Abstract: The present study investigated various characteristics concerning photosynthetic source capacity and biomass production of intact soybean plants grown under daily 10 h photoperiod as controls or under continuous light without darkness as CL plants from sowing. It was shown that while total photosynthetic source capacity and total dry matter production were larger in CL plants than in control plants, photosynthetic rate per unit leaf area and dry matter production of reproductive organs (pods) were smaller in CL plants than in control plants. It was also shown that CL plants had similar leaf intercellular CO2 concentration despite having lower leaf stomatal conductance and had lower activation state of Rubisco and similar content of leaf Rubisco as compared to control plants. With leaf microsomal membranes, three phosphatase activities, which have been found to increase under limitation of inorganic phosphate, were shown to be higher in CL plants than in control plants. Content of starch, a major photosynthetic end product in leaf was higher in CL plants than in control plants, showing that CL plants were subjected to a surplus source, an imbalance of photosynthetic source/sink balance. There are previous findings that inorganic phosphate stimulates the activation of Rubisco by promoting the binding of activator CO2 to the uncarbamylated inactive Rubisco. There is also evidence for the accumulation of starch in leaf under limitation of inorganic phosphate. Therefore, these results suggest that under continuous light that gives a surplus source, although plant total photosynthetic capacity and total biomass production are likely to increase, the efficiency of plant photosynthetic matter production is likely to decrease because of a lowered activation state of Rubisco that is likely to result from the limitation of inorganic phosphate. It is thus suggested that for the efficient improvement of plant biomass production, well-balanced improvement of source and sink would be essential.
INTRODUCTION
Plants are living organisms that acquire exterior resources, produce various substances such as chemical energy and reserve substances and grow. Since in plants photosynthates are fundamental substances for making chemical energy and various cellular components, it is evident that changes in the photosynthetic source capacity are an important factor affecting a variety of physiological functions in plants. It is speculated that an increase in plant photosynthetic source capacity would result in an increase in plant biomass production. Increasing the plant biomass production by increasing the plant photosynthetic source capacity may be a powerful way for overcoming the food and energy problems on earth, which may hereafter increase. Recent studies demonstrate that plant biomass production can be increased by increasing the photosynthetic source capacity. For example, studies using transgenic plants have shown that increasing the activities of Calvin cycle enzymes (sedoheptulose-1,7-bisphosphatase, aldolase and transketolase) or the activity of CO2 (HCO3–) transport at leaf plasma membranes increases the leaf photosynthetic rate and biomass production (Raines, 2003, 2006). Because plant leaf photosynthetic rate does not reach the saturation rate at the present atmospheric CO2 concentration, treating plants with CO2 at the concentration higher than the present atmospheric CO2 concentration may be an available way for examining whether increasing the plant photosynthetic source capacity increases the plant biomass production. A number of studies have shown that plant photosynthetic source capacity and biomass production are actually increased by high CO2 treatment (Reuveni and Bugbee, 1997; Onoda, 2007). However, the increase in photosynthetic source capacity caused by high CO2 diminishes during the period of treatment, because of a decrease in photosynthetic rate (Sage et al., 1989; Reuveni and Bugbee, 1997; Paul and Foyer, 2001; Onoda, 2007). Besides the high CO2 treatment, exposure of plants to continuous light may be an available way for examining whether increasing the plant photosynthetic source capacity increases the plant biomass production. It is inferred that exposing plants to continuous light extends the photosynthetic period and increases the photosynthetic source capacity and then, increases the biomass production. Information is still limited particularly on the characteristics such as leaf photosynthetic rate, leaf intercellular CO2 concentration, leaf Rubisco content, activation state of Rubisco and dry matter production concerning photosynthetic source capacity and biomass production of intact higher plants grown under continuous light. The information, together with the information on the effect of improving the Calvin cycle enzymes or plasma membrane CO2 transport, should be useful not only for examining the regulatory mechanism of plant photosynthetic rate under changes in photosynthetic source/sink balance but also for examining how plant photosynthetic source capacity should be improved to increase plant biomass production. In the present study, soybean plants were used and the plants were grown under daily photoperiod of 10 h as controls or under continuous light as CL plants from sowing. Then, the leaf photosynthetic rate and the other characteristics concerning photosynthetic source capacity and biomass production were investigated in both control and CL plants to examine the regulatory mechanism of photosynthetic rate and the way of improvement of plant photosynthetic source capacity to the improvement of plant biomass production.
MATERIALS AND METHODS
Experimental site: Experiments were conducted in 2007 at the laboratories belonging to the department of Biofunctional Science of Hirosaki University located in Aomori Prefecture of Japan.
Plant materials and growth conditions: Soybean (Glycine max L. Merr. cv. Tsurunoko) seeds were sown in plastic pots (13.5 cm in height, 12.5 cm in diameter) that contained almost equal volumes of vermiculite and sand and were grown in the same type of two growth chambers (Koitotoron, HNL type, Koito Industries, Ltd., Tokyo). Plants in one growth chamber were grown under daily light-dark cycles of 10 h light (24°C) and 14 h darkness (17°C) and those in another growth chamber were grown under continuous light without darkness. Light was supplied with incandescent lamps at an intensity of 80 μmol photons m–2 sec–1 (400-700 nm) on the pots. Nutrients were supplied twice a week with a 1,000-fold diluted solution of Hyponex (6-10-5 Type, N: P: K = 6: 10: 5, Hyponex Co., Osaka, Japan) and tap water was supplied in sufficient amounts.
Photosynthetic rate: Photosynthetic rate and stomatal conductance per unit leaf area and leaf intercellular CO2 concentration were measured at a light intensity of 800 μmol photons m–2 sec–1, air flow rate of 200 mL min–1, air temperature of 24°C, relative humidity of 60% and CO2 concentration of 350 μl L–1 using a photosynthetic analyzer (Cylus-1, Koito Industries, Ltd., Tokyo, Japan). Three trifoliate leaves developed in the middle of plant aboveground were used for the measurements. In a preliminary experiment, the leaves showed higher photosynthetic rates than other trifoliate leaves.
Other analyses: Dry weights of plant organs were measured after organs were dried for 2 days at 75°C. Leaf area was measured with an automatic area meter (AAM-7, Hayashidenko Co., Tokyo). Leaf discs (1.79 cm2 per one leaf disc) were collected from the three trifoliate leaves developed in the middle of plant aboveground on day 49 after seed sowing after measuring leaf photosynthetic rate, stomatal conductance and leaf intercellular CO2 concentration. The remaining leaves including the remaining part of leaves from which leaf discs were taken and all other organs including roots were dried for 2 days at 75°C and the dry weights were measured. The dry weight of leaves from which leaf discs were taken was corrected by measuring the dry weight of a small sample of the leaf discs. Leaf discs other than those used for analyzing the dry weight were immediately frozen in liquid nitrogen and stored at -80°C until used for the other analyses described below.
Leaf Rubisco content was determined as described by Makino et al. (1986).
The initial and total activities of Rubisco in leaf extract were determined at 25°C essentially as described by Cheng and Fuchigami (2000). For the initial activity, 20 μL of a leaf extract obtained by homogenizing a leaf disc with ice-old buffer [100 mM HEPES-KOH (pH 7.8), 2 mL] was added to a cuvette containing 1.98 mL of assay medium [100 mM Bicine-KOH (pH 8.2), 20 mM MgCl2, 20 mM NaHCO3, 5 mM creatin phosphate, 1 mM ATP, 0.2 mM NADH, 20 units creatin kinase, 20 units 3-phosphoglycerate kinase and 20 units glyceraldehyde-3-phosphate dehydrogenase], immediately followed by the addition of ribulose 1,5-bisphosphate (RuBP, final concentration 0.6 mM), then mixed well. Time taken from the homogenization of leaf disc sample to assay of initial activity was less than 1 min. For the total activity, RuBP was added 5 min later (which gave the highest total activity), after 20 μL of the leaf disc extract was immediately combined with the assay medium. The change in absorbance at 340 nm was monitored using a spectrophotometer (Model U-2000, Hitachi Co., Tokyo, Japan).
The amount of protein-bound RuBP in leaf extract was determined essentially as described by Brooks and Portis (1988). A leaf extract (800 μL) obtained by homogenizing a leaf disc with an ice-cold buffer [100 mM HEPES-KOH (pH 7.8), 1 mL] was centrifuged (100 g, 1 min, 4°C) after loading onto a column containing Sephadex G-50 (bed volume before centrifugation, 4 mL) that had been equilibrated with the same buffer. The eluent (500 μL) from the column lacking free RuBP was centrifuged (10,000 g, 10 min, 4°C) after mixing with an acidic solution (5.5 M HClO4, 50 μL) to precipitate protein in the eluent. The resulting supernatant was centrifuged (10,000 g, 10 min, 4°C) after neutralizing to pH 5.6 with K2CO3 and RuBP in the supernatant was determined in the assay medium for determining Rubisco activity using purified spinach Rubisco (0.5 units).
For the determination of three phosphatase activities, leaf microsomal membranes were prepared from leaf discs collected from the three trifoliate leaves developed in the middle of plant aboveground in a separate experiment. Approximately 2 g of the leaf discs was homogenized in ice-cold buffer solution [250 mM sorbitol, 25 mM HEPES-BTP (pH 7.6), 5 mM EDTA-BTP (pH 7.6), 10 mM NaF, 2.5 mM Na2S2O5, 4 mM DTT, 1 mM PMSF and 0.2%(w/v) bovine serum albumin, 16 mL] and the homogenate was filtered through four layers of gauze. The filtrate was centrifuged (10,000 g, 20 min, 4°C) and the resulting supernatant was centrifuged (80,000 g, 60 min, 4°C) to obtain the leaf microsomal membrane pellet. The pellet was suspended in ice-cold buffer solution [250 mM sorbitol, 20 mM HEPES-BTP (pH 7.2), 0.5 mM DTT] and stored at -80°C. The nonspecific phosphatase activity of the microsomal membranes was analyzed as the difference in the activities determined in the presence and absence of molybdate (5 mM Na2MoO4) in assay medium containing ATP [50 mM HEPES-BTP (pH 7.2), 3 mM MgSO4, 3 mM ATP, 0.3 mM EGTA-BTP (pH 7.2), 50 mM KCl, 0.1%(w/v) Brij 58 and 20 μg of membranes] at 28°C (Sze, 1985). The tonoplast H+-ATPase activity and H+-PPase activity of the microsomal membranes were determined in assay medium containing ATP [50 mM HEPES-BTP (pH 7.2), 3 mM MgSO4, 3 mM ATP, 0.3 mM EGTA-BTP (pH 7.2), 50 mM KCl, 0.1%(w/v) Brij 58 and 20 μg of membranes] and the medium containing PPi [50 mM HEPES-BTP (pH 7.2), 0.5 mM Na2P2O7, 1.5 mM MgSO4, 0.3 mM EGTA-BTP (pH 7.2), 50 mM KNO3, 0.1%(w/v) Brij 58 and 20 μg of membranes], respectively, at 28°C in the presence of various inhibitors [molybdate (5 mM Na2MoO4, nonspecific phosphatase inhibitor), vanadate (0.1 mM Na3VO4, plasma membrane H+-ATPase inhibitor) and azide (1 mM NaN3, mitochondrial H+-ATPase inhibitor)] (Sze, 1985). The liberated Pi was determined by the method of Heinonen and Lahti (1981). Protein was determined by the method of Bradford (1976).
Leaf starch content was determined as described by Sawada et al. (1999).
Sample number: Four plant samples were used for analyzing the leaf photosynthetic rate, stomatal conductance, leaf intercellular CO2 concentration, dry weights of organs and the three phosphatase activities. For the other individual analyses, four leaf disc samples were used. The leaf disc samples were selected at random from frozen leaf discs that had been collected from the four plant samples.
RESULTS AND DISCUSSION
The photosynthetic rate and stomatal conductance per unit leaf area and leaf intercellular CO2 concentration were measured on day 28 and 49 after sowing. Both days, the photosynthetic rate and stomatal conductance in CL plants were significantly lower than those in control plants (Table 1). In contrast, the leaf intercellular CO2 concentration in CL plants did not differ significantly from that in control plants both days (Table 1). These results implicate that the supply of CO2 through stomata and the use of CO2 in leaf photosynthetic cells in CL plants were almost equally smaller than those in control plants during experimental period.
Despite the lower photosynthetic rate in CL plants, biomass of CL plants was larger than that of control plants. On day 28 after sowing, the dry weights of leaves, stems and roots in CL plants were 3.8, 4.2 and 3.4-fold larger than those in control plants. The total dry weight in CL plants was 3.7-fold larger than that in control plants (Table 1). The total leaf area in CL plants was 2.3-fold larger than that in control plants (Table 1). When the value of photosynthetic rate per unit leaf area x total leaf area x daily photoperiod (during which plants could perform photosynthesis) was calculated, the value in CL plants was about 2-fold larger than that in control plants. On day 49 after sowing, similar results were obtained. The dry weights of leaves, stems and roots in CL plants were 3.1, 4.4 and 4.1-fold larger than those in control plants. The total dry weight in CL plants was 3.0-fold larger than that in control plants (Table 1). The total leaf area in CL plants was 2.7-fold larger than that in control plants (Table 1). The value of photosynthetic rate per unit leaf area x total leaf area x daily photoperiod in CL plants was about 1.5-fold larger than that in control plants. These results show that although photosynthetic rate per unit leaf area was lower in CL plants than in control plants, total photosynthetic source capacity and total biomass production were larger in CL plants than in control plants during experimental period. Reproductive organs, i.e., pods could be analyzed on day 49 after sowing. The dry weight of pods in CL plants was significantly smaller than that in control plants (Table 1).
| Table 1: | Leaf photosynthetic rate, leaf stomatal conductance, leaf intercellular CO2 concentration, dry weights of organs and total leaf area analyzed in soybean plants grown under daily photoperiod of 10 h (control plants) or under continuous light without darkness (CL plants) on day 28 and 49 after sowing |
| Each value indicates mean±SD (n = 4). (a) μmol CO2 m–2 sec–1. (b) mmol m–2 sec–1. (c) μL L–1. (d) g plant–1. (e) m–2. *p<0.01/**p<0.05 (t-test) when compared to control plants. The leaf intercellular CO2 concentration did not differ significantly (p>0.05, t-test) between control and CL plants | |
| Table 2: | Leaf Rubisco content, activation ratio (percentage of initial activity to total activity) of Rubisco in leaf extract, amount of protein-bound RuBP in leaf extract, leaf microsomal three phosphatase activities and leaf starch content analyzed in soybean plants grown under daily photoperiod of 10 h (control plants) or under continuous light without darkness (CL plants) on day 49 after sowing |
| Each value indicates mean±SD (n = 4). (a) g m–2. (b) μmol CO2 m–2 sec–1. (c) %. (d) nmol RuBP mg protein–1. (e) μmol Pi mg protein–1 h–1. *p<0.01/**p<0.05 (t-test) when compared to control plants. The leaf Rubisco content did not differ significantly (p>0.05, t-test) between control and CL plants | |
Leaf photosynthetic rate is thought to be an essential basis for plant biomass production. The results of leaf photosynthetic rate, stomatal conductance and leaf intercellular CO2 concentration in control and CL plants showed that CL plants had lower CO2 fixation rate in leaf photosynthetic cells as compared to control plants. To examine the mechanism responsible for the lower CO2 fixation rate in leaf photosynthetic cells of CL plants, leaf Rubisco content, activation ratio (percentage of initial activity to total activity) of Rubisco in leaf extract and the amount of protein-bound RuBP in leaf extract were analyzed with plant samples on day 49 after sowing. In a previous study investigating the light activation of Rubisco in Arabidopsis thaliana, the amount of protein-bound RuBP in leaf extract was found to correlate negatively with the activation ratio of Rubisco in leaf extract (Brooks and Portis, 1988). There are also findings from in vitro studies using purified spinach Rubisco that RuBP easily binds the uncarbamylated inactive Rubisco and can disturb the activation of Rubisco through the binding of activator CO2 to the uncarbamylated inactive Rubisco (Jordan and Chollet, 1983). The leaf Rubisco content in CL plants did not differ significantly from that in control plants (Table 2). In contrast, the activation ratio of Rubisco in CL plants was significantly lower than that in control plants (Table 2). The amount of protein-bound RuBP in CL plants was significantly higher than that in control plants (Table 2). These results suggest that the lower CO2 fixation in leaf photosynthetic cells of CL plants is likely to be attributed to a lower activation state of Rubisco in leaf. In in vitro studies using purified spinach Rubisco, inorganic phosphate has been found to stimulate the activation of Rubisco by promoting the binding of activator CO2 to the uncarbamylated inactive Rubisco (Bhagwat, 1981; McCurry et al., 1981; Anwaruzzaman et al., 1995). It may be that under continuous light, limitation of inorganic phosphate occurred within the leaf chloroplasts of CL plants, where Rubisco exists and as a result, the lower activation state of Rubisco occurred. At present, there is no appropriate method(s) for analyzing the concentration of inorganic phosphate within the leaf chloroplasts of intact higher plants at real times under light. However, there have been findings that nonspecific phosphatase activity or tonoplast H+-ATPase and H+-PPase activities increase under limitation of inorganic phosphate (Duff et al., 1994; Palma et al., 2000; Baldwin et al., 2001; Ohnishi et al., 2007). The nonspecific phosphatase activity and tonoplast H+-ATPase and H+-PPase activities were roughly analyzed with leaf microsomal membranes prepared from control and CL plants using inhibitors. All of the three phosphatase activities were found to be significantly higher in the membranes from CL plants than in the membranes from control plants (Table 2). Leaf starch content was also analyzed. There is evidence for the accumulation of starch in leaf under limitation of inorganic phosphate (Champigny, 1985; Foyer and Spencer, 1986; Fredeen et al., 1989). Leaf starch content in CL plants was significantly higher than that in control plants (Table 2). These results suggest that indeed, the above-mentioned possibility is likely. The result of higher content of starch, a major photosynthetic end product in leaf in CL plants implicates that CL plants were subjected to a surplus source, an imbalance of photosynthetic source/sink balance, under continuous light, as compared to control plants.
All the results obtained in the present study suggest that under continuous light that gives a surplus source, although plant total photosynthetic capacity and total biomass production are likely to increase, the efficiency of plant photosynthetic matter production is likely to decrease because of a lowered activation state of Rubisco that is likely to result from the limitation of inorganic phosphate.
Besides the exposure to continuous light, which was carried out in the present study, high CO2 treatment or sink removal has also been shown to cause a surplus source. High CO2 treatment has been shown to increase the total photosynthetic source capacity, total biomass production and the content of leaf photosynthetic end product such as starch and decrease the photosynthetic rate per unit leaf area and the activation ratio of Rubisco in leaf extract (Sage et al., 1989; Reuveni and Bugbee, 1997; Paul and Foyer, 2001; Onoda, 2007). Sink removal such as pod removal or flower bud removal has been shown to decrease the photosynthetic rate per unit leaf area and the activation ratio of Rubisco in leaf extract and increase the leaf photosynthetic end product (Mondal et al., 1978; Sawada et al., 1995; Nakano et al., 2000; Kasai et al., 2008). Although, in high CO2 treatment, whether limitation of inorganic phosphate occurs has not been examined, it was observed that, when sink (pod) removal was conducted with soybean plants, which decreased the activation ratio of Rubisco in a previous study (Kasai et al., 2008), the three phosphatase activities (nonspecific phosphatase activity and tonoplast H+-ATPase and H+-PPase activities) were all higher in leaf microsomal membranes from the sink-removed plants than in the membranes from control plants (data not shown). It is considered that decreases in photosynthetic rate per unit leaf area and activation ratio of Rubisco and in addition, limitation of inorganic phosphate, although the last is still putative, may be the common phenomena that occur under conditions that give a surplus source.
With leaf hyperchloric acid extracts, insignificant decrease of leaf inorganic phosphate content in CL plants relative to control plants was observed (not shown). In the present study, insignificant difference in leaf Rubisco content between control and CL plants was also observed, while there is evidence for the decrease in leaf Rubisco content under limitation of mineral nutrient such as inorganic phosphate (Lauer et al., 1989; Osaki et al., 1993; Rao et al., 1995). It is thought that the limitation of inorganic phosphate might have occurred especially within the leaf chloroplasts of CL plants, where Rubisco exists. The present study used the leaf discs from near expanded or fully expanded leaves for various analyses. In such leaves, the vacuole occupies a large volume of the cells (Deepesh, 2000). Data from studies with Chara implicate that the vacuole contains the significant amount of inorganic phosphate and that the concentrations of inorganic phosphate in different subcellular compartments (cytoplasm and vacuole) do not necessarily show the similar degree of change (Mimura, 1999). As already mentioned, there does not seem to be appropriate method(s) for analyzing the concentration of inorganic phosphate within the leaf chloroplasts of intact higher plants under light. Development of a new method(s) is required.
Although it is not study with intact higher plants, studies with single-rooted soybean leaves showed that exposure of leaves to continuous light decreased the photosynthetic rate per unit leaf area and the activation ratio of Rubisco in leaf extract, increased the content of photosynthetic end product such as starch and caused a small decrease in leaf inorganic phosphate content (Sawada et al., 1986, 1990). However, in the studies, characteristics such as leaf Rubisco content, leaf intercellular CO2 concentration, the amount of protein-bound RuBP in leaf extract and the three phosphatase activities were not investigated. To search, for example, what is the main factor(s) involved in the regulation of photosynthetic rate under continuous light (that gives a surplus source), investigating the characteristics is important. The present study investigated various characteristics concerning photosynthetic source capacity and biomass production including the above with intact higher plants.
It was a surprising result that CL plants developed less reproductive organs, i.e., pods, as compared to control plants. It was observed visibly that while CL plants developed about two or three times more and larger flowers than control plants, a lot of the flowers in CL plants were abscised soon after flowering. With soybean plants, similar phenomena of the abscission of developed flowers by prolonged daily photoperiods exceeding 12 h have been found (Mann and Jaworski, 1970; Thomas and Raper, 1977; Cure et al., 1982). However, the detailed mechanism still remains unknown. It is speculated that a surplus source, an imbalance of photosynthetic source/sink balance might be involved in the phenomenon of flower abscission in CL plants. As described in the Introduction, changes in the photosynthetic source capacity are thought to be an important factor affecting a variety of physiological functions in plants.
Results obtained in the present study implicate that in plants, excessive source relative to sink would decrease the efficiency of photosynthetic matter production. It is thus suggested that for the efficient improvement of plant biomass production, well-balanced improvement of source and sink would be essential.