2. Measurements of CO2 assimilation rates and chlorophyll fluorescence of intact leaves

Leaf lettuce (Lactuca sativa L. crispa 'Bio Saradana', Nakahara Seed Product Co., Ltd., Fukuoka, Japan) seeds were sown on watered sponge blocks (10 10 20 mm). After germination, 20 seedlings that had grown uniformly with three leaves were each transplanted into a polyvinyl pot (90 mm diameter, 80 mm depth) filled with vermiculite and grown for 30 days by bottom irrigation with commercial liquid fertilizer (OAT Agrio Co., Ltd., Tokyo, Japan, EC 1.3 mS cm<sup>1</sup> , pH 6.0). The air temperature and relative humidity throughout the cultivation were maintained at 22C and 60%, respectively. The photosynthetic photon flux density (PPFD) was 200 μmol m<sup>2</sup> s <sup>1</sup> supplied by LEDs lamps (Legu LED, HRD Co., Ltd. Tootori, Japan) providing a peak wavelength of red (660 nm) and blue (455 nm) with a 16-h day length (Figure 2). Fourteen irradiation treatments were examined to determine the effect of wide-range frequencies of pulsed lighting (20, 10, 4, 2, 1.3, 1 kHz, and 500, 50, 5, 2.5, 1.3, 1, 0.5 Hz at 50% duty ratio) and continuous lighting. After measurements of leaf photosynthetic parameters at the end of culture period, all plants were sampled and fresh weights of leaves and roots and total leaf area were recorded.

and total leaf area increased significantly by up to 20% at high frequencies (20–1.3 kHz) compared to low frequencies (500–0.5 Hz) and compared to continuous illumination. Meanwhile, pulse illumination had little effect on root growth because of its underground development without direct exposure to pulsed light. Leaf thickness was thinner under pulsed light, but the dependence on the frequency was not consistent. The growth estimated by total leaf area per plant of Arabidopsis thaliana was previously reported to increase by pulsed light with a frequency at 2.5 kHz and 45% duty cycle compared to continuous light under an average

Figure 3. Growth characteristics (top and root fresh weights, total leaf area and specific leaf weight) of plants grown under continuous lighting (black column) and different pulsed lighting (empty columns) conditions at 200 μmol m<sup>2</sup> s

for 20 days. Different lowercase letters represent significant differences (P ≤ 0.05) among lighting treatments and bars on

Figure 4. Leaf lettuce hydroponically grown under different pulsed lighting for 20 days.

<sup>1</sup> supplied by LEDs [13]. It was explained that the growth increased

Growth and Photosynthesis under Pulsed Lighting http://dx.doi.org/10.5772/intechopen.75519

1

21

intensity of 20 μmol m<sup>2</sup> s

column represent SE (n = 10).

Figure 2. Pulsed lighting by using LEDs under growth conditions. Flashing pulse cycles were 20, 10, 4, 2, 1.3, 1 kHz, and 500, 50, 5, 2.5, 1.3, 1, 0.5 Hz, respectively, with 50% duty ratio (light on period: light off period = 1:1) and 200 μmol m�<sup>2</sup> s �1 effective PPFD at the top canopy of plants. As an example, 5 Hz pulsed or continuous lighting scheme during 1 s is shown in Figure 2a by using a LEDs lighting system which irradiates two narrow peak wavelengths of red (660 nm) and blue (455 nm) with 4:1 of light intensity (Figure 2b).

The CO2 uptake rates of a fully expanded mature single attached leaf were measured under varied PPFD and pulsed light conditions supplied by a same type of red-blue LEDs light source used for growing plants at constant leaf temperature, 22�C; leaf vapor pressure deficit, 1.0 kPa; and ambient CO2 concentration, 400 μmol mol�<sup>1</sup> using an LI-6400 portable photosynthesis system (Li-Cor, Lincoln, NE, USA). The apparent quantum yield (Ø) was estimated as a parameter of the best-fitted non-rectangular hyperbola for the photosynthetic responses to PPFD. Chlorophyll fluorescence has a more rapid response than CO2 uptakes, which enable photochemical and non-photochemical quenching to be measured during each flash. This can reveal the extent to which the transthylakoid ΔpH gradient builds up with irradiance in continuous and flashing light. For each light treatment, parameters of chlorophyll fluorescence from a fully expanded mature single attached leaf were measured by using a FluorPen FP 100 (Photon Systems Instruments, Drasov, Czech Republic): maximum quantum yield (Fv/Fm) in a dark-adapted leaf, effective quantum yield (Fv'/Fm<sup>0</sup> ) in an actinic light-adapted leaf with each pulse cycle.

#### 3. Plant growth under pulsed light

The growth of lettuce leaves was affected by pulsed illumination as light source for hydroponic cultivation under controlled environmental conditions (Figures 3, 4). Shoot fresh weight and total leaf area increased significantly by up to 20% at high frequencies (20–1.3 kHz) compared to low frequencies (500–0.5 Hz) and compared to continuous illumination. Meanwhile, pulse illumination had little effect on root growth because of its underground development without direct exposure to pulsed light. Leaf thickness was thinner under pulsed light, but the dependence on the frequency was not consistent. The growth estimated by total leaf area per plant of Arabidopsis thaliana was previously reported to increase by pulsed light with a frequency at 2.5 kHz and 45% duty cycle compared to continuous light under an average intensity of 20 μmol m<sup>2</sup> s <sup>1</sup> supplied by LEDs [13]. It was explained that the growth increased

Figure 3. Growth characteristics (top and root fresh weights, total leaf area and specific leaf weight) of plants grown under continuous lighting (black column) and different pulsed lighting (empty columns) conditions at 200 μmol m<sup>2</sup> s 1 for 20 days. Different lowercase letters represent significant differences (P ≤ 0.05) among lighting treatments and bars on column represent SE (n = 10).

Figure 4. Leaf lettuce hydroponically grown under different pulsed lighting for 20 days.

The CO2 uptake rates of a fully expanded mature single attached leaf were measured under varied PPFD and pulsed light conditions supplied by a same type of red-blue LEDs light source used for growing plants at constant leaf temperature, 22�C; leaf vapor pressure deficit, 1.0 kPa; and ambient CO2 concentration, 400 μmol mol�<sup>1</sup> using an LI-6400 portable photosynthesis system (Li-Cor, Lincoln, NE, USA). The apparent quantum yield (Ø) was estimated as a parameter of the best-fitted non-rectangular hyperbola for the photosynthetic responses to PPFD. Chlorophyll fluorescence has a more rapid response than CO2 uptakes, which enable photochemical and non-photochemical quenching to be measured during each flash. This can reveal the extent to which the transthylakoid ΔpH gradient builds up with irradiance in continuous and flashing light. For each light treatment, parameters of chlorophyll fluorescence from a fully expanded mature single attached leaf were measured by using a FluorPen FP 100 (Photon Systems Instruments, Drasov, Czech Republic): maximum quantum yield (Fv/Fm) in a

Figure 2. Pulsed lighting by using LEDs under growth conditions. Flashing pulse cycles were 20, 10, 4, 2, 1.3, 1 kHz, and 500, 50, 5, 2.5, 1.3, 1, 0.5 Hz, respectively, with 50% duty ratio (light on period: light off period = 1:1) and 200 μmol m�<sup>2</sup> s

20 Photosynthesis - From Its Evolution to Future Improvements in Photosynthetic Efficiency Using Nanomaterials

effective PPFD at the top canopy of plants. As an example, 5 Hz pulsed or continuous lighting scheme during 1 s is shown in Figure 2a by using a LEDs lighting system which irradiates two narrow peak wavelengths of red (660 nm) and blue

The growth of lettuce leaves was affected by pulsed illumination as light source for hydroponic cultivation under controlled environmental conditions (Figures 3, 4). Shoot fresh weight

) in an actinic light-adapted leaf with each

�1

dark-adapted leaf, effective quantum yield (Fv'/Fm<sup>0</sup>

3. Plant growth under pulsed light

(455 nm) with 4:1 of light intensity (Figure 2b).

pulse cycle.

at higher maximum light intensity of pulsed light than that of continuous light. The growth of lettuce increased by 23% at a pulse frequency of 2.5 kHz (50% duty ratio) under an average intensity of 50 μmol m<sup>2</sup> s <sup>1</sup> supplied by LEDs [14].

seemed to be higher at high frequency of pulsed measuring light. The quantum yield is well understood, and the maximum rate can be related to the electron transport capacity of the leaf. It may still influence the Pn-PPFD response via the transthylakoid ΔpH gradient, which slows

The maximum PSII quantum yield (Fv/Fm) showed no difference in a wide range of pulse frequency (10 kHz–0.5 Hz) except for a significant decrease at 0.2 Hz (Figure 7), indicating that the function of PSII component might be affected by exposing leaves to slow flash light during their development. The effective PSII quantum yield induced in pulse light (Fv'/Fm<sup>0</sup>

decreased at only 0.2 Hz, indicating that there is no relationship between Fv'/Fm<sup>0</sup> and high frequency of pulsed light over 0.5 Hz. Similar tendencies were shown in qP and in calculated parameters (ØPSII, ETR). Decreases in these parameters indicate lowered efficiency of light energy utilization by the plant grown under pulsed light at low frequency (0.2 Hz). On the other hand, NPQ, which relates the distribution of light energy into non-photochemical processes by heat dissipation, increased at low frequencies below 1 Hz compared to NPQ mea-

In leaves of tomato plants grown under pulsed light based-LEDs at a wide range of frequencies (100 kHz–0.1 Hz) with 50% duty cycle, the frequencies had both positive and negative

Figure 6. Relationships between photosynthetic rates and PPFD during measurements under different pulsed lighting cycles or continuous lighting of leaves grown under non-pulsed lighting conditions. Ø indicates the apparent quantum

yield (the initial slope of response curve). The dotted vertical line shows the growth PPFD (200 μmol m�<sup>2</sup> s

) also

23

Growth and Photosynthesis under Pulsed Lighting http://dx.doi.org/10.5772/intechopen.75519

> �1 ).

down electron transport by restricting plastoquinone reoxidation.

sured at high frequencies above 5 Hz and at continuous light (Figure 8).

#### 4. Photosynthetic capacity measured at pulsed light

The photosynthetic rates (Pn) of mature leaves grown under continuous light were measured at pulsed light with various frequencies and at continuous light with two intensities (80 and 200 μmol m<sup>2</sup> s 1 ). There were no significant differences between pulsed light with high frequencies (20 kHz–50 Hz) and continuous light, but Pn measured at low frequencies (2.5– 0.1 Hz) gradually decreased to 75% of Pn measured at continuous light. Pn showed no difference between pulse and continuous measuring light when it was measured at low light intensity (80 μmol m<sup>2</sup> s 1 ) (Figure 5). Pn measured at 80 μmol m<sup>2</sup> s <sup>1</sup> showed no difference between leaves grown under pulsed light and leaves grown under continuous light, but leaves grown under pulsed light showed no decrease in Pn at 200 μmol m<sup>2</sup> s 1 . On the other hand, leaves grown under continuous light significantly decreased compared to pulse-irradiated leaves at lower frequencies than 2 Hz. Declined rates of Pn might be due to low scattering light in thicker leaves grown under continuous light compared than leaves grown under pulsed light as shown in Figure 3. For Pn-PPFD response curve (Figure 6) measured with leaves unfolded under continuous light, light-saturated Pn decreased by lowering pulse frequency of measuring light, resulting in low light saturation. The apparent quantum yield (Ø)

Figure 5. Photosynthetic rates of leaves grown under continuous light (left, broken curve) and under continuous or pulsed light (right, bar graph). PPFD during growing period was 200 μmol m<sup>2</sup> s <sup>1</sup> with or without pulsed lighting. Measurement conditions of photosynthesis were 80 and 200 μmol m<sup>2</sup> s <sup>1</sup> PPFD with continuous and with different frequencies of pulse lighting under 400 μmol mol<sup>1</sup> CO2. \* and ns show the significant and non-significant differences between two growth conditions.

seemed to be higher at high frequency of pulsed measuring light. The quantum yield is well understood, and the maximum rate can be related to the electron transport capacity of the leaf. It may still influence the Pn-PPFD response via the transthylakoid ΔpH gradient, which slows down electron transport by restricting plastoquinone reoxidation.

at higher maximum light intensity of pulsed light than that of continuous light. The growth of lettuce increased by 23% at a pulse frequency of 2.5 kHz (50% duty ratio) under an average

The photosynthetic rates (Pn) of mature leaves grown under continuous light were measured at pulsed light with various frequencies and at continuous light with two intensities (80 and

frequencies (20 kHz–50 Hz) and continuous light, but Pn measured at low frequencies (2.5– 0.1 Hz) gradually decreased to 75% of Pn measured at continuous light. Pn showed no difference between pulse and continuous measuring light when it was measured at low light

) (Figure 5). Pn measured at 80 μmol m<sup>2</sup> s

between leaves grown under pulsed light and leaves grown under continuous light, but leaves

leaves grown under continuous light significantly decreased compared to pulse-irradiated leaves at lower frequencies than 2 Hz. Declined rates of Pn might be due to low scattering light in thicker leaves grown under continuous light compared than leaves grown under pulsed light as shown in Figure 3. For Pn-PPFD response curve (Figure 6) measured with leaves unfolded under continuous light, light-saturated Pn decreased by lowering pulse frequency of measuring light, resulting in low light saturation. The apparent quantum yield (Ø)

Figure 5. Photosynthetic rates of leaves grown under continuous light (left, broken curve) and under continuous or

frequencies of pulse lighting under 400 μmol mol<sup>1</sup> CO2. \* and ns show the significant and non-significant differences

pulsed light (right, bar graph). PPFD during growing period was 200 μmol m<sup>2</sup> s

Measurement conditions of photosynthesis were 80 and 200 μmol m<sup>2</sup> s

between two growth conditions.

). There were no significant differences between pulsed light with high

<sup>1</sup> showed no difference

<sup>1</sup> with or without pulsed lighting.

<sup>1</sup> PPFD with continuous and with different

. On the other hand,

1

<sup>1</sup> supplied by LEDs [14].

22 Photosynthesis - From Its Evolution to Future Improvements in Photosynthetic Efficiency Using Nanomaterials

4. Photosynthetic capacity measured at pulsed light

grown under pulsed light showed no decrease in Pn at 200 μmol m<sup>2</sup> s

intensity of 50 μmol m<sup>2</sup> s

200 μmol m<sup>2</sup> s

intensity (80 μmol m<sup>2</sup> s

1

1

The maximum PSII quantum yield (Fv/Fm) showed no difference in a wide range of pulse frequency (10 kHz–0.5 Hz) except for a significant decrease at 0.2 Hz (Figure 7), indicating that the function of PSII component might be affected by exposing leaves to slow flash light during their development. The effective PSII quantum yield induced in pulse light (Fv'/Fm<sup>0</sup> ) also decreased at only 0.2 Hz, indicating that there is no relationship between Fv'/Fm<sup>0</sup> and high frequency of pulsed light over 0.5 Hz. Similar tendencies were shown in qP and in calculated parameters (ØPSII, ETR). Decreases in these parameters indicate lowered efficiency of light energy utilization by the plant grown under pulsed light at low frequency (0.2 Hz). On the other hand, NPQ, which relates the distribution of light energy into non-photochemical processes by heat dissipation, increased at low frequencies below 1 Hz compared to NPQ measured at high frequencies above 5 Hz and at continuous light (Figure 8).

In leaves of tomato plants grown under pulsed light based-LEDs at a wide range of frequencies (100 kHz–0.1 Hz) with 50% duty cycle, the frequencies had both positive and negative

Figure 6. Relationships between photosynthetic rates and PPFD during measurements under different pulsed lighting cycles or continuous lighting of leaves grown under non-pulsed lighting conditions. Ø indicates the apparent quantum yield (the initial slope of response curve). The dotted vertical line shows the growth PPFD (200 μmol m�<sup>2</sup> s �1 ).

Figure 7. Chlorophyll fluorescent parameters of leaves grown under non-pulsed continuous lighting (horizontal line) and under different pulsed lighting cycles (○). Fv/Fm and Fv'/Fm<sup>0</sup> were measured with dark-adapted or exposing leaves to 500 μmol m�<sup>2</sup> s �<sup>1</sup> PPFD (saturated irradiance with or without pulse cycles) to estimate photochemical (qP) and nonphotochemical (NPQ) quenching.

effects on the chlorophyll fluorescence parameters. Relative to continuous light, the pulse frequencies at 0.1, 1, 100 Hz and 1 kHz were reported to be optimal for growth, productivity and energy consumption [15]. However, earlier report suggested that tomato plants could use intermittent light (in kHz frequencies) as effectively as they use continuous light [16]. Flashing light has been useful as an experimental technique to extract additional information from photosynthesis measurements. The complex web of reactions in photosynthesis have different response times, so that fluxes through some reactions can be much faster than others resulting in fluctuating pool sizes. Furthermore, each reaction process seems to occur very rapidly in nanosecond to millisecond rates in the light reaction compared to seconds to minute rates in the dark reaction (Figure 9). When the light is delivered in pulse short enough to only allow a single turnover of PSII, the absorbed quanta are used with maximal efficiency until they

exceed the surface density of PSII [16]. Providing brighter flashes does not result in any additional photochemistry because the reaction centers do not reopen during the lifetime of the quanta in the pigment array. Consequently, it is possible to quantify the number of functional PSII reaction centers per unit leaf area in intact leaves [17]. With longer pulses of light, photochemical efficiency is reduced because a proportion of reaction centers are closed. However, photochemical efficiency expressed per open reaction center is still maximally efficient. Once a transthylakoid ΔpH gradient develops, photochemical efficiency of open centers

Figure 9. Photochemical reactions of photosynthesis are connected by an electron transport chain (ETC) between Photosystem I (PSI) and II (PSII), and ATP synthase embedded in thylakoid membrane in chloroplast. ETC composes of PSII reaction center (P680), plastquinone (PQ), cytochrome b6f complex (b6f), plastocyanin (PC), PSI reaction center (P700), ferredoxin (Fd), ferredoxin-NADP<sup>+</sup> reductase (FNR). Each estimated electron transfer rate is an example from Refs. [3, 4].

Figure 8. Photochemical reactions of photosynthesis of leaf lettuce are affected by exposing leaves to slow flash pulsed strong lighting (<1 Hz). An instantaneous response of PS by enhancing heat dissipation from leaves exposed to natural

Growth and Photosynthesis under Pulsed Lighting http://dx.doi.org/10.5772/intechopen.75519 25

sunfleck in forest floor where plants are protected against photoinhibition.

begins to decline [18].

Figure 8. Photochemical reactions of photosynthesis of leaf lettuce are affected by exposing leaves to slow flash pulsed strong lighting (<1 Hz). An instantaneous response of PS by enhancing heat dissipation from leaves exposed to natural sunfleck in forest floor where plants are protected against photoinhibition.

Figure 9. Photochemical reactions of photosynthesis are connected by an electron transport chain (ETC) between Photosystem I (PSI) and II (PSII), and ATP synthase embedded in thylakoid membrane in chloroplast. ETC composes of PSII reaction center (P680), plastquinone (PQ), cytochrome b6f complex (b6f), plastocyanin (PC), PSI reaction center (P700), ferredoxin (Fd), ferredoxin-NADP<sup>+</sup> reductase (FNR). Each estimated electron transfer rate is an example from Refs. [3, 4].

effects on the chlorophyll fluorescence parameters. Relative to continuous light, the pulse frequencies at 0.1, 1, 100 Hz and 1 kHz were reported to be optimal for growth, productivity and energy consumption [15]. However, earlier report suggested that tomato plants could use intermittent light (in kHz frequencies) as effectively as they use continuous light [16]. Flashing light has been useful as an experimental technique to extract additional information from photosynthesis measurements. The complex web of reactions in photosynthesis have different response times, so that fluxes through some reactions can be much faster than others resulting in fluctuating pool sizes. Furthermore, each reaction process seems to occur very rapidly in nanosecond to millisecond rates in the light reaction compared to seconds to minute rates in the dark reaction (Figure 9). When the light is delivered in pulse short enough to only allow a single turnover of PSII, the absorbed quanta are used with maximal efficiency until they

Figure 7. Chlorophyll fluorescent parameters of leaves grown under non-pulsed continuous lighting (horizontal line) and under different pulsed lighting cycles (○). Fv/Fm and Fv'/Fm<sup>0</sup> were measured with dark-adapted or exposing leaves to

24 Photosynthesis - From Its Evolution to Future Improvements in Photosynthetic Efficiency Using Nanomaterials

�<sup>1</sup> PPFD (saturated irradiance with or without pulse cycles) to estimate photochemical (qP) and non-

500 μmol m�<sup>2</sup> s

photochemical (NPQ) quenching.

exceed the surface density of PSII [16]. Providing brighter flashes does not result in any additional photochemistry because the reaction centers do not reopen during the lifetime of the quanta in the pigment array. Consequently, it is possible to quantify the number of functional PSII reaction centers per unit leaf area in intact leaves [17]. With longer pulses of light, photochemical efficiency is reduced because a proportion of reaction centers are closed. However, photochemical efficiency expressed per open reaction center is still maximally efficient. Once a transthylakoid ΔpH gradient develops, photochemical efficiency of open centers begins to decline [18].

leaves grown under continuous light when they were measured under pulsed light. It is suggested that photosynthetic adaptations to intermittent radiation might have occurred during

affected the growth of lettuce leaves under controlled environment. The photosynthetic performances showed differences between leaves developed under pulsed light and leaves developed under continuous light, when the CO2 uptake rates and chlorophyll fluorescence parameters were measured at lower frequencies (<2 Hz). In the pulsed light technique, it is important to determine both optimal frequency and duty ratio for plants to attain the most efficient use of harvested light. The reason why growth was enhanced under pulsed light at high frequencies has not been resolved by analyzing photosynthetic performances in this study. Further research is required for detecting the pool size of PIs in leaves during their exposure to intermittent radiation. We propose that the pulsed lighting technique by using LEDs could become a useful for the production of leafy vegetables controlled plant factory

)

�1

Growth and Photosynthesis under Pulsed Lighting http://dx.doi.org/10.5772/intechopen.75519

) positively

27

leaf development, and that the adapted leaves can more efficiently use flashing light.

Pulsed light at high frequencies (2–20 kHz, 50% duty ratio, 200 μmol m�<sup>2</sup> s

5. Conclusions

systems in the near future.

photosystem I & II (PSI & PSII)

quantum efficiency of PSII (ØPSII)

photosynthetic photon flux density (PPFD)

maximum quantum yield of PSII in dark-adapted state (Fv/Fm)

photochemical quenching as an estimate of open PSII reaction centers (qP)

Graduate School of Agricultural Science, Kobe University, Kobe, Japan

effective quantum yield of PSII induced in light (Fv'/Fm<sup>0</sup>

non-photochemical quenching by heat dissipation (NPQ)

Address all correspondence to: kanechi@kobe-u.ac.jp

Abbreviations

Author details

Michio Kanechi

The photosynthetic responses to sudden changes in light conditions (lightflecks) were studied and the CO2 uptake rate found to be maintained at a certain level for several seconds after a sudden decrease in light intensity [4, 1]. When photosynthetic rates have been measured with flashing light, considerable rate increases have been observed at high irradiances in the absence [19] or presence [20] of continuous background light. This has been attributed to post-illumination CO2 fixation consuming the pools of RuBP [21] as well as triose phosphate that requires some extra ATP synthesis from the ΔpH gradient to convert it to RuBP [22]. If the time between flashes is sufficiently long, the ΔpH gradient is unable to build up as it is dissipated by the post-illumination CO2 fixation's demand for ATP synthesis. This means that electron transport can occur more rapidly during the pulses of light than in continuous light because plastoquinone reoxidation is not restricted by the ΔpH gradient. If a sufficiently large ΔpH gradient exists at high irradiances even with high CO2 partial pressures in leaf, then one would expect that flashing light could further enhance the photosynthetic rate. Stitt [23] observed a 30% increase in the rate at 1500 μmol m<sup>2</sup> s <sup>1</sup> with spinach when long flashing cycles (10s) were used in an oxygen electrode with 5% CO2, the leaf was kept for 10 s at each light condition. Roden and Pearcy [2] found that the efficiency of post-illumination CO2 fixation only declined once the intervening dark period exceeded about 1 s. Kriedemann et al. [19] also showed that fluctuating light with 200 ms dark intervening periods enhanced photosynthesis. Electron transport during the flash reduces NADPH and builds up the pool of RuBP and triose phosphate. These pools, termed assimilatory power by Laisk et al. [21] enable postillumination CO2 fixation to occur and were equivalent to 5 s of photosynthesis in sunflower leaves at 100–200 μmol m<sup>2</sup> s <sup>1</sup> PPFD. Sharkey et al. [22] showed the rapid buildup and consumption of these pools during and after lightflecks. For Phaseolus leaves grown in sunlight, the RuBP pool was 5 μmol m<sup>2</sup> and the total post-illumination CO2 fixation was 12 μmol m<sup>2</sup> when triose phosphate was included. The latter requires additional ATP synthesis that comes from the proton pool stored in the thylakoid lumen [22]. Steady-state pool sizes of 100 μmol m<sup>2</sup> for RuBP have been measured in Raphanus leaves [24], which are certainly adequate to cope with the maximum of 5 μmol m<sup>2</sup> observed here per flash. The balance between Rubisco activity and electron transport rate is effectively increased by the ratio of intervening time to flash length up to the limit set by the pool sizes of RuBP and triose phosphate. Therefore, in flashing light, the dependence of electron transport rate on CO2 should be small. Thus, photosynthetic intermediates (PIs) were quickly produced by photochemical reactions during lightflecks and consumed thereafter in the CO2 fixation occurred during next dark or dim light period. An actual estimation of PIs content is difficult under pulsed light, especially at high frequencies. A kinetic model to estimate Pn was developed by considering that photosynthetic intermediates were pooled during light periods and then consumed by partial photosynthetic reactions during dark periods [25]. According to this model, they quantitatively estimated the effects of pulsed light frequency and duty ratio on photosynthetic rates of cos lettuce leaves. The estimated Pn was lower, especially under pulsed light at lower frequencies and did not exceed Pn under continuous light. Accordingly, they concluded that, compared with a constant PPFD, fluctuation in PPFD can theoretically be disadvantageous to photosynthesis, even though the time-averaged PPFD are identical. In this study, lettuce leaves grown under pulsed light at low frequencies (2–0.5 Hz) maintained higher Pn compared to leaves grown under continuous light when they were measured under pulsed light. It is suggested that photosynthetic adaptations to intermittent radiation might have occurred during leaf development, and that the adapted leaves can more efficiently use flashing light.
