D. from four determinations made in two separate experiments.
J.L. Embry, E.A. Nothnagel/ Plant Sci. 95 (1994) 141-152
have been previously reported [5] for induced senescence of excised first leaves of P. miliaceum. In the present work, relative total chlorophyll content per leaf was used as an indicator of the timecourse of senescence (Table 3). The chlorophyll a to chlorophyll b ratio in thylakoid membrane fractions from excised P. miliaceum leaves exhibited a slight upward drift until a substantial drop occurred very late in senescence (Table 3). The total chlorophyll to total carotenoid ratio gradually decreased throughout indu~:ed senscence. Changes in the chlorophyll fluorescence excitation spectrum during the time-course of induced senescence (Fig. 5) were generally 10% or less until very late in senescence (Fig. 5E) when a significant
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+12[ C
I
-121
400
'
.
'
.
.
/
.
,
,
'
'
500 600 W A V E L E N G T H (nm)
/ /
700
Fig. 5. Excitation of chlorophyll fluorescence in thylakoid membranes from excised P. miliaceum leaves in various stages of dark-induced senescence. Corresponding pigment ratios are presented in Table 3. Spectra are excitation difference spectra with the spectrum of thylakoids from freshly excised leaves subtracted from spectra of thylakoids from leaf segments excised and incubated for: (A) 6 h; (B) 16 h; (C) 48 h: (D) 72 h; (El 120 h. Spectra are averages from two experiments.
149
reduction of excitation occurred in the 430-500 nm region. A change at the red wavelengths, similar to that observed during droughtaccelerated senescence (Fig. 2), was also evident at this late stage of senescence (Fig. 5E). 4. Discussion Panicum miliaceum plants undergoing droughtaccelerated senescence exhibit the decreasing chlorophyll a to chlorophyll b and total chlorophyll to total carotenoid ratios (Table l) that are typical of other senescing plants [2-6]. One method to assess the effects of these pigment changes on photosynthetic light-harvesting is to examine the excitation, or action, spectrum for the generation of chlorophli fluorescence from broken thylakoids in the presence of 3-(3,4-dichlorophenyl)-l,l-dimethylurea. At 25°C most of this fluorescence arises from chlorophyll a in the photosystem II reaction center, although a small contribution from photosystem I cannot be ruled out when fluorescence emission is measured at 734 nm as in the present work [20,22,23]. The shape of the fluorescence excitation spectrum reflects the combined effectiveness of all of the pigments in the antenna system that absorb incident light and transfer excitation energy to the photosynthetic reaction center [10,11,15-17,19,21]. The data of Fig. 2 show that the excitation spectrum changes during the course of droughtaccelerated senescence. The most important aspect of these observations is that the changes in fluorescence excitation are remarkably small (less than 16%) compared to the changes in pigment ratios (up to nearly two-fold, Table 1). In particular, no remarkable increase in excitation occurs in the 400-500 nm wavelength range that is characteristic of carotenoid absorption [13]. No appreciable excitation enhancement is evident even at 487 nm, which is a wavelength at which the carotenoid contribution to light-harvesting is relatively prominent [10,11]. This observation shows that the relative importance of lightharvesting by carotenoids does not increase during senescence, in spite of a decrease in the chlorophyll to carotenoid content ratio. Within the 600-700 nm range of red light,
150
chlorophyll b absorbance in thylakoid membranes is strongest in a band peaking around 650 nm, while chlorophyll a absorbance is most prominent in a band peaking between 670 and 680 nm [10,11,29]. Thus, the change centered at 670 nm that develops in the excitation difference spectrum during senescence (Fig. 2) indicates that fluorescence excitation through chlorophyll a is decreasing relative to fluorescence excitation through chlorophyll b. This change in fluorescence excitation is consistent with the decreasing chlorophyll a to chlorophyll b content ratio that was observed in this (Table 1) and many previous [2-6,30] studies of leaf senescence. The mechanism of these changes in relative pigment content and fluorescence excitation likely involves the relative stability of the various pigment-protein complexes of the thylakoid membrane. Within the thylakoid membrane, chlorophyll b is predominantly associated with light-harvesting complex II [30]. Work by KuraHotta et al. [31] has shown that the decrease in the chlorophyll a to chlorophyll b ratio with leaf age is paralleled by a decrease in the reaction center to light-harvesting complex II ratio. This and other work has led to the conclusion that the decrease in the chlorophyll a to chlorophyll b ratio is due to a more rapid loss of reaction center proteins, especially those associated with photosystem I, than light-harvesting complex proteins during senescence [2]. Studies of the turnover of the various polypeptide chains of the pigment-protein complexes during senescence have indicated that the relatively prolonged abundance of some complexes can be due either to slow degradation, as in the case of light-harvesting complex I1, or to continued synthesis, as in the case of the D-1 polypeptide of photosystem II [32,33]. The changes in fluorescence excitation around 670 nm in Fig. 2 might then be due to prolonged abundance of light-harvesting complex II. This interpretation is especially appealing since, under low and moderate light levels, light-harvesting complex 1I is predominantly associated with photosystem II [30], the source of most chlorophyll fluorescence at 25°C. It is also important to consider that uncoupling of excitation energy transfer might occur during senescence,
ZL. Embrv, E.4. Nothnagel/Plant SoL 95 (1994) 141-152
however, in which case the proportion of chlorophyll fluorescence emitted directly from the antenna system would be increased. Since fluorescence emission from isolated light-harvesting complex I1 extends only very weakly to 734 nm [34], the measurements at this wavelength in the present work would have likely missed most of the fluorescence emitted directly from the antenna system. It remains possible, however, that some of the observed change in fluorescence excitation could be due to uncoupling of energy transfer. Although the small relative increase in fluorescence excitation around 535 nm that was observed during drought-accelerated senescence (Fig. 2) seemed to be confirmed by measurements of electron transport activity (Table 2), the mechanism of this increase remains obscure. Also unclear is why a far larger increase in excitation in this wavelength region occurred in a few experiments (Fig. 3) but not in others. It remains possible that the putative chlorophyll catabolites (Fig. 4) that seem to be responsible for the large band in Fig. 3C are also responsible for this small increase at 535 nm in Fig. 2, This hypothesis would seem to require that a small portion of the light energy absorbed by the chlorophyll catabolites is able to drive electron transport, a rather surprising conclusion, if correct. On the other hand, Thomas et al. [28] have shown that at least the initial catabolites of chlorophyll a remain associated with the light-harvesting complex II. Although the mechanism of this 535 nm increase in excitation remains uncertain, the results presented in Fig. 4 point to the need for caution when using fluorescence excitation alone to assess photosynthetic light-harvesting. In the case of induced senescence of excised leaf segments, a small increase in excitation in the 450-520 nm range is evident in the first 48 h after excision (Figs. 5A-C). This increase in excitation occurs before the decline in total chlorophyll to total carotenoid content ratio is very far advanced, however, and is then reversed by a much larger decrease in 430-500 nm excitation that occurs by late senescence (Fig. 5E). This large negative band may be due to light loss to carotenoids or other pigments that do not effectively transfer excitation energy to the reaction centers.
151
J.L. Embry, E.A. Nothnagel/Plant Sci. 95 (1994) 141-152
The changes in the 660-680 nm region of the excitation spectra for induced senescence (Fig. 5) are generally consistent with the observed changes in the chlorophyll a to chlorophyll b content ratio (Table 3). A very slight indication of enhanced light-harvesting by chlorophyll a is evident until late in senescence (Fig. 5E) when a significant reversal in the shape of the difference spectrum occurs and signals enhanced light-harvesting by chlorophyll b. The central result of this work is the demonstration that only small (usually < 15%) changes occur in the shape of the chlorophyll excitation spectrum while as much as 90% of the total chlorophyll is lost during senescence. No evidence of a significant increase in relative light-harvesting by carotenoid pigments is observed, even when the chlorophyll to carotenoid content ratio is significantly decreased. These observations tend to confirm the notion [2] that disassembly of the photosynthetic system during senescence is a coordinated process wherein loss of function occurs by units rather than by degrees. More specifically, loss of chlorophyll from a pigment/protein complex seems to signal total loss of function for that complex, i.e., loss of chlorophyll is not compensated by increased light-harvesting by carotenoid pigments.
5
6
7
8
9
10
II
12
13
5. Acknowledgements 14
This study was supported by an Intramural Research Grant (5-547570-19900) from the University of California, Riverside.
15
6. References 16 I
2
3
4
K.V. Thimann, The senescence of leaves, in: K.V. Thimann (Ed.), Senescence in Plants, CRC Press, Boca Raton, Florida, 1980, pp. 85-115. S. Gepstein, Photosynthesis, in: L.D. Nooden and A.C. Leopold (Eds.), Senescence and Aging in Plants, Academic Press, San Diego, 1988, pp. 85-109. A. Grover, S.C. Sabat and P.R. Mohanty, Relative sensitivity of various spectral forms of photosynthetic pigments to leaf senescence in wheat (Triticum aestivum L.). Photosynth. Res., 10 (1986) 223-229. H. Gut, C. Rutz, P. Matile and H . L Thomas, Leaf senescence in a non-yellowing mutant of Festuca pratensis, degradation of carotenoids. Physiol. Plant., 70 (1987) 659-663.
17
18
19
J.L. Embry and E.A. Nothnagel, Leaf development and senescence in Panicum miliaceum L., a cereal with a short seed-to-seed cycle. Plant Sci., 55 t1988) 129-136. J.J. Guiamet, E. Schwartz, E. Pichersky and L,D. Nooden, Characterization of cytoplasmic and nuclear mutations affecting chlorophyll and chlorophyll-binding proteins during senescence in soybean. Plant Physiol., 96 (1991) 227-231. D. Steinmuller and M. Tevini, Composition and function of plastoglobuli, 1. Isolation and purification from chloroplasts and chromoplasts. Planta, 163 (1985) 201-207. M. Tevini and D. Steinmuller, Composition and function of plastoglobuli, 11. Lipid composition of leaves and plastoglobuli during beech leaf senescence. Planta, 163 (1985) 91-96. W.W. Adams, III, K. Winter, U. Schreiber and P. Schramel, Photosynthesis and chlorophyll fluorescence characteristics in relationship to changes in pigment and element composition of leaves of Platanus occidentalis L. during autumnal leaf senescence. Plant Physiol., 93 (1990) 1184-1190. D. Siefermann-Harms. Carotenoids in photosynthesis., I. Location in photosynthetic membranes and lightharvesting function. Biochim. Biophys. Acta, 811 (1985) 325-355. D. Siefermann-Harms, The light-harvesting and protective functions of carotenoids in photosynthetic membranes. Physiol. Plant., 69 (1987) 561-568. B. Demmig, K. Winter, A. Kruger and F.-C. Czygan. Photoinhibition and zeaxanthin formation in intact leaves, a possible role of the xanthophyll cycle in the dissipation of excess light energy. Plant Physiol.. 84 (1987) 218-224. H.K. Lichtenthaler, Chlorophylls and carotenoids. pigments of photosynthetic biomembranes. Methods Enzymol., 148 (1987) 350-382. V.A. Sineshchekov, F.F. Litvin and M. Das, Chlorophyll a and carotenoid aggregates and energy migration in monolayers and thin films. Photochem. Photobiol.. 15 (1972) 187-197. W.L. Butler, Chloroplast development, energy transfer and structure. Arch. Biochem. Biophys., 92 (1961) 287-295. M. Koizumi, H. Kano, N. Katsura and K. Inada, The effect of chlorophyll content on changes of photochemical reactions in intact kidney bean leaves. Plant Cell Environ., 13 (1990) 65-71. L.I. Fradkin, A.G. Samoilenko and A.A. Shlyk, Development of energy transfer from carotenoids to chlorophyll a during the Shibata shift. Doklady Biophys., 281 (1985) 100-103. Q. Li and E.A. Nothnagel, Fluorescence and delayed light emission from mesophyll and bundle sheath cells in leaves of normal and salt-treated Panicum mi/iaceum. Plant Physiol., 89 (1989) 211-219. J.C. Goedheer, Fluorescence action spectra of algae and bean leaves at room and at liquid nitrogen temperatures. Biochim. Biophys. Acta, 102 (1965) 73-89.
152 20 21
22
23
24
25
26
27
J.L. Emho', E.A. Nothnagel/Phmt Sci. 95 (1994) 141-152 J.C. Goedheer, Fluorescence in relation to photosynthesis. Annu. Rev. Plant Physiol., 23 (1972) 87-I 12. C.W. Vertucci, J.L. Ellenson and A.C. Leopold, Chlorophyll fluorescence characteristics associated with hydration level in pea cotyledons. Plant Physiol., 79 (19851 248-252. M.F. Hipkins and N.R. Baker. Spectroscopy. in: M.F. Hipkins and N.R. Baker (Eds.), Photosynthesis and Energy Transduction, A Practical Approach, IRL Press, Oxford, 1986, pp. 51-101. M.P. Percival and N.R. Baker, Comparison of chlorophyll fluorescence emission characteristics of wheat leaf tissue and isolated thylakoids as a function of excitation wavelength. Plant Cell Environ., 8 (1985) 41-48. E.A. Nothnagel, Quantum counter for correcting fluorescence excitation spectra at 320 to 800 nm wavelengths. Anal. Biochem., 163 (1987) 224-237. P. Matile, S. Ginsburg, M. Schellenberg and H. Thomas, Catabolites of chlorophyll in senescent leaves. J. Plant Physiol., 129 (1987) 219-228. P. Matile, S. Ginsburg, M. Schellenberg and H. Thomas, Catabolites of chlorophyll in senescing barley leaves are localized in the vacuoles of mesophyll cells. Proc. Natl. Acad. Sci., USA, 85 (1988) 9529-9532. P. Matile, T. Duggelin, M. Schellenberg, D. Rentsch, K. Bortlik, C. Peisker and H. Thomas, How and why is chlorophyll broken down in senescent leaves. Plant Physiol. Biochem., 27 (1989) 595-604.
28
29
30
31
32
33
34
H. Thomas, K. Bortlik, D. Rentsch, M. Schellenberg and P. Matile, Catabolism of chlorophyll in vivo, significance of polar chlorophyll catabolites:in a non-yellowing senescence mutant of Festuca pratensis Huds. New Phytol., 111 (1989) 3-8. K. Sauer and M. Calvin, Absorption spectra of spinach quantasomes and bleaching of the pigments. Biochim. Biophys. Acta, 64 (1962) 324-339. K. Gounaris, J. Barber and J.L. Harwood, The thylakoid membranes of higher plant chloroplasts. Biochem. J., 237 (1986) 313-326. M. Kura-Hotta, K. Satoh and S. Katoh, Relationship between photosynthesis and chlorophyll content during leaf senescence of rice seedlings. Plant Cell Physiol., 28 (1987) 1321-1329. D.R. Roberts, J.E. Thompson, E.B. Dumbroff, S. Gepstein and A.K. Mattoo, Differential changes in the synthesis and steady-state levels of thylakoid proteins during bean leaf senescence. Plant Mol. Biol., 9 (19871 343-353. N.J. Bate, N.A. Straus and J.E. Thompson, Expression of chloroplast photosynthesis genes during leaf scnesccncc. Physiol. Plant., 80 (1990) 217-225. D. Siefermann-Harms, Fluorescence properties of isolated chlorophyll protein complexes, in: H.K. Lichtenthaler (Ed.), Applications of Chlorophyll Fluorescence, Kluwer Academic Publishers, Boston, 1988, pp. 45-54.