In recent years, global warming has caused various abnormal weather conditions, such as high temperatures, droughts, and a lack of sunlight, which greatly affect crop cultivation. To address these problems, the concept of an indoor plant cultivation system has been proposed. Numerous studies on indoor plant cultivation have been conducted worldwide, and one of the most important environmental factors is light and light sources.

Light is an important environmental factor with a powerful influence on plant growth and development from seed germination to flowering and fruiting [1]. Higher plants utilize a complex set of photoreceptor proteins to sense ambient red/far-red, blue/ultraviolet A, and ultraviolet B. And plants use the light energy sensed by these photoreceptors to perform photosynthesis and growth [2-3]. Thus, light is an essential environmental factor for plant growth and various properties of light affect plant growth. Therefore, it is important to study the light sources suitable for indoor plant cultivation. Over the past 20 years, light-emitting diodes (LEDs) have been used as light sources in plant cultivation systems that require fully controlled environmental conditions [4]. This is because LEDs are generally considered to emit light at wavelengths that are more effective for photosynthesis and photomorphogenesis than other light sources. Although many studies have been reported on irradiating LED light, all light used in studies to date has been steady light [5-10].

We are studying plant cultivation with a light source that we call an EDL. EDLs are light sources with rapid temporal changes in intensity at μs intervals. EDLs also have an extremely low photosynthetic photon flux density (< 0.1 μmol photons/m²/s). We thought that EDL would not affect photosynthesis because they are very dark lights. However, when we used EDLs in combination with white LED light sources, growth-promoting effects were observed in various plants [11-15]. Therefore, the purpose of this study is to elucidate this phenomenon, by cultivating Persicaria tinctoria and investigating the effects of EDL irradiation on plant growth.

Persicaria tinctoria (indigo plant), the subject of this study, is an annual plant belonging to the family Polygonaceae that grows wild from Southeast Asia to China and Japan. The fresh leaves of indigo plants contain many specific metabolites, such as indican, a precursor of indigo dye, polyphenols with antioxidant properties, and trypsin with antibacterial properties. Therefore, it has long been used as a raw material for herbal medicines and blue dyes [16-18]. Among the specific metabolites of indigo plants, indican, a precursor of blue dye, is known to be stored in the leaves [19]. Therefore, simultaneous examination of plant growth and the amount of indican is possible to investigate the effects on translocation and metabolic pathways within the indigo plants. Thus, this study aims to determine the effects of EDL irradiation on plant growth and specific metabolite production by examining the EDL-irradiated indigo plants.

Materials and Reagents

All reagents were of special grade and were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) or Nacalai Tesque (Kyoto, Japan). Seeds of P. tinctoria used in this study were obtained from the Agriculture, Forestry, and Fisheries Comprehensive Technical Support Center (Tokushima Prefecture, Japan).

LED Fluorescent Light And EDL

LED fluorescent light (visible-light emission) was used as the primary source of photosynthetic light. Figure 1 shows the emission spectra of the fluorescent LED and the EDL used in the experiment. The strong emission peak at 446 nm corresponded to the light used to excite the phosphors. The width at half-maximum of the emission peak was 10 nm.

Figure 1: Emission spectrum of (a) fluorescent light and (b) extremely dark light (EDL).

Figure 2: A schematic of time variation of extremely dark light (EDL). In this study, ΔT and ΔT₁ were 20 μs and 2 μs, respectively.

Figure 2 shows a temporal change of EDL, where ΔT, ΔT₁, and ΔT₂ represent one cycle time, increasing time, and decreasing time, respectively. In this study, ΔT, ΔT₁, and ΔT₂ were less than a few tens of microseconds. The photosynthetic photon flux density (PPFD) of LED and EDL was set to 130 and 0.01 μmol photons/m²/s at the plant surface, respectively.

Indoor Cultivation System of Indigo Plants

Indigo plants were cultivated hydroponically indoors. First, a sponge was placed on a metal tray filled with water and seeded. The seedlings were then irradiated with LED for 20 days. After seedling growth, seedlings with the same plant height and number of developed cotyledons were selected and replanted using the Aqua Cultivation Kit (SANEI, 40 x 39.6 x 26.3 cm, Japan).

Cultivation experiments were conducted starting on the day of replanting in an LED fluorescent light-only plot (control plot) and an LED fluorescent light and EDL plot (experimental plot). Both plots were light-shielded. Kyowa Hyponica Liquid Fertilizer (Kyowa, Japan) was used as the liquid fertilizer for hydroponics, and temperature and humidity were maintained at 22 ±1 ? and 60±10% for 63 days, respectively.

Figure 3 shows the daily irradiation profiles. During the light period, fluorescent light was continuously irradiated for 12 h. During the dark period, the EDL was continuously irradiated for eight hours, immediately after the LED fluorescent light irradiation was completed.

Figure 3: Light irradiation profile in 1-day cycle in (a) control plot (LED only) and (b) experimental plot (EDL after LED).

Quantitative Analysis of Weight and Metabolite in Fresh Leaf

The number and weight of the indigo plant specimens were measured within three hours of harvest. Indican was extracted from fresh leaves using hot water within three hours of harvest and quantitatively analyzed using high-performance liquid chromatography (Shimadzu, Kyoto) with a TSK gel ODS-120H column (4.6 mm x 15 cm; Tosoh, Tokyo) and an APD-10AVP UV/Vis detector. The eluent used was a mixture of milliQ water and methanol, and its flow rate was 1.0 mL/min with an injection volume of 10 μL. The wavelength detection was set to 220 nm and the column oven temperature was set at 40 ?. Calibration curves for the high-performance liquid chromatography analysis were measured in advance using indigo standard chemicals. The significance of the differences between the datasets was verified using a t-test.

Effects of EDL Irradiation on Biomass Production

Figures 4 and 5 show photographs of the indigo plants in the control and experimental plots, respectively. Figure 4 indicates that the leaves and stems of the indigo plants grew well, suggesting that cultivation kits, liquid fertilizers, and LEDs, as primary sources of photosynthetic light, are sufficient for the cultivation of indigo plants.

Indigo plants in the experimental plot exhibited greater overall width and height than those in the control plot. Additionally, the stems in the experimental plots were thicker and firmer, and the leaves were wider and denser than those in the control plots. Thus, EDL irradiation appeared to promote the growth of indigo plants.

Figure 4: Appearance of P. tinctoria in control plot (without EDL irradiation).

Figure 5: Appearance of P. tinctoria in experimental plot (with EDL irradiation).

Figure 6 shows the total weight, fresh leaf weight, and number of leaves of the indigo plants in the control and experimental plots. The total weight of indigo plants and the weight of fresh leaves in the experimental plot were 2.4 times and 2.3 times that of the control plot, respectively, and increased at almost the same rate. Therefore, we inferred that stem growth in the experimental plots was promoted at approximately the same rate. In contrast, the number of leaves in the experimental plot was 1.8 times that in the control plot and did not increase as much as the weight. Thus, the weight of each leaf was 1.3 times greater than that of the control plot, suggesting that EDL irradiation promotes the growth of fresh leaves.

Figure 6: Characterization of P. tinctoria cultivated by LED with EDL: (a) Total wet weight, (b) Wet weight of fresh leaves, (c) Wet weight of stem, (d) Number of leaves.

Although blue LEDs have been reported to have an impact on plant growth [5-9], we have confirmed that blue EDL (446 nm) also had a growth-promoting effect. This is evidence that indigo plants sensed the EDL despite the extremely low PPFD.

Effects of EDL Irradiation on Indican Production and Metabolic Pathway

Figure 7 shows the indican weights of control and experimental plots per unit weight of fresh leaves. There was no significant difference in the indican weight per unit weight of fresh leaves in the presence or absence of EDL irradiation (P >0.05). This suggests that indican synthesis is promoted as the fresh leaf weight increases.

Figure 7: The total amount of indican in (a) 50 g indigo leaf and (b) one indigo plant. The amount of indican was measured by HPLC 3 hours after harvest. Error bars indicate SD (n=6). Significant differences: *p<0.05 vs. the value for LED.

As indican is reported to exist only in fresh leaves [19], the indican weight per plant can be determined from the weight of the leaves and the weight of indican per unit weight of the leaves. Consequently, we found that the total weight of indican contained per plant was significantly increased by EDL irradiation, increasing 2.3 times. In other words, indican synthesis improved with increasing fresh leaf weight owing to EDL irradiation for the same number of days.

Since the ratio of this increase in indican was almost the same as the ratio of the increase in the weight of fresh leaves, the concentration of indican per fresh leaf was unchanged. This indicates that leaf weight gain and indican production occurred at the same rate, suggesting that the metabolic pathway was altered further upstream by EDL.

The Mechanism by Which EDL Irradiation Increased the Amount of Indican

Indigo, obtained from the leaves of indigo plants, is a well-known dye. Indicans remained in the vacuole and accumulated in the leaf cells; indigo was produced from indican when the indigo plant cells were destroyed (Figure 8).

Figure 8: Glycosides in P. tinctoria and the pigments produced by enzymatic hydrolysis. Indican stays in the vacuole and accumulates in the cells of indigo leaves, and indigo is produced from indican when the cells of indigo plant are destroyed.

Indican is enzymatically synthesized only from indoxyl and UDP-glucose (UDP-Glc) in the cells of the indigo plant [19] and is shown by the following reaction equation:

Indoxyl + UDP-Glc → Indican + UDP

The synthesis rate of indican is k, where k is the rate constant.

where and are proportional to the concentrations of indoxyl and UDP-Glc, respectively. As indoxyl is considered a very unstable compound and a transient intermediate product, the accumulated concentration of indoxyl is extremely low [19]. In contrast, UDP-Glc is present in plants at a constant concentration [20] because it functions as an important substrate in the synthesis of sucrose and polysaccharides, and as a Glc donor in many glycosylation reactions. If the concentration of indoxyl is sufficiently low in the UDP-Glc solution, the change in the UDP-Glc concentration due to the reaction is negligible. Therefore, the rate of indican synthesis can be approximated as follows:

However, k_app refers to k [UDP-Glc], indicating that its magnitude is determined by the concentration of UDP-Glc. Therefore, the rate of indican synthesis is proportional to the concentration of UDP-Glc. Changes in indican levels in leaves indicate changes in the amount of UDP-Glc. The results for indican weight shown in Figure 7 indicate that EDL irradiation increased the amount of UDP-Glc accumulated in plants.

Indicans are present only in leaves and are more abundant in younger leaves [19]. UDP-Glc is also produced from glucose via glucose-1-phosphate, a substrate used to synthesize cellulose molecules. Since cellulose is a component of plant cell walls, increased UDP-Glc accumulation may indicate an increase in indican and leaf growth promotion, which is biomass. Since photosynthesis occurs primarily in the leaves of plants, leaf growth enhancement represents both acceleration and an increase in photosynthesis. Therefore, the increases in fresh leaves and indican are related. In other words, the enhanced photosynthesis induced by EDL irradiation may have promoted leaf growth, stimulated the production of UDP-Glc, which is necessary for leaf growth, and enhanced the synthesis of indican, thereby increasing leaf indican accumulation (Figure 9).

Figure 9: The scheme of photosynthesis and indican production in P. tinctoria. EDL irradiation enhanced photosynthesis and the production of UDP-Glc. As a result, leaf growth and synthesis of indican were promoted.

In short, indigo plants can clearly sense the EDL, and an increase in the amount of indican due to EDL irradiation indicates that the rate of photosynthesis accelerated.

Characteristics of EDL On Photosynthesis

In conventional indoor plant cultivation, plants are exposed to high-intensity light or large amounts of light to promote photosynthesis. However, the EDL that produced the plant-promoting effect in this study was extremely weak, with a PPFD of 0.01 μmol photons/m²/s. Since the number of photons in the EDL is 1/10000 of that in the LED used for photosynthesis, it is highly unlikely that the amount of photosynthesis was directly increased by the photons in the EDL themselves. We confirm that no change in growth occurred when continuous LED light of 0.01 μmol photons/m²/s, which is the same intensity and PPFD as EDLs, was used (data not shown).

However, EDL is not just weak light, but fluctuates in intensity over time, and illuminates for only 2 μs in 20 μs with an intensity equivalent to the LED used for photosynthesis (shown in Figure 2). Therefore, the EDL affected the plant because of the intensity of the instantaneous light and not because of the number of photons. In other words, it is assumed that an increase in biomass and the amount of indican occurred because the plant sensed a certain intensity of instantaneous light, which stimulated photosynthesis and increased the amount of glucose (Figure 10). Thus, EDL could have acted as a signal to enhance photosynthesis.

Scheme of Enhanced Photosynthesis in Indigo Plants by EDL Irradiation

In plants, the light required for photosynthesis is collected by the light-harvesting antenna (LHA), which is a chlorophyll aggregate. The amount of chlorophyll in LHA increases or decreases slowly depending on sunlight conditions; it decreases during the light period and increases during the dark period of the day [21]. Furthermore, higher plants have sensors for developmental stage, light environment, photosynthetic rate, and respiration rate, and they precisely control chlorophyll concentration according to various growth environments [21]. Therefore, plants constantly regulate their chlorophyll levels to ensure that light is required for photosynthesis.

Figures 4 and 5 show that indigo plants change in growth when LEDs were used in combination with EDLs, compared with that in LEDs alone. From these results, there is no doubt that indigo plants sense EDLs, which may cause changes in chlorophyll levels. In parallel with this study, the chlorophyll content of the indigo leaves was measured using a SPAD analytical sensor (Konika Minolta, SPAD-502), and an average increase of 10% in SPAD value was observed by the EDL irradiation. This fact indicates that the amount of chlorophyll in the leaves increased by the EDL irradiation.

As mentioned previously, the number of photons of EDLs is extremely low, making them unsuitable for photosynthesis. However, the light intensity of the EDL varies with time, showing intensity comparable to that of LEDs used as a light source for photosynthesis for a very short time (2 μs). Thus, we speculate that this causes indigo plants to attempt to initiate photosynthesis by the instantaneous light intensity of EDL irradiation but are unable to do so. Therefore, we hypothesized that indigo plants perceive that they are starving for light, and as a result, increase the amount of chlorophyll to capture more light.

For example, in mammals, including humans, DNA issues a hemoglobin-increase command when blood oxygen levels decrease. This stress response ensures the amount of oxygen in the body in response to oxygen deprivation [22-24]. Using this mechanism, athletes strengthen their cardiopulmonary function by training at high altitudes where oxygen levels are low. We reasoned that plants are sensitive to changes in light because light is an essential element for their survival, just as oxygen is for humans. Our idea of this contrast between the responses of humans and plants is shown in Figure 11.

Figure 11: Countermeasures in face of a survival crisis for human and plant.

As shown in Figure 3, EDL irradiation was conducted during the dark period. Since it is well known that the chlorophyll content of LHA increases during the dark period [24], it is not surprising that the chlorophyll content increases further after EDL irradiation. When indigo plants, which have increased chlorophyll content owing to the illusion of starvation, are exposed to LED irradiation as a photosynthetic light source during the light period, the photosynthetic rate increases because of increased light-harvesting efficiency. As a result, the growth of the indigo plants is promoted and the accumulation of indican, which is derived from glucose produced by photosynthesis, is increased.

Therefore, the action of the EDL irradiation used in this study on plants was determined by two factors: light intensity and time-varying. If the light intensity remains low, it is a normal dark period; no increase in chlorophyll content occurs. To create the illusion of starvation, high intensity of light is necessary for photosynthesis. On the other hand, if the light intensity remains high, it is a normal light period; the number of photons required for photosynthesis is obtained, and no photosynthesis-promoting effect is obtained. To reduce the number of photons per μs so that photosynthesis is not possible, it is necessary to repeat with rapid temporal changes in intensity at μs intervals. Thus, the EDL used in this study is the light with an intensity that promoted photosynthesis and time-varying that promoted starvation in indigo plants.

We investigated the effects of EDL irradiation on the growth and specific metabolite production of indigo plant. Our results reveal that the EDL (0.01 μmol photons/m²/s) promoted the growth: the weight of fresh leaves and the indican metabolite were both increased by 2.3 times to the control. This suggests that EDL enhances photosynthesis because indican is a compound of glucose and indoxyl.

The mechanism of photosynthesis promotion by the EDL irradiation is discussed from the perspective of the plant response to the crisis of survival. We hypothesize that photosynthesis starvation caused by weak but periodic EDL acts as a trigger for chlorophyll synthesis and that sophisticated differential detection of photosynthesis functions in plants.

Acknowledgements

The authors thank K. Miwa, K. Takemura, and M. Takagi for their support. One of the authors (H. K.) thanks Uto Y., Murai K., and Amano S. for providing information on the cultivation and chemical analysis techniques of P. tinctoria.

Funding Information

This study was supported by JSPS KAKENHI (grant number J15K14637). The results were obtained from a project subsidized by the New Energy and Industrial Technology Development Organization and the Ministry of Economy, Trade, and Industry. Research on the high-speed cultivation of Persicaria tinctoria was financially supported by a project from the Promotion of Regional Industries and Universities in Tokushima Prefecture.

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