Photosynthetic Shutdown in Chlorella NC64A Paramecium bursaria

Plant Physiol. (1995) 108: 1431-1438

Photosynthetic Shutdown in Chlorella NC64A Associated with the lnfection Cycle of Paramecium bursaria

Chlorella Vi r u s - 1

Ceorge G. R. Seaton, Kit Lee, and Jan Rohozinski*

Research School of Biological Sciences, lnstitute of Advanced Studies, The Australian National University, Canberra, ACT 0200, Australia (G.G.R.S., J.R.); and School of Biological Sciences,

University of Nebraska, Lincoln, Nebraska 68588-01 18 (K.L.)

l h e effects of the algal virus Paramecium bursaria Cblorella virus-1 on the photosynthetic physiology of its host, Cblorella NC64A, was studied by observing changes in Chl fluorescence quenching and O2 exchange. Metabolic changes were calibrated against electron microscopic analysis of the morphological changes that occur during the infection cycle. I t takes approximately 1 0 h from attachment of the virus to final lysis of the host cell, so a complete infection cycle can be observed continuously in one experiment. During the early stages of the infection cycle many rapid changes occurred in the host cell’s metabolism and these were reflected in changes of photosynthetic and respiratory rates. The dramatic inhibition of photosynthesis in Cblorella NC64A cells by P. bursaria Chlorella virus-1 has facilitated the use of fluorescence quenching as an accurate measure of the first phase of viral infection (attachment and penetration of the host cell) and the extent to which a population of host cells is infected. Effects of temperature and cation requirement of the infection cycle are described. The relevance of our observations to the events observed during viral infection of higher plants is discussed.

Photosynthetic efficiency reflects the metabolic and physiological status of chloroplast-containing cells. Envi- ronmental stresses that directly or indirectly affect the functioning of PSII can lead to characteristic changes in fluorescence quenching. This phenomenon has been used for large-scale analysis of photosynthetic efficiency in veg- etation using sunlight- or laser-induced fluorescence detected from the ground or aircraft (Krause and Weis, 1991). Chl fluorescence has been used to assess the photosynthetic performance of field crops in the United Kingdom (Baker et al., 1989), the United States (Schroeter et al., 1992), and Sweden (Oquist and Huner, 1991). However, before fluorescence can be used to study the effect of viral pathogens on a large-scale a model of the effects of the viral replication cycle on photosynthesis needs to be created.

Infection of plants with viruses often leads to a variety of leaf symptoms such as vein clearing, light green or dark green mosaics, and light green or yellow mottling, a11 of which imply alteration of photosynthetic function and changes in the balance of photosynthetic and nonphotosyn-

* Corresponding author; e-mail [email protected]; fax 61- 6 -279 - 8056.

thetic pigments in the infected leaf cells. Direct evidence that photosynthetic function is affected by virus infection has come from fluorescence studies of plants infected with tobacco mosaic virus (Hodgson et al., 1989; Reinero and Beachy, 1989; Osmond et al., 1990; Van Kooten et al., 1990; Balachandran et al., 1994) and peanut green mosaic virus (Naidu et al., 1984a, 198413). There are, however, many limitations to studying the effects of virus infection on photosynthesis in higher plants. These include the inability to obtain a synchronous infection in leaf cells, which results in the cells within a leaf showing symptoms of various stages of the virus infection cycle. Thus, even when sys- tematically infected leaves are used for studying the effects of viral infection, early events of the infection cycle that occur in the leaf initials and during leaf expansion cannot be easily studied.

Many of these limitations can be overcome by using a single-celled alga as a model system for leaf cells in higher plants. The Cklorella strain NC64A is an ex-symbiont from Paramecium bursaria and has a11 the morphological features of eukaryotic plant cells except a vacuole (reviewed by Van Etten et al., 1991). Cklorella NC64A is host to a group of viruses (Van Etten et al., 1983a) assigned to the family Phycodnaviridae (Francki et al., 1991), which are polyhe- dral, 150 to 190 nm in diameter (Van Etten et al., 1982), and have a linear nonpermuted double-stranded DNA genome of about 330 kb with covalently closed hairpin ends (Girton and Van Etten, 1987; Rohozinski et al., 1989; Zhang et al., 1994). The type member of this virus group, PBCV-1, was used to study the effect of virus infection on photosynthetic quantum efficiency in algal cells. Cklorella cells and their associated viruses offer many unique advantages over using leaves or protoplasts from higher plants to study plant- virus interaction at the physiological and biochemical levels. These advantages include the ability to obtain

Abbreviations: Aqp, change in qp; At, change in time; Fm, max- imal fluorescence when a11 PSII reaction centers are closed during actinic illumination; F,, initial fluorescence; F’,, minimal fluorescence when a11 PSII reaction centers are open after removal of actinic light; PBCV-1, P. bursaria Chlorella virus-1; pfu, plaque forming units; QA, primary electron accepting plastoquinone of PSII; qN, nonphotochemical quenching; qp, photochemical quenching.

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1432 Seaton et al. Plant Physiol. Vol. 108, 1995

synchronous infection of a large number of cells, a rapid infection cycle, and ease of manipulation because of Cklor- ella cells’ resistance to mechanical damage.

MATERIALS A N D METHODS

Alga and Virus Culture

Production and purification of PBCV-1 and the growth of its host, CIzlorella NC64A, on modified Bold’s basal medium was done as previously described (Van Etten et al., 198310). Cells were grown at 18°C under a constant photon flux density of 50 pmol m-’ sP1 in the absence of tetracycline to ensure fully functional chloroplasts. As cultures ap- proached the stationary phase, variable fluorescence/max- imal fluorescence was monitored as a measure of photosynthetic competence. Fresh cultures of algal cells containing about 2 X 107 cells mL-’ were harvested by centrifugation at 1000 rpm for 4 min in a Heltich (Tuttlin- gen, Germany) model30RF centrifuge using a model 1424 swinging bucket rotor. Cells were subsequently resus- pended in air-saturated distilled water at a concentration of 8 X 107 cells mL-’ and used for fluorescence measurements. Virus preparations were titered and the number of pfu were calculated according to the method of Van Etten et al. (1983a).

Photosynthesis Measurements

Simultaneous changes in Chl fluorescence and O, evolution were recorded using a Hansatech (Kings Lynn, Nor- folk, UK) DW3 liquid-phase chamber with O, electrode (Sl) and control box (CB1-D) coupled to a PAM 101/103 modulated Chl fluorimeter with polyfurcated fiberoptic and KL1500 electronic flash lamp (H. Walz, Effeltrich, Ger- many). When measured alone, Chl fluorescence was recorded using a DW2/2 chamber (Hansatech) coupled to a PAM 101 /103 fluorimeter (H. Walz). The automated methods of Seaton and Walker (1992) based on those described by Schreiber (1986) and Schreiber et al. (1986) were used for data acquisition. The fluorescence nomenclature used is that of Schreiber and Bilger (1993). The liquid-phase chamber was loaded with 16.5 or 2 mL of cells, depending on the chamber used, in air-saturated water containing 1.5 mM MgCl, (unless otherwise stated) and held at 25°C (unless otherwise stated) with a mode19100 refrigerated circulator (Fisher Scientific) during measurement. All salt and temperature treatments were started before illumination. Prior to infection the cells were allowed to equilibrate for 20 min illuminated with red light at 20 pmol photons mP2 sP1 supplied by a light-emitting diode source (Hansatech LCl- LH36-LS2/FO). Virus infection was achieved using a saturating level of virus (0.75 pfu/cell).

The experiment involving the simultaneous measurement of O, rates and fluorescence was designed to main- tain a relatively constant O, level in the chamber (DW 3) under computer control, thus preventing either supersatu- ration or anaerobiosis. Prior to measurement the O, concentration in the chamber was reduced to approximately 50% of that of air-saturated water. During the first 4 h postinfection, when photosynthetic O, evolution rates

were high, the cells were exposed to alternating light and dark periods to measure dark respiration. Dark intervals during this period were not less than 4.5 min, permitting the measurement of F’, without far-red light. After 4 h, the net O, evolution rate in the light was approximately zero; therefore, continuous illumination was used to monitor the terminal stages of infection, except following a saturating pulse when a 20-s dark period was used for F’ , estimation. During the latter stages of the experiment (8-10 h), when the net O, exchange rate was negative, the chamber was periodically opened and bubbled with air to prevent anaerobiosis.

EM

Samples for EM were prepared and treated using the method developed by K. Lee (Meints et al., 1981). Sections were examined with a Phillips (Eindhoven, The Nether- lands) 201 transmission electron microscope at 60 kV.

Pigment Analysis

Cell pigments were measured from 0.5-mL aliquot samples removed from the chamber and sequentially extracted in 150 pL of methanol, followed by 150 pL of 100% acetone and then 200 p L of 80% acetone. Pigments were measured using the HPLC method and solvents A and B described by Gilmore and Yamamoto (1991). Extracted samples were separated on a Spherisorb ODSl column (Alltech Associ- ates, Sydney, Australia) at a flow rate of 1 mL min-l with a 20-pL injection volume.

RESULTS A N D DISCUSSION

Virus Effects on qp

q, is inversely related to the reduction status of QA. Assay conditions were chosen so that only about 5% of the QA pool was initially reduced (qF - 0.95), thus maximizing the sensitivity of the system. The basic response of qp to virus infection involves a short period during which q, is maintained at the high, preinfection level followed by a rapid decrease to a steady minimum value (Fig. 1). The initial period is interpreted as a lag phase, representing the time taken for attachment and penetration of the host cell by the virus. The subsequent rapid decrease in qF, reflecting a rapid increase in the reduction status of QA, parallels the establishment of virus infection within the host. Two parameters have been selected to describe events occurring during the early part of the infection cycle. The first parameter is At (Fig. l), which is the time taken from addition of virus to the algal cell suspension to the start of the second phase of the infection cycle. By observing the effect of various treatments on A t we can study vira1 attachment and penetration. The second parameter is Aq, (Fig. l), which is the difference between q p at the time of infection and the q, level after the initial decrease has stabilized. In any single batch of host cells, Aqp is dependent on the number of cells infected. When saturating levels of virus are used, Aq, reflects the response of the second phase of

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Virus-Associated Photosynthetic Shutdown in Chlorella 1433

-a 1.0 o- v

0.8 .- 1 o (I:

2 0.6 o-

-4 0.4 -

I I I I I

8 0.2

o I I I I I

O 10 20 30 40 50 60 0.0 I

Time from inoculation (minutes)

Figure 1. Detailed analysis of changes in the q, during the 1st h of viral infection. Alga1 cells were incubated at 25°C in the presence of 1.5 mM MgCI, in a volume of 2 mL and a cell concentration of 8 X 107 cells mL-’. A saturating amount of virus (0.7 pfu cell-’) was added at O time, after the cells had been preincubated for approximately 20 min in red light at 20 ymol quanta m-2 s-’. Changes in fluorescence were recorded using a Hansatech liquid-phase chamber (DW2/2) and auxiliary equipment coupled to a PAM 101 modulated Chl fluorimeter (Walz) using the methods of Seaton and Walker (1 992). Parameters used to indicate the various stages of the infection cycle (Aq, and At) are indicated. The virus attachment and penetration phase of the infection cycle corresponds to At, and the second phase of rapid virus-induced changes in cell metabolism are indicated by Aq,.

virus infection to environmental parameters, e.g. temperature (see below).

Direct Relationship between Virus and Aqp

To verify that the observed Aq, is directly related to virus infection, the relationship between the amount of virus added and Aqp had to be established (Fig. 2). Aq, is maxi- mized when saturating levels of virus are added to NC64A cells. Use of subsaturating levels of virus results in lower AqF values, since uninfected cells show no change in q,. Thus, the observed Aq, is proportional to the number of cells actually infected. The Aq, response is a curve because more than one virus particle can attach per cell and saturation is observed at a multiple of infection of approximately 0.63 pfu per cell. In fact one would expect infectivity to follow a Poisson distribution (Ellis and Delbriick, 1939) with saturation at about 3 pfu per cell. This experiment suggests that the plaque assay is inherently inefficient and the number of viable virions is underestimated by the plaque assay. Similar inefficiencies in the plaque assay of bacteriophages have been reported, with the efficiency of plating varying from 0.3 to 0.5 (Ellis and Delbriick, 1939). No significant change in A t was observed over the virus concentration range used (data not presented). Further evidence that the change in q, is related to virus and not some other component was obtained by adding inactivated virus to the cell culture. When virus disrupted by heating or freezing was added, no (or little) Aq, was observed (Fig. 3A).

Changes in Light Utilization, O, Balance, and Cell Morphology during the lnfection Cycle

The relationship between photosynthetic quantum efficiency and virus infection was elucidated by correlating the photosynthetic response of the algal cells with cytological changes observed during the complete infection cycle. Si- multaneous changes in modulated Chl fluorescence quenching parameters and O, rates were observed for up to 12 h following infection of NC64A cells with saturating levels of virus (Figs. 1 and 4).

Since PBCV-1 attaches rapidly and irreversibly to the algal cell wall (Van Etten et al., 1991), it was assumed that viral infection of the cells is essentially synchronous. A detailed analysis of the photosynthetic response of the algal cells during the first 60 min following virus infection is shown in Figure 1. There is a lag phase during which no change in q, is observed, followed by a rapid decrease after which a steady minimum value is obtained. The depression in q, could be associated with the change from host protein and nucleic acid synthesis to synthesis of virus-encoded protein and nucleic acid and not merely the result of cell wall penetration.

Long-term changes in q,, fluorescence, F‘,, qN, and O, rate are shown in Figure 4. The infection cycle can be divided into two time periods with respect to these parameters. The early phase, which occurs from the time of virus infection (time = O) to 3 to 4 h postinfection, and late events, which occur from 4 h postinfection to cell lysis at 10 to 12 h postinfection.

Once infection is established there is a rapid decrease in qP followed by a steady state, which is maintained up to 3 to 4 h. The qp decrease is inversely proportional to the observed change in fluorescence (Fig. 4), which increases rapidly and remains steady up to about 4 h postinfection. This increase in fluorescence indicates an ”inefficiency” in electron transport on the acceptor side of PSII, resulting in the reduction of the QA pool. Associated with the qp decrease (fluorescence increase) is an increase in F’, (Fig. 4B), suggesting a reduced efficiency of exciton trapping by PSII

2 0.2 U

0.1

0.04 I I I I 1 0.0 0.2 0.4 0.6 0.8 1 .o

PFU cell-’ Figure 2. Relationship between the amount of virus added and Aq,. Experimental conditions were as described in Figure 1 . Virus titer expressed as pfu was calculated using the method of Van Etten et al. (1 983a).




o 0.2 s a,

methyl viologen added 40 min postinfection. - 0.2 - -

B

reaction centers from the light-harvesting pigments. Clearly the increased reduction of Q, has a concomitant effect on the trapping ability of PSII reaction centers. Al- though this could be explained by a virus-mediated inhibition of energy transfer from the antenna pigments to the PSII reaction center, we believe that the F' , increase is a direct result of the increased Q, reduction as described by the reversible exiton-radical pair equilibrium model of charge separation (Trissl et al., 1993; Dau, 1994). Also, the kinetics of qp reduction and the increase in both F ' , and F ' , are essentially identical, suggesting that one process is involved.

The long-term slow decrease in fluorescence that occurs during the later stages of infection (after 4 h postinfection) could be attributed to either a relieving of the inefficiency in PSII acceptor side electron transport or an increased dissipation of absorbed photons thermally rather than as fluorescence (Fig. 4B). The latter is supported given that qF remains low, reflecting a maintained QA reduction level, and there is a related quenching in F ' , and F',. In addition, although in the early stages F', increases, the final F ' , level is comparable to that of the dark-adapted F,, indicating that increased quenching rather than a relief of the inefficiency causes QA reduction. This could be caused by one of two things: first, increased antenna quenching of fluorescence due to a photoinhibitory response (Walters and Hor- ton, 1991) or, second, breakdown of the integrity of the photosynthetic apparatus due to virus intervention in repair/maintenance processes. This is reflected in the slow, steady increase in the level of qN from approximately 3 h postinfection, indicating increased thermal dissipation in the antenna pigments (Fig. 4B). The increase in qN is not due to xanthophyll conversion (Demmig-Adams and Ad- ams, 1992; Ruban et al., 1993), because there is no significant increase in the de-epoxidation state, nor can fluorescence quenching be attributed to wholesale Chl photo- oxidation, since the total Chl content decreases by only 6%

viologen added 6 min before infection; c, E

The final concentration of methyl viologen was 0.5 mM. C and D, Effects of cations and bicar- O 20 40 60 bonate on the infection cycle as measured by changes in qp. Concentrations used were 1.5

Unless otherwise indicated experimental condi-

A o,o

-NaCI -MgCI, -

mM MgCI,, 4 mM NaCI, and 5 mM NaCO,.

tions were as described in Figure 1. -

+NaCI

+MgCI,

0.2 - -

0.0 C

l5 1 10

0.0 O 30 60 90 120 150 1 .o

80 ?.

NaHCO, - virus 0.8 -

0.6

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-

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.-

.- - 2 Y

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O 2 4 6 8 1 0 1 2 1 4

Time (hours)

Figure 4. Analysis of virus-induced changes in photosynthesis during the complete virus replication cycle. Experimental conditions were as described in Figure 1, and 16.5 mL of cells were used in a DW3 chamber. A, Changes in qp (A), net O, evolution and consumption rate (A), and dark respiration rate (O). B: F (the actinic light-induced modulated Chl fluorescence), V; F0, +; Ffm, V; qN, O.



Virus-Associated Photosynthetic Shutdown in Cblorella 1435

from 4 to 10 h. PBCV-1 infection leads to a rapid degradation of the chloroplast and nuclear genome, which results in the loss of chloroplast- and nuclear-encoded protein synthesis (Van Etten et al., 1991). This loss of plant genetic information could possibly lead to an inability to repair the PSII core. Of particular importance are the chloroplast- encoded D1 and D2 proteins, which are normally rapidly turned over during photosynthesis and which form the reaction center (Geiken et al., 1992; Critchley and Russell, 1994). The presence of inactive PSII cores could result in an increase in the component of qN that relaxes slowly a ta rate that is dependent on protein synthesis (Walters and Hor- ton, 1991).

These changes in fluorescence quenching are reflected in changes in O, evolution (Seaton and Walker, 1992) and respiration. The rapid decrease in qp is directly related to a corresponding shutdown of photosynthesis and decrease in the rate of photosynthetic O, evolution (Fig. 4A). Respi- ration rate doubles between the healthy and infected state (Fig. 4A), and by 4 h postinfection the respiration rate exceeds the photosynthetic rate. At 10 h postinfection respiration decreases so that the net rate of change of O, level in the incubation medium increases to zero as the cells die and lysis occurs.

The large decline in qP following infection correlates with an increase in the reduction of QA, presumably because of photosynthetic limitations involving electron transport and/or carbon metabolism rather than a direct interaction between virus gene products and the photosystems. Fur- ther evidence supporting indirect interference with electron flow is gained by the addition of the PSI artificial electron acceptor methyl viologen. If methyl viologen is added to the medium after the qp decline following infection, q p recovers slowly and, if added prior to infection, the initial decline in qp is prevented (Fig. 3B). This indicates that electron transport from water to NADP via PSII and PSI is not directly affected by viral infection and the photosystems remain essentially intact.

To correlate changes in photosynthetic function and events that occur within virus-infected cells during the infection cycle, cells infected with virus were sampled throughout the infection cycle, fixed, and studied by transmission EM. Healthy cells show typical features of a eukaryotic cell with a membrane-bound nucleus and large cup-shaped chloroplast (Fig. 5A). At 5 min postinfection, capsids can be found in close proximity to the cell wall (Fig. 5B). By 15 min after infection, the virus particles are closely associated with the cell wall (Fig. 5C) and digestion of the cell wall is in progress (Van Etten et al., 1991). At 19 min (Fig. 5D) postinfection, some capsids are empty, indicating that the viral core material has been transferred into the host cells and the infection cycle has been initiated. After establishment of virus infection there is little change in the host cells; however, the nucleus is usually located peripherally (Fig. 5E). Aggregations of membranes within the cytoplasm are visible, indicating the formation of virus assembly centers. At 3 h, virus assembly centers are clearly detectable and empty capsids are present in the cytoplasm (Fig. 5, F and G). By 4.5 h, empty and filled capsids are

accumulating in the cytoplasm (Fig. 5G). Accumulation of virus in the host cells continues through 6 to 9 h (Fig. 5, H and I). Occasionally, degradation of the chloroplast inter- na1 structure can be encountered at 9 h. Cell lysis occurs between 10 and 12 h postinfection, and the cell sample fixed at 11.5 h shown in Figure 5J consists of broken cell debris and released virus particles.

The observed sequence of events clearly demonstrates that the lag phase shown in Figure 1 represents the time taken for the virus to attach to the host cell, penetrate the cell wall, and transfer viral core material into the host cell (Fig. 5, A to D). Following transfer, there is a large decrease in qP, and this decrease occurs at the same time as the induction of virus-directed RNA synthesis (Schuster et al., 1986) and detection of virus-induced DNA polymerase activity (Rohozinski and Van Etten, 1989). Newly synthe- sized viral DNA can be detected 1 h postinfection, with a large accumulation occurring by 4 h postinfection (Van Etten et al., 1984). The accumulation of viral DNA in the host cells is thus associated with the steady low level of photosynthetic function observed between the bottom of the qp decrease and approximately 4 h postinfection (Fig. 4A). During this time the virus assembly centers are established (Fig. 5, E and F), with virus assembly being established between 3 and 4.5 h postinfection (Fig. 5G).

The late phase of virus infection, which is characterized by a slow, steady decrease in fluorescence and qP (Fig. 4A), is associated with accumulation of virus within the host cells, which results in major changes in the cytoplasm as the cells fill with virus (Fig. 5, H and I). This process continues until approximately 10 h after infection, at which time respiration ceases (Fig. 4A) and cell lysis occurs (Fig. 5J).

lnfection by PBCV-1 Requires Metal lons

Previous work has shown that PBCV-1 does not attach to purified alga1 cell walls in the absence of metal ions (Meints et al., 1988). Figure 3C shows that when PBCV-1 is added to the host cell in the absence of metal ions no change in qp is observed, indicating the failure of the virus to establish infection. However, in the presence of 1.5 mM MgCI, or 4 mM NaCl, virus infection is established and changes in qp are observed (Fig. 3 0 . These data are consistent with those previously reported (Meints et al., 1988), and failure to establish infection in the absence of metal ions can be attributed to the inability of the virus to attach to the host cell.

Elevated CO, Does Not Prevent PBCV-1 -Mediated QA Reduction

Since air-saturated water was used as the incubation medium for our experiments, it is feasible that CO, could become rate limiting for carbon fixation, slowing photosynthetic electron transport and resulting in a depressed qP. This seems unlikely, however, because long-term uninfected controls do not demonstrate any qP change. The addition of 4 mM sodium bicarbonate (NaHCO,) had no substantial effect on the qF level in uninfected cells or on




Figure 5. Cytological changes that occur dur-ing the infection cycle. Cells were inoculatedwith a saturating level of virus and monitored forchanges in photosynthesis as shown in Figure 2.A small sample of cells was taken at varioustimes, fixed and embedded in plastic, sectioned,and examined by EM. A, Preinfection: healthycells showing nucleus (N) and chloroplast (Ch).Bar scale = 454 nm. B, At 5 min postinfection:virus particles associate with cells. Bar scale =83 nm. C, At 15 min: virus and cell in closeassociation, digestion of the cell wall occurs,and the inner core of the virus is transferred intothe cell. Bar scale = 83 nm. D, At 19 min: emptycapsid associated with the cell. Bar scale = 83nm. E, At 1.5 h: nucleus is peripherally locatedin the cytoplasm. Bar scale = 454 nm. F, At 3 h:viroplasm (V) is formed in the cytosol and emptycapsids begin to form. Bar scale = 454 nm. G,At 4.5 h: viroplasm is well formed and somefilled and empty capsids are present. Bar scale =135 nm. H, At 6 h: viroplasm is very large andthe cytoplasm is filling with virus particles. Barscale = 454 nm. I, At 9 h: cytoplasm is almostcompletely filled with virus particles. Bar scale= 454 nm. J, At 11.5 h: cell lysis and release ofvirus particles. Bar scale = 454 nm.



Virus-Associated Photosynthetic Shutdown in Chlorella

"."

the maximum Aqp in infected cells (Fig. 3D). This indicates that the observed change in qp is related to virus infection and CO, is not limiting in our system. The fact that there is sufficient CO, to sustain photosynthesis in the water that we used for our incubation medium suggests that the NC64A may have a C0,-concentrating mechanism similar to that reported for Chlamydomonas reinhardtii (Badger et al., 1978) and cyanobacteria (Miller et al., 1988).

O

Temperature Effects on lnfection lnitiation (At ) and Establishment (Aq,)

The effect of temperature on biochemical reaction rates within plant cells can directly alter the interaction between a virus and its host. A plant cell's response to viral infection, as measured by Aqp at different temperatures, is therefore a composite response to changes in the balance of photosynthesis, photorespiration, and metabolic changes induced by the viral replication cycle. An example of where this could cause complications with our observations is the case of Rubisco, where temperature affects the balance between photosynthetic carbon reduction (photosynthesis) and photorespiratory carbon oxidation (photorespiration). The effect of temperature sensitivity of Rubisco on qp was minimized by using nonsaturating light fluxes in our experiments so that only the effects of virus infection on 9,, relative to temperature were observed.

The insensitivity of qp to temperature change is indicated by the uninfected cells showing essentially similar qp values (again approximately 0.95) at various temperatures before infection (data not shown). The duration of the lag phase is temperature sensitive, with increasing temperature accelerating the rate of attachment and establishment of infection between virus and alga (Fig. 6A). This decrease in At with increasing temperature is biphasic in nature, suggesting that a component involved in the early attachment and penetration phase of tlie infection cycle under- goes a conformational change between 21 and 24°C. This is clearly demonstrated in an Arrhenius plot (Fig. 6B). Once infection is established, metabolic events that are observed as a rapid change in qF, the extent of which we measure as A9p, are relatively insensitive to temperature over the 12 to 24°C range. This point is illustrated by plotting Aqp against temperature (Fig. 6A). At 27°C and above, Aqp decreases significantly, indicating that events occurring during the establishment of infection within the host cell are also temperature sensitive and are correlated with the break in the At curve.

CONCLUSIONS

We report what to our knowledge is the first direct demonstration of early-event dynamics of virus interfer- ente with photosynthesis function. Virus infection leads to changes in electron transport and/or carbon metabolism. Evidently, viral encoded products do not interact directly with electron transport processes because qp can be re- stored by the artificial electron transport acceptor methyl viologen. We hypothesize that viral replication may mo- nopolize cytoplasmic carbon and/or phosphate pools, thus

a z

n n I A

1437

60

45

D - h

3 30 7

s 8 -

15

1 03/Temperature ('K)

Figure 6 . A, Effect of temperature on the virus infection cycle. Ex- perimental conditions were as described in Figure 1 . The time taken for attachment and penetration of the virus is given by At (B) and the photosynthetic response to initiation of the virus infection cycle within the cell is given by Aqp (O). B, Arrhenius plot of the relationship between A t and temperature.

interfering with the capacity of the alga to carry out normal photosynthesis (Walker and Sivak, 1986). A possible sink for phosphate is viral nucleic acid synthesis, which results in a 3- to 10-fold increase in the leve1 of DNA within the host cell5 h after infection (Van Etten et al., 1991). Reduced qP levels have previously been reported for young Nicotiana tabacum leaves, systemically infected with tobacco mosaic virus (Hodgson et al., 1989). Thus, our observations may have general implications for the effect of viruses in higher plant leaves because photoinhibition and loss of Chl could occur under conditions of moderate light fluxes due to virus-mediated overreduction of Qa.

ACKNOWLEDCMENTS

We thank V. Hurry for pigment analyses and C.B. Osmond for discussion and critica1 review of this manuscript.

Received January 7, 1995; accepted April 13, 1995. Copyright Clearance Center: 0032-0889/95/108/ 1431 /OS.




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Photosynthetic Shutdown in Chlorella NC64A Paramecium bursaria