American Journal of Botany 84(6): 823–829. 1997.
EFFECTS
OF VIRUS INFECTION AND GROWTH
IRRADIANCE ON FITNESS COMPONENTS AND PHOTOSYNTHETIC PROPERTIES OF
EUPATORIUM MAKINOI (COMPOSITAE)1 SACHIKO FUNAYAMA,2,4 KOUKI HIKOSAKA,3,5 TETSUKAZU YAHARA2,6 2
AND
Department of Biology, College of Arts and Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153, Japan; and 3 Department of Botany, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
We examined the effects of geminivirus infection on fitness components and on photosynthetic properties of the host plant, Eupatorium makinoi, grown at two irradiance levels in a natural-light greenhouse. Under the low-light condition (13% full sunlight), more than a half of the infected plants died during the 9-mo experiment, while most of uninfected plants survived. Growth rate was also lowered by infection. At high light (50% full sunlight), by contrast, virus infection did not cause mortality despite slight decrease in growth rate. Flowering occurred only at high light, and reproductive outputs of the plants were markedly reduced by the infection. Infected leaves had distinct yellow variegations and, when compared with uninfected leaves, they showed (1) comparable light-saturated photosynthetic rate per unit area, but (2) lower initial slope of light-response curve of photosynthesis on an incident irradiance basis. The lower initial slope was mainly due to reduction of light-harvesting chlorophyll-protein complexes in the variegated parts. Since the differences in plant performance, depending both on infection and on growth irradiance, were largely explained by the differences in growth rate and/or plant size, the reduced photosynthetic production in the infected plants would be a major factor explaining the inferior performance of the host plants. Key words: Compositae; Eupatorium makinoi; fitness; geminivirus; light-harvesting chlorophyll a/b binding protein; pathogen; photosynthesis; shading.
Interactions between plants and pathogens in natural ecosystems have been attracting increasing attention. Through studies with model systems including Linum– Melampsora (e.g., Jarosz and Burdon, 1992), Senecio– Puccinia (e.g., Paul and Ayres, 1986a, b, 1987), Amphicarpaea–Synchytrium (e.g., Parker, 1986), and Silene– Ustilago (e.g., Alexander and Antonovics, 1988), it is now well established that pathogens often have the critical effects on fitness of individual plants, persistence of plant populations, and on diversity of plant communities (for reviews, Burdon, 1987; Augspurger, 1988; Dobson and Crawley, 1994; Jarosz and Davelos, 1995). In these model systems, the pathogens are fungi, which utilize photosynthates and other metabolites of the host plants, while there also are many other systems in which the pathogens are viruses (e.g., MacClement and Richards, 1956; Kelley, 1993). Since viruses require host functions for their replication as well as photosynthates and other metabolites, the interaction between plants and
viruses would be closer than those between plants and fungi. However, only few ecological studies have been conducted with plant viruses (Harper, 1990). This is probably due to a technical limitation: the detection of virus infection usually requires sophisticated serological or molecular biological procedures. We have been conducting a series of studies with the Eupatorium–tobacco leaf curl virus (TLCV) system. Unlike any other plant–virus systems, the infection of TLCV is easily detected as yellow variegation. Thus, this system is most suitable for ecological studies. In a previous paper, we reported that TLCV infection impaired survivorship and growth of E. makinoi (Yahara and Oyama, 1993). This conclusion was drawn from a close observation of one field population on the floor of an artificial forest of a Japanese cedar, Cryptomeria japonica. In this field, relative photon flux densities above E. makinoi plants are 1–20% (S. Funayama et al., unpublished data). However, infected E. makinoi plants were observed in various light environments from the open field to the shaded understory. Moreover, many studies with plant– fungi systems indicated that shading increases host mortality, disease severity, and/or fungal transmission (Augspurger, 1984; Augspurger and Kelly, 1984; Matson and Waring, 1984; Jarosz and Burdon, 1988; Jarosz and Levy, 1988; Wennstro¨m and Ericson, 1990; Fowler and Clay, 1995). Thus, it is necessary to examine effects of irradiance levels on interactions of E. makinoi and TLCV. The adverse effect of TLCV infection on the host plants could be smaller at high irradiance. How TLCV infection affects E. makinoi plants is ob-
1 Manuscript received 7 June 1996; revision accepted 19 November 1996. The authors thank Dr. K. Clay, Dr. M. A. Parker, and Dr. I. Terashima for helpful comments on an earlier manuscript, Dr. E. Kasuya for a program, and Mr. K. Ohashi for his help in the greenhouse experiments. This work is supported by grant number 05257203 from the Ministry of Education and a grant from the Sumitomo Foundation. 4 Author for correspondence and current address: c/o Dr. Ichiro Terashima, Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305 Japan (e-mail:
[email protected]). 5 Current address: Biological Institute, Graduate School of Science, Tohoku University, Aoba, Sendai, 980-77 Japan. 6 Current address: Department of Biology, Faculty of Science, Kyushu University, Hakozaki, Fukuoka, 812-81 Japan.
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scure. However, the yellow variegation, caused by infection, inevitably affects photosynthesis. It is even probable that the virus impairs plant performance through limiting photosynthetic production. There are many studies on photosynthesis of virus-infected crops and cultivated plants (e.g., Matthews, 1991 for a review; Marler, Mickelbart, and Quitugua, 1993; Balachandran, Osmond, and Makino, 1994). However, effects of infection vary from one system to another. Moreover, no such studies have been conducted with wild plants. The objectives of the present study were as follows: (1) to clarify whether effects of virus infection on host fitness are changed by light environment and (2) to examine photosynthetic properties of TLCV-infected leaves with the symptom of yellow variegation. MATERIALS AND METHODS The host plant—Eupatorium makinoi Kawahara et Yahara (5E. chinense var. oppositifolium in Yahara and Oyama, 1993; Yahara, Watanabe, and Kawahara, 1995) is a short-lived perennial, which is widely distributed in Japan, Korea, and China (Kawahara, Yahara, and Watanabe, 1989a; Yahara, Watanabe, and Kawahara, 1995). Populations of this species are distinguished by mating systems associated with polyploidy levels; sexual plants are diploids while agamospermous plants are polyploids (Watanabe, 1986; Yahara, 1990). Sexual and agamospermous plants are distinguishable by their morphologies (Kawahara, Yahara, and Watanabe, 1989b). In Japan, agamospermous plants grow in habitats of various light conditions from disturbed mountain paths to shaded understories of temperate forests. Eupatorium makinoi plants set flowers from August to September, and set achenes, containing one seed, from late September to October. Achenes have pappus hairs and are wind-dispersed. The plants do not reproduce by means of clonal growth, and it is easy to identify genets in the field. In the present study, we used plants from an agamospermous population on Mt. Futago, Kanagawa, Japan (358N, 1398E). These plants were tetraploids (4n 5 40; unpublished data). The pathogen—Tobacco leaf curl virus (TLCV) is a geminivirus, which is transmitted by two species of whitefly (Bemicia tabaci and B. lonicerae) (Osaki and Inouye, 1979) and never transmitted through seeds. It was firstly identified as a disease of tobacco and tomato plants (Osaki and Inouye, 1981). Eupatorium makinoi and Lonicera japonica are known as wild hosts (Osaki and Inouye, 1979; Osaki, Kobatake, and Inouye, 1979). TLCV causes yellow variegations along veins of infected E. makinoi. Infected E. makinoi used in this experiment showed narrow yellow variegations (, 5 mm). Symptoms appear in 10–14 d after the graft-inoculation of TLCV (S. Funayama et al., unpublished data). Experimental design in the greenhouse—In September 1993, 120 plants of E. makinoi, 60 infected by TLCV and 60 uninfected, were collected from a population on Mt. Futago. The aboveground parts were cut off and the roots were washed well to remove soil. These roots were planted in pots (20 cm diameter, 20 cm height) filled with red soil, and kept outdoors during the winter. In February 1994, the roots of all individuals were recovered from the pots, washed well, desiccated to surface dryness, and weighed. These were replanted in larger pots (30 cm diameter, 30 cm height, one individual per pot). On April 1994, equal numbers of infected and uninfected plants were placed under two light conditions of 13% (low light) and 50% (high light) of full sunlight in a natural-light greenhouse of the University of Tokyo. Irradiance levels were adjusted with shading cloths. In order to
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exclude the effects of the initial size, the means and variances of fresh masses of roots measured in February for four groups were adjusted to 3.82 6 2.37, 3.90 6 2.28, 3.89 6 2.75, and 3.93 6 2.82 g (all expressed as the mean 6 1 SD) for high-light/infected, high-light/uninfected, lowlight/infected, and low-light/uninfected blocks, respectively. Air temperature was not controlled. Pots were moved within the block once a week until the end of October to exclude positional effects. Each plant was fertilized with 200 mL of 1/500 strength of a commercial fertilizer (Hyponex, Murakami Bussan Co., Tokyo) once a week during the experiment. The working solution contained 2.3 mmol/L ammonium, 0.23 mmol/L nitrate, 2.1 mmol/L water-soluble phosphate, 2.6 mmol/L potassium, and trace amount of micronutrients. A fungicide (Morestan, Bayer, Tokyo) and/or an insecticide (Ortoran, Takeda, Tokyo) were sprayed, when necessary. There was no trace of yellow variegation in the plants designated as uninfected plants throughout the experiment. Plant mortality, growth, and reproduction—Plants were harvested at the end of the growing season in November 1994. The number of plants surviving was counted. For the plants with flowers, the number of heads was counted and seed masses were measured. At harvest, the root systems (which mean all the underground parts) were carefully washed with water. The individual plants were separated into roots, leaves, stems, and reproductive parts. Fresh masses of roots were measured for all individuals. Then, all parts of plants were dried for 72 h at 708C and measured. The relative growth rate (RGR) of the root during the growing season (9 mo) was calculated as, RGR 5 ln WNov 2 ln WFeb in grams per gram per 9 mo, where WFeb and WNov refer to fresh masses of the root in February and November, respectively. This calculation assumes an equal root/shoot mass ratio. Effects of virus infection on photosynthetic properties—Plants were grown in the natural-light greenhouse as described above. Leaves of the plants grown under the high-light condition were used for measurements. Chlorophyll fluorescence—Chlorophyll fluorescence was measured at room temperature using a pulse amplitude-modulated fluorometer (PAM-101, Walz, Effeltrich). Fluorescence from dark-adapted leaves (which incubated in the dark for more than 30 min) was measured with weak measuring beam (F0) and, then, maximum fluorescence under a strong 1-s flash (6000 mmol quanta·m22·s21) provided by a halogen lamp (Fm) was determined. Fv/Fm was calculated as (Fm 2 F0)/Fm. Photosynthetic rate—The rate of CO2 exchange of the attached leaf was determined with an open gas-exchange system at the leaf temperature of 24.58 6 18C (range) (for detail, see Hikosaka, 1996). At first, the rate of photosynthesis at 1260 mmol quanta·m22·s21 and at ambient CO2 partial pressure of 35 Pa was measured and, then irradiance was decreased in a stepwise manner. At the end, the rate of dark respiration was determined. Leaf area was determined using an image scanner (GT6000, Epson, Tokyo) and a microcomputer (LC475, Apple, CA). Apparent green area of the infected leaf was also roughly estimated with the photocopy of the leaf by the same method. Determination of chlorophyll and nitrogen—After measurement of photosynthesis, the leaf was washed with distilled water and six discs of 1 cm diameter per leaf were punched out. Residual parts were stored at 2808C until determination of ribulose 1,5-bisphosphate carboxylase/ oxygenase (RuBPCase). Chlorophyll was extracted from three of these discs with N, N-dimethylformamide and determined according to Porra, Thompson, and Kriedmann (1989). Other three discs were dried at 708C for 72 h for the measurements
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TABLE 1. Effects of virus infection on the survivorship and growth of E. makinoi grown under high- and low-light conditions. Means 6 1 SD are shown. High light Uninfected (N 5 29)
100 9.80 6 5.74 3.00 6 2.12 1.83 6 0.82 1.19 6 0.39 0.586 6 0.046
100 15.0 6 6.3 4.51 6 2.04 2.23 6 0.53 1.24 6 0.28 0.493 6 0.073
Characteristic
Final survivorship (%)a Plant biomass (g)b Plant biomass/fresh massFebc RGR [g·g21·(9 mo)21)]c Root/shootc Leaf/shootc
Low light
Infected (N 5 29)
— ** ** * NS ***
Infected (N 5 8)
Uninfected (N 5 28)
46.7 0.856 6 0.616 0.19 6 0.09 20.930 6 0.344 2.21 6 1.90 0.387 6 0.118
93.3 1.27 6 0.85 0.33 6 0.10 20.077 6 0.293 1.32 6 0.40 0.514 6 0.121
*** NS ** *** NS *
a
Fisher’s exact probability test. Student’s t test. Mann-Whitney U test. * P , 0.05; ** P , 0.01; *** P , 0.001; NS, not significant. b c
of dry mass and nitrogen content. Total nitrogen content of the leaf was measured with an NC analyzer (NC-80, Shimadzu, Kyoto). Determination of RuBPCase and polypeptide composition—The frozen leaf was homogenized in 100 mmol/L Na-phosphate buffer (pH 7.5) containing 0.4 mol/L sorbitol, 10 mmol/L ascorbate, 2 mmol/L MgCl2, 10 mmol/L NaCl, 1 mmol/L phenylmethyl-sulfonyl fluoride, 5 mmol/L iodoacetic acid, and 1% (mass/volume) soluble polyvinylpyrrolidone. Content of RuBPCase was determined according to Makino, Mae, and Ohira (1986) with a slight modification (Hikosaka, 1996). Polypeptides were separated from the homogenate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Laemmli, 1970), and the gel was scanned with a gel densitometer (Shimadzu, Kyoto) after staining with Coommasie Brilliant Blue R-250. Statistical analysis—Frequency data were examined by Fisher’s exact probability test. We used Student’s t test for comparing two sampleswith equal variations, and Mann-Whitney U test in comparing samples with unequal variations. We tested for a difference in a slope of the regression using analysis of covariance. To examine the correlation between two variables, Pearsons’s correlation coefficient was calculated and tested by Fisher’s r to z. All computations but analysis of covariance were made using Statview version 4.02 (Abacus Concepts, CA). Analysis of covariance was conducted using a laboratory-made program (programmed by Dr. E. Kasuya, Kyushu University).
RESULTS Survivorship and growth—At high light, no plants died during the experimental period (Table 1). Plant biomass of infected plants was about two-thirds of that of uninfected plants (P , 0.01), and the ratio of the final plant dry mass measured in November to the initial fresh mass measured in February was lower in infected plants TABLE 2. Effects of virus infection on the reproduction of E. makinoi grown under high-light conditions. Means 6 1 SD are shown. Characteristic
Frequency of flowered plantsa Seed mass/shoot massb Flower heads number/ shoot massb a
Infected (N 5 6)
Uninfected (N 5 17)
20.7% 0.0093 6 0.0057
58.6% 0.0403 6 0.0152
** ***
9.75 6 4.59
23.06 6 8.75
***
Fisher’s exact probability test. Mann-Whitney U test. ** P , 0.01; *** P , 0.001; NS not significant. b
than in uninfected plants (P , 0.01). Mean RGRs of root in infected and uninfected plants were 1.83 and 2.23 g/g for 9 mo, respectively, and the difference was significant (P , 0.05). Infection did not significantly affect root/ shoot mass ratio. However, the leaf/shoot mass ratio was significantly greater in infected plants than in uninfected plants (P , 0.001). Under the low-light condition, final survivorship of infected plants was 46.7%, which was markedly lower than 93.3% for the uninfected plants (Table 1, P , 0.001). The data other than the survivorship in Table 1 are for the plants that were alive at the harvest. RGR data indicated that roots of most infected plants decreased to about one-third of the original level, but those of uninfected plants were almost unchanged (P , 0.001). The final plant biomass/initial fresh mass of root was also significantly lower in infected plants than in uninfected plants (P , 0.01). Although the difference in biomass between infected and uninfected plants was statistically insignificant probably because of large variation, the mean biomass was considerably greater for uninfected plants. The root/shoot mass ratio was not significantly altered by the infection. The leaf/shoot mass ratio was smaller in infected plants than in uninfected plants (P , 0.05). For infected plants, survivorship was not significantly related to the initial root mass (P 5 0.54, Mann-Whitney U test). However, among uninfected plants, dead plants were the smallest ones. RGR of the root of the infected plants decreased with the reduction in the initial size (P , 0.001, Fisher’s r to z; r 5 0.944). For uninfected plants, RGR was not dependent on initial root mass (P 5 0.81, Fisher’s r to z; r 5 0.048). Reproduction—Twenty-three plants (39.7%) set flowers under the high-light condition, but no plant did so at low light (Table 2). Analyses described in Table 2 are accordingly only for the plants grown under the high light condition. The frequency of flowered plants was only 20.7% for infected plants, while 58.6% of uninfected plants set flowers (P , 0.01). In both groups, however, there was a clear tendency toward size-dependent flowering (Fig. 1). There were significant differences in biomass between plants that flowered and that those did not (P , 0.05 for infected, and P , 0.001 for uninfected, according to Mann-Whitney U test). There was also a tendency that the proportion of reproductive organs in-
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Fig. 2. Light-response curve of photosynthetic rate per unit area of E. makinoi leaves with and without TLCV infection. The inset represents photosynthetic rates at low photon flux densities. v, TLCV-infected leaves; V, uninfected leaves. Means 6 1 SD (N 5 4) are shown.
Fig. 1. Frequency distributions of plant biomass of TLCV-infected (top panel) and uninfected (bottom panel) plants in relation to flowering. Total numbers of both infected and uninfected plants are 29. M, flowered; g, not flowered.
creased with plant size (data not shown). The ratios of head number/shoot mass and seed mass/shoot mass were calculated to cancel out these size-dependent effects. These ratios were significantly lower in infected plants than in uninfected plants (head/shoot, P , 0.001; seed/ shoot, P , 0.001). Photosynthetic rate—Figure 2 shows irradiance-response curves of the photosynthetic rate per unit leaf area in infected and uninfected leaves. The initial slope of light-response curve of photosynthesis in infected leaves was significantly smaller than that of uninfected leaves (Table 3, P , 0.01). However, the light-saturated rate of photosynthesis (Pmax) between infected and uninfected leaves did not differ significantly (Table 3). In a separate experiment with plants grown under other irradiance levels, infected leaves also showed smaller initial slopes than uninfected leaves, but their Pmax levels were similar (data not shown). Photosynthetic components—As factors affecting light absorption of leaves, leaf chlorophyll content and the green area of infected leaves were measured (Table 3). Total chlorophyll content of infected leaves was almost half that of uninfected leaves (P , 0.001). The green area in infected leaves was 64 6 13% of the total leaf area. When photosystem II is damaged, Fv/Fm and the maximum quantum yield of photosynthesis decrease in parallel (Bjo¨rkman and Demmig, 1987). To examine whether the quantum yield of leaves was lowered due to the dam-
age to PS II, we examined Fv/Fm in infected and uninfected leaves. Fv/Fm was greater than 0.80 in both infected and uninfected leaves, and there was virtually no difference between them (Table 3). Pmax is strongly correlated with the content of leaf nitrogen and chloroplast proteins, such as ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase), H1-ATPase, and cytochrome f (Bjo¨rkman, 1981; Terashima and Evans, 1988). As expected, the differences in RuBPCase and nitrogen contents per leaf area between infected and uninfected leaves were insignificant (Table 3). The chlorophyll a/b ratio in infected leaves was significantly higher than in uninfected leaves (Table 3, P , 0.01). Leaf mass per area (LMA) was not significantly different between infected and uninfected leaves (Table 3). Polypeptide analysis—Polypeptides of leaf homogenates were analyzed by one-dimensional SDS polyacrylamide gel electrophoresis. Densitograms of the gel with the polypeptides from the identical area of infected and uninfected leaves are compared in Fig. 3. The amount of light-harvesting chlorophyll a/b-binding proteins (LHC II) in infected leaves was markedly smaller than that in TABLE 3. Photosynthetic properties in TLCV-infected and uninfected E. makinoi. Means 6 1 SD are shown. Characteristic
Initial slope (mol CO2/ mol incident quanta)a Pmax (mmol CO2·m22·s21)b Green leaf area/total leaf area (%) Chl a 1 b (mmol/m2)b Chl a/bb Fv/Fmb RuBPCase (g/m2)b Nitrogen content (g/m2)b Leaf mass per area (g/m2)b a
Infected (N 5 4)
Uninfected (N 5 4)
0.038 8.1 6 0.8
0.050 8.1 6 1.5
** NS
6 6 6 6 6 6
100 0.402 6 0.040 3.04 6 0.10 0.807 6 0.003 0.74 6 0.15 0.827 6 0.064
— *** ** NS NS NS
17.5 6 2.8
NS
64 0.204 3.62 0.817 0.91 0.879
13 0.034 0.25 0.008 0.19 0.119
19.0 6 3.9
Analysis of covariance. Student’s t test. ** P , 0.01; *** P , 0.001; NS not significant. b
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Fig. 3. Densitograms of leaf polypeptides separated by SDS polyacrylamide gel electrophoresis. Polypeptides were stained with Coommasie Brilliant Blue R-250. The amounts of the extracts applied were adjusted so that the polypeptides from the same leaf areas are compared. Solid line, polypeptides from TLCV-infected leaves; dotted line, uninfected leaves. Figure abbreviations: RuBPCase, ribulose-1,5-bisphosphate carboxylase/oxygenase; LS, large subunit; SS, small subunit; LHC II, light-harvesting chlorophyll a/b-binding proteins.
uninfected leaves. The amounts of other polypeptides, including large and small subunits of RuBPCase, were almost identical between infected and uninfected leaves. DISCUSSION Effects of virus infection on the fitness components— A remarkable finding of this study is that the virus infection did not lower the survivorship of the plants under the high-light condition (Table 1). Biomass and RGR of roots were smaller in the infected plants even under the high-light condition. It is, however, notable that infected plants did increase the mass of roots, when the light environment was favorable. On the other hand, damage caused by virus infection at low light was severe (Table 1). These results obtained for the plants at low light are consistent with the conclusion of Yahara and Oyama (1993) that TLCV infection had negative effects on survivorship in E. makinoi. Since their observation was made also on the shaded population of E. makinoi on the floor of Japanese cedar forest, it is probable that the mortality due to virus infection occurs only in the shaded environment. Our present study, thus, clarified a general feature that TLCV infection does not always exert fatal effects on host plants, and the survivorship of infected plants strongly depends on light environment. Actually, an on-going long-term study with several populations in the field showed that infected plants survive well in open habitats (S. Funayama et al., unpublished data).
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The death of infected plants under the low-light condition may be due to their negative growth rate. When compared under the same light condition, uninfected plants photosynthesize more than the infected plants (Fig. 2). Thus, it is probable that light energy in the present 13%-sunlight block might be just sufficient to allow most of the uninfected plants to attain the critical growth rate that enables plants to be viable, but insufficient for the infected plants. Plants with a negative growth rate because of infection would be more susceptible to other stresses (see Paul, 1990 for a review). Although, at high light, virus infection caused neither death of the plants nor very marked reduction of their growth, it exerted prominent effects on reproduction. The frequency of plants that flowered was significantly lowered by infection (Table 2). This is largely due to the tendencies that infected plants were smaller (Table 1), and flowering was size-dependent (Fig. 1). However, for the plants of the same size, the reproductive output in infected plants was still less than that in uninfected plants, which indicates the involvement of physiological changes of the infected plants. Firstly, the lower photosynthetic rate in infected leaves (Fig. 2) may be responsible. If infected and uninfected plants of the same plant biomass go into the reproductive phase at the same time, then the infected plants cannot develop reproductive parts as much as the uninfected plants because of smaller photosynthetic production. A second possibility is impaired translocation. Translocation is often inhibited in diseased plants (e.g., Leal and Lastra, 1984; Burdon, 1987 for a review), and, in several plant–virus systems, it has been revealed that virus movement proteins inhibit the translocation process of the host plants (e.g., Olesinski et al., 1995). Since TLCV probably localizes in phloem (Osaki and Inouye, 1979), inhibition of phloem transport would be probable. An increased leaf/shoot mass ratio (Table 1) and the greater starch contents in the leaves (data not shown) in the infected plants at high light may result from limited translocation of the photosynthates to the reproductive parts. From an ecological point of view, smaller reproductive allocation in the infected plants is interesting. For the virus, the reduction in reproductive allocation of hosts or parasitic castration (Baudoin, 1975; Clay, 1991) is adaptive, because TLCV cannot transmit through seeds. On the other hand, the increased leaf/shoot ratio in infected plants may be favorable also for the host plants, because such allocation can compensate for the reduction in photosynthetic activity through the increase in leaf area. We did not measure the leaf area directly, but the comparable LMA (Table 3) and the increased leaf/shoot ratio (Table 1) indicate greater leaf area in the infected plants. To elucidate whether reproduction is suppressed by virus or by a plant itself for compensation, detailed physiological studies are needed. The effects of virus infection on photosynthesis—In TLCV-infected leaves of E. makinoi, the light-saturated rate of photosynthesis (Pmax) was similar to that of uninfected leaves, while the initial slope of light-response curve of photosynthesis was smaller (Fig. 2, Table 3). Moreover, the marked decrease in light-harvesting chlo-
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rophyll a/b binding proteins (LHC II) was found in infected leaves (Fig. 3). The initial slope of light-response curve of photosynthesis is expressed as the product of light absorption of leaves and quantum yield of photosynthesis on absorbed quantum basis. Since light absorption of leaves is dependent on their chlorophyll content (Gabrielsen, 1948; Bjo¨rkman, 1981), smaller chlorophyll content in the infected leaves (Table 3) led to smaller initial slope through reducing absorptance. The lower initial slope can be also caused by the lower quantum yield of photosynthesis on absorbed quantum basis, which is mostly caused by the damage to photosystem II (but see Sonoike, 1996). However, this possibility was ruled out because both infected and uninfected leaves showed Fv/Fm values higher than 0.80 (Table 3) (Schreiber, Bilger, and Neubauer, 1993). When quantum yield was expressed on an absorbed quantum basis, it was slightly lower in infected leaves (S. Funayama et al., unpublished data). This may be caused by the unequal energy distribution between PS I and PS II (Andrews, Fryer, and Baker, 1995). However, this difference is small. The difference in the initial slope on an incident quantum basis is, therefore, largely attributed to the difference in absorptance. Leaf nitrogen and RuBPCase contents were also comparable between infected and uninfected leaves (Table 3), which supports the fact that Pmax in infected leaves was similar to that in uninfected leaves. Since a substantial amount of RuBPCase was found in the yellow area of the infected leaves (data not shown), it is probable that the yellow area in infected leaves has the maximum photosynthetic activity comparable to that in the green area of leaves. Since these photosynthetic tendencies all agree with those of chlorophyll b-less mutants (Highkin, Boardman, and Goodchild, 1969), it may be that photosynthetic properties of variegated parts of E. makinoi leaves are similar to those of chlorophyll b-less mutants. Some of these photosynthetic tendencies have been also reported for the leaves of Tolmiea menziesii T. and G. infected by tomato bushy stunt virus and cucumber mosaic virus. The infected T. menziesii is native in western United States, and is cultivated as an ornamental with variegated leaves (Platt, Henriques, and Rand, 1979). There are two plausible mechanisms for the reduction in LHC II in TLCV-infected leaves. Firstly, chlorophyll synthesis may be suppressed. It is well known that relative amount of LHC II to other protein–chlorophyll complexes tends to decrease with the reduction in total chlorophyll content (e.g., Shimada et al., 1990) and that apoprotein of LHC II is unstable without chlorophyll molecules (Terao and Katoh, 1989). Secondly, end products of photosynthesis, such as glucose and sucrose, may inhibit the transcription of LHC II gene (lhcb). This phenomenon is known as sugar repression (for a review; Sheen, 1994). The two hypotheses are not mutually exclusive. Ecological meanings of reduction in LHC II are also unknown. However, for the virus, exploitation of resources that would otherwise form LHC II may be a smart strategy to coexist with its host plant because LHC II is the sole photosynthetic component that can be lost without fatal effects. High growth rates of many chloro-
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