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Studies on peanut green mosaic virus infected peanut (Arachis hypogaea L.) leaves. I. Photosynthesis and photochemical reactions
Physiological
Plant Pathology
( 1984) 25, 181-I
90
Studies on peanut green mosaic virus infected peanut (Arachk hypogaea L.) leaves. I. Photosynthesis and photochemical reactions R. A. NAIDU, *M. KRISHNAN,
*P. RAMANUJAM,
*A. GNANAM and M. V. NAWDU
Department of Botany, S. V. University, Tirupati 517502, India. *Department of Plant Sciences, School of Biological Sciences, Madurai (Accepted for publication
Kamaraj
University,
Madurai
625 021, India
June 1984)
Changes in chlorophyll levels, in photosynthetic rates,and in photochemical reactions were studied in peanut plants infected with peanut green mosaic virus at three different stages of systemic infection: (1) early infection; (2) the stage of maximum symptom expression and (3) after the recovery phase of systemic disease development. Chlorophyll content per unit leaf area was reduced at all three stages of infection with the greatest reduction coinciding with the phase of maximum symptom expression. Net photosynthesis was also reduced over a range of light intensities, with the maximum reduction occurring at the highest light intensities. Infection inhibited photosrjtem II activity to a significantly greater extent, particularly at infection stage 2, than it did photosystem I activity. Non-cyclic photophosphorylation was also inhibited to a much greater extent than cyclic photophosphorylation. Chlorophyll a fluorescence measurements showed a reduced plastoquinone pool size. Evidence is presented to show that the reduced electron transport caused by infection is partly due to the reduction in chlorophyll levels, particularly chlorophyll a but also to direct inhibition of photosystem II, mainly at the plastoquinone level.
INTRODUCTION
Most virus infections affect the photosynthetic rates of the host to some extent. In some cases the rates show a dramatic reduction when estimated on a leaf area basis [S, 7, 9, 10, 19, 221. However, in a few cases infection leads to an apparent increase when the photosynthetic rates are measured at high light intensities and related to total chlorophyll although the rates may still be similar to those of uninfected plants when calculated on a leaf area basis [28]. The apparent increase appears to be due to a reduction in the levels of light harvesting chlorophylls. The photosynthetic process involves a series of complex partial reactions whose operation depends upon the structural integrity of the chloroplast. Viruses may thus affect photosynthesis in a variety of ways and the present study was carried out to determine the mechanisms by which peanut green mosaic virus [24] reduced the photosynthetic rates in its host. Abbreviations used in text: Asc, sodium ascorbate; DCDMQ 2,5-dimethoxy-3,6dichloro-p-benzoquinone; DCMU, 3-(3,4-dichlorophenyl)-l,l-dimethyl urea; DCPIP, 2,6 dichlorophenol indophenol; DPC, diphenyl carbazide; MV, methyl viologen; PGMV, peanut green mosaic virus; PMS, phenaxine methosulphate; PQ plastoquinone; Q, quinone; TCA, trichloroacetic acid. 00484059/84/050181+
11 $03.00/O
0
1984 Academic
Press Inc.
(London)
Limited
R. A. Naidu
182
et a/.
It involved studies of changes in the rates of CO, uptake during the various stages of infection from the development of severe symptoms which occur soon after infection, through to the recovery stage, which occurs when the virus has become systemically established and attempts to relate the changed rates with changes in chlorophyll levels and in electron transport. MATERIALS
AND
METHODS
Host Plant Peanut (Armhis hypogaea L. var. TMV2) seeds were sown in clay pots (6” diam) containing garden soil mixed with farmyard manure (3 : 1 ratio). Plants were dusted with carborundum (800 mesh) and inoculated at the five leaf stage with virus. The inoculum was prepared from PGMV-infected peanut leaves by grinding them in 0.01 M phosphate buffer (pH 7.0) containing 0.02 M bmercaptoethanol. Control plants were treated with buffer alone. All the inoculated and control leaves were washed with distilled water 1 h after inoculation. Analyses were carried out at three different stages ofsystemic disease development. Stage l-14 days after inoculation when the newly developed and partially unfolded seventh, eighth and ninth leaves showed chlorotic spots.,,, Stage 2-21 days after inoculation when the seventh, eighth and ninth leaves were completely unfolded and showed extensive chlorotic rings and patches (severe symptom phase). Stage 3-28 days after inoculation when the seventh, eighth and ninth leaves had recovered and showed mild mosaic mottling only (recovery phase). Chlorophyll determinations Chlorophyll was extracted and estimated in 80% acetone according to the method of Amon [I]. Measurementsof net photosynth& The rate of photosynthetic CO, uptake by intact leaves was measured at 28 “C in a closed system using an infrared gas analyser (series 225, Analytical Development Co. Ltd, UK). A single seventh, eighth or ninth leaf, while still attached to the plant, was enclosed in a plexiglass chamber and illuminated from both sides with 100 W reflector flood lamps. To eliminate heat radiation 3 cm water filters were used. Light intensity was determined at the position of the leafsample with a LI-188 Quantum/Radiometer (Lambda Inst. Corp., USA). Carbon dioxide uptake was allowed to reach a steady state at each light intensity prior to measurement. After the measurements were made the leaflets were excised, and the area of each leaflet was measured using a LI-COR Model 3100 leaf area meter. Isolation of chloroplasts Deribbed leaves were cut into small pieces, homogenized for two periods of 15 s with a 5 s interval in between, in ice cold 30 mM phosphate buffer (pH 7*5), containing 10 mM NaCl, 5 mM MgCl,, 330 mM sorbitol, and 0.1 oh BSA using a Sorvall Omnimixer operated at 60% voltage. The homogenate was strained through eight layers of muslin cloth and centrifuged at 3000g for 1 min. The supematant was discarded and the
Peanut
green mosaic virus infected
chloroplast phosphate chloroplast pended in chlorophyll
peanut
leaves, I
183
pellet gently suspended in a resuspension medium consisting of 30 mM buffer pH 7.5, containing 10 rnM NaCl and 5 mM MgCl,. The broken suspension was centrifuged at 1OOOOg for 10 min and the pellet susthe resuspension medium. This suspension was normally adjusted to a concentration of 1 mg ml-l.
Electron transport
Hill reaction rates were determined by following the decrease in absorbance of DCPIP at 610 nm. Photosystem II activity, in terms of 0, evolved, was measured by following electron transport from water to ferricyanide, or to DCDMQ at 25 “C using a Clark type 0, electrode. Photosystem I activity was determined by measuring 0, uptake during electron transport from reduced DCPIP to methyl viologen in the presence of DCMU. Illumination was provided by two 100 W reflector flood lights and the light was passed through an 8 cm length water path to prevent a temperature rise in the reaction chamber. The photon flux density in the reaction chamber was 250 pE m-2 s-l. Photophosphorylation
The rates of non-cyclic and cyclic photophosphorylation by isolated chloroplasts were determined using the method of Whatley & Amon [2.5]. The reactions were terminated by adding 0.3 ml of 20% TCA (w/v). After centrifitgation at 1200g for 15 min the supematants were assayed for esterified 32P as described by Losada & Amon [Z3]. Chlorophyll
Kinetics described grinding multiplier
a fruorescence
of chlorophyll a fluorescence induction in isolated cells was followed as by Kulandaivelu & Daniel1 [II]: The cells were isolated by mechanical as described by Gnanam & Kulandaivelu [5]. The signal from the photowas directly displayed on the storage oscilloscope.
RESULTS
Total amounts of chlorophyll, of chlorophylls a and b together with the ratios of chlorophyll a/b at different stages of disease development are given per unit leaf area in Table 1. Total chlorophyll content of infected leaves was reduced by 17%, 36% and 9% respectively at infection stages 1, 2 and 3, compared to the healthy controls. Chlorophyll a levels were reduced more than chlorophyll b levels resulting in a reduced chlorophyll a/b ratio, particularly at infection stage 2. Light saturation curves for net photosynthesis in both healthy and virus infected leaves are shown in Fig. 1. In both cases CO, uptake was saturated at 180 uE m-2 s-r. Infection significantly reduced net photosynthesis over a wide range of irradiances at infection stage 2. This reduction was minimal at infection stage 1 and at infection stage 3 no reduction was observed. The net photosynthetic rates at saturating light intensities (180 pE m-2 s-l) in both healthy and infected leaves are shown in Table 2. Infected leaves showed 18%, 31 y. and 2% reduction in net photosynthesis at stages 1, 2 and 3 respectively, over that of controls. Changes in the photochemical reactions are shown in Table 3. The DCPIP photoreduction with water as the electron donor was lower in infected leaves than in
R. A. Naidu
184 1
TABLE
Total chlorophyll,
chlorophylls a and b and chlorophyll a/b ratio of healthy and peanut green mosaic m’m infected peanut leaves at d$mnt stages of infection
Total Infection Control Infection
stage
stage
Control Infection
chlorophyll CM cm-7
stage 3
(a) $24 $20 56. q aEach
bvalues
value
12
: F g
6
f I0
a
Chlorophyll be cm+)
24.9 * 1.7
80
(-39.1) 25.5 * 2.5 23.0 & 2.1 (-9.8)
(-)
40 Light
3.2 i 0.2 3.0 f 0.4
1.5 1.7
3.2 + 0.2 2.7 f 0.3
1.8
3.3 f 0.3 3.3 f 0.4
1.9
over the uninfected
controls.
(c)
(b)
0
a/b
(-9.1)
is the mean & SE of five estimations. represent percentage reduction
120 60200240
Chlorophyll ratio
(-13.9) 8.0 k 5.9 * (-26.3) 7.7 k 7.0 *
4
40
b
7.9 + 1.6 6.8 * 2.0
20.3 5 2.1 (-18.5) 25.6 & 1.9 15.6 * 1.5
in parentheses
L26 N
,i
Chlorophyll (Pk? cm-*1
32.8 * 2.P 27.2 & 1.5 (--17.l)b 33.5 & 14 21.5 * 14 (- 35.8) 33.5 f 1.9 30.5 f 2.1 (-8.9)
1
stage 2
Control Infection
et al.
80
120 160200240
intensity
(pE
me2 C’)
l!Yf!
0
40
80
120 160 200
240
FIG. 1. Rate of CO, uptake by healthy (0) and peanut green mosaic virus infected (0) peanut leaves as a function of light intensity at three infection stages: (a) infection stage 1 showing chlorotic spots; (b) infection stage 2 showing severe symptoms; (c) infection stage 3 after recovery from severe symptoms.
controls by 17% and 42% at infection stages 1 and 2, respectively. Diphenyl carbazide, when used as an electron donor for photosystem II, was ineffective in restoring the Hill activity to control levels in the infected samples. The rate of photosystem II activity measured as 0, evolution with ferricyanide as the electron acceptor was comparable with the rate of DCPIP photoreduction. However, when DCDMQwas used as the electron acceptor, photosystem II activity was reduced by 6% and 10% at infection stages 1 and 2, respectively. Photosystem I activity was reduced by 9% and 15% at the same two stages of infection. Isolated chloroplasts from healthy and virus infected leaves at infection stage 3 showed similar rates of photochemical activities. The effect of virus infection on the formation of ATP in both non-cyclic and cyclic photophosphorylation is shown in Table 4. ATP formation through non-cyclic
Peanut
green mosaic virus infected
peanut
TABLE N&photosynthesis
at saturating
Control Infected
2
light intensity (180 fl m-8 s-l) infected peanut leaves
CO, Sample
Stage
1
22.4 f 2.4a 18.4 h 3.1 (--1%g)b
185
leaves, I
uptake
in healthy and peanut green mosaic virus
(mg dm-*
h-l)
Stage 2
Stage 3
22.5 + 1.8 15.6 h 1.3 (-30.7)
22.9 + 3.2 22.5 i 2.6 (-1.8)
“Each value is the mean f SE of three separate experiments. bvalues in parentheses show percentage decrease (-) over the controls.
photophosphorylation was reduced by 26% and 54% and through cyclic photophosphorylation by 5% and 16% at infection stages 1 and 2 respectively. A similar severe reduction in ferricyanide mediated photoreduction in coupled non-cyclic photophosphorylation raction was observed during infection stages 1 and 2. However, at infection stage 3 photophosphorylation processes were no longer affected. The stoichiometry of non-cyclic photophosphorylation was similar at all stages of infection and was more or less equal to that in healthy leaves. The kinetics of fluorescence induction in isolated cells showed an initial rapid rise (F,) followed by a gradual rise leading to a steady state (F,) level (Fig. 2). Infection did not affect F,, but it did produce a marked increase in the variable phase of fluorescence induction. In the presence of DCMU, the steady state fluorescence level increased significantly. The AF (fluorescence level in the presence of DCMU minus fluorescence level in the absence of DCMU), which reflects relative photosynthetic efficiency [II, 201 was reduced by 17% and 50%, respectively, at infection stages 1 and 2. When sodium dithionite was added to the DCMU treated cells, the fluorescence level showed a further increase in all the samples. This was due to the direct action of sodium dithionite on Q [4, 8, 141. However the percentage increase induced by the sodium dithionite over the DCMU level was similar for both healthy and infected samples at all the three stages of infection. The normalized area above the chlorophyll a fluorescence induction curve, which is proportional to the concentration of the total IQ pool [3], was reduced over that of the controls by 22% and 52% respectively at infection stages 1 and 2 but by infection stage 3 there was no difference between infected and healthy controls (Fig. 3). DISCUSSION
In the present study, we have found a direct correlation between chlorophyll levels and the rate of net photosynthesis in virus infected leaves (Table 1, Table 2, Fig. 1) with the lowest levels of both occurring at infection stage 2. However, by infection stage 3 both the chlorophyll content and the net photosynthetic rates had returned to control levels. Similar reductions in photosynthetic rates have been found in
H,O (F mol0,
H,O + (p mol0,
(iii)
(iv)
h-l)
h-l)
h-l)
h-l)
h-l)
3.5
193.7 f
3.5
160.2 + 7.4
122.9 f
172.4 + 5.6
129.8 * 8.6”
Infected
2.5
175.6 f 4.9 (-9.3)
151.2 38.6 (-5.6)
96.2 f (-21.7)
120.6 + 10.4 (-30.1)
Control
Stage
isolatedfrom
3.7
195.0 * 5.3
162.2 i
120.6 + 7.0
170.8 f 8.7
125.5 * 4.0
in chloroplarts
108.3 i 5.2 (-16.6)”
1
activities
3
64.0 f (-46.9)
5.9
79.9 * 6.4 (-53.2)
72.2 5 9.8 (-42.5)
Infected
166.0 * 3.4 (-14.9)
145.7 + 5.1 (-10.2)
2
3.3
193.4 & 4.6
159.7 + 4.2
123.7 i
171.3k4.3
125.4 + 5.9
Control
stage
plants at di$crent stages of infection
Infected
5.1
195.3 + 3.0 (t- 1.0)
(+2q
162.9 & 2.9
121.6 h4.6 (-1.7)
168.3 + 6.0 (-1.8)
122.3 i (-2.5)
3
For DCPIP photoreduction, the reaction mixture (3 ml) contained 30 rnM phosphate buffer (pH 7.5), 10 rnM NaCl, 5 rnM MgCl,, 1 p gramicidin, 50 FM DCPIP and chloroplasts equivalent to 2&25 pg chlorophyll. Diphenyl carbazide (DPC) at a final concentration of 50~~ was used as an artificial electron donor. For ferricyanide/DCDMQ mediated 0, evolution, DCPIP was replaced with 1 rnM ferricyanide or 0.5 rnM DCDMQ When photosystem I driven 0, uptake was measured, 1 rnM methyl viologen (MV), 1 rnM sodium ascorbate (Ax) and 50 PM DCPIP were added to the reaction mixture. 5 PM DCMU was added to block electron flow from photosystem II. aEach value is the mean *SE of three separate experiments. bValues in parentheses show percentage increase (+) or decrease (-) over uninfected controls.
Photosystem I DCPIP-Asc MV (p mol0, uptake mg Chl-’
DCDMQ evolved mg Chlm’
ferricyanide evolved mg Chl-’
DPC --L DCPIP (p mol DCPIP red mg Chl-’
(ii)
Photosystem II (i) H,O --+ DCPIP (p mol DCPIP red mg Chl-’
Control
stage
Effect of peanut green mosaic virus on photochemical
TABLE
?
P
103.4
103.0 * 9.2
2
3
* 10.9
(+@2)
103.2
49.0 & 7.8 (52.6)
71.8 i5.8 (-26.4)
Infected
h-l)
13.9
h 9.4
+ 11.1
126.6 i
126.1
121.0
Control
(+@‘4
125.9 (-52.7)* 10.7
59.6 (-26.6)f 8.6
88.8 * 9.9
Infected
Ferricyanide reduction (F mol red mg Chl-’ h-t)
in chloroplasts
4
plants
0.81
0.82
0.81
Conrrol
at dz@rent
0.82
0.82
0.81
Infected
Stoichiometry (ATP : 2 e-)
isolatedfrom
258.6
258.2
257.4
5 8.8
+ 10.9
+ 13.5
Control
243.7 & 9.7 (-5.3) 217.3 *8.4 (-15.8) 257.7 i 10.2 (+0.5)
Infected
Cyclic photophosphorylation (u mol ATP mg Chl-’ h-i)
stages of infection
the reaction mixture contained chloroplasts equivalent to 25 pg Chl, 33 rnsr aFor non-cyclic photophosphorylation, Tris-HCl buffer pH 8.2,5 mt.r MgCl, 2 rnM ADP, 10 rnM NaCl, 2 rnM KsHssPO, and 5 mt+r ferricyanide in a total volume of 3 ml. ferricyanide The reduction was carried out at 25 “C, light intensity 250 uE mm2 s- 1, for 5 min. For cyclic photophosphorylation was replaced with 5 rnM PMS. bEach value is the mean i SE of three separate experiments. CValues in parentheses represent percentage increase (+) or decrease (-) over uninfected controls.
+ 12.6
97.6 _t 10.4b
Control
1
Infection stage
Non-cyclic photophosphorylation (u mol ATP mg Chl-’
Effect of peanut green mosaic virus on photophospho y&ion
TABLE
188
R. A. Naidu
b
et a/.
b 0
a
ON
b
ON
H
FIG. 2. Kinetics of fluorescence induction in peanut green mosaic virus infected (VI) peanut cells at infection stages 1 (VI,), 2 (VI,) and 3 (VI,) and in healthy (H) peanut cells. (a) No additions; (b) +lOp~ DCMU; (c) +I0 FM DCMU and a few crystals of sodium dithionite.
lnfectm
The
stages
FIG. 3. Relative plastoquinone pool size in peanut green mosaic virus infected peanut leaves. pool size is the area above the induction curve (shadowed area) as shown in the inset.
infected with tobacco ringspot virus [29], in sugar beet infected with sugar beet yellows virus [Cl, peach infected with rosette and decline viruses [22] and tomato infected with tomato aspermy virus [9]. Since the number of chloroplasts per cell did not decrease at any stage of infection [23], the reduced rates of photosynthesis must be due in part at least to the reduced chlorophyll levels in the chloroplasts, particularly of chlorophyll a [25]. Infection reduced the activity of photosystem II to a greater extent than that of
tobacco
Peanut
green mosaic virus infected
peanut
leaves, I
189
photosystem I (Table 3) and the reduced activity of photosystem II was most evident at infection stage 2. Using DPC instead ofwater, as an electron donor for photosystem II in the Hill reaction, did not overcome the virus induced loss in activity at either infection stage 1 or 2. Thus the donor side of photosystem II does not appear to be affected. This conclusion is supported by observations on chlorophyll II fluorescence which showed an increased yield in samples from both infection stages 1 and 2 (Fig. 2). Chlorophyll fluorescence is a sensitive indicator of photosynthetic energy conversion [27] and any inhibition on the water splitting system would reduce chlorophyll a fluorescence whereas a block on the acceptor side of photosystem II would increase it [2]. The observed increase in fluorescence in infected samples indicates reduced electron flow from the acceptor side of photosystem II towards photosystem I. Photosystem II mediated electron transport was also monitored in the presence of ferricyanide and DCDMQ in order to investigate the acceptor side of photosystem II. When ferricyanide was used as an electron acceptor the rate of photosystem II was reduced to a greater extent than when DCDMQ was used (Table 3). Since DCDMQcan accept electrons from a position preceding PQin the electron transport chain [21], whereas ferricyanide can accept electrons from components after PQ [12, 161, the recovery of photosystem II activity in the presence of DCDMQindicates that PQis the probable site of inhibition of electron transport in severely diseased leaves at infection stage 2. This is supported by the fluorescence measurements which indicated a reduced pool size of PQ (Fig. 3). Since the sodium dithionite induced increase in fluorescence level in the presence of DCMU was to the same level in both healthy and infected samples at all infection stages (Fig. 2) Q the primary acceptor of photosystem II, appears to be intact in virus infected leaves. Non-cyclic photophosphorylation was inhibited more than cyclic photophosphorylation (Table 4) at infection stage 2, but the fact that the stoichiometry of noncyclic photophosphorylation (ratio of ATP formed per 2 e- transferred from water to ferricyanide) is the same for both healthy and infected leaves indicates that non-cyclic photophosphorylation is not uncoupled. It is more probable that the reduction in PQ levels is responsible for the reduction in the rates of non-cyclic photophosphorylation and electron transport in the severely infected leaves. In summary, this study indicates that virus infection leads to inhibition of electron transport at the PQ level and this inhibition must be partly responsible for the reduced photosynthesis since it would lead to a reduction in the generation of both ATP and NADPH. In addition, however, the reduced levels of chlorophylls, particularly of chlorophyll a must also contribute to the reduction in photosynthesis. R. A. Naidu is grateful to U.G.C. (New Delhi) for financial assistance. The authors thank Drs G. Kulandaivelu and Salil Bose for their help. REFERENCES 1. ARNON,
D.
I. (1949).
Plant Physiology
Copper
enzymes
in isolated
chloroplasts:
Polyphenol
oxidases
in Beta vulgaris.
24, l-l 5.
2. BUTLER, W. L. (1977). Chlorophyll fluorescence as a probe for electron transfer and energy transfer. In Encyclopaedia Plant physiology New Series, Vol. 5, Ed. by A. Trebst & M. Avron, pp. 14%167. Springer-Verlag, Berlin. 3. CAHEN, D., MALKIN, D., SHOCKAT, S. & OHAD, I. (1976). Development of photosystem II complex during greening of Chlamydomonar reinhardi y-l. Plant Physiology 58, 257-267.
190
R. A. Naidu
et al.
4. DUYSENS, L. N. M. & SWEERS, H. E. (1963). Mechanism of two photochemical reactions in algae as studied by means of fluorescence. In S&dies oa Microalgae and Photosynthetic Bactcria, Ed. by Japanese Society Plant Physiology, pp. 353-372. University of Tokyo Press, Tokyo. 5. GNANAM, A. & KULANDAIVELU, G. (1969). Photosynthetic studies with leaf cell suspensions from higher plants. Plant physiology 44, 1451-1456. 6. HALL, A. E. & LOOMIS, R. S. (1972). Photosynthesis and respiration by healthy and beet yellow virus-infected sugar beets (Beta vulgaris L.). Crop Scimcc 12,56&572. 7. HALL, A. E. & LOOMIS, R. S. (1972). An explanation for the difference in photosynthetic capabilities of healthy and beet yellows infected sugar beets (Beta vulgar& L.). Plantphysiology 50,576-580. 8. HORTON, P. & CROZE, E. (1979). Characterization of two quenchers of chlorophyll fluorescence with different midpoint oxidation-reduction potentials in chloroplasts. Biochimica et Biophysics Acta 545, 188-201. 9. HUNTUR, C. S. & PEAT, W. E. (1973). The effect of tomato aspermy virus on photosynthesis in the young tomato plant. Physiological Plant Pathology 13,517-524. 10. JENSEN, S. G. (1972). Metabolism and carbohydrate composition in barley yellow dwarf virus infected wheat. Phytopathology 62,587-592. 11. KULANDAWELU, G. & DANIELL, H. (1980). Dichlorophenyl dimethylurea (DCMU) induced increase in chlorophyll a fluorescence intensity-An index of photosynthetic oxygen evolution in leaves, chloroplasts and algae. Physiologia Plantarum 48, 385-388. 12. LIEN, S. & BANNISTIXR, T. T. (1971). Multiple sites on DCIP reduction by sonicated oat chloroplasts: Role of plastocyanin. Biochimica et Biophysics Acta 245,465-481. 13. LOSADA, M. & ARNON, D. I. (1964). Enzyme systems in photosynthesis. In Modern Methods of Plant Analysis, Vol. 7, Ed. by K. Peach, B. 0. Sanwel & M. V. Tracey, pp. 569-615. Springer-Verlag, Berlin. 14. MALKIN, S. & BARBER, J. (1979). On the function of the fluorescence quenchers in chloroplasts and their relation to the primary electron acceptor of photosystem II. Archives of Biochemistry and Biophysics 193, 169178. 15. NAIDU, R. A., KRISHNAN, M., NA~UDU, M. V. & GNANAM, A. (1984). Studies on peanut green mcsaic virus infected peanut (Arachis hypogaea L.) leaves. II. Chlorophyll-protein complexes and polypeptide composition of thylakoid membranes. Physiological Plant Pathology 25, 191-198. 16. OUITRAKUL, R. & IZAWA, S. (1973). Electron transport and photophosphorylation in chloroplasts as a function of the electron acceptor. II. Acceptor specific inhibition by KCN. Biochimica et Biophysics Acta 305, 105-118. 17. PAPAGEORGIOU, G. (1975). Chlorophyll fluorescence: an intrinsic probe of photosynthesis. In Bioencrgetics ofPhotosynthesis, Ed. by Govindjee, pp. 319-371. Academic Press, New York. 18. F’LATT, S. G., HENRIQUES, F. & RAND, L. (1979). Effects ofvirus infection on the chlorophyll content, photosynthetic rate and carbon metabolism of Tolmiea menziesii. Physiological Plant Pathology 15, 351-366. 19. ROBERTS, D. A. & CORBETT, M. K. (1965). Reduced photosynthesis in tobacco plants infected with tobacco ring spot, virus. Phytopathology 55,37C-37 1. 20. SAMUELSSON, G. & OQUIST, G. (1977). A method for studying photosynthetic capacities of unicellular algae based on in vivo chlorophyll fluorescence. Physiologia Plantawn 40, 315-319. 21. SAROJINI, G. & DANIELL, H. (1981). Site of action of 2,5-Dimethoxy-3,6-Dichloro-p-Benzoquinone in the photosynthetic electron transport chain. Z-itschriftfir Naturforschung 36c, 65C661. 22. SMITH, P. R. & NEALES, T. F. (1977). Analysis of the effects of virus infection on the photosynthetic properties of peach leaves. Australian Journal of Plant Physiology 4, 723-732. 23. SREENIVASULU, P. & NAWDU, M. V. (1978). Anatomy and pigment changes in chlorotic spot virus (GCSV) infected groundnut leaves (Arachis hypogaea L.). Indian Journal of Expcrimcntal Biology 16, 525-527. 24. SREENIVWULU, P., IIZUKA, N., RAJESHWARI, R., REDDY, D. V. R. & NA~~JDU, M. V. (1981). Peanut green mosaic virus-a member of the potato virus Y group infecting groundnut (Arachis hypogaea) in India. Annals of Applied Biology 98,255-260. 25. WHATLEY, F. R. & ARNON, D. I. (1963). Photosynthetic phosphorylation in plants. In Methodc in Enzymology, Vol. 6, Ed. by S. P. Colowick & N. 0. Kaplan, pp. 308-313. Academic Press, New York.
Plant Pathology
( 1984) 25, 181-I
90
Studies on peanut green mosaic virus infected peanut (Arachk hypogaea L.) leaves. I. Photosynthesis and photochemical reactions R. A. NAIDU, *M. KRISHNAN,
*P. RAMANUJAM,
*A. GNANAM and M. V. NAWDU
Department of Botany, S. V. University, Tirupati 517502, India. *Department of Plant Sciences, School of Biological Sciences, Madurai (Accepted for publication
Kamaraj
University,
Madurai
625 021, India
June 1984)
Changes in chlorophyll levels, in photosynthetic rates,and in photochemical reactions were studied in peanut plants infected with peanut green mosaic virus at three different stages of systemic infection: (1) early infection; (2) the stage of maximum symptom expression and (3) after the recovery phase of systemic disease development. Chlorophyll content per unit leaf area was reduced at all three stages of infection with the greatest reduction coinciding with the phase of maximum symptom expression. Net photosynthesis was also reduced over a range of light intensities, with the maximum reduction occurring at the highest light intensities. Infection inhibited photosrjtem II activity to a significantly greater extent, particularly at infection stage 2, than it did photosystem I activity. Non-cyclic photophosphorylation was also inhibited to a much greater extent than cyclic photophosphorylation. Chlorophyll a fluorescence measurements showed a reduced plastoquinone pool size. Evidence is presented to show that the reduced electron transport caused by infection is partly due to the reduction in chlorophyll levels, particularly chlorophyll a but also to direct inhibition of photosystem II, mainly at the plastoquinone level.
INTRODUCTION
Most virus infections affect the photosynthetic rates of the host to some extent. In some cases the rates show a dramatic reduction when estimated on a leaf area basis [S, 7, 9, 10, 19, 221. However, in a few cases infection leads to an apparent increase when the photosynthetic rates are measured at high light intensities and related to total chlorophyll although the rates may still be similar to those of uninfected plants when calculated on a leaf area basis [28]. The apparent increase appears to be due to a reduction in the levels of light harvesting chlorophylls. The photosynthetic process involves a series of complex partial reactions whose operation depends upon the structural integrity of the chloroplast. Viruses may thus affect photosynthesis in a variety of ways and the present study was carried out to determine the mechanisms by which peanut green mosaic virus [24] reduced the photosynthetic rates in its host. Abbreviations used in text: Asc, sodium ascorbate; DCDMQ 2,5-dimethoxy-3,6dichloro-p-benzoquinone; DCMU, 3-(3,4-dichlorophenyl)-l,l-dimethyl urea; DCPIP, 2,6 dichlorophenol indophenol; DPC, diphenyl carbazide; MV, methyl viologen; PGMV, peanut green mosaic virus; PMS, phenaxine methosulphate; PQ plastoquinone; Q, quinone; TCA, trichloroacetic acid. 00484059/84/050181+
11 $03.00/O
0
1984 Academic
Press Inc.
(London)
Limited
R. A. Naidu
182
et a/.
It involved studies of changes in the rates of CO, uptake during the various stages of infection from the development of severe symptoms which occur soon after infection, through to the recovery stage, which occurs when the virus has become systemically established and attempts to relate the changed rates with changes in chlorophyll levels and in electron transport. MATERIALS
AND
METHODS
Host Plant Peanut (Armhis hypogaea L. var. TMV2) seeds were sown in clay pots (6” diam) containing garden soil mixed with farmyard manure (3 : 1 ratio). Plants were dusted with carborundum (800 mesh) and inoculated at the five leaf stage with virus. The inoculum was prepared from PGMV-infected peanut leaves by grinding them in 0.01 M phosphate buffer (pH 7.0) containing 0.02 M bmercaptoethanol. Control plants were treated with buffer alone. All the inoculated and control leaves were washed with distilled water 1 h after inoculation. Analyses were carried out at three different stages ofsystemic disease development. Stage l-14 days after inoculation when the newly developed and partially unfolded seventh, eighth and ninth leaves showed chlorotic spots.,,, Stage 2-21 days after inoculation when the seventh, eighth and ninth leaves were completely unfolded and showed extensive chlorotic rings and patches (severe symptom phase). Stage 3-28 days after inoculation when the seventh, eighth and ninth leaves had recovered and showed mild mosaic mottling only (recovery phase). Chlorophyll determinations Chlorophyll was extracted and estimated in 80% acetone according to the method of Amon [I]. Measurementsof net photosynth& The rate of photosynthetic CO, uptake by intact leaves was measured at 28 “C in a closed system using an infrared gas analyser (series 225, Analytical Development Co. Ltd, UK). A single seventh, eighth or ninth leaf, while still attached to the plant, was enclosed in a plexiglass chamber and illuminated from both sides with 100 W reflector flood lamps. To eliminate heat radiation 3 cm water filters were used. Light intensity was determined at the position of the leafsample with a LI-188 Quantum/Radiometer (Lambda Inst. Corp., USA). Carbon dioxide uptake was allowed to reach a steady state at each light intensity prior to measurement. After the measurements were made the leaflets were excised, and the area of each leaflet was measured using a LI-COR Model 3100 leaf area meter. Isolation of chloroplasts Deribbed leaves were cut into small pieces, homogenized for two periods of 15 s with a 5 s interval in between, in ice cold 30 mM phosphate buffer (pH 7*5), containing 10 mM NaCl, 5 mM MgCl,, 330 mM sorbitol, and 0.1 oh BSA using a Sorvall Omnimixer operated at 60% voltage. The homogenate was strained through eight layers of muslin cloth and centrifuged at 3000g for 1 min. The supematant was discarded and the
Peanut
green mosaic virus infected
chloroplast phosphate chloroplast pended in chlorophyll
peanut
leaves, I
183
pellet gently suspended in a resuspension medium consisting of 30 mM buffer pH 7.5, containing 10 rnM NaCl and 5 mM MgCl,. The broken suspension was centrifuged at 1OOOOg for 10 min and the pellet susthe resuspension medium. This suspension was normally adjusted to a concentration of 1 mg ml-l.
Electron transport
Hill reaction rates were determined by following the decrease in absorbance of DCPIP at 610 nm. Photosystem II activity, in terms of 0, evolved, was measured by following electron transport from water to ferricyanide, or to DCDMQ at 25 “C using a Clark type 0, electrode. Photosystem I activity was determined by measuring 0, uptake during electron transport from reduced DCPIP to methyl viologen in the presence of DCMU. Illumination was provided by two 100 W reflector flood lights and the light was passed through an 8 cm length water path to prevent a temperature rise in the reaction chamber. The photon flux density in the reaction chamber was 250 pE m-2 s-l. Photophosphorylation
The rates of non-cyclic and cyclic photophosphorylation by isolated chloroplasts were determined using the method of Whatley & Amon [2.5]. The reactions were terminated by adding 0.3 ml of 20% TCA (w/v). After centrifitgation at 1200g for 15 min the supematants were assayed for esterified 32P as described by Losada & Amon [Z3]. Chlorophyll
Kinetics described grinding multiplier
a fruorescence
of chlorophyll a fluorescence induction in isolated cells was followed as by Kulandaivelu & Daniel1 [II]: The cells were isolated by mechanical as described by Gnanam & Kulandaivelu [5]. The signal from the photowas directly displayed on the storage oscilloscope.
RESULTS
Total amounts of chlorophyll, of chlorophylls a and b together with the ratios of chlorophyll a/b at different stages of disease development are given per unit leaf area in Table 1. Total chlorophyll content of infected leaves was reduced by 17%, 36% and 9% respectively at infection stages 1, 2 and 3, compared to the healthy controls. Chlorophyll a levels were reduced more than chlorophyll b levels resulting in a reduced chlorophyll a/b ratio, particularly at infection stage 2. Light saturation curves for net photosynthesis in both healthy and virus infected leaves are shown in Fig. 1. In both cases CO, uptake was saturated at 180 uE m-2 s-r. Infection significantly reduced net photosynthesis over a wide range of irradiances at infection stage 2. This reduction was minimal at infection stage 1 and at infection stage 3 no reduction was observed. The net photosynthetic rates at saturating light intensities (180 pE m-2 s-l) in both healthy and infected leaves are shown in Table 2. Infected leaves showed 18%, 31 y. and 2% reduction in net photosynthesis at stages 1, 2 and 3 respectively, over that of controls. Changes in the photochemical reactions are shown in Table 3. The DCPIP photoreduction with water as the electron donor was lower in infected leaves than in
R. A. Naidu
184 1
TABLE
Total chlorophyll,
chlorophylls a and b and chlorophyll a/b ratio of healthy and peanut green mosaic m’m infected peanut leaves at d$mnt stages of infection
Total Infection Control Infection
stage
stage
Control Infection
chlorophyll CM cm-7
stage 3
(a) $24 $20 56. q aEach
bvalues
value
12
: F g
6
f I0
a
Chlorophyll be cm+)
24.9 * 1.7
80
(-39.1) 25.5 * 2.5 23.0 & 2.1 (-9.8)
(-)
40 Light
3.2 i 0.2 3.0 f 0.4
1.5 1.7
3.2 + 0.2 2.7 f 0.3
1.8
3.3 f 0.3 3.3 f 0.4
1.9
over the uninfected
controls.
(c)
(b)
0
a/b
(-9.1)
is the mean & SE of five estimations. represent percentage reduction
120 60200240
Chlorophyll ratio
(-13.9) 8.0 k 5.9 * (-26.3) 7.7 k 7.0 *
4
40
b
7.9 + 1.6 6.8 * 2.0
20.3 5 2.1 (-18.5) 25.6 & 1.9 15.6 * 1.5
in parentheses
L26 N
,i
Chlorophyll (Pk? cm-*1
32.8 * 2.P 27.2 & 1.5 (--17.l)b 33.5 & 14 21.5 * 14 (- 35.8) 33.5 f 1.9 30.5 f 2.1 (-8.9)
1
stage 2
Control Infection
et al.
80
120 160200240
intensity
(pE
me2 C’)
l!Yf!
0
40
80
120 160 200
240
FIG. 1. Rate of CO, uptake by healthy (0) and peanut green mosaic virus infected (0) peanut leaves as a function of light intensity at three infection stages: (a) infection stage 1 showing chlorotic spots; (b) infection stage 2 showing severe symptoms; (c) infection stage 3 after recovery from severe symptoms.
controls by 17% and 42% at infection stages 1 and 2, respectively. Diphenyl carbazide, when used as an electron donor for photosystem II, was ineffective in restoring the Hill activity to control levels in the infected samples. The rate of photosystem II activity measured as 0, evolution with ferricyanide as the electron acceptor was comparable with the rate of DCPIP photoreduction. However, when DCDMQwas used as the electron acceptor, photosystem II activity was reduced by 6% and 10% at infection stages 1 and 2, respectively. Photosystem I activity was reduced by 9% and 15% at the same two stages of infection. Isolated chloroplasts from healthy and virus infected leaves at infection stage 3 showed similar rates of photochemical activities. The effect of virus infection on the formation of ATP in both non-cyclic and cyclic photophosphorylation is shown in Table 4. ATP formation through non-cyclic
Peanut
green mosaic virus infected
peanut
TABLE N&photosynthesis
at saturating
Control Infected
2
light intensity (180 fl m-8 s-l) infected peanut leaves
CO, Sample
Stage
1
22.4 f 2.4a 18.4 h 3.1 (--1%g)b
185
leaves, I
uptake
in healthy and peanut green mosaic virus
(mg dm-*
h-l)
Stage 2
Stage 3
22.5 + 1.8 15.6 h 1.3 (-30.7)
22.9 + 3.2 22.5 i 2.6 (-1.8)
“Each value is the mean f SE of three separate experiments. bvalues in parentheses show percentage decrease (-) over the controls.
photophosphorylation was reduced by 26% and 54% and through cyclic photophosphorylation by 5% and 16% at infection stages 1 and 2 respectively. A similar severe reduction in ferricyanide mediated photoreduction in coupled non-cyclic photophosphorylation raction was observed during infection stages 1 and 2. However, at infection stage 3 photophosphorylation processes were no longer affected. The stoichiometry of non-cyclic photophosphorylation was similar at all stages of infection and was more or less equal to that in healthy leaves. The kinetics of fluorescence induction in isolated cells showed an initial rapid rise (F,) followed by a gradual rise leading to a steady state (F,) level (Fig. 2). Infection did not affect F,, but it did produce a marked increase in the variable phase of fluorescence induction. In the presence of DCMU, the steady state fluorescence level increased significantly. The AF (fluorescence level in the presence of DCMU minus fluorescence level in the absence of DCMU), which reflects relative photosynthetic efficiency [II, 201 was reduced by 17% and 50%, respectively, at infection stages 1 and 2. When sodium dithionite was added to the DCMU treated cells, the fluorescence level showed a further increase in all the samples. This was due to the direct action of sodium dithionite on Q [4, 8, 141. However the percentage increase induced by the sodium dithionite over the DCMU level was similar for both healthy and infected samples at all the three stages of infection. The normalized area above the chlorophyll a fluorescence induction curve, which is proportional to the concentration of the total IQ pool [3], was reduced over that of the controls by 22% and 52% respectively at infection stages 1 and 2 but by infection stage 3 there was no difference between infected and healthy controls (Fig. 3). DISCUSSION
In the present study, we have found a direct correlation between chlorophyll levels and the rate of net photosynthesis in virus infected leaves (Table 1, Table 2, Fig. 1) with the lowest levels of both occurring at infection stage 2. However, by infection stage 3 both the chlorophyll content and the net photosynthetic rates had returned to control levels. Similar reductions in photosynthetic rates have been found in
H,O (F mol0,
H,O + (p mol0,
(iii)
(iv)
h-l)
h-l)
h-l)
h-l)
h-l)
3.5
193.7 f
3.5
160.2 + 7.4
122.9 f
172.4 + 5.6
129.8 * 8.6”
Infected
2.5
175.6 f 4.9 (-9.3)
151.2 38.6 (-5.6)
96.2 f (-21.7)
120.6 + 10.4 (-30.1)
Control
Stage
isolatedfrom
3.7
195.0 * 5.3
162.2 i
120.6 + 7.0
170.8 f 8.7
125.5 * 4.0
in chloroplarts
108.3 i 5.2 (-16.6)”
1
activities
3
64.0 f (-46.9)
5.9
79.9 * 6.4 (-53.2)
72.2 5 9.8 (-42.5)
Infected
166.0 * 3.4 (-14.9)
145.7 + 5.1 (-10.2)
2
3.3
193.4 & 4.6
159.7 + 4.2
123.7 i
171.3k4.3
125.4 + 5.9
Control
stage
plants at di$crent stages of infection
Infected
5.1
195.3 + 3.0 (t- 1.0)
(+2q
162.9 & 2.9
121.6 h4.6 (-1.7)
168.3 + 6.0 (-1.8)
122.3 i (-2.5)
3
For DCPIP photoreduction, the reaction mixture (3 ml) contained 30 rnM phosphate buffer (pH 7.5), 10 rnM NaCl, 5 rnM MgCl,, 1 p gramicidin, 50 FM DCPIP and chloroplasts equivalent to 2&25 pg chlorophyll. Diphenyl carbazide (DPC) at a final concentration of 50~~ was used as an artificial electron donor. For ferricyanide/DCDMQ mediated 0, evolution, DCPIP was replaced with 1 rnM ferricyanide or 0.5 rnM DCDMQ When photosystem I driven 0, uptake was measured, 1 rnM methyl viologen (MV), 1 rnM sodium ascorbate (Ax) and 50 PM DCPIP were added to the reaction mixture. 5 PM DCMU was added to block electron flow from photosystem II. aEach value is the mean *SE of three separate experiments. bValues in parentheses show percentage increase (+) or decrease (-) over uninfected controls.
Photosystem I DCPIP-Asc MV (p mol0, uptake mg Chl-’
DCDMQ evolved mg Chlm’
ferricyanide evolved mg Chl-’
DPC --L DCPIP (p mol DCPIP red mg Chl-’
(ii)
Photosystem II (i) H,O --+ DCPIP (p mol DCPIP red mg Chl-’
Control
stage
Effect of peanut green mosaic virus on photochemical
TABLE
?
P
103.4
103.0 * 9.2
2
3
* 10.9
(+@2)
103.2
49.0 & 7.8 (52.6)
71.8 i5.8 (-26.4)
Infected
h-l)
13.9
h 9.4
+ 11.1
126.6 i
126.1
121.0
Control
(+@‘4
125.9 (-52.7)* 10.7
59.6 (-26.6)f 8.6
88.8 * 9.9
Infected
Ferricyanide reduction (F mol red mg Chl-’ h-t)
in chloroplasts
4
plants
0.81
0.82
0.81
Conrrol
at dz@rent
0.82
0.82
0.81
Infected
Stoichiometry (ATP : 2 e-)
isolatedfrom
258.6
258.2
257.4
5 8.8
+ 10.9
+ 13.5
Control
243.7 & 9.7 (-5.3) 217.3 *8.4 (-15.8) 257.7 i 10.2 (+0.5)
Infected
Cyclic photophosphorylation (u mol ATP mg Chl-’ h-i)
stages of infection
the reaction mixture contained chloroplasts equivalent to 25 pg Chl, 33 rnsr aFor non-cyclic photophosphorylation, Tris-HCl buffer pH 8.2,5 mt.r MgCl, 2 rnM ADP, 10 rnM NaCl, 2 rnM KsHssPO, and 5 mt+r ferricyanide in a total volume of 3 ml. ferricyanide The reduction was carried out at 25 “C, light intensity 250 uE mm2 s- 1, for 5 min. For cyclic photophosphorylation was replaced with 5 rnM PMS. bEach value is the mean i SE of three separate experiments. CValues in parentheses represent percentage increase (+) or decrease (-) over uninfected controls.
+ 12.6
97.6 _t 10.4b
Control
1
Infection stage
Non-cyclic photophosphorylation (u mol ATP mg Chl-’
Effect of peanut green mosaic virus on photophospho y&ion
TABLE
188
R. A. Naidu
b
et a/.
b 0
a
ON
b
ON
H
FIG. 2. Kinetics of fluorescence induction in peanut green mosaic virus infected (VI) peanut cells at infection stages 1 (VI,), 2 (VI,) and 3 (VI,) and in healthy (H) peanut cells. (a) No additions; (b) +lOp~ DCMU; (c) +I0 FM DCMU and a few crystals of sodium dithionite.
lnfectm
The
stages
FIG. 3. Relative plastoquinone pool size in peanut green mosaic virus infected peanut leaves. pool size is the area above the induction curve (shadowed area) as shown in the inset.
infected with tobacco ringspot virus [29], in sugar beet infected with sugar beet yellows virus [Cl, peach infected with rosette and decline viruses [22] and tomato infected with tomato aspermy virus [9]. Since the number of chloroplasts per cell did not decrease at any stage of infection [23], the reduced rates of photosynthesis must be due in part at least to the reduced chlorophyll levels in the chloroplasts, particularly of chlorophyll a [25]. Infection reduced the activity of photosystem II to a greater extent than that of
tobacco
Peanut
green mosaic virus infected
peanut
leaves, I
189
photosystem I (Table 3) and the reduced activity of photosystem II was most evident at infection stage 2. Using DPC instead ofwater, as an electron donor for photosystem II in the Hill reaction, did not overcome the virus induced loss in activity at either infection stage 1 or 2. Thus the donor side of photosystem II does not appear to be affected. This conclusion is supported by observations on chlorophyll II fluorescence which showed an increased yield in samples from both infection stages 1 and 2 (Fig. 2). Chlorophyll fluorescence is a sensitive indicator of photosynthetic energy conversion [27] and any inhibition on the water splitting system would reduce chlorophyll a fluorescence whereas a block on the acceptor side of photosystem II would increase it [2]. The observed increase in fluorescence in infected samples indicates reduced electron flow from the acceptor side of photosystem II towards photosystem I. Photosystem II mediated electron transport was also monitored in the presence of ferricyanide and DCDMQ in order to investigate the acceptor side of photosystem II. When ferricyanide was used as an electron acceptor the rate of photosystem II was reduced to a greater extent than when DCDMQ was used (Table 3). Since DCDMQcan accept electrons from a position preceding PQin the electron transport chain [21], whereas ferricyanide can accept electrons from components after PQ [12, 161, the recovery of photosystem II activity in the presence of DCDMQindicates that PQis the probable site of inhibition of electron transport in severely diseased leaves at infection stage 2. This is supported by the fluorescence measurements which indicated a reduced pool size of PQ (Fig. 3). Since the sodium dithionite induced increase in fluorescence level in the presence of DCMU was to the same level in both healthy and infected samples at all infection stages (Fig. 2) Q the primary acceptor of photosystem II, appears to be intact in virus infected leaves. Non-cyclic photophosphorylation was inhibited more than cyclic photophosphorylation (Table 4) at infection stage 2, but the fact that the stoichiometry of noncyclic photophosphorylation (ratio of ATP formed per 2 e- transferred from water to ferricyanide) is the same for both healthy and infected leaves indicates that non-cyclic photophosphorylation is not uncoupled. It is more probable that the reduction in PQ levels is responsible for the reduction in the rates of non-cyclic photophosphorylation and electron transport in the severely infected leaves. In summary, this study indicates that virus infection leads to inhibition of electron transport at the PQ level and this inhibition must be partly responsible for the reduced photosynthesis since it would lead to a reduction in the generation of both ATP and NADPH. In addition, however, the reduced levels of chlorophylls, particularly of chlorophyll a must also contribute to the reduction in photosynthesis. R. A. Naidu is grateful to U.G.C. (New Delhi) for financial assistance. The authors thank Drs G. Kulandaivelu and Salil Bose for their help. REFERENCES 1. ARNON,
D.
I. (1949).
Plant Physiology
Copper
enzymes
in isolated
chloroplasts:
Polyphenol
oxidases
in Beta vulgaris.
24, l-l 5.
2. BUTLER, W. L. (1977). Chlorophyll fluorescence as a probe for electron transfer and energy transfer. In Encyclopaedia Plant physiology New Series, Vol. 5, Ed. by A. Trebst & M. Avron, pp. 14%167. Springer-Verlag, Berlin. 3. CAHEN, D., MALKIN, D., SHOCKAT, S. & OHAD, I. (1976). Development of photosystem II complex during greening of Chlamydomonar reinhardi y-l. Plant Physiology 58, 257-267.
190
R. A. Naidu
et al.
4. DUYSENS, L. N. M. & SWEERS, H. E. (1963). Mechanism of two photochemical reactions in algae as studied by means of fluorescence. In S&dies oa Microalgae and Photosynthetic Bactcria, Ed. by Japanese Society Plant Physiology, pp. 353-372. University of Tokyo Press, Tokyo. 5. GNANAM, A. & KULANDAIVELU, G. (1969). Photosynthetic studies with leaf cell suspensions from higher plants. Plant physiology 44, 1451-1456. 6. HALL, A. E. & LOOMIS, R. S. (1972). Photosynthesis and respiration by healthy and beet yellow virus-infected sugar beets (Beta vulgaris L.). Crop Scimcc 12,56&572. 7. HALL, A. E. & LOOMIS, R. S. (1972). An explanation for the difference in photosynthetic capabilities of healthy and beet yellows infected sugar beets (Beta vulgar& L.). Plantphysiology 50,576-580. 8. HORTON, P. & CROZE, E. (1979). Characterization of two quenchers of chlorophyll fluorescence with different midpoint oxidation-reduction potentials in chloroplasts. Biochimica et Biophysics Acta 545, 188-201. 9. HUNTUR, C. S. & PEAT, W. E. (1973). The effect of tomato aspermy virus on photosynthesis in the young tomato plant. Physiological Plant Pathology 13,517-524. 10. JENSEN, S. G. (1972). Metabolism and carbohydrate composition in barley yellow dwarf virus infected wheat. Phytopathology 62,587-592. 11. KULANDAWELU, G. & DANIELL, H. (1980). Dichlorophenyl dimethylurea (DCMU) induced increase in chlorophyll a fluorescence intensity-An index of photosynthetic oxygen evolution in leaves, chloroplasts and algae. Physiologia Plantarum 48, 385-388. 12. LIEN, S. & BANNISTIXR, T. T. (1971). Multiple sites on DCIP reduction by sonicated oat chloroplasts: Role of plastocyanin. Biochimica et Biophysics Acta 245,465-481. 13. LOSADA, M. & ARNON, D. I. (1964). Enzyme systems in photosynthesis. In Modern Methods of Plant Analysis, Vol. 7, Ed. by K. Peach, B. 0. Sanwel & M. V. Tracey, pp. 569-615. Springer-Verlag, Berlin. 14. MALKIN, S. & BARBER, J. (1979). On the function of the fluorescence quenchers in chloroplasts and their relation to the primary electron acceptor of photosystem II. Archives of Biochemistry and Biophysics 193, 169178. 15. NAIDU, R. A., KRISHNAN, M., NA~UDU, M. V. & GNANAM, A. (1984). Studies on peanut green mcsaic virus infected peanut (Arachis hypogaea L.) leaves. II. Chlorophyll-protein complexes and polypeptide composition of thylakoid membranes. Physiological Plant Pathology 25, 191-198. 16. OUITRAKUL, R. & IZAWA, S. (1973). Electron transport and photophosphorylation in chloroplasts as a function of the electron acceptor. II. Acceptor specific inhibition by KCN. Biochimica et Biophysics Acta 305, 105-118. 17. PAPAGEORGIOU, G. (1975). Chlorophyll fluorescence: an intrinsic probe of photosynthesis. In Bioencrgetics ofPhotosynthesis, Ed. by Govindjee, pp. 319-371. Academic Press, New York. 18. F’LATT, S. G., HENRIQUES, F. & RAND, L. (1979). Effects ofvirus infection on the chlorophyll content, photosynthetic rate and carbon metabolism of Tolmiea menziesii. Physiological Plant Pathology 15, 351-366. 19. ROBERTS, D. A. & CORBETT, M. K. (1965). Reduced photosynthesis in tobacco plants infected with tobacco ring spot, virus. Phytopathology 55,37C-37 1. 20. SAMUELSSON, G. & OQUIST, G. (1977). A method for studying photosynthetic capacities of unicellular algae based on in vivo chlorophyll fluorescence. Physiologia Plantawn 40, 315-319. 21. SAROJINI, G. & DANIELL, H. (1981). Site of action of 2,5-Dimethoxy-3,6-Dichloro-p-Benzoquinone in the photosynthetic electron transport chain. Z-itschriftfir Naturforschung 36c, 65C661. 22. SMITH, P. R. & NEALES, T. F. (1977). Analysis of the effects of virus infection on the photosynthetic properties of peach leaves. Australian Journal of Plant Physiology 4, 723-732. 23. SREENIVASULU, P. & NAWDU, M. V. (1978). Anatomy and pigment changes in chlorotic spot virus (GCSV) infected groundnut leaves (Arachis hypogaea L.). Indian Journal of Expcrimcntal Biology 16, 525-527. 24. SREENIVWULU, P., IIZUKA, N., RAJESHWARI, R., REDDY, D. V. R. & NA~~JDU, M. V. (1981). Peanut green mosaic virus-a member of the potato virus Y group infecting groundnut (Arachis hypogaea) in India. Annals of Applied Biology 98,255-260. 25. WHATLEY, F. R. & ARNON, D. I. (1963). Photosynthetic phosphorylation in plants. In Methodc in Enzymology, Vol. 6, Ed. by S. P. Colowick & N. 0. Kaplan, pp. 308-313. Academic Press, New York.