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Photosynthesis and chloroplast functioning in leaves of barley infected with brown rust
Physiological
Plant Pa.thology
(1983)
23, 411419
Photosynthesis and chloroplast functioning of barley infected with brown rust I. AHMAD, J.F. FARRAR andR. School of Plant Biology,
University
(Accepted for publication
July
in leaves
WHITBREAD
College of North
Wales,
Bangor,
Gwynedd
LL57
.?lJW
1983)
The rate of net photosynthesis is reduced in leaves of barley infected with brown rust. This reduction is not due to reduced carbon dioxide fixation per chloroplast, but is ascribed to a decrease in the number of functional chloroplasts. Chloroplasts isolated from diseased leaves show increased contents of starch and phosphorus, and unaltered rates of ferricyanidedependent oxygen evolution, as compared with controls. Fluxes through the phosphate translocator on the chloroplast inner envelope are shown to be higher in diseased leaves. The kinetics of in oivo chlorophyll fluorescence induction are described, and it is shown that whilst they change with leaf age in control leaves, in diseased leaves the juvenile kinetics are retained. These data are interpreted as showing that each surviving chloroplast in diseased leaves is functioning at least as well as those from healthy leaves.
INTRODUCTION
Like many foliar diseases, rust infection causes a reduction in rates of net photosynthesis [7, 9, 181. Several causes for this reduction have been proposed : destruction of green leaf area [20], increased fungal respiration [23], increased host respiration [see 71 and inhibition of the light reactions of photosynthesis [6]. Whilst these are not necessarily mutually exclusive, there is no assessment of their individual contributions to the decline in net photosynthesis. However, it has been suggested that gross photosynthesis per unit chlorophyll-and thus per chloroplast-is enhanced in rusted tissue [20], and this would seem incompatible with partial inhibition of photophosphorylation and reductant generation. Further, a suggestion that carbon fixation by chloroplasts is reduced as a result of lowered host cytoplasmic inorganic phosphate (Pi) levels [27] would also be incompatible with the idea of increased gross photosynthesis by each surviving chloroplast. This paper seeks to examine the functioning of chloroplasts of leaves of barley infected with brown rust, both in viva and in vitro, to test the hypothesis that rust infection causes impairment of chloroplast metabolism, and to see if such impairment could be the whole or partial explanation for the rust-induced decrease in net photosynthesis. MATERIALS
AND
METHODS
Plants of barley [Hodwn distichum (L.) L am. cv. Maris Mink] were grown and when 10 days old their fully-expanded first leaves were infected with urediniospores of brown rust (Puccinia horde’ Otth.), as described previously [20]. 00484059/83/060411
+ 09 303.00/O
0
1983 Academic
Press Inc.
(London)
Limited
412
I. Ahmad
et al.
Leaf gas exchange was measured as before [ZO] with the addition of an A.D.C. infra-red gas analyser for water vapour. The temperature of the leaf surface was measured with copper-constantan thermocouples, which were also used in a thermocouple psychrometer to measure the water vapour content of the air supplied to the leaf. For measurement of in vivo chlorophyll fluorescence, intact plants were removed from the growth chamber at times between 5 and 8 h into the photoperiod and held in the dark at 23 “C for 1 h. Leaves were then detached and immediately placed with the middle of the upper surface under the fluorescence probe of a fluorimeter (Richard Brancker Associates model S.F. 10) and irradiated with red light (maximum emission at 670 nm) at 5.9 pm01 quanta m-2 s-l for 100 s whilst recording the fluorescence at wavelengths longer than 710 nm. The typical fluorescence curve, analysed following references [17, 29,211, is shown in Fig. 1. Isolated chloroplasts were prepared by a method similar to that used by Plesnicar & Bendall [ZZ]. At 4 “C leaves were cut into small pieces and disintegrated with a homogenizer for 3-5 s in a medium containing (in mol m-3) sucrose, 400; HEPES, 50; NaCl, 10; MgCl,, 1; EDTA, 1; cysteine, 4.1; 0.25% bovine serum albumin (BSA) (Fraction V) ; 1y0 (w/w) polyethylene glycol (PEG) (average mol. wt. 4000)) adjusted to pH 8.0. The homogenate was squeezed through two layers of muslin. The filtrate was centrifuged at 100 r min-l to remove urediniospores, and the plastids after sedimenting by centrifuging at 4000 rmin-1 for 5 min, washed and resuspended in a medium similar to the above, but with PEG and cysteine omitted and BSA at 1.25%. The total time for extraction was about 15 min. When examined by phase contrast all the chloroplasts appeared as Class A (highly reflective, bright opaque appearance, presence of a halo). However, determination by ferricyanide-dependent oxygen evolution before and after an osmotic shock showed that there were only 50% Class A chloroplasts [16].
0
20
40
60
00
100
Time(s)
FIG. 1. The kinetics following pre-darkening induction process in the duction period, attained F,,, variable component within 5 ms of the onset
of in uivo fluorescence induction. Red light is turned on at zero time the leaf for 1 h. The labelling OIDPSMT refers to components of the scheme of references 1.21, 261. F,,. maximal fluorescence during inat either I or P; F,,, stationary level of fluorescence attained at T; of fluorescence (= F,,, - F,,) ; F,, initial level of fluorescence, attained uf illumination.
Photosynthesis
in rust-infected
413
barley
The total chlorophyll in chloroplast preparations was measured following reference [Z]. The Hill reaction rate of isolated chloroplasts was determined by measuring ferricyanide-dependent oxygen evolution following reference [26] using an OX-15259 Clark oxygen electrode in conjunction with a Gilson K-1C oxygraph. Phosphorus in the preparation was determined by the ammonium molybdate method using stannous chloride as reducing agent [I] and starch was determined as glucose in amyloglucosidase-treated extracts [8]. The number of chloroplasts in the preparation was counted using a haemocytometer. Values presented in figures and tables are the means of three to five replicates, together with the standard error of the mean. RESULTS Gas exchange of intact leaves
Rates of net photosynthesis and transpiration fell following inoculation with brown rust, with the fall in transpiration lagging about 2 days behind that of net photosynthesis. A large and significant difference in net photosynthesis of diseased leaves was seen 6 days after infection. By the day of sporulation, net photosynthesis was about one-third, and transpiration one-half, of control rates (Fig. 2).
A 0
2
4
6
8
12
2
Days after
inoculation
IO
4
6
8
IO
12
FIG. 2. Net photosynthesis (a) of control (0) and rust-infected (0) first leaves of barley, with time after inoculation, and transpiration (b) of rust-infected leaves (expressed as o/0 control values; mea” rate for controls, 1.35 &to.16 mmol water m-* s-l). Conditions: 2O”C, 210 pmol quanta m-2 s-l over the waveband 400-700 nm; aerodynamic resistance 0.15 s cm-‘; vapour pressure deficit, 810 Pa. The vertical bars denote one standard error each side of the mean, and the arrow indicates the day of sporulation. TABLE
1
Phosphorus content [mmol P (g chlorophyll)-‘], Hill reaction rate [pal 0, (g chlorophyll)ml s-l] and starch content [mmol glucose (g chlorophyll) -I] of chloroplasts isolated from Jirst leaves of barley 8 days after infection with brown rust Treatment
Phosphorus
Hill
reaction
Control Rusted
1.02 k 0.06 2.83 & 0.39
123.5 + 10.7 160.2 + 11.8
Starch 24.8 f 4.8 79.4 * 20.7
5.76 3.82
1.19 x IO” 0.79 x 10”
Control Rusted
9.3 8.3
Reference
Gross (p&d [ZO]
3.1 2.8
(3) x 0.33
(4) Gross photosynthesis (pm01 DHAP m-* leaf s-l)
through the phosphate trandocator
(3) photosynthesis co, rnd leaf s-l)
of thejlux
2
\
I
0.54 0.73
(4)1(2)
DHAP translocation ( pmol m -* chloroplast envelope s-l)
rate
\
,
chloroplast
0.54 0.73
of triose
(5) Assuming 1 :1 exchange of Pi and DHAP
Pi translator (pm01 Pi m-* envelope s-l)
The flux is calculated from the area across which it occurs, this being the total surface area of chloroplasts, and dividing this into the amount phosphate leaving the chloroplast in unit time, this being taken as one-third the rate of gross photosynthesis expressed in moles of carbon dioxide. Calculations for the phosphate-DHAP translocator on the chloroplast inner envelope are made for the day of sporulation, 8 days after infection.
(1) x Area of ohlate spheroid with axes of 1 and 2.5 pm
area
Measured
Chloroplast surface (m* m-* leaf)
origin
no.
Chloroplast (m -* leaf)
(2)
Parameter
(1)
Calculation
TABLE
Photosynthesis
in rust-infected
415
barley
on chlorqtdads in vitro Chloroplasts isolated from infected leaves on the day of sporulation showed rates of ferricyanide-dependent oxygen evolution as high as those from healthy leaves (Table 1). Their starch and phosphorus contents were both higher on this day (Table 1). The number of chloroplasts per unit area in diseased leaves was two-thirds of that in controls (Table 2).
Measurments
In vivo chlorophyllfiorescewe The time course of fluorescence was examined at various times following inoculation. Typical curves are shown in Fig. 3. It can be seen that the rusted leaves maintained an essentially similar pattern of fluorescence throughout the 13 days of the experiment. By contrast, the control leaves showed a steady rise in magnitude of the peak P at about 15 s after induction, until this eventually exceeded peak I in height. These changes are quantified in Fig. 4, where two parameters (normalized fluorescence rise and normalized quenching) derived from such curves are plotted. As leaves aged, normalized fluorescence rise showed no significant change in rusted leaves, but a large fall in controls. Similarly normalized quenching capacity dropped to one-third of its initial value in control leaves but was maintained in leaves infected with brown rust until 12 days after infection. Differences between control and rusted leaves were not apparent until 8 days after infection. The low peak occurring at about 60 s after irradiation began was detectable in both control and rusted leaves until 7 days after inoculation; thereafter this peak (labelled M in Fig. 1) was absent.
(0)
(b)
8 7
I 50
I
I
I 100
I
I
50 Time
I
Control, In day of moculotlon
I
100
I
0
I
50
I
1
100
(s)
FIG. 3. Fluorescence induction curves for control (a) and rust-infected (b) first leaves of barley at various times after infection with brown rust. The vertical axis is in arbitrary units; see the legend to Fig. 1 for identifications for parts of these curves.
I. Ahmad
416
0
4
8
et al.
12 Days after
moculatlon
FIG. 4. Changes in (a) normalized fluorescence rise, F, [ = (F,,, - F,,)/F,] and (b) normalized quenching capacity, F, [ = (F,,, - F,,)/F,,] of control (0) and infected (0) first leaves of barley with time after inoculation with brown rust. The vertical bars denote one standard error each side of the mean.
DISCUSSION The depression in net photosynthesis reported here is similar to that reported previously for rust disease (see references in Introduction). It is not explicable in terms of altered water relations, since the reduction in transpiration occurred after the reduction in photosynthesis. Whether transpiration falls due to increased resistance to water flux in the leaf, or to lowered leafwater potential, any effects these mechanisms might have on carbon dioxide fixation will be superimposed on a reduction in net photosynthesis which is already large. It seems clear that the major fall in photosynthesis is due to destruction of chloroplasts within developing lesions, since the fall in photosynthesis roughly parallels a fall in leaf chlorophyll content [ZU], and we report here a much lower chloroplast number per unit area. An analytical approach, considering carbon dioxide fluxes in photosynthesis as a diffusion process, has shown that allowing for such destruction of photosynthetic machinery there may still be effects at the level of mesophyll resistance-that gross photosynthesis per unit of chlorophyll may be increased by rust infection [ZO, and see Table 21. This contrasts with other suggestions that the effects of rusts in depressing photosynthesis are largely located in the chloroplast. We have shown in this paper that chloroplasts freshly isolated from rusted leaves do not show impaired rates of ferricyanide-dependent oxygen evolution (the Hill reaction), lending credence to the idea that chloroplasts in rusted leaves can operate as well as control chloroplasts. Similarly, Wynn [.28] showed no reduction in photophosphorylation by chloroplasts isolated from rust-infected oats. The findings of Buchanan et al. [6] are apparently quite different: that non-cyclic electron transport is inhibited up to 45% in chloroplasts isolated from infected leaves. These workers themselves caution against the generalizing of results between diseases, or even different stages in the development of a single host-pathogen combination. A quite different explanation for reduction in gross carbon dioxide fixation per chloroplast is that if biotrophic fungi accumulate Pi from host tissue, the concentration the of Pi in the cytoplasm of the host will fall [27]. Th is will reduce the flux through
Photosynthesis
in rust-infected
barley
417
phosphate translocator at the chloroplast inner membrane, due to lowered availability of Pi for import. Less reduced carbon will therefore be exported from the chloroplast, and, as a result, the rate of photosynthesis will fall and carbon within the chloroplast will be diverted to starch rather than used in the formation of dihydrooxyacetone phosphate (DHAP) for export. This attractive hypothesis is not strongly supported by the present data, as although we find the predicted and usual increase in chloroplast starch level [see 271 we also find increased levels of phosphorus in chloroplasts from rusted leaves, hardly compatible with the idea of phosphorus-starved chloroplasts. It is possible that our extracted chloroplasts were contaminated with polyphosphate bodies [3], giving an artificially high phosphorus content, but careful washing did not change their phosphorus status. Perhaps also applicable to starchenriched diseased leaves is the hypothesis that large starch granules increase the diffusion path for carbon dioxide into chloroplasts, and that this is the direct cause of decreased gross photosynthesis [Z]. However, neither this nor the phosphatestarvation hypothesis are consistent with the suggestion of increased photosynthesis per surviving chloroplast. In passing, it can be calculated that the flux through the phosphate translocator is greater in diseased than in healthy leaves (Table 2), and that it is approaching the highest rates recorded for anion transport across membranes [24]. We suggest that such high rates may only be sustainable if the Pi concentration is high enough inside the chloroplast to permit high DHAP concentrations to be realized, which could also result in increased starch synthesis, as suggested by Whipps & Lewis [27], without a concomitant decrease in carbon dioxide fixation. ,411 of the above evidence is, of course, open to criticism. That individual chloroplasts perform better in rusted than in control leaves, and that there is a greater Aux through the phosphate translocator, are deductions from gas-exchange measurements. That chloroplasts from rusted leaves show Hill reaction rates comparable to those from controls, and that their phosphorus content is increased, could be attributed to changes during their isolation. Clearly, a direct, in vivo, inspection of chloroplast functioning is needed. Here we present the results of such an inspection on intact leaves using chlorophyll fluorescence kinetics. The secondary fluorescence rise, the S-M -IT transition, shows no differences between control and rusted leaves. This rise may be linked to the onset of photosynthetic carbon metabolism, and can be abolished by agents that sequester Pi [2rS]. The lack of a rust-dependent abolition of the M peak therefore argues against the hypothesis that cytoplasmic Pi is lowered by rust infection. Changes in fluorescence kinetics were seen for the earlier peaks at I and P, and further discussion concerns parameters derived from them. We follow Miranda et a/. [17] in deriving the fluorescence rise (F,,, ~ F,) and fluorescence quenching F,J, as they have physiological meaning; they are normalized on F, as F mm fluorescence is partially dependent on chlorophyll content, and then termed F, (fluorescence rise) and F, (fluorescence quenching), respectively. Most of the fluorescence emanates from chlorophyll in photosystem II [1.5, 211. Thus F, indicates the reduction of electron acceptors between photosystems II and I, which produces a reduced electron flow through the photosystem II reaction centre and thus an increase in variable fluorescence [17]. It, therefore, is a direct indicator of the state of photosystem II activity.
I. Ahmad
418
et al.
F, has three major causes. First it arises from the reoxidation of the primary electron acceptor ofphotosystem II, Q[.5,27] ; the rate of reoxidation of Qis dependent on the rate of electron flow from the secondary electron acceptors of photosystem II through photosystem I to NADP [27]. Second, the fluorescence is also quenched because of the high energy status of thylakoid membranes [Z.%151. The third cause of F, is the phosphorylation of chloroplast membrane proteins by ATP [4, 20, 12, 261. The latter two causes result from the creation of a light-induced electrical potential difference, and proton and Mg2+ concentration gradients, across the thylakoid membranes [1.5,2 11. Thus F, is a direct indicator of the ability of thylakoids to generate electrochemical gradients across their membranes and stimulate ATP production [17]. Roth F, and F, remained steady in rusted leaves during the experiment. On the other hand, in control leaves both fell considerably below the levels in rusted leaves. Thus there is a strong indication that while in control leaves the activity of photosystem II, the ability of thylakoids to generate electrochemical gradients across their membranes, and ATP production, are decreasing with age, in rusted leaves the photophysiological processes do not show any inhibitory effect. It is as if chloroplasts in rusted leaves retain their juvenility, whilst those in control leaves begin to age. This is support for the idea that green island formation does not involve re-greening, but simply the retardation of senescence in areas adjacent to the biotroph. It may not be necessary to involve any special effect of the fungus on chloroplast senescence; it may simply be that the sustained higher carbon fluxes through individual chloroplasts in diseased leaves results in maintained juvenility. It is also strong support for the idea that chloroplasts in rusted leaves are functioning at least as well as those in healthy leaves, and that reduced gross photosynthesis is simply due to destruction of chloroplasts. All the data in this paper refer to average values: it is quite possible that the chloroplast population in diseased leaves is highly heterogeneous, some chloroplasts showing grossly impaired metabolism. Clearly, more precise examination of discrete areas in and around a lesion would be desirable. The widespread belief that a general increase in membrane permeability follows infection with a biotroph would also seem in need of reassessment, and this forms the subject of a forthcoming paper. I.A. gratefully acknowledges the receipt of a Sir William Roberts scholarship. REFERENCES I. ALLEN, S. 2.
3. 4.
5.
E., GRIMSHAW, H. M., PARKINSON,J. A. & QUARMBY, C. (1974). Chemical Analysis of Ecological Materials. Blackwell Scientific Publications, Oxford. ARNON, D. I. (1949). Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgnris. Plant Physiology 24, I-15. BENNETT,J. & Scorn, K. J. (1971). Inorganic polyphosphates in the wheat stem rust fungus and in rust-infected wheat leaves. Physiological Plant Pathology 1, 185-198. BENNETT,J., STEINBACK, K. E. & ARNTZEN, C. J. (1980). Chloroplast photoproteins: regulation of excitation energy transfer by phosphorylation of thylakoid membrane polypeptides. Proceedings of the National Academy of Sciences 77, 5253-5257. BRADBURY, M. & BAKER, N. R. (1981). Analysis of the slow phases of the in viva chlorophyll fluorescence induction curve. Changes in the redox state of photosystem II electron acceptors and fluorescence emission from photosystem I and II. Biochimica et Biophysics Acta 635, 542-551.
Photosynthesis
8. 9.
10. 11. 12. 13. 14.
15.
16. 17. 18. 19. 20.
21. 22. 23. 24.
25. 26. 27.
28.
barley
419
B. B., HUTCHINSON, S. W., MAGYAROSY, A. C. & MONTALBIM, P. (1981). Photosynthesis in healthy and diseased plants. In Effects of Disease on the Physiology of the Growing Plant, Ed. by P. G. Ayres, pp. 13-28. Cambridge University Press, Cambridge. DALY, J. M. (1976). The carbon balance of diseased plants. In Encyclopedia of Plant Physiology, Vol. 4, Ed. by R. Heitefuss & P. H. Williams, pp. 450-479. Springer-Verlag, Berlin. FARRAR, J. F. (1980). Allocation of carbon to growth, storage and respiration in the vegetative barley plant. Plant, Cell B Environment 3, 97-105. HABESHAW, D. (1979). The effect of foliar pathogens on the leaf photosynthetic carbon dioxide uptake of barley. In Photosynthesis and Plant Development, Ed. by R. Marcelle, H. Clijsters & M. van Pouche, pp. 355-373. Dr W. Junk, The Hague. HORTON, P. & BLACK, M. T. (1980). Activation of adenosine 5-triphosphate induced quenching of chlorophyll fluorescence by reduced plastoquinone. FEBS Letters 119, 141-144. HORTON, P. & BLACK, M. T. (1981). Light dependent quenching of chlorophyll fluorescence in pea chloroplasts induced by adenosine 5i-triphosphate. Biochimica et Biophysics Ada 635,53-62. HORTON, P., ALLEN, J. F., BLACK, M. T. & BENNETT, J. (1981). Regulation of phosphoregulation of chloroplast membrane polypeptides by the redox state of plastoquinone. FEBS Letters 125, 193-6. KRAUSE, G. H. (1973). The high-energy state of the thylakoid system as indicated by chlorophyll fluorescence and chloroplast shrinkage. Biochimica et Biophysics Acta 292, 715-28. KRAUSE, G. H., BRIANTIS, J. M. & VERNOTTE, C. (1980). Two mechanisms of reversible fluorescence quenching in chloroplasts. In Proceedings of the 5th International Congress on Photosynthesis, International Science Services, Jerusalem. LAVOREL, J. & ETIENNE, A. L. (1977). In vivo chlorophyll fluorescence. In Primary Processes of Photosynthesis, Ed. by J. Barber, pp. 203-268. Elsevier, Amsterdam. LILLEY, R. McC., FITZGERALD, M. P., RIENITS, K. G. & WALKER, D. A. (1975). Criteria of intactness and the photosynthetic activity of spinach chloroplast preparations. New Phytologist 75, l-10. MIRANDA, V., BAKER, N. R. & LONG, S. P. (1981). Limitations of photosynthesis in different regions of the Zea mays leaf. New Phytologist 89, 179-80. MITCHELL, D. T. (1979). Carbon dioxide exchange by infected first leaf tissues susceptible to wheat stem rust. Transactions of the British Mycological Society 7’2, 6>68. NESTERENKO, T. V. & SID’KO, F. YA. (1980). Induction of fluorescence in wheat leaves during their ontogenesis. Soviet Plant Physiology 27,26246. OWERA, S. A. P., FARRAR, J. F. & WHITBRWD, R. (1981). Growth and photosynthesis in barley infected with brown rust. Physiological Plant Pathology 18, 79-90. PAPAGEORGIOU, G. (1975). Chlorophyll fluorescence: an intrinsic probe of photosynthesis. In Bioenergetics of Photosynthesis, Ed. by Govindjee, pp. 3 19-37 1. Academic Press, London. PLESNICAR, M. & BENDALL, D. S. (1973). The photochemical activities and electron carriers of developing barley leaves. Biochemical Journal 136,80>812. RAGGI, V. (1980). Correlation of CO* compensation point (7) with photosynthesis and respiration in rust-affected bean leaves. Physiological Plant Pathology 16, 19-24. RAVEN, J. A. (1976). Transport in algal cells. In Encyclopedia of Plant Physiology, Vol. 2A, Ed. by U. Luttge and M. G. Pitman, pp. 1299187. Springer-Verlag, Berlin. THORNE, J. H. & KOLLER, H. R. (1974). Influence of assimilate demand on photosynthesis, diffusive resistances, translocation, and carbohydrate levels of soybean leaves. Plant Physiology 54, 20 l-207. WALKER, D. A. (1981). Secondary fluorescence kinetics of spinach leaves in relation to the onset of photosynthetic carbon assimilation. Planta 153,273-278. WHIPPS, J. M. & LEWIS, D. H. (1981). Patterns of translocation, storage and interconversion of carbohydrates. In Effects of Disease on the Physiology of the Growing Plant, Ed. by P. G. Ayres, pp. 4783. Cambridge University Press, Cambridge. WYNN, W. K. (1963). Photosynthetic phosphorylation by chloroplasts isolated from rust-infected oats. Phytopathology 53, 1376-l 377.
6. BUCHANAN,
7.
in rust-infected
Plant Pa.thology
(1983)
23, 411419
Photosynthesis and chloroplast functioning of barley infected with brown rust I. AHMAD, J.F. FARRAR andR. School of Plant Biology,
University
(Accepted for publication
July
in leaves
WHITBREAD
College of North
Wales,
Bangor,
Gwynedd
LL57
.?lJW
1983)
The rate of net photosynthesis is reduced in leaves of barley infected with brown rust. This reduction is not due to reduced carbon dioxide fixation per chloroplast, but is ascribed to a decrease in the number of functional chloroplasts. Chloroplasts isolated from diseased leaves show increased contents of starch and phosphorus, and unaltered rates of ferricyanidedependent oxygen evolution, as compared with controls. Fluxes through the phosphate translocator on the chloroplast inner envelope are shown to be higher in diseased leaves. The kinetics of in oivo chlorophyll fluorescence induction are described, and it is shown that whilst they change with leaf age in control leaves, in diseased leaves the juvenile kinetics are retained. These data are interpreted as showing that each surviving chloroplast in diseased leaves is functioning at least as well as those from healthy leaves.
INTRODUCTION
Like many foliar diseases, rust infection causes a reduction in rates of net photosynthesis [7, 9, 181. Several causes for this reduction have been proposed : destruction of green leaf area [20], increased fungal respiration [23], increased host respiration [see 71 and inhibition of the light reactions of photosynthesis [6]. Whilst these are not necessarily mutually exclusive, there is no assessment of their individual contributions to the decline in net photosynthesis. However, it has been suggested that gross photosynthesis per unit chlorophyll-and thus per chloroplast-is enhanced in rusted tissue [20], and this would seem incompatible with partial inhibition of photophosphorylation and reductant generation. Further, a suggestion that carbon fixation by chloroplasts is reduced as a result of lowered host cytoplasmic inorganic phosphate (Pi) levels [27] would also be incompatible with the idea of increased gross photosynthesis by each surviving chloroplast. This paper seeks to examine the functioning of chloroplasts of leaves of barley infected with brown rust, both in viva and in vitro, to test the hypothesis that rust infection causes impairment of chloroplast metabolism, and to see if such impairment could be the whole or partial explanation for the rust-induced decrease in net photosynthesis. MATERIALS
AND
METHODS
Plants of barley [Hodwn distichum (L.) L am. cv. Maris Mink] were grown and when 10 days old their fully-expanded first leaves were infected with urediniospores of brown rust (Puccinia horde’ Otth.), as described previously [20]. 00484059/83/060411
+ 09 303.00/O
0
1983 Academic
Press Inc.
(London)
Limited
412
I. Ahmad
et al.
Leaf gas exchange was measured as before [ZO] with the addition of an A.D.C. infra-red gas analyser for water vapour. The temperature of the leaf surface was measured with copper-constantan thermocouples, which were also used in a thermocouple psychrometer to measure the water vapour content of the air supplied to the leaf. For measurement of in vivo chlorophyll fluorescence, intact plants were removed from the growth chamber at times between 5 and 8 h into the photoperiod and held in the dark at 23 “C for 1 h. Leaves were then detached and immediately placed with the middle of the upper surface under the fluorescence probe of a fluorimeter (Richard Brancker Associates model S.F. 10) and irradiated with red light (maximum emission at 670 nm) at 5.9 pm01 quanta m-2 s-l for 100 s whilst recording the fluorescence at wavelengths longer than 710 nm. The typical fluorescence curve, analysed following references [17, 29,211, is shown in Fig. 1. Isolated chloroplasts were prepared by a method similar to that used by Plesnicar & Bendall [ZZ]. At 4 “C leaves were cut into small pieces and disintegrated with a homogenizer for 3-5 s in a medium containing (in mol m-3) sucrose, 400; HEPES, 50; NaCl, 10; MgCl,, 1; EDTA, 1; cysteine, 4.1; 0.25% bovine serum albumin (BSA) (Fraction V) ; 1y0 (w/w) polyethylene glycol (PEG) (average mol. wt. 4000)) adjusted to pH 8.0. The homogenate was squeezed through two layers of muslin. The filtrate was centrifuged at 100 r min-l to remove urediniospores, and the plastids after sedimenting by centrifuging at 4000 rmin-1 for 5 min, washed and resuspended in a medium similar to the above, but with PEG and cysteine omitted and BSA at 1.25%. The total time for extraction was about 15 min. When examined by phase contrast all the chloroplasts appeared as Class A (highly reflective, bright opaque appearance, presence of a halo). However, determination by ferricyanide-dependent oxygen evolution before and after an osmotic shock showed that there were only 50% Class A chloroplasts [16].
0
20
40
60
00
100
Time(s)
FIG. 1. The kinetics following pre-darkening induction process in the duction period, attained F,,, variable component within 5 ms of the onset
of in uivo fluorescence induction. Red light is turned on at zero time the leaf for 1 h. The labelling OIDPSMT refers to components of the scheme of references 1.21, 261. F,,. maximal fluorescence during inat either I or P; F,,, stationary level of fluorescence attained at T; of fluorescence (= F,,, - F,,) ; F,, initial level of fluorescence, attained uf illumination.
Photosynthesis
in rust-infected
413
barley
The total chlorophyll in chloroplast preparations was measured following reference [Z]. The Hill reaction rate of isolated chloroplasts was determined by measuring ferricyanide-dependent oxygen evolution following reference [26] using an OX-15259 Clark oxygen electrode in conjunction with a Gilson K-1C oxygraph. Phosphorus in the preparation was determined by the ammonium molybdate method using stannous chloride as reducing agent [I] and starch was determined as glucose in amyloglucosidase-treated extracts [8]. The number of chloroplasts in the preparation was counted using a haemocytometer. Values presented in figures and tables are the means of three to five replicates, together with the standard error of the mean. RESULTS Gas exchange of intact leaves
Rates of net photosynthesis and transpiration fell following inoculation with brown rust, with the fall in transpiration lagging about 2 days behind that of net photosynthesis. A large and significant difference in net photosynthesis of diseased leaves was seen 6 days after infection. By the day of sporulation, net photosynthesis was about one-third, and transpiration one-half, of control rates (Fig. 2).
A 0
2
4
6
8
12
2
Days after
inoculation
IO
4
6
8
IO
12
FIG. 2. Net photosynthesis (a) of control (0) and rust-infected (0) first leaves of barley, with time after inoculation, and transpiration (b) of rust-infected leaves (expressed as o/0 control values; mea” rate for controls, 1.35 &to.16 mmol water m-* s-l). Conditions: 2O”C, 210 pmol quanta m-2 s-l over the waveband 400-700 nm; aerodynamic resistance 0.15 s cm-‘; vapour pressure deficit, 810 Pa. The vertical bars denote one standard error each side of the mean, and the arrow indicates the day of sporulation. TABLE
1
Phosphorus content [mmol P (g chlorophyll)-‘], Hill reaction rate [pal 0, (g chlorophyll)ml s-l] and starch content [mmol glucose (g chlorophyll) -I] of chloroplasts isolated from Jirst leaves of barley 8 days after infection with brown rust Treatment
Phosphorus
Hill
reaction
Control Rusted
1.02 k 0.06 2.83 & 0.39
123.5 + 10.7 160.2 + 11.8
Starch 24.8 f 4.8 79.4 * 20.7
5.76 3.82
1.19 x IO” 0.79 x 10”
Control Rusted
9.3 8.3
Reference
Gross (p&d [ZO]
3.1 2.8
(3) x 0.33
(4) Gross photosynthesis (pm01 DHAP m-* leaf s-l)
through the phosphate trandocator
(3) photosynthesis co, rnd leaf s-l)
of thejlux
2
\
I
0.54 0.73
(4)1(2)
DHAP translocation ( pmol m -* chloroplast envelope s-l)
rate
\
,
chloroplast
0.54 0.73
of triose
(5) Assuming 1 :1 exchange of Pi and DHAP
Pi translator (pm01 Pi m-* envelope s-l)
The flux is calculated from the area across which it occurs, this being the total surface area of chloroplasts, and dividing this into the amount phosphate leaving the chloroplast in unit time, this being taken as one-third the rate of gross photosynthesis expressed in moles of carbon dioxide. Calculations for the phosphate-DHAP translocator on the chloroplast inner envelope are made for the day of sporulation, 8 days after infection.
(1) x Area of ohlate spheroid with axes of 1 and 2.5 pm
area
Measured
Chloroplast surface (m* m-* leaf)
origin
no.
Chloroplast (m -* leaf)
(2)
Parameter
(1)
Calculation
TABLE
Photosynthesis
in rust-infected
415
barley
on chlorqtdads in vitro Chloroplasts isolated from infected leaves on the day of sporulation showed rates of ferricyanide-dependent oxygen evolution as high as those from healthy leaves (Table 1). Their starch and phosphorus contents were both higher on this day (Table 1). The number of chloroplasts per unit area in diseased leaves was two-thirds of that in controls (Table 2).
Measurments
In vivo chlorophyllfiorescewe The time course of fluorescence was examined at various times following inoculation. Typical curves are shown in Fig. 3. It can be seen that the rusted leaves maintained an essentially similar pattern of fluorescence throughout the 13 days of the experiment. By contrast, the control leaves showed a steady rise in magnitude of the peak P at about 15 s after induction, until this eventually exceeded peak I in height. These changes are quantified in Fig. 4, where two parameters (normalized fluorescence rise and normalized quenching) derived from such curves are plotted. As leaves aged, normalized fluorescence rise showed no significant change in rusted leaves, but a large fall in controls. Similarly normalized quenching capacity dropped to one-third of its initial value in control leaves but was maintained in leaves infected with brown rust until 12 days after infection. Differences between control and rusted leaves were not apparent until 8 days after infection. The low peak occurring at about 60 s after irradiation began was detectable in both control and rusted leaves until 7 days after inoculation; thereafter this peak (labelled M in Fig. 1) was absent.
(0)
(b)
8 7
I 50
I
I
I 100
I
I
50 Time
I
Control, In day of moculotlon
I
100
I
0
I
50
I
1
100
(s)
FIG. 3. Fluorescence induction curves for control (a) and rust-infected (b) first leaves of barley at various times after infection with brown rust. The vertical axis is in arbitrary units; see the legend to Fig. 1 for identifications for parts of these curves.
I. Ahmad
416
0
4
8
et al.
12 Days after
moculatlon
FIG. 4. Changes in (a) normalized fluorescence rise, F, [ = (F,,, - F,,)/F,] and (b) normalized quenching capacity, F, [ = (F,,, - F,,)/F,,] of control (0) and infected (0) first leaves of barley with time after inoculation with brown rust. The vertical bars denote one standard error each side of the mean.
DISCUSSION The depression in net photosynthesis reported here is similar to that reported previously for rust disease (see references in Introduction). It is not explicable in terms of altered water relations, since the reduction in transpiration occurred after the reduction in photosynthesis. Whether transpiration falls due to increased resistance to water flux in the leaf, or to lowered leafwater potential, any effects these mechanisms might have on carbon dioxide fixation will be superimposed on a reduction in net photosynthesis which is already large. It seems clear that the major fall in photosynthesis is due to destruction of chloroplasts within developing lesions, since the fall in photosynthesis roughly parallels a fall in leaf chlorophyll content [ZU], and we report here a much lower chloroplast number per unit area. An analytical approach, considering carbon dioxide fluxes in photosynthesis as a diffusion process, has shown that allowing for such destruction of photosynthetic machinery there may still be effects at the level of mesophyll resistance-that gross photosynthesis per unit of chlorophyll may be increased by rust infection [ZO, and see Table 21. This contrasts with other suggestions that the effects of rusts in depressing photosynthesis are largely located in the chloroplast. We have shown in this paper that chloroplasts freshly isolated from rusted leaves do not show impaired rates of ferricyanide-dependent oxygen evolution (the Hill reaction), lending credence to the idea that chloroplasts in rusted leaves can operate as well as control chloroplasts. Similarly, Wynn [.28] showed no reduction in photophosphorylation by chloroplasts isolated from rust-infected oats. The findings of Buchanan et al. [6] are apparently quite different: that non-cyclic electron transport is inhibited up to 45% in chloroplasts isolated from infected leaves. These workers themselves caution against the generalizing of results between diseases, or even different stages in the development of a single host-pathogen combination. A quite different explanation for reduction in gross carbon dioxide fixation per chloroplast is that if biotrophic fungi accumulate Pi from host tissue, the concentration the of Pi in the cytoplasm of the host will fall [27]. Th is will reduce the flux through
Photosynthesis
in rust-infected
barley
417
phosphate translocator at the chloroplast inner membrane, due to lowered availability of Pi for import. Less reduced carbon will therefore be exported from the chloroplast, and, as a result, the rate of photosynthesis will fall and carbon within the chloroplast will be diverted to starch rather than used in the formation of dihydrooxyacetone phosphate (DHAP) for export. This attractive hypothesis is not strongly supported by the present data, as although we find the predicted and usual increase in chloroplast starch level [see 271 we also find increased levels of phosphorus in chloroplasts from rusted leaves, hardly compatible with the idea of phosphorus-starved chloroplasts. It is possible that our extracted chloroplasts were contaminated with polyphosphate bodies [3], giving an artificially high phosphorus content, but careful washing did not change their phosphorus status. Perhaps also applicable to starchenriched diseased leaves is the hypothesis that large starch granules increase the diffusion path for carbon dioxide into chloroplasts, and that this is the direct cause of decreased gross photosynthesis [Z]. However, neither this nor the phosphatestarvation hypothesis are consistent with the suggestion of increased photosynthesis per surviving chloroplast. In passing, it can be calculated that the flux through the phosphate translocator is greater in diseased than in healthy leaves (Table 2), and that it is approaching the highest rates recorded for anion transport across membranes [24]. We suggest that such high rates may only be sustainable if the Pi concentration is high enough inside the chloroplast to permit high DHAP concentrations to be realized, which could also result in increased starch synthesis, as suggested by Whipps & Lewis [27], without a concomitant decrease in carbon dioxide fixation. ,411 of the above evidence is, of course, open to criticism. That individual chloroplasts perform better in rusted than in control leaves, and that there is a greater Aux through the phosphate translocator, are deductions from gas-exchange measurements. That chloroplasts from rusted leaves show Hill reaction rates comparable to those from controls, and that their phosphorus content is increased, could be attributed to changes during their isolation. Clearly, a direct, in vivo, inspection of chloroplast functioning is needed. Here we present the results of such an inspection on intact leaves using chlorophyll fluorescence kinetics. The secondary fluorescence rise, the S-M -IT transition, shows no differences between control and rusted leaves. This rise may be linked to the onset of photosynthetic carbon metabolism, and can be abolished by agents that sequester Pi [2rS]. The lack of a rust-dependent abolition of the M peak therefore argues against the hypothesis that cytoplasmic Pi is lowered by rust infection. Changes in fluorescence kinetics were seen for the earlier peaks at I and P, and further discussion concerns parameters derived from them. We follow Miranda et a/. [17] in deriving the fluorescence rise (F,,, ~ F,) and fluorescence quenching F,J, as they have physiological meaning; they are normalized on F, as F mm fluorescence is partially dependent on chlorophyll content, and then termed F, (fluorescence rise) and F, (fluorescence quenching), respectively. Most of the fluorescence emanates from chlorophyll in photosystem II [1.5, 211. Thus F, indicates the reduction of electron acceptors between photosystems II and I, which produces a reduced electron flow through the photosystem II reaction centre and thus an increase in variable fluorescence [17]. It, therefore, is a direct indicator of the state of photosystem II activity.
I. Ahmad
418
et al.
F, has three major causes. First it arises from the reoxidation of the primary electron acceptor ofphotosystem II, Q[.5,27] ; the rate of reoxidation of Qis dependent on the rate of electron flow from the secondary electron acceptors of photosystem II through photosystem I to NADP [27]. Second, the fluorescence is also quenched because of the high energy status of thylakoid membranes [Z.%151. The third cause of F, is the phosphorylation of chloroplast membrane proteins by ATP [4, 20, 12, 261. The latter two causes result from the creation of a light-induced electrical potential difference, and proton and Mg2+ concentration gradients, across the thylakoid membranes [1.5,2 11. Thus F, is a direct indicator of the ability of thylakoids to generate electrochemical gradients across their membranes and stimulate ATP production [17]. Roth F, and F, remained steady in rusted leaves during the experiment. On the other hand, in control leaves both fell considerably below the levels in rusted leaves. Thus there is a strong indication that while in control leaves the activity of photosystem II, the ability of thylakoids to generate electrochemical gradients across their membranes, and ATP production, are decreasing with age, in rusted leaves the photophysiological processes do not show any inhibitory effect. It is as if chloroplasts in rusted leaves retain their juvenility, whilst those in control leaves begin to age. This is support for the idea that green island formation does not involve re-greening, but simply the retardation of senescence in areas adjacent to the biotroph. It may not be necessary to involve any special effect of the fungus on chloroplast senescence; it may simply be that the sustained higher carbon fluxes through individual chloroplasts in diseased leaves results in maintained juvenility. It is also strong support for the idea that chloroplasts in rusted leaves are functioning at least as well as those in healthy leaves, and that reduced gross photosynthesis is simply due to destruction of chloroplasts. All the data in this paper refer to average values: it is quite possible that the chloroplast population in diseased leaves is highly heterogeneous, some chloroplasts showing grossly impaired metabolism. Clearly, more precise examination of discrete areas in and around a lesion would be desirable. The widespread belief that a general increase in membrane permeability follows infection with a biotroph would also seem in need of reassessment, and this forms the subject of a forthcoming paper. I.A. gratefully acknowledges the receipt of a Sir William Roberts scholarship. REFERENCES I. ALLEN, S. 2.
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6. BUCHANAN,
7.
in rust-infected