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AlboPepper - Drought Proof Urban Gardening

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Grow Lights 101: Best Plant Growth Spectrum Color? White vs Red/Blue LED + What is PAR & ePAR Light?

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Jessy Christopher-Tham 🇲🇾 on Instagram: "Read the caption ✨ Will the lighting condition affect the plant DNA? #happymonsteramonday Light helps plants grow and do their photosynthesis thing, turning light energy into plant energy. It doesn't mess with the plant's DNA directly, but it can influence how certain traits show up. So, it's like light shaping their vibe but not messing with the genetic code. In summary, light affects the functioning and behavior of plants, including gene expression, but it doesn't directly modify the DNA sequence of the plant. Here's an example of the same #monsteraalbovariegata in three separate lighting conditions. The first one was at the other end of the balcony where it received the most morning light; the second photo was moved to the middle of the aroid wall, and the third photo was moved to the beginning of the wall recently due to a mishap. The one I'm holding is the propagation of the same mother plant from the first photo, the second, and the third. It is being placed where it receives a good amount of light. *Edited - Based on my experience with plants over the years on my balcony condition, I've found that lighting plays a crucial role in fostering plant growth. Sturdy support poles are equally essential, particularly for climbing plants. Relying solely on plant food/fertilizer isn't sufficient for achieving optimal growth. ✨Anyone noticed that IG is messing with photos too? It is overexposed (see photo no. 3)."

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jcchris on January 21, 2024: "Read the caption ✨ Will the lighting condition affect the plant DNA? #happymonsteramonday Ligh..."

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Yan Li 1,2,3,4 na1,

Guofeng Xin 1 na1,

Chang Liu 5,

Qinghua Shi 1,2,3,4,

Fengjuan Yang 1,3,4 &

...

Min Wei 1,2,3,4

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BMC Plant Biology 20, Article number: 318 (2020) Cite this article

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Abstract

Background

The red (R) and blue (B) light wavelengths are known to influence many plant physiological processes during growth and development, particularly photosynthesis. To understand how R and B light influences plant photomorphogenesis and photosynthesis, we investigated changes in leaf anatomy, chlorophyll fluorescence and photosynthetic parameters, and ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) and Calvin cycle-related enzymes expression and their activities in sweet pepper ( Capsicum annuum L.) seedlings exposed to four light qualities: monochromatic white (W, control), R, B and mixed R and B (RB) light with the same photosynthetic photon flux density (PPFD) of 300 μmol/m ² ·s.

Results

The results revealed that seedlings grown under R light had lower biomass accumulation, CO ₂ assimilation and photosystem II (PSII) electron transportation compared to plants grown under other treatments. These changes are probably due to inactivation of the photosystem (PS). Biomass accumulation and CO ₂ assimilation were significantly enriched in B- and RB-grown plants, especially the latter treatment. Their leaves were also thicker, and photosynthetic electron transport capacity, as well as the photosynthetic rate were enhanced. The up-regulation of the expression and activities of Rubisco, fructose-1, 6-bisphosphatase (FBPase) and glyceraldehyde-phosphate dehydrogenase (GAPDH), which involved in the Calvin cycle and are probably the main enzymatic factors contributing to RuBP (ribulose-1, 5-bisphosphate) synthesis, were also increased.

Conclusions

Mixed R and B light altered plant photomorphogenesis and photosynthesis, mainly through its effects on leaf anatomy, photosynthetic electron transportation and the expression and activities of key Calvin cycle enzymes.

Background

Light is one of the most important environmental factors affecting plant growth and development [ 1]. Using light rather than chemicals to control plant architecture can reduce the environmental impacts [ 2]. Light affects the photosynthetic characteristics of seedlings by regulating chloroplast and anatomy development, and through its influence on key enzyme activities and the related expression of genes involved in the Calvin cycle, etc. [ 3, 4, 5, 6].

Photosynthesis is the green engine that powers life on Earth, as it is the only biological process that allows plants, etc., to convert light energy into chemical energy [ 7]. Improving photosynthesis is critical to maintaining sufficient dry biomass accumulation. It is well known that in addition to light intensity and photoperiod, light quality, namely, light color or wavelength, exerts a significant effect on regulating plant growth and photosynthesis [ 8, 9, 10, 11, 12]. Specific light qualities have precise effects on plants. For example, blue (B) and red (R) light are the most effectively utilized wavelengths during plant photosynthesis because the absorption spectra of the photosynthetic pigments mainly focus on the B (400-500 nm) and R (600-700 nm) light spectra. Therefore, their utility and regulatory mechanisms have always been important areas of research [ 13, 14].

A few studies have used R and B light to examine the effects of light quality on anatomy, photosynthesis and morphology of plants. In general, R light plays an important role in controlling the functions of the chloroplast, stem and petiole growth and the reproductive system [ 15, 16]. B light affects plant growth, leaf expansion, photomorphogenesis, stomatal opening, photosynthesis and pigment accumulation [ 17, 18]. Furthermore, it is shown that plants grown under B light have higher stomatal conductance, lager chlorophyll (Chl) a/ b, greater photosystem (PS) activity and photosynthetic electron transport ability, higher levels of ribose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activity and expression of genes related to Calvin cycle than those plants grown under R light [ 19, 20].

The Calvin cycle which occurs during the process of photosynthesis consists of light-independent redox reactions which happens in the stroma of chloroplasts and exerts a key effect on photosynthetic carbon fixation. The efficiency of carbon assimilation is affected by the regeneration rate of ribulose-1, 5-bisphosphate (RuBP). Rubisco is a key enzyme in plant photosynthesis that controls both carbon dioxide and carbon fixation [ 21]. This set of reactions is catalyzed by Rubisco as well as other corresponding key enzymes and finally converts carbon dioxide and water into organic sugars. According to the previous researches, light quality exerts an impact on photosynthetic property by regulating the expression of these genes related [ 22, 23].

It has also been shown that monochromatic R or B light does not satisfy normal plant growth requirements and the absence of one of the two light qualities creates photosynthetic inefficiencies [ 24]. Various studies have found that mixed R and B light is an effective lighting source that improves plant development and a suitable proportion of R and B light accelerates photosynthesis and the growth of tomato, cucumber and sweet pepper, etc. [ 24, 25, 26]. Leaf anatomy may directly influence light capture by its leaf thickness as well as by the differentiation of palisade and spongy mesophyll. Earlier report showed that leaf thickness increased when R light was supplemented with B light [ 27]. Furthermore, Klein [ 28] and Naznin [ 26] found that mixed R and B light led to higher Chl a, b and total Chl levels, an improved electron transport rate (ETR) and an early onset of non-photochemical quenching (NPQ), all of which lead to increases in photosynthetic efficiency. Therefore, mixed R and B light is now used in research studies and commercial horticulture because of their effective photosynthetic wavelengths at the leaf level [ 29, 30]. Despite these achievements, the specific photosynthesis processes in plants affected by mixed R and B light remains largely unknown.

The popularity of sweet pepper ( Capsicum annuum L.) for fresh market consumption or in ready-to-eat food has risen significantly during the past decades and these peppers are mostly produced in protected environments [ 31]. Mixed R and B light has an apparent influence on the growth and physiology of pepper plants [ 26, 32, 33]. Gaining a more complete mechanistic picture of how plants adapt and respond to R and B light quality is important since light quality plays important roles in growth and physiology. In addition, a better understanding of the leaf anatomy, CO ₂ assimilation and photosynthetic electron transport that influence responses to R and B light can improve the photosynthetic efficiency and assist in developing better methods to evaluate plant responses to light quality. Recently, light-emitting diodes (LEDs), which are light sources that have a high photosynthetic efficiency, have been successfully used in scientific research and protected horticulture [ 34, 35, 36]. Our previous studies have found that a suitable proportion of mixed R and B light (light intensity of R:B = 3:1, RB) accelerated pepper seedlings' photosynthesis and growth. The objective of this study was to examine how R and/or B light sources affected pepper seedling photomorphogenesis, photosynthetic characteristics, as well as the transcriptional and translation levels of key enzymes in the Calvin cycle.

Results

Plant morphology and biomass accumulation under different light treatments

A visual overview of the influence of monochromic and mixed R and B light on morphology of sweet pepper seedlings at 28 day (d) after treatment (DAT) was shown in Fig. 1 and Supplementary Fig. 2 and the differences among different treatments were significant. The plant shoot dry weight (DW) under RB was significantly increased compared with W ( P < 0.05), and it was also higher than that under other treatments, whereas, R light produced the lowest DWs (Fig. 2a). The root DWs showed similar trends under all the treatments (Fig. 2 b).

Effects of different light treatments on plant morphology of sweet pepper seedlings at 28 day after treatment. W, white light; R, monochromatic R light; B, monochromatic B light; RB, mixed R and B light of 3:1

Effects of different light treatments on ( a) shoot dry weight and ( b) root dry weight of sweet pepper seedlings at 28 day after treatment. Data are presented as means ± SE, n = 3. Different letters indicate significant differences between values ( p  < 0.05). W, white light; R, monochromatic R light; B, monochromatic B light; RB, mixed R and B light of 3:1

Leaf anatomy under different light treatments

Table 1 and Fig. 3 showed that R and B light had a significant effect on the anatomical structure of pepper leaves. Leaf thickness was the highest under RB, followed by B and W, while the thinnest leaves were found under R light. Furthermore, compared to W, the thickness of palisade mesophyll tissue (PT), spongy mesophyll tissue (SPT) and the upper epidermis were significantly greater under RB treatment ( P < 0.05). These three parameters increased by 26, 19 and 22%, respectively, but they were significantly reduced by R light. Thinner lower epidermal thicknesses were found under R, whereas the epidermis tended to be thicker under RB although they were not significantly different from W. The effect on the PT and SPT ratio was not strong ( P  > 0.05) and the thinnest cell layers occurred under R.

Table 1 Effects of different light treatments on leaf anatomy of sweet pepper seedlings at 28 day after treatment

Effects of different light treatments: ( a) white light; ( b) monochromatic R light; ( c) monochromatic B light; ( d) mixed R and B light of 3:1 on leaf sectioning anatomy of sweet pepper seedlings at 28 day after treatment. Images of leaf sectioning anatomy are at the same magnification. The images were taken at 200 × magnification. EP, epidermis cell; PT, palisade mesophyll tissue; SPT, spongy mesophyll tissue

Photosynthetic light- and CO₂-response curves under different light treatments

Both of the net photosynthetic rate (Pn) of the leaves increased rapidly along with the increment in PPFD (Fig. 4a) and CO ₂ concentration (Fig. 4b) at the initial stage, after that, their increasing tendency gradually became stable. The highest Pn-PPFD response curve value was detected under RB, followed by B and W, whereas R produced the lowest value. Furthermore, different light treatments produced similar trends for Pn-CO ₂. The apparent quantum efficiency (AQY), light saturation point (LSP), light-saturated maximum (Pn max), carboxylation efficiency (CE) and CO ₂ saturation point (CSP) levels and the maximum RuBP regeneration rate were significantly higher under RB ( P < 0.05) than those under W, whereas, the light compensation point (LCP) and CO ₂ compensation point (CCP) values were decreased under this treatment (Table 2 and Table 3).

Effects of different light treatments on ( a) photosynthetic light- and ( b) CO ₂-response curves of sweet pepper seedlings at 28 day after treatment. Pn, net photosynthetic rate; PPFD, photosynthetic photon flux density; W, white light; R, monochromatic R light; B, monochromatic B light; RB, mixed R and B light of 3:1. □ W; ● R; △ B; ◆ RB

Table 2 Effects of different light treatments on photosynthetic light-response curve parameters of sweet pepper seedlings at 28 day after treatment

Table 3 Effects of different light treatments on photosynthetic CO₂-response curve parameters of sweet pepper seedlings at 28 day after treatment

Chlorophyll a fluorescence and the chlorophyll fluorescence transients under different light treatments

The effects of R and B light on the pepper seedling Chl fluorescence parameters were shown in Fig. 5. F v/ F m, which represents the greatest light conversion efficiency or the maximum quantum yield of PS II, was significantly higher under RB and B than that under W and there were no significant differences between RB and B treatments (Fig. 5a). Furthermore, this parameter significantly declined under R ( P < 0.05). Φ PSII represents the actual conversion efficiency of PS II or the actual quantum yield and it showed a similar reaction to the four light quality treatments (Fig. 5b). F' v/ F' m indicates how efficiency the excitation energy is captured by open photosystem II (PSII) reaction centers and it was enhanced in RB-grown seedlings, followed by W and B, and there were no significant differences among these three treatments ( P > 0.05) (Fig. 5c). However, seedlings grown under R light had significantly lower F' v/ F' m values ( P  < 0.05), and no significant difference was found between R and B treatments.

Effects of different light treatments on chlorophyll fluorescence parameters: ( a) F v/ F m, maximum photochemical efficiency of PSII; ( b) Φ PSII, actual PSII photochemical efficiency; ( c) F' v/ F' m, maximum photochemical efficiency of PSII under light adaptation of sweet pepper seedlings at 28 day after treatment. Data are presented as means ± SE, n = 3. Different letters indicate significant differences between values ( p  < 0.05). W, white light; R, monochromatic R light; B, monochromatic B light; RB, mixed R and B light of 3:1

The typical polyphasic Chl a fluorescence transient (OJIP) increased at different experimental time points were shown in Fig. 6a-d. In general, the results indicated that the W, B and RB treatments decreased the amplitude of the OJIP curves compared with R, mainly at the J and I step, whereas they were higher under R light. There was no obvious difference in the maximal amplitude of the O and P steps among the treatments ( P > 0.05). In order to further study the mechanisms behind the observed changes, the JIP-test was used for the fluorescence induction transients (Fig. 7a-h). Most JIP-test parameters (e.g., the general electron carrier of the reaction center (S m), the potential for energy conservation from photons absorbed by PSII to the reduction of the intersystem electron acceptors (PI ABS), the potential for energy conservation from photons absorbed by PSII to the reduction of PSI end acceptors (PI total), the quantum yield for reduction of end electron acceptors at the PSI acceptor side ( Φ Ro) and the efficiency/probability with which an electron from the intersystem electron carriers is transferred to reduce end electron acceptors at the PSI acceptor side ( δ Ro)) were significantly elevated by B and RB compared with W ( P < 0.05), but the R light produced relatively lower values. Additionally, the fraction of PSII Chl a molecules that function as reaction centers (RC/ABS), the dissipated energy in the reaction center (DI o/RC) and the maximum trapped energy exciton per active PSII reaction center (TR o/RC) in the leaves under R were significantly greater than those under other treatments ( P  < 0.05).

Effects of different light treatments on chlorophyll a fluorescence transient (OJIP) of sweet pepper seedlings at different experimental periods. ( a), ( b), ( c), and ( d) were at 7, 14, 21, and 28 day after treatment, respectively. W, white light; R, monochromatic R light; B, monochromatic B light; RB, mixed R and B light of 3:1

Effects of different light treatments on JIP-test parameters: ( a) RC/ABS, fraction of PSII Chl a molecules that function as reaction centers; ( b) S m, general electronic carrier of the reaction center; ( c) DI o/RC, dissipated energy in the reaction center; ( d) TR o/RC, maximum trapped energy exciton per active PSII reaction center; ( e) PI ABS, potential for energy conservation from photons absorbed by PSII to the reduction of the intersystem electron acceptors; ( f) PI total, potential for energy conservation from photons absorbed by PSII to the reduction of PSI end acceptors; ( g) Φ Ro, quantum yield for reduction of end electron acceptors at the PSI acceptor side; ( h) δ Ro, efficiency/probability with which an electron from the intersystem electron carriers is transferred to reduce end electron acceptors at the PSI acceptor side of sweet pepper seedlings at different experimental periods. Data are presented as means ± SE, n = 3. Different letters indicate significant differences between values ( p  < 0.05). W, white light; R, monochromatic R light; B, monochromatic B light; RB, mixed R and B light of 3:1. □ W; ● R; △ B; ◆ RB

Calvin cycle enzymes activity under different light treatments

Rubisco, FBPase, fructose-1, 6-bisphosphate aldolase (FBA), glyceraldehyde-phosphate dehydrogenase (GAPDH) and transketolase (TK) are key enzymes in the Calvin cycle. The results showed that the Rubisco activities increased initially and then decreased with the duration of different light quality treatments increased (Fig. 8a-e). Seedlings under B and RB had significantly higher Rubisco activities than W-grown seedlings ( P < 0.05) with 65 and 36% increases, respectively, at 28 DAT (Fig. 8). In contrast, R-grown plants had a significantly lower activity levels (15% less) than W-grown plants.

Effects of different light treatments on activities of Calvin cycle-related enzymes: ( a) Rubisco, ribulose-1, 5-bisphosphate carboxylase/oxygenase; ( b) FBPase, fructose-1, 6-bisphosphatase; ( c) FBA, fructose-1, 6-bisphosphate aldolase; ( d) GAPDH, glyceraldehyde-phosphate dehydrogenase; ( e) TK, transketolase from sweet pepper seedlings at different experimental periods. Data are presented as means ± SE, n = 3. Different letters indicate significant differences between values ( p  < 0.05). FW, fresh weight; W, white light; R, monochromatic R light; B, monochromatic B light; RB, mixed R and B light of 3:1. □ W; ● R; △ B; ◆ RB

Sharp increases in FBPase activity were observed in pepper seedlings under the different light treatments. The FBPase activities reached their highest levels at 21 DAT and then decreased over the following days (Fig. 8b). Activities of this enzyme in plants under B light remained significantly higher than those under other treatments from 7 to 21 DAT ( P < 0.05), but there was no significant difference between W and B at 28 DAT ( P > 0.05). Significantly lower activities were observed under R light than those under other treatments during the experimental period. The FBA activities in plants treated with W and R light increased slowly during the experimental period (Fig. 8c), whereas, they rapidly increased in the RB and B treatments after 14 DAT, which indicated that the enzyme activity in the RB and B treatments was greater than in the W and R treatments. The GAPDH activities decreased in plants under all treatments, but the W and RB light applications alleviated the reduction (Fig. 8d). The TK activities were similar under all the treatments during the experimental period, except that the GAPDH and TK activities were significantly lower under the R-treatment than those under other treatments (Fig. 8 e).

Gene expression under different light treatments

The RT-PCR method was used to analyze the relative expression levels of FBA, FBPase, GAPDH and TK genes involved in the Calvin cycle after pepper seedling exposure to different light qualities for 28 d. Figure 9a-d showed that the transcriptional levels of these genes varied significantly depending on the light qualities supplied and similar variation patterns were obtained for FBA, FBPase and GAPDH under different treatments. Generally, compared to W, seedlings under RB showed significantly increased expression levels of these three genes, whereas exposure to R light resulted in decreased gene transcription. Additionally, the relative expression level of TK was up-regulated in B-treated seedlings, followed by RB and W, but R produced the lowest TK levels.

Effects of different light treatments on expression of ( a) FBA; ( b) FBPase; ( c) GAPDH; ( d) TK from sweet pepper seedlings at 28 day after treatment. Data are presented as means ± SE, n = 3. Different letters indicate significant differences between values ( p  < 0.05). W, white light; R, monochromatic R light; B, monochromatic B light; RB, mixed R and B light of 3:1

Discussion

During the process of light-controlled growth, it is stated that photoreceptors modulate light-responsive nuclear genes by perceiving and interpreting incident light and transduce signals. In the light spectra, R and B wavelengths can strongly affect plant photosynthesis, physiological metabolism and morphology as the main spectral wavelengths [ 37, 38, 39]. In this study, the photomorphogenesis and photosynthetic characteristics of sweet pepper seedlings were significantly influenced by the light qualities. Biomass is an important indicator of seedling quality. In this study, the seedling DW under RB was significantly greater than those under other treatments, which suggested that this spectrum was optimal because it promoted plant development and drove photosynthesis by increasing Chl a and total Chl contents in the seedlings [ 33, 40]. Previous studies also found that mixed R and B light could promote fresh weight (FW) and DW in many other plant species, such as chrysanthemum, upland cotton and tomato [ 41, 42, 43]. The biomass of pepper seedling was significantly increased under RB compared with other treatments and this was probably due to the enlarged leaf area (LA) [ 44] and changes to the leaf anatomy.

Light is absorbed by chloroplasts when it passes through the PT and SPT, which are both important photosynthetic tissues. In our study, RB treatment greatly increased the PT, SPT, as well as upper and lower epidermis thickness, which led to thicker leaves, and this was consistent with the results of Arena et al. [ 45] and Liu et al. [ 46]. The vertically elongated PT cells minimized light scattering, which allowed deeper penetration into the chloroplasts, while the changes to the SPT cells enhanced light capture by scattering the light [ 47]. This improved the photosynthetic structure, which should increase the light capture and absorbance capacities, and contribute to better photosynthetic light acclimation. In addition, leaf thickness plays a key role in determining space availability for chloroplast development [ 48]. The RB treatment increased leaf thickness, which enhanced the chloroplast ultrastructure [ 49]. The results suggested that a larger LA and increased leaf, as well as PT and SPT cells thickness improved light interception by the pepper seedlings. and this could be another important reason why RB was able to improve photosynthetic efficiency. Furthermore, the thinner leaves recorded under R light can be explained as a reaction to radiation stress on plant development and metabolic processes, as suggested by Macedo et al. [ 50].

The ability to do well out of the increments in optical energy and CO ₂ of plants is reflected by the light- and CO ₂-response curves, which provides interesting opinions on the mechanisms based on light capture and CO ₂ fixation. In this study, Pn-PPFD under the different light qualities was significantly lower than Pn-CO ₂. This might be due to a CO ₂ concentration limitation. The AQY and CE values showed the initial slopes of the light- and CO ₂-response curves, respectively. They stand for the ability to obtain low levels of light energy and CO ₂ of plants. Our results confirmed a previous study [ 51], which showed that mixed R and B light promoted AQY and CE, and that these increases led to a rise in Pn max and maximized the RuBP regeneration rate. The RB light led to significant increases in AQY, CE, Pn max and the maximum RuBP regeneration rate. This indicates that mixed R and B light exerts an synergistic effect on increasing photosynthetic capacity [ 52]. The LSP values, which reflect the plant ability to use the highest light intensity level, were also significantly higher under RB. This showed that RB improved the ability of the leaves to utilize mixed light qualities. Furthermore, the LCP and CCP values were significantly decreased under RB, which showed that this treatment improved photosynthetic performance and light energy utilization efficiency. These results indicated that the energy conversion of mixed R and B light into chemical energy by the leaves was very efficient, as this fraction of visible light had, by far, the highest quantum yield for CO ₂ fixation compared with other light treatments [ 53].

Light qualities can regulate photosynthesis by affecting the formation of different types of chloroplast proteins and electron transport between light systems [ 54]. Chl fluorescence can partly reflect the photosynthetic ability of plants [ 55] and the efficiency of PSII photochemistry ( Φ PSII) can be used to reveal the physiological state of plants [ 56]. Our results showed that there was a reduction in Φ PSII in pepper seedlings after exposure to the RB treatment. F v/ F m represents the maximal efficiency of the excitation energy captured by the PSII reaction centers and the significantly higher value observed in RB-treated seedlings indicated that resistance to photoinhibition was up-regulated under this treatment [ 57]. Additionally, the higher F' v/ F' m and Φ PSII levels under RB treatment showed that mixed R and B light increased the openness and electron transport efficiency of PSII, which meant that more electrons could be absorbed, captured and transported.

There is a correlation relationship between the J-step, I-step and IP phases of Chl fluorescence transients and the redox states of quinone electron acceptor (Q A), plastoquinone and the end acceptors at the side of PSI electron acceptor [ 58, 59]. The finding that R-treated leaves increased the J- and I-step suggested that electron transport at both the donor and acceptor sides of PSII was inhibited. Therefore, CO ₂ assimilation was decreased by the imbalance of excitation energy distribution between PSI and PSII. Monochromatic B and mixed R and B light induced a decrease in all the OJIP steps during the experimental period compared with other treatments, which altered both the donor and acceptor sides of PSII and affected electron transport [ 60]. These changes maintained electron transportation on both the donor and acceptor sides. Furthermore, we found that RB increased S m, PI ABS, PI total, Φ Ro and δ Ro, but decreased RC/ABS, DI o/RC and TR o/RC (Fig. 7), which less damaged the photochemical and non-photochemical redox reactions, enhanced the ability of electron transport and sped up ATP synthesis and RuBP regeneration [ 61].

In C3 plants, the Calvin cycle is the predominant pathway for CO ₂ assimilation [ 62]. Rubisco is a representative and unique enzyme in the Calvin cycle and other Calvin cycle enzymes, including FBPase, FBA, GADPH and TK, play an important part in modulating this pathway [ 63, 64]. As a significant environmental signal, light provokes gene expression and regulates related enzyme activities during the growth of plants. How light adjusts the expressions and activities of enzymes in photosynthesis was examined by several researches [ 52, 65]. These previous studies were verified by the present study. The Rubisco activity in B- and RB-treated plants was significantly higher than those in the plants treated with other light wavelengths. This finding suggested that the application of B or RB could increase carbon assimilation and RuBP regeneration in the Calvin cycle. It was also found that under R light, photosynthetic rate has decreased as the number of Rubisco activities and the transcriptional levels of most genes in the Calvin cycle reduced. This result was consistent with an earlier observation and implied that the inhibition of CO ₂ carboxylation in the Calvin cycle and PSII slow down as a result of the impaired activity of Rubisco activase, which removes inhibitors bound to Rubisco, are probably responsible for the decreased CO ₂ assimilation rate in R-grown seedlings compared with other light treatments [ 36, 66]. Furthermore, according to a previous research, the stomatal factor regulating the availability of RuBP differentially, and CO ₂ may participate in adjusting gene expression because there is a high correlation between the expression levels of the genes examined and the changes in stomatal conductance [ 36].

The FBA and FBPase activities directly affect photosynthetic efficiency and carbon accumulation [ 67]. Furthermore, a previous study showed that a significantly decrease in TK activity led to a significant reduction in RuBP regeneration and significantly inhibited the plant photosynthetic rate [ 68]. In our study, the activities of these enzymes under B and RB and the relative expression of their associated genes, except for FBA and TK, were significantly elevated, which promoted RuBP regeneration and increased Pn [ 67, 68]. Chloroplast GAPDH is a key enzyme involved in the carbon reduction process during photosynthesis [ 69] and the greater GAPDH expression level under RB light in the present study may be due to the increased demand for carbon flux [ 70], suggesting that maintenance of active GAPDH expression in the carbon reduction process could be an important factor contributing to superior photosynthesis under RB light [ 71]. Changes in activities of FBA and TK as well as their expression under all treatments were not positively correlated, suggesting that transcript abundance is poorly linked to de novo protein synthesis due to profound regulation at the level of translation Oelze et al. [ 72]. Moreover, the different patterns of gene expression and activity are probably correlated with regulatory factors other than light quality, but this needs further investigation.

Conclusions

Light quality is an important environmental factor that regulates the plant photomorphogenesis and photosynthetic characteristics. In conclusion, sweet pepper growth, development and photosynthesis are precisely controlled and genetically regulated by light quality. The results indicated that photosynthesis in seedlings under R light was inhibited by the decreased photosynthetic electron transport capacity, which caused a reduction in CO ₂ assimilation. This led to down-regulation of Calvin cycle associated gene expressions and their related enzymatic activities. However, the use of monochromatic B and mixed R and B light, especially the latter, could enhance the activity of the PSII reaction center and improve photosynthesis and the expression and activities of Calvin cycle-related enzymes, including Rubisco, FBPase and GAPDH, which are probably the main enzymatic factors contributing to RuBP synthesis. Therefore, mixed R and B light may provide more suitable light conditions for the growth of sweet pepper seedlings.

Methods

Plant material and climate conditions

The experiment was performed from June to October, 2016 in a Chinese solar greenhouse (CSG) and an artificial climate chamber (ACC, Zhejiang Qiushi Environment Co., Zhejiang, China) at the Horticultural Research Center, Shandong Agricultural University, P. R. China. After immersing sweet pepper ( Capsicum annuum L. cv. Hongqijian) seeds (Jinan Weili Seeds Co., Ltd., Shandong, China) in water for 15 min at the temperature of 55 °C and soaking it in cold water (4 °C) for 24 h. The seeds were sown into 50-cell plug trays (54.0 × 30.0 × 4.4 cm) filled with a mixture of peat (Floragard Seed 2, Floragard Co., Oldenburg, Germany) and vermiculite (2:1, v/v) in the CSG. All seedlings were watered daily with half-strength Yamazaki's pepper nutrient solution. Three weeks later, when their second true leaf had fully expanded, the seedlings were transplanted into plastic pots (8 cm long, 8 cm wide and 10 cm deep, one seedling per pot) containing the same substrate and watered with full-strength nutrient solution. Then, 480 seedlings in total were chosen, transferred into the ACC and cultured while receiving four kinds of light quality treatments for 28 d. Each light treatment was repeated three times in the same ACC and there were 40 plants for per replication per treatment. Five plants were randomly sampled at 7, 14, 21 and 28 DAT from each replication each treatment and were subjected to morphological and biochemical analyses. There was ventilation in the controlled environment, so the CO ₂ level was the same as the CO ₂ level of atmosphere outside. The relative humidity (RH) was kept at 70 ± 10%, with a 12 h photoperiod and a temperature of 26 ± 1 °C during the daytime and 18 ± 1 °C at night.

Light treatments

All the mixed LEDs had a uniform spectrum for R and B light and were designed by Chunying Optoelectronics Technology Co., Ltd., Guangdong, China. The cultivation rack in the ACC was a steel frame structure with an LED light source placed at the top. The different treatments were insulated from one another by silver shading material. The plants were grown under the following light conditions: monochromatic B light with a maximum intensity at 457 nm, R light or mixed R and B light (3:1, RB: 75% R light witht a wavelength of 657 nm and 25% B light with a wavelength of 457 nm) has a maximum intensity at 657 nm. There was a multi-wavelength W light treatment as control (Supplementary Fig. 1). The light intensity, expressed as PPFD at the canopy level, was set at 300 μmol/m ² ·s, which was measured using a quantum sensor (LI-250, LI-COR Inc., Lincoln, NE, USA) and maintained by adjusting the distance of the LEDs from the canopies. The LEDs was approximately 10 cm far away from the canopy. A spectroradiometer (Unispec-SC Spectral Analysis System, PP Systems Inc., Haverhill, MA, USA) was used to measure the spectral photon flux density distributions (SPDs) of the LEDs.

Biomass analysis

Five seedlings, including leaves and roots, were removed from each replication each treatment at 28 DAT and dried in an oven at 105 °C for 30 min. The oven temperature was changed to 75 °C and the plants were dried to a constant weight. Then, the DWs of leaves and roots were measured using an electronic balance (precision: ± 0.1 g, Model LA16001S, Sartorius Co., Hamburg, Germany).

Leaf anatomy

Leaf anatomy was measured on the fully expanded second leaves from five pepper seedlings at a similar position for each replication each treatment [ 46] on 28 DAT. Leaf segments of 5 mm × 5 mm were taken from the central leaf blade next to the main vein, fixed with formalin-acetic acid-alcohol (FAA) fixative, dehydrated in an alcohol and xylene series, embedded in paraffin, cross-sectioned to a thickness of 10 μm, and stained with red-solid green. The total thickness of the whole leaf and the thickness of the upper epidermis, lower epidermis, PT and SPT were measured under a transmission light microscope (DP71, Olympus Inc., Tokyo, Japan). Images were collected using a digital camera (Camedia C4040, Olympus Inc., Tokyo, Japan) and analyzed by AnalySIS 5.0 (Olympus Inc., Tokyo, Japan).

Photosynthetic light- and CO₂-response curves

Between 09:00 am and 14:00 pm, the measurement of photosynthetic light-response curves and CO ₂-response curves was made on the second leaf fully-unfolded using a portable photosynthesis systems machine (LI-6400XT, Li-COR, Lincoln, NE, USA) at 28 DAT. The measurement technique was based on a modified method described by Pan et al. [ 52]. In the leaf chambers, the temperature was 26 ± 1 ∘C, air relative humidity was 65 ± 5% and the flow rate was 300 μmol/s. The measurement of light-response curves was made under different graded PPFD series of 1800, 1500, 1200, 1000, 800, 600, 400, 300, 200, 150, 100, 50, 20 and 0 μmol/m ²·s. When the CO ₂-response curve measurements were taken, the light intensity and CO ₂ concentration of the leaf cuvette were set to 1000 μmol/m ²·s and 400 μmol/mol, respectively, for 30 min. After reaching a steady state, the curves of CO ₂ response were measured by a CO ₂ mixer under a graded Ci value series of 400, 300, 200, 100, 50, 100, 200, 300, 400, 600, 800, 1000, 1200, 1500 and 1800 μmol·CO ₂/mol. The leaf chamber spends 120 to 180 s in adjusting its new microclimate each time. According to a previous report, three times of measurement were made for each curve, which was suitable for a non-linear regression equation [ 73, 74], so that the LCP, LSP, Pn max, CCP, CSP and the maximum RuBP regeneration rate. The starting slope of the curve of light response was the AQY, and the starting slope of the curve of CO ₂ response was the CE.

Chlorophyll fluorescence and chlorophyll fluorescence transients

The Chl fluorescence measurements were performed using a portable pulse modulation fluorometer (FMS-II, Hansatech Instruments Ltd., King's Lynn, Norfolk, UK). The second fully expanded leaves of five seedlings from each replication each treatment were dark adapted for 20 min, and the F o (original fluorescence yield) and F m (maximum fluorescence yield) were determined. Then, the leaves were put under natural light for 1 h, and the measurements of F' o, F' m and F s values was made under the activating light of 800 μmol/m ²·s. With the saturation pulse intensity of 3000 μmol/m ²·s and the duration of 0.8 s, F' o and F' m respectively stand for the minimum and maximum fluorescence yields of an illuminated leaf, which were measured by applying the method of saturation pulse. F s means the steady fluorescence yield. The maximum photochemical efficiency of PSII was calculated using F v/ F m = ( F m - F o) / F m, actual PSII photochemical efficiency was calculated using ( Φ PSII) = ( F' m - F s) / F' m and maximum photochemical efficiency of PSII under light adaptation was calculated using ( F' v/ F' m) = ( F' m - F' o) / F' m.

A plant efficiency analyzer (Handy PEA, Hansatech Instruments Ltd., King's Lynn, Norfolk, UK) was used to measure the OJIP on the second leaves. Strasser's method was employed to calculate the JIP-test formulae and glossary of terms [ 75, 76]. The following derivative parameters were determined according to Lin et al. [ 61] and Miao et al. [ 30]: RC/ABS, S m, DI o/RC, TR o/RC, PI ABS, PI total, Φ Ro and δ Ro.

Calvin cycle enzymes activity

After being sampled at 7, 14, 21 and 28 DAT, the second leaves selected from top 15 plants of each treatment were used to determine the enzyme activities. Leaf tissue (0.5 g) was homogenized in 4 mL of ice-cold extraction buffer: (25 mM Hepes (K⁺), pH 7.5, 10 mM MgSO ₄, 5 mM dithiothreitol (DTT), 1 mM Na ₂EDTA, 1 mM phenylmethanesulfonyl fluoride (PMSF), 5% (w/v) insoluble polyvinylpyrrolidone (PVP) and 0.05% (v/v) Triton X-100). The homogenate was filtered through muslin cloth and centrifuged at 14,000×g for 5 min at 4 °C. The supernatant was used as the enzyme extract for the enzyme activity assays [ 77].

An ELISA kit (Shanghai Yanji Biological Technology Ltd., Shanghai, China) was employed to determine the Rubisco (EC 4.1.1.39), FBPase (EC 3.13.11), FBA (EC 4.1.2.13), GAPDH (EC 1.2.1.12) and TK (EC 2.2.1.1) activities, and the extraction approach for these enzymes were modified based on Rao and Terry [ 78] and Wang et al. [ 36]. After grounding the frozen leaf samples (0.5 g) to fine powder in a liquid nitrogen with a mortar and pestle, the powder was put into a centrifuge tube and extracted to the precool extraction buffer (5 mL). The centrifugation of enzyme extraction solution was made at 12,000×g for 15 min at the temperature of 4 °C. The activity assay of Calvin cycle enzymes used the supernatant. Afterwards, a microplate absorbance reader (Bio-Tek ELX800, Bio-Tek Instruments, Winooski, VT, USA) was used to determine the activities of the Calvin cycle enzymes under an absorbance of 450 nm based on the instructions of the manufacturer.

The measurement of the protein concentration of each enzyme extraction solution was made based on Bradford [ 79]. The results of the measurement were showed as U/g of protein.

Gene expression

Quick RNA Isolation Kit was used to extract total RNA according to the supplier's instructions (Huayueyang Biotech Co., Ltd., Beijing, China). A ReverTra Ace qPCR RT-Kit (Toyobo Bio-Technology, Co., Ltd., Osaka, Japan) was applied to make reverse transcription. Real-time PCR was employed to conduct the gene expression analysis with 18S rRNA as an internal control. The thermal cycler procedure was cycled once for 2 min at the temperature of 94 °C and cycled for 40 times at the temperature of 94 °C for 10 s, 60 °C for 20 s and 72 °C for 30 s. The method described in Livak and Schmittgen was used to analyze relative gene expressions [ 80]. The specific gene primers used for real-time PCR analysis of the genes involved in the PS complexes are shown in Supplementary Table 1.

Data analysis

The experiment had a totally random design. Values presented are the mean ± standard deviation (SD) of three replicates. One-way variance analysis (ANOVA) was employed to analyze the data, and the differences between the means were tested by Duncan's multiple range test ( P  < 0.05). The charts were created using Origin (version 8.5, Microcal Software Inc., Northampton, MA, USA).

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Abbreviations

R:

Red

B:

Blue

W:

White

RB:

Mixed red and blue light

PPFD:

Photosynthetic photon flux density

Chl:

Chlorophyll

ETR:

Electron transport rate

NPQ:

Non-photochemical quenching

LED:

Light-emitting diode

LA:

Leaf area

CSG:

Chinese solar greenhouse

ACC:

Artificial climate chamber

D:

Day

DAT:

Day after treatment

RH:

Relative humidity

SPD:

Spectral photon flux density distribution

DW:

Dry weight

FAA:

Formalin-acetic acid-alcohol

EP:

Epidermis cell

PT:

Palisade mesophyll tissue

SPT:

Spongy mesophyll tissue

Pn:

Net photosynthetic rate

LCP:

Light compensation point

LSP:

Light saturation point

Pnmax :

Light-saturated maximum

CCP:

CO ₂ compensation point

CSP:

CO ₂ saturation point

AQY:

Apparent quantum efficiency

CE:

Carboxylation efficiency

F v/F m :

Maximum photochemical efficiency of PSII

Φ PSII :

Actual PSII photochemical efficiency

F'v/F'm :

Maximum photochemical efficiency of PSII under light adaptation

OJIP:

Chl a fluorescence transient

RC/ABS:

Fraction of PSII Chl a molecules that function as reaction centers

Sm :

General electronic carrier of the reaction center

DIo/RC:

Dissipated energy in the reaction center

TRo/RC:

Maximum trapped energy exciton per active PSII reaction center

PIABS :

Potential for energy conservation from photons absorbed by PSII to the reduction of the intersystem electron acceptors

PItotal :

Potential for energy conservation from photons absorbed by PSII to the reduction of PSI end acceptors

Φ Ro :

Quantum yield for reduction of end electron acceptors at the PSI acceptor side

δ Ro :

Efficiency/probability with which an electron from the intersystem electron carriers is transferred to reduce end electron acceptors at the PSI acceptor side

RuBP:

Ribulose-1, 5-bisphosphate

Rubisco:

Ribulose-1, 5-bisphosphate carboxylase/oxygenase

FBPase:

Fructose-1, 6-bisphosphatase

FBA:

Fructose-1, 6-bisphosphate aldolase

GAPDH:

Glyceraldehyde-phosphate dehydrogenase

TK:

Transketolase

DTT:

Dithiothreitol

PMSF:

Phenylmethanesulfonyl fluoride

PVP:

Polyvinylpyrrolidone

References

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Acknowledgements

We acknowledge all the members of the research team for their assistance in the field and laboratory work. We thank International Science Editing ( http://www.internationalscienceediting.com) for editing this manuscript.

Funding

This work was supported by China Agriculture Research System (CARS-23-C04), the National Natural Science Foundation of China (31401921), the Natural Science Foundation of Shandong Province (ZR2014-CQ029), Science and Technology Innovation Team of Shandong Agriculture University-Facility Horticulture Advantages Team (SYL2017YSTD07) and the National Key Research and Development Program (2016YFB0302403). The funding bodies were not involved in the design of the study, collection, analysis and interpretation of data, and in writing the manuscript.

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Author notes

Yan Li and Guofeng Xin contributed equally to this work.

Authors and Affiliations

1. College of Horticultural Science and Engineering, Shandong Agricultural University, Tai'an, China

Yan Li, Guofeng Xin, Qinghua Shi, Fengjuan Yang & Min Wei

2. Scientific Observing and Experimental Station of Environment Controlled Agricultural Engineering in Huang-Huai-Hai Region, Ministry of Agriculture, Tai'an, China

Yan Li, Qinghua Shi & Min Wei

3. Shandong Collaborative Innovation Center of Fruit & Vegetable Quality and Efficient Production, Tai'an, China

Yan Li, Qinghua Shi, Fengjuan Yang & Min Wei

4. State Key Laboratory of Crop Biology, Tai'an, 271018, China

Yan Li, Qinghua Shi, Fengjuan Yang & Min Wei

5. Entomology and Nematology Department, University of Florida, 1881 Natural Area Dr, Gainesville, FL, USA

Chang Liu

Contributions

WM conceived and designed research. XGF conducted experiments and analyzed data. LY analyzed data and wrote the manuscript. LC, SQH and YFJ modified the paper. All authors have read and approved the manuscript.

Corresponding author

Correspondence to Min Wei.

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Li, Y., Xin, G., Liu, C. et al. Effects of red and blue light on leaf anatomy, CO ₂ assimilation and the photosynthetic electron transport capacity of sweet pepper ( Capsicum annuum L.) seedlings. BMC Plant Biol 20, 318 (2020). https://doi.org/10.1186/s12870-020-02523-z
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# data[].attributes.attachment_link.title
Effects of red and blue light on leaf anatomy, CO2 assimilation and the photosynthetic electron transport capacity of sweet pepper (Capsicum annuum L.) seedlings - BMC Plant Biology

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Background The red (R) and blue (B) light wavelengths are known to influence many plant physiological processes during growth and development, particularly photosynthesis. To understand how R and B light influences plant photomorphogenesis and photosynthesis, we investigated changes in leaf anatomy, chlorophyll fluorescence and photosynthetic parameters, and ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) and Calvin cycle-related enzymes expression and their activities in sweet pepper (Capsicum annuum L.)

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Horticultural Physiology Laboratory, Department of Horticulture, University of Georgia, Athens, GA, United States

Red and blue light are traditionally believed to have a higher quantum yield of CO ₂ assimilation ( QY, moles of CO ₂ assimilated per mole of photons) than green light, because green light is absorbed less efficiently. However, because of its lower absorptance, green light can penetrate deeper and excite chlorophyll deeper in leaves. We hypothesized that, at high photosynthetic photon flux density ( PPFD), green light may achieve higher QY and net CO ₂ assimilation rate ( A n) than red or blue light, because of its more uniform absorption throughtout leaves. To test the interactive effects of PPFD and light spectrum on photosynthesis, we measured leaf A n of "Green Tower" lettuce ( Lactuca sativa) under red, blue, and green light, and combinations of those at PPFDs from 30 to 1,300 μmol⋅m ⁻²⋅s ⁻¹. The electron transport rates ( J) and the maximum Rubisco carboxylation rate ( V c,max) at low (200 μmol⋅m ⁻²⋅s ⁻¹) and high PPFD (1,000 μmol⋅m ⁻²⋅s ⁻¹) were estimated from photosynthetic CO ₂ response curves. Both QY m,inc (maximum QY on incident PPFD basis) and J at low PPFD were higher under red light than under blue and green light. Factoring in light absorption, QY m,abs (the maximum QY on absorbed PPFD basis) under green and red light were both higher than under blue light, indicating that the low QY m,inc under green light was due to lower absorptance, while absorbed blue photons were used inherently least efficiently. At high PPFD, the QY inc [gross CO ₂ assimilation ( A g)/incident PPFD] and J under red and green light were similar, and higher than under blue light, confirming our hypothesis. V c,max may not limit photosynthesis at a PPFD of 200 μmol m ⁻² s ⁻¹ and was largely unaffected by light spectrum at 1,000 μmol⋅m ⁻²⋅s ⁻¹. A g and J under different spectra were positively correlated, suggesting that the interactive effect between light spectrum and PPFD on photosynthesis was due to effects on J. No interaction between the three colors of light was detected. In summary, at low PPFD, green light had the lowest photosynthetic efficiency because of its low absorptance. Contrary, at high PPFD, QY inc under green light was among the highest, likely resulting from more uniform distribution of green light in leaves.

Introduction

The photosynthetic activity of light is wavelength dependent. Based on McCree's work ( McCree, 1971, 1972), photosynthetically active radiation is typically defined as light with a wavelength range from 400 to 700 nm. Light with a wavelength shorter than 400 nm or longer than 700 nm was considered as unimportant for photosynthesis, due to its low quantum yield of CO ₂ assimilation, when applied as a single waveband ( Figure 1). Within the 400-700 nm range, McCree (1971) showed that light in the red region (600-700 nm) resulted in the highest quantum yield of CO ₂ assimilation of plants. Light in the green region (500-600 nm) generally resulted in a slightly higher quantum yield than light in the blue region (400-500 nm) ( Figure 1; McCree, 1971). The low absorptance of green light is partly responsible for its low quantum yield of CO ₂ assimilation. Within the visible spectrum, green leaves have the highest absorptance in the blue region, followed by red. Green light is least absorbed by green leaves, which gives leaves their green appearance ( McCree, 1971; Zhen et al., 2019).

Figure 1. The normalized action spectrum of the maximum quantum yield of CO ₂ assimilation for narrow wavebands of light from ultra-violet to far-red wavelengths ( McCree, 1971). Redrawn using data from Sager et al. (1988).

Since red and blue light are absorbed more strongly by photosynthetic pigments than green light, they are predominantly absorbed by the top few cell layers, while green light can penetrate deeper into leaf tissues ( Nishio, 2000; Vogelmann and Evans, 2002; Terashima et al., 2009; Brodersen and Vogelmann, 2010), thus giving it the potential to excite photosystems in deeper cell layers. Leaf photosynthesis may benefit from the more uniform light distribution throughout a leaf under green light. Absorption of photons by chloroplasts near the adaxial surface may induce heat dissipation of excess excitation energy in those chloroplasts, while chloroplasts deeper into the leaf receive little excitation energy ( Sun et al., 1998; Nishio, 2000). Blue and red photons, therefore, may be used less efficiently and are more likely to be dissipated as heat than green photons.

The misconception that red and blue light are used more efficiently by plants than green light still occasionally appears ( Singh et al., 2015), often citing McCree's action spectrum or the poor absorption of green light by chlorophyll extracts. The limitations of McCree's action spectrum were explained in his original paper: the quantum yield was measured under low photosynthetic photon flux density ( PPFD), using narrow waveband light, and expressed on an incident light basis ( McCree, 1971), but these limitations are sometimes ignored. The importance of green light for photosynthesis has been well established in more recent studies ( Sun et al., 1998; Nishio, 2000; Terashima et al., 2009; Hogewoning et al., 2012; Smith et al., 2017).

From those studies, one trend has emerged that has not received much attention: there is an interactive effect of light quality and intensity on photosynthesis ( Sun et al., 1998; Evans and Vogelmann, 2003; Terashima et al., 2009). At low PPFD, green light has the lowest QY inc (quantum yield of CO ₂ assimilation on incident light basis) because of its low absorptance; at high PPFD, on the other hand, red and blue light have a lower QY inc than green light, because of their high absorptance by photosynthetic pigments, which shifts much of the light absorption closer to the upper leaf surface. This reduces both the quantum yield of CO ₂ assimilation in cells in the upper part of a leaf and light availability in the bottom part of a leaf.

The interactive effect between light quality and intensity was illustrated in an elegant study that quantified the differential quantum yield, or the increase in leaf CO ₂ assimilation per unit of additional light ( Terashima et al., 2009). The differential quantum yield was measured by adding red or green light to a background illumination of white light of different intensities. At low background white light levels, the differential quantum yield of red light was higher than that of green light, due to the low absorptance of green light. But as the background light level increased, the differential quantum yield of green light decreased more slowly than that of red light, and was eventually higher than that of red light ( Terashima et al., 2009). The red light was absorbed efficiently by the chloroplasts in the upper part of leaves. With a high background level of white light, those chloroplasts already received a large amount of excitation energy from white light and up-regulated non-photochemical quenching (NPQ) to dissipate excess excitation energy as heat, causing the additional red light to be used inefficiently. Green light, on the other hand, was able to reach the chloroplasts deeper in the mesophyll and excited those chloroplasts that received relatively little excitation energy from white light. Therefore, with high background white light intensity, additional green light increased leaf photosynthesis more efficiently than red light ( Terashima et al., 2009).

In this paper, we present a comprehensive study to explore potential interactive effect of light intensity and light quality on C ₃ photosynthesis and underlying processes. We quantified the photosynthetic response of plants to blue, green, and red light over a wide PPFD range to better describe how light intensity and waveband interact. In addition, we examined potential interactions among blue, green, and red light, using light with different ratios and intensities of the three narrow waveband lights. To get a better understanding of the biochemical reasons for the effects of light spectrum and intensity on CO ₂ assimilation, we constructed assimilation - internal leaf CO ₂ ( C i) response curves ( curves) under blue, green, and red light, as well as combinations of the three narrow waveband lights at both high and low PPFD. We hypothesized that effects of different light spectra would be reflected in the electron transport rate ( J) required to regenerate consumed ribulose 1,5-bisphosphate (RuBP), rather than the maximum carboxylation rate of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) ( V c,max).

Materials and Methods

Plant Material

Lettuce "Green Towers" plants were grown from seed in 1.7 L round pots filled with soilless substrate (Fafard 4P Mix, Sun Gro Horticulture, Agawam, MA, United States). The plants were grown in a growth chamber (E15, Conviron, Winnipeg, Manitoba, Canada) at 23.2 ± 0.8°C (mean ± SD), under white fluorescent light with a 14-hr photoperiod, vapor pressure deficit (VPD) of 1.20 ± 0.43 kPa and a PPFD of 200-230 μmol⋅m ⁻²⋅s ⁻¹ at the floor level, and ambient CO ₂ concentration. Plants were sub-irrigated when necessary with a nutrient solution containing 100 mg⋅L ⁻¹ N, made with a complete, water-soluble fertilizer (Peter's Excel 15-5-15 Cal-Mag fertilizer, Everris, Marysville, OH, United States).

Leaf Absorptance, Transmittance, and Reflectance

Leaf absorptance was determined using a method similar to that of Zhen et al. (2019). Three plants were randomly selected. A newly expanded leaf from each plant was illuminated with a broad-spectrum halogen bulb (70W; Sylvania, Wilmington, MA, United States) for leaf transmittance measurement. Transmittance was measured with a spectroradiometer (SS-110, Apogee, Logan, UT, United States). The halogen light spectrum was taken as reference measurement with the spectroradiometer placed directly under the halogen bulb in a dark room. Then, a lettuce leaf was placed between the halogen bulb and spectroradiometer, with its adaxial side facing the halogen bulb and transmitted light was measured. Leaf transmittance was then calculated on 1 nm resolution. Light reflectance of the leaves was measured using a spectrometer with a leaf clip (UniSpec, PP systems, Amesbury, MA, United States). Light absorptance was calculated as 1− r e f l e c t a n c e− t r a n s m i t t a n c e. We verified that this method results in similar absorptance spectra as the use of an integrating sphere. Absorptance of each of the nine light spectra used in this study were calculated from the overall leaf absorptance spectrum and the spectra of the red, green, and blue LEDs.

Leaf Photosynthesis Measurements

All gas exchange measurements were made with a leaf gas exchange system (CIRAS-3, PP Systems). Light was provided by the LEDs built into the chlorophyll fluorescence module (CFM-3, PP Systems). This module has dimmable LED arrays of different colors, with peaks at 653 nm [red, full width at half maximum (FWHM) of 17 nm], 523 nm (green, FWHM of 36 nm), and 446 nm (blue, FWHM of 16 nm). Nine different combinations of red, green, and blue light were used in this study ( Table 1). Throughout the measurements, the environmental conditions inside the cuvette were controlled by the leaf gas exchange system. Leaf temperature was 23.0 ± 0.1°C, CO ₂ concentration was 400.5 ± 4.1 μmol⋅mol ⁻¹, and the VPD of air in the leaf cuvette was 1.8 ± 0.3 kPa (mean ± SD).

Table 1. List of light spectrum abbreviations and their spectral composition.

Photosynthesis - Light Response Curves

To explore photosynthetic efficiency of light with different spectra, we constructed light response curves for lettuce plants using each light spectrum. Lettuce plants were exposed to 10 PPFD levels ranging from 30 to 1,300 μmol⋅m ⁻²⋅s ⁻¹ (30, 60, 90, 120, 200, 350, 500, 700, 1,000, and 1,300 μmol⋅m ⁻²⋅s ⁻¹) in ascending orders for light response curves. Photosynthetic measurements were taken on 40-66 days old lettuce plants. Lettuce plants were taken out of the growth chamber and dark-adapted for 30 min. Starting from the lowest PPFD, one newly expanded leaf was exposed to all nine spectra. Net CO ₂ assimilation rate ( A n) of the leaf was measured using the leaf gas exchange system. Under each light spectrum, three A n readings were recorded at 10 s intervals after readings were stable (about 4-20 min depending on PPFD after changing PPFD and spectrum). The three A n readings were averaged for analysis. After A n measurements under all nine light spectra were taken, the leaf was exposed to the next PPFD level and A n measurements were taken with the light spectra in the same order, until measurements were completed at all PPFD levels. Throughout the light response curves, C i decreased with increasing PPFD, from 396 ± 10 μmol⋅mol ⁻¹ at a PPFD of 30 μmol⋅m ⁻²⋅s ⁻¹ to 242 ± 44 μmol⋅mol ⁻¹ at a PPFD of 1,300 μmol⋅m ⁻²⋅s ⁻¹. To account for the potential effect of plants and the order of the spectra on assimilation rates, the order of the different spectra was re-randomized for each light response curve, using a Latin square design with plant and spectrum as the blocking factors. Data were collected on nine different plants.

Regression curves (exponential rise to maximum) were fitted to the data for each light spectrum and replication (plant):

Photosynthesis - Internal CO₂ Response () Curves

To explore the underlying physiological mechanisms of assimilation responses to different light spectra, we constructed curves. Typically, curves are collected under saturating PPFD. We collected curves at two PPFDs (200 and 1,000 μmol⋅m ⁻²⋅s ⁻¹) to explore interactive effects of light spectrum and PPFD on the assimilation rate. At a PPFD of 200 μmol⋅m ⁻²⋅s ⁻¹, red light has the highest A n and green light the lowest A n, while at PPFD of 1,000 μmol⋅m ⁻²⋅s ⁻¹, red and green light resulted in the highest A n and blue light in the lowest A n.

We used the rapid response (RACiR) technique that greatly accelerates the process of constructing curves ( Stinziano et al., 2017). We used a Latin square design, similar to the light response curves. curves were measured under the same nine spectra used for the light response curves. Nine lettuce plants were used as replicates. For each curve, CO ₂ concentration in the leaf cuvette started from 0 μmol⋅mol ⁻¹, steadily ramping to 1,200 μmol⋅mol ⁻¹ over 6 min. A reference measurement was also taken at the beginning of each replication with an empty cuvette to correct for the reaction time of the leaf gas exchange system. Post-ramp data processing was used to calculate the real A and C i with the spreadsheet provided by PP systems, which yielded the actual curves with C i range of about 100-950 μmol mol ⁻¹. Throughout the data collection, leaf temperature was 24.4 ± 1.3°C and VPD in the cuvette was 1.4 ± 0.2 kPa.

Data Analysis

The QY m,inc, QY m,abs, and A g,max were analyzed with ANOVA to determine the effects of light spectrum using SAS (SAS University Edition; SAS Institute, Cary, NC, United States). A n, QY inc, and QY abs at each PPFD level and V c,max and J estimated from curves were similarly analyzed with ANOVA using SAS. A n at different PPFD levels were analyzed with regression analysis to detect interactive effect of blue, green, and red light on leaf assimilation rates using the fractions of red, blue, and green light as explanatory variables (JMP Pro 15, SAS Institute).

Results

Leaf Absorptance

A representative spectrum of light absorptance, reflectance and transmittance of a newly fully expanded lettuce leaf is shown in Figure 2. In the blue region, 400-500 nm, the absorptance by "Green Towers" lettuce leaves was high and fairly constant, averaging 91.6%. The leaf absorptance decreased as the wavelength increased from 500 to 551 nm where the absorptance minimum was 69.8%. Absorptance increased again at longer wavelengths, with a second peak at 666 nm (92.6%). Above 675 nm, the absorptance decreased steadily to <5% at 747 nm ( Figure 2). The absorptance spectrum of our lettuce leaves is similar to what McCree (1971) obtained for growth chamber-grown lettuce, with the exception of slightly higher absorptance in the green part of the spectrum in our lettuce plants. Using this spectrum, the absorptance of the blue, green, and red LED lights were calculated to be 93.2 ± 1.0%, 81.1 ± 1.9% and 91.6 ± 1.1%, respectively. Absorptance of all nine spectra was calculated based on their ratios of red, green, and blue light ( Table 2).

Figure 2. Light absorptance, reflectance, and transmittance spectrum of a newly fully expanded "Green Towers" lettuce leaf.

Table 2. Light absorptance and transmittance of new fully expanded "Green towers" lettuce leaves under nine light spectra.

Light Quality and Intensity Effects on Photosynthetic Parameters

Light response curves of lettuce under all nine spectra are shown in Figure 3, with regression coefficients in Supplementary Table 1. It is worth noting that a few plants showed photoinhibition under 100B (decrease in A n with PPFD > 1,000 μmol⋅m ⁻²⋅s ⁻¹). Those data were excluded in curve fitting for light response curves to better estimate asymptotes. Photoinhibition was not observed under other spectra.

Figure 3. Net assimilation ( A n) - light response curves of "Green Towers" lettuce under nine light spectra. Error bars represent the standard deviation ( n = 9). Inserts show A n against PPFD of 30-90 μmol⋅m ⁻²⋅s ⁻¹s to better show the initial slopes of curves. The composition of the nine light spectra is shown in Table 1. The light spectra in the graphs are (A) 100B, 100G, and 100R; (B) 100B, 80B20G, 20B80G, and 100G; (C) 100G, 80G20R, 20G80R, and 100R; and (D) 20B80R, 16B20G64R, and 100G.

The QY m,inc of lettuce plants was 22 and 27% higher under red light (74.3 mmol⋅mol ⁻¹) than under either 100G (60.8 mmol⋅mol ⁻¹) or 100B (58.4 mmol⋅mol ⁻¹), respectively ( Figure 4A and Supplementary Table 1). Spectra with a high fraction of red light (64% or more) resulted in a high QY m,inc ( Figure 4A), while 80G20R resulted in an intermediate QY m,inc ( Figure 4A). To determine whether differences in QY m,inc were due to differences in absorptance or in the ability of plants to use the absorbed photons for CO ₂ assimilation, we also calculated QY m,abs. On an absorbed light basis, 100B light still resulted in the lowest QY m,abs (62.7 mmol⋅mol ⁻¹) and red light resulted in the highest QY m,abs (81.1 mmol⋅mol ⁻¹) among narrow waveband lights ( Figure 4B). Green light resulted in a QY m,abs (74.9 mmol⋅mol ⁻¹) similar to that under red light, but significantly higher than that of blue light ( Figure 4B). We did not find any interactions (synergism or antagonism) between lights of different colors, with all physiological responses under mixed spectra being similar to the weighted average of responses under single colors. Thus, for the rest of the results we focus on the three narrow waveband spectra.

Figure 4. Maximum quantum yield of CO ₂ assimilation of "Green Towers" lettuce based on incident ( QY m,inc) (A) and absorbed light ( QY m,abs) (B) under nine different light spectra. Values are calculated as the initial slope of the light response curves of corresponding light spectra (see Figure 3). Bars with the same letter are not statistically different ( p ≤ 0.05). Error bars represent the standard deviation ( n = 9). The composition of the nine light spectra is shown in Table 1.

Among the three narrow waveband lights, 100G resulted in the highest A g,max (20.0 μmol⋅m ⁻²⋅s ⁻¹), followed by red (18.9 μmol⋅m ⁻²⋅s ⁻¹), and blue light (17.0 μmol⋅m ⁻²⋅s ⁻¹) ( Figure 5 and Supplementary Table 1). As with and QY m,abs, combining two or three colors of light resulted in an A g,max similar to the weighted averages of individual light colors.

Figure 5. Maximum gross assimilation rate ( A g,max) of "Green Towers" lettuce under different light spectra, calculated from the light response curves. Bars with the same letter are not statistically different ( p ≤ 0.05). Error bars represent standard deviation ( n = 9). The composition of the nine light spectra is shown in Table 1.

QY inc initially increased with increasing PPFD and peaked at 90-200 μmol⋅m ⁻²⋅s ⁻¹, then decreased at higher PPFDs ( Figure 6A). The QY inc under 100R was higher than under either green or blue light at low PPFD (≤300 μmol⋅m ⁻²⋅s ⁻¹). Although 100G resulted in lower QY inc than 100B at low PPFD (≤300 μmol⋅m ⁻²⋅s ⁻¹), the decrease in QY inc under 100G with increasing PPFD was slower than that with 100B or 100R. Above 500 μmol m ⁻² s ⁻¹, the QY inc with 100G was comparable to the QY inc with 100R, and higher than with 100B ( Figure 6A). The QY abs with 100R was higher than that with either 100G or 100B at PPFDs from 60 to 120 μmol⋅m ⁻²⋅s ⁻¹ ( p < 0.05). The QY abs with 100G was similar to 100B at low PPFD, but decreased slower than that with either 100R or 100B as PPFD increased. At PPFD ≥ 500 μmol⋅m ⁻²⋅s ⁻¹, QY abs was lowest under 100B among the three monochromatic lights ( p < 0.05) ( Figure 6B).

Figure 6. The quantum yield of CO ₂ assimilation of "Green Towers" lettuce as a function of incident ( QY inc) (A) and absorbed PPFD ( QY abs) (B) under blue, green, and red LED light. Error bars represent the standard deviation ( n = 9).

The differential QY, which quantifies the increase in CO ₂ assimilation per unit of additional PPFD, decreased with increasing PPFD. The differential QY with 100R was higher than those with 100B and 100G at low PPFD. At a PPFD of 30 μmol⋅m ⁻²⋅s ⁻¹, the differential QY was 70.5 mmol⋅mol ⁻¹ for 100R, 59.4 mmol⋅mol ⁻¹ for 100G, and 55.8 mmol⋅mol ⁻¹ for 100B ( Figure 7). However, the differential QY with 100R decreased rapidly with increasing PPFD and was lower than the differential QY with 100G at high PPFD ( Figure 7). At high PPFD, the differential QY with 100G was highest among three monochromatic light ( Figure 7). For instance, at a PPFD of 1,300 μmol⋅m ⁻²⋅s ⁻¹, the differential QY with 100G was 1.09 mmol⋅mol ⁻¹, while those with 100B and 100R were 0.64 mmol⋅mol ⁻¹ and 0.46 mmol⋅mol ⁻¹, respectively ( Figure 7).

Figure 7. The differential quantum yield of CO ₂ assimilation ( differential QY) of "Green Towers" lettuce under blue, green, and red LED light as a function of the PPFD. The differential QY is the increase in net assimilation per unit additional PPFD and was calculated as the first derivate of the light response curves ( Figure 3). The insert shows the differential quantum yield plotted at PPFDs of 1,000-1,300 μmol m ⁻² s ⁻¹s to better show differences at high PPFD (note the different y-axis scale).

Effect of Light Spectrum and Intensity on J and V c,max

J of lettuce leaves at low PPFD was lowest under 100G (47.4 μmol⋅m ⁻²⋅s ⁻¹), followed by 100B (56.1 μmol⋅m ⁻²⋅s ⁻¹), and highest under 100R (64.1 μmol⋅m ⁻²⋅s ⁻¹) ( Figure 8A). At high PPFD, on the other hand, J of leaves exposed to 100G (115.3 μmol⋅m ⁻²⋅s ⁻¹) and 100R (112.1 μmol⋅m ⁻²⋅s ⁻¹) were among the highest, while J of leaves under 100B was the lowest (97.0 μmol⋅m ⁻²⋅s ⁻¹) ( Figure 8A). At high PPFD, V c,max of leaves under blue light (59.3 μmol⋅m ⁻²⋅s ⁻¹) was lower than V c,max of leaves under 16B20G64R light (72.1 μmol⋅m ⁻²⋅s ⁻¹), but none of the other treatments differed significantly ( Figure 8). When PPFD increased from 200 to 1,000 μmol⋅m ⁻²⋅s ⁻¹, J under green light increased by 143%, while J under blue and red light increased by 73% and 75%, respectively ( Figure 8A). J and V c,max at high PPFD were strongly correlated ( R ² = 0.82) ( Supplementary Figure 3).

Figure 8. Electron transport rate ( J) at PPFDs of 200 (left bars) and 1,000 μmol m ⁻² s ⁻¹ (right bars) (A) and maximum Rubisco carboxylation rate ( V c,max) at a PPFD of 1,000 μmol m ⁻² s ⁻¹ (B) of "Green Towers" lettuce, as estimated from curves under different light spectra. Bars with the same letter are not statistically different ( p ≤ 0.05). Error bars represent the standard deviation ( n = 9). The light composition of the nine light spectra is shown in Table 1.

Discussion

Interactive Effect of Light Spectrum and PPFD on Photosynthesis

There was an interactive effect of light spectrum and PPFD on photosynthetic properties of lettuce. Under low light conditions (≤200 μmol⋅m ⁻²⋅s ⁻¹), the QY inc of lettuce leaves under green light was lowest among blue, green, and red light ( Figure 6A), due to its lower absorptance by lettuce leaves. After accounting for absorptance, green photons were used at similar efficiency as blue photons, while red photons were used most efficiently ( Figure 6B). The QY m,abs under green and red light were higher than under blue light ( Figure 4B). At high PPFD, green and red light had similar quantum yield, higher than that of blue light, both on an absorbed and incident light basis ( Figure 6A). Multiple factors contributed to the interactive effect of light spectrum and PPFD on quantum yield and photosynthesis.

Light Absorptance and Non-Photosynthetic Pigments Determine Assimilation at Low PPFD

QY m,inc with blue and green light was lower than with red light ( Figure 4A), consistent with McCree's action spectrum ( McCree, 1971). But when taking leaf absorptance into account, QY m,abs was similar under green and red light and lower under blue light ( Figure 4B). Similarly, at low PPFD (≤200 μmol⋅m ⁻²⋅s ⁻¹), QY inc of lettuce leaves was highest under red, intermediate under blue, and lowest under green light. When accounting for leaf absorptance, QY abs under red light remained highest and QY abs under both green and blue light were similar at low PPFD ( Figure 6A). Consistent with our data, previous studies also documented that, once absorbed, green light can drive photosynthesis efficiently at low PPFD ( Balegh and Biddulph, 1970; McCree, 1971; Evans, 1987; Sun et al., 1998; Nishio, 2000; Terashima et al., 2009; Hogewoning et al., 2012; Vogelmann and Gorton, 2014). For example, the QY m,abs of spinach ( Spinacia oleracea) and cabbage ( Brassica oleracea L.) was highest under red light, followed by that under green light and lowest with blue light. But on incident light basis, QY m,inc of under green light was lower than under red or blue light ( Sun et al., 1998).

Both our data ( Figure 4B) and those of Sun et al. (1998) show that QY m,abs with blue light is lower than that with red and green light, indicating that blue light is used intrinsically less efficiently by lettuce. Blue light, and, to a lesser extent, green light is absorbed not just by chlorophyll, but also by flavonoids and carotenoids ( Sun et al., 1998). Those pigments can divert energy away from photochemistry and thus reduce the QY abs under blue light. Flavonoids (e.g., anthocyanins) are primarily located in the vacuole and cannot transfer absorbed light energy to photosynthetic pigments ( Sun et al., 1998). Likewise, free carotenoids do not contribute to photochemistry ( Hogewoning et al., 2012). Carotenoids in light-harvesting antennae and reaction centers channel light energy to photochemistry, but with lower transfer efficiency than chlorophylls ( Croce et al., 2001; de Weerd et al., 2003a, b; Wientjes et al., 2011; Hogewoning et al., 2012). Therefore, absorption of blue light by flavonoids and carotenoids reduces the quantum yield of CO ₂ assimilation. Thus, even with the high absorptance of blue light by green leaves, QY m,abs of leaves under blue light was the lowest among the three monochromatic lights ( Figure 4B). It is likely that the lower QY abs under green light than that under red light was also due to absorption of green light by carotenoids and flavonoids ( Hogewoning et al., 2012). At high PPFD, absorption of blue light by flavonoids and carotenoids still occurs, but this is less of a limiting factor for photosynthesis, since light availability is not limiting under high PPFD.

Light Dependence of Respiration and Rubisco Activity May Reduce the Quantum Yield at Low PPFD

At PPFDs below 200 μmol⋅m ⁻²⋅s ⁻¹, the QY inc and QY abs of lettuce showed an unexpected pattern in response to PPFD ( Figure 6). Unlike the quantum yield of PSII, which decreases exponentially with increasing PPFD ( Weaver and van Iersel, 2019), QY inc and QY abs increased initially with increasing PPFD ( Figure 6). A similar pattern was previously observed by Craver et al. (2020) in petunia ( Petunia × hybrida) seedlings. This pattern could result from light-dependent regulation of respiration ( Croce et al., 2001), alternative electron sinks such as nitrate reduction ( Skillman, 2008; Nunes-Nesi et al., 2010), or Rubisco activity ( Campbell and Ogren, 1992; Zhang and Portis, 1999). In our calculations, we assumed that the leaf respiration in the light was the same as R d. However, leaf respiration in the light is lower than in the dark, in a PPFD-dependent manner ( Brooks and Farquhar, 1985; Atkin et al., 1997), which can lead to overestimation of A g with increasing PPFD. When we accounted for this down-regulation of respiration, using the model by Müller et al. (2005) to correct A g, , and QY abs, we found that depression of respiration by light did not explain the initial increase in QY inc and QY abs we observed ( Supplementary Figure 4). Alternative electron sinks in the chloroplasts that are upregulated in response to light can explain the low , and QY abs at low PPFD, because they compete with the Calvin cycle for reducing power (ferredoxin/NADPH). Such processes include photorespiration ( Krall and Edwards, 1992), nitrate assimilation ( Nunes-Nesi et al., 2010), sulfate assimilation ( Takahashi et al., 2011) and the Mehler reaction ( Badger et al., 2000) and their effect on , and QY abs would be especially notable under low PPFD ( Supplementary Figure 5).

Upregulation of Rubisco activity by Rubisco activase in the light may also have contributed to the increase in QY inc and QY abs at low PPFD ( Campbell and Ogren, 1992; Zhang and Portis, 1999). In the dark, 2-carboxy-D-arabinitol-1-phosphate (CA1P) or RuBP binds strongly to the active sites of Rubisco, preventing carboxylation activity. In the light, Rubisco activase releases the inhibitory CA1P or RuBP from the catalytic site of Rubisco, in a light-dependent manner ( Campbell and Ogren, 1992; Zhang and Portis, 1999; Parry et al., 2008). At PPFD < 120 μmol⋅m ⁻²⋅s ⁻¹, low Rubisco activity may have limited photosynthesis.

Light Distribution Within Leaves Affects QY at High PPFD

Except for the initial increase at low PPFD, both QY inc and QY abs decreased with increasing PPFD. QY inc decreased slower under green than under red or blue light ( Figure 6A). At a PPFD ≥ 500 μmol⋅m ⁻²⋅s ⁻¹, QY inc under green light was higher than that under blue light ( Figure 6A). Accordingly, A n under blue light was lower than under green and red light at PPFDs above 500 μmol⋅m ⁻²⋅s ⁻¹ ( Figure 3A). The lower QY inc under blue light than under green and red light at high PPFD can be explained by disparities in the light distribution within leaves.

Blue and red light were strongly absorbed by lettuce leaves (93.2 and 91.6%, respectively), while green light was absorbed less (81.1%) ( Table 2). Similar low green absorptance was found in sunflower ( Helianthus annuus L.), snapdragon ( Antirrhínum majus L.) ( Brodersen and Vogelmann, 2010), and spinach ( Vogelmann and Han, 2000). In leaves of those species, absorption of red and blue light peaked in the upper 20% of leaves, and declined sharply further into the leaf. Absorption of red light decreased slower with increasing depth than that of blue light ( Vogelmann and Han, 2000; Brodersen and Vogelmann, 2010). Green light absorption peaked deeper into leaves, and was more evenly distributed throughout leaves, because of low absorption of green light by chlorophyll ( Vogelmann and Han, 2000; Brodersen and Vogelmann, 2010). The more even distribution of green light within leaves, as compared to red and blue light, can explain the interactive effects between PPFD and light spectrum on leaf photosynthesis. It was estimated that less than 10% of blue light traveled through the palisade mesophyll and reached the spongy mesophyll in spinach, while about 35% of green light and 25% of red light did so ( Vogelmann and Evans, 2002). It was also estimated that chlorophyll in the lowermost chloroplasts of spinach leaves absorbed about 10% of green and <2% of blue light, compared to chlorophyll in the uppermost chloroplasts ( Vogelmann and Evans, 2002; Terashima et al., 2009).

The more uniform green light distribution within leaves may be a key contributor to higher leaf level QY inc under high PPFD because less heat dissipation of excess light energy is needed ( Nishio, 2000; Terashima et al., 2009). Reaction centers near the adaxial leaf surface receive more excitation energy under blue, and to a lesser extent under red light, than under green light, because of the differences in absorptance. Consequently, under high intensity blue light, NPQ is up-regulated in the chloroplasts near the adaxial leaf surface to dissipate some of the excitation energy ( Sun et al., 1998; Nishio, 2000), lowering the QY inc under blue light. Since less green light is absorbed near the adaxial surface, less heat dissipation is required. When incident light increased from 150 to 600 μmol⋅m ⁻²⋅s ⁻¹, the fraction of whole leaf CO ₂ assimilation that occurred in the top half of spinach leaves remained the same under green light (58%), but decreased from 87 to 73% under blue light. This indicates more upregulation of heat dissipation in the top of the leaves under blue, than under green light ( Evans and Vogelmann, 2003). On the other hand, the bottom half of the leaves can still utilize the available light with relatively high QY inc, since the amount of light reaching the bottom half is relatively low, even under high PPFD ( Nishio, 2000). By channeling more light to the under-utilized bottom part of leaves, leaves could achieve higher QY inc even under high intensity green light. In our study, high QY inc under green light and low QY inc under blue light at high PPFD ( Figure 6) can be thus explained by the large disparities in the light environment in chloroplasts from the adaxial to the abaxial side of leaves due to differences in leaf absorptance. Similarly, differential QY of lettuce leaves was highest under green light and lower under blue and red light at high PPFD (>300 μmol⋅m ⁻²⋅s ⁻¹) ( Figure 7), also potentially because of the more uniform distribution of green light and the uneven distribution of blue and red light in leaves.

Along the same line, A n of lettuce leaves was the lowest under blue light at PPFD > 500 μmol⋅m ⁻²⋅s ⁻¹ ( Figure 3). Also, A n of lettuce leaves approached light saturation at lower PPFDs under blue and red light, than under green light ( Figure 3A). Under blue, green, and red light, lettuce leaves reached 95% of A n,max at PPFDs of 954, 1,110 and 856 μmol⋅m ⁻²⋅s ⁻¹, respectively. This can be seen more clearly in the differential QY at high PPFD ( Figure 7). At a PPFD of 1,300 μmol⋅m ⁻²⋅s ⁻¹, green light had a differential QY of 1.09 mmol⋅mol ⁻¹, while that of red and blue light was only 0.46 and 0.69 mmol⋅mol ⁻¹, respectively ( Figure 7). Green light also resulted in a higher A g,max (22.9 μmol⋅m ⁻²⋅s ⁻¹) than red and blue light (21.8 and 19.3 μmol⋅m ⁻²⋅s ⁻¹, respectively) ( Figure 5). As discussed before, the high A g,max under green light resulted from the more uniform light distribution under green light, allowing deeper cell layers to photosynthesize more. Previous research similarly found that at high PPFD (>500 μmol⋅m ⁻²⋅s ⁻¹), A n of both spinach and cabbage were lower under blue light than under white, red and green light ( Sun et al., 1998). Overall, under high PPFD, the differences in light distribution throughout a leaf are important to quantum yield and assimilation rate, since it affects NPQ up-regulation ( Sun et al., 1998; Nishio, 2000). However, light distribution within a leaf is less important at low than at high PPFD, because upregulation of NPQ increases with increasing PPFD ( Zhen and van Iersel, 2017).

Light Spectrum Affects J, but Not V c,max

We examined the effect of light quality and intensity on J and V c,max ( Figure 8). For the light-dependent reactions, the interactive effect between light spectra and PPFD found for CO ₂ assimilation and quantum yield was also observed for J ( Figure 8A). At low PPFD (200 μmol⋅m ⁻²⋅s ⁻¹), green light resulted in the lowest J and red light in the highest J among single waveband spectra. But at a PPFD of 1,000 μmol⋅m ⁻²⋅s ⁻¹, red and green light resulted in the highest J and blue light in the lowest J ( Figure 8A), similar to the differences in A g.

There was no clear evidence of Rubisco limitations to photosynthesis at a PPFD of 200 μmol⋅m ⁻²⋅s ⁻¹, so the rate of the light-dependent reactions likely limited photosynthesis. This is corroborated by the strong correlation between A g and J at a PPFD of 200 μmol⋅m ⁻²⋅s ⁻¹. Although Rubisco limitations to photosynthesis were observed at a PPFD of 1,000 μmol⋅m ⁻²⋅s ⁻¹, there were no meaningful differences in V c,max in response to light spectrum, in contrast to J ( Figure 8).

When PPFD increased 5×, from 200 to 1,000 μmol⋅m ⁻²⋅s ⁻¹, there was only a 1.7 to 2.4× increase in J, indicating a lower at higher PPFD. This matches the lower QY inc and the asymptotic increase in A n in response to increasing PPFD ( Figure 3). The relative increase of J under green light (143%) was greater than that under both blue and red light (73 and 75%, respectively) as PPFD increased. This similarly can be attributed to a more uniform energy distribution of green light among reaction centers throughout a leaf and weaker upregulation of non-photochemical quenching with increasing green light intensity ( Sun et al., 1998; Nishio, 2000; Evans and Vogelmann, 2003), as discussed before.

There was a strong correlation between J and A g under the nine light spectra at both PPFD levels ( Figure 9A). QY abs and are similarly strongly correlated ( Figure 9B). Unlike J, V c,max was largely unaffected by light spectra ( Figure 8B) and was not correlated with A g (data not shown). There was, however, a strong correlation between J and V c,max at a PPFD of 1,000 μmol⋅m ⁻²⋅s ⁻¹ ( R ² = 0.82, Supplementary Figure 3), suggesting that J and V c,max are co-regulated. Similarly, Wullschleger (1993) noted a strong linear relationship between J and V c,max across 109 C ₃ species. The ratio between J and V c,max in our study (1.5-2.0) similar to the ratio found by Wullschleger (1993). These results suggest that the interactive effect of light spectra and PPFD resulted from effects on J, which is associated with light energy harvesting by reaction centers, rather than from V c,max.

Figure 9. The correlation between gross CO ₂ assimilation rate ( A g) estimated from light response curves and electron transport rate ( J) estimated from curves (A), and between the quantum yield of CO ₂ assimilation ( QY abs) and the quantum yield of electron transport on an absorbed light basis [] (B), under low PPFD (200 μmol m ⁻² s ⁻¹) and high PPFD (1,000 μmol m ⁻² s ⁻¹) under nine light spectra averaged over nine "Green Towers" lettuce plants. The color scheme representing the nine spectra is the same as Figure 8.

No Interactive Effects Among Blue, Green, and Red Light

The Emerson enhancement effect describes a synergistic effect between lights of different wavebands (red and far-red) on photosynthesis ( Emerson, 1957). McCree (1971) attempted to account for interactions between light with different spectra when developing photosynthetic action spectra and applied low intensity monochromatic lights from 350 to 725 nm with white background light to plants. His results showed no interactive effect between those monochromatic lights and white light ( McCree, 1971). We tested different ratios of blue, green, and red light and different PPFD s, and similarly did not find any synergistic or antagonistic effect of different wavebands on any physiological parameters measured or calculated.

Importance of Interactions Between PPFD and Light Quality and Its Applications

The interactive effect between PPFD and light quality demonstrates a remarkable adaptation of plants to different light intensities. By not absorbing green light strongly, plants open up a "green window," as Terashima et al. (2009) called it, to excite chloroplasts deeper into leaves, and thus facilitating CO ₂ assimilation throughout the leaf. While red light resulted in relatively high QY inc, QY abs and A n at both high and low PPFD ( Figures 3, 6), it is still mainly absorbed in the upper part of leaves ( Sun et al., 1998; Brodersen and Vogelmann, 2010). Green light can penetrate deeper into leaves ( Brodersen and Vogelmann, 2010) and help plants drive efficient CO ₂ assimilation at high PPFD ( Figures 3, 5).

Many early photosynthesis studies investigated the absorptance and action spectrum of photosynthesis of green algae, e.g., Haxo and Blinks (1950) or chlorophyll or chloroplasts extracts, e.g., Chen (1952). Extrapolating light absorptance of green algae and suspension of chlorophyll or chloroplast to whole leaves from can lead to an underestimation of absorptance of green light by whole leaves and the belief that green light has little photosynthetic activity ( Moss and Loomis, 1952; Smith et al., 2017). Photosynthetic action spectra developed on whole leaves of higher plants, however, have long shown that green light effectively contributes to CO ₂ assimilation, although with lower QY inc than red light ( Hoover, 1937; McCree, 1971; Inada, 1976; Evans, 1987). The importance of green light for photosynthesis was clearly established in more recent studies, emphasizing its role in more uniformly exciting all chloroplasts, which especially important under high PPFD ( Sun et al., 1998; Nishio, 2000; Terashima et al., 2009; Hogewoning et al., 2012; Smith et al., 2017). The idea that red and blue light are more efficient at driving photosynthesis, unfortunately, still lingers, e.g., Singh et al. (2015).

Light-emitting diodes (LEDs) have received wide attention in recent years for use in controlled environment agriculture, as they now have superior efficacy over traditional lighting technologies ( Pattison et al., 2018). LEDs can have a narrow spectrum and great controllability. This provides unprecedented opportunities to fine tune light spectra and PPFD to manipulate crop growth and development. Blue and red LEDs have higher efficacy than white and green LEDs ( Kusuma et al., 2020). By coincidence, McCree's action spectrum ( Figure 1; McCree, 1971) also has peaks in the red and blue region, although the peak in the blue region is substantially lower than the one in the red region. Therefore, red and blue LEDs are sometimes considered optimal for driving photosynthesis. This claim holds true only under low PPFD. Green light plays an important role in photosynthesis, as it helps plants to adapt to different light intensities. The wavelength-dependent absorptance of chlorophylls channels green light deeper into leaves, resulting in more uniform light absorption throughout leaves and providing excitation energy to cells further from the adaxial surface. Under high PPFD, this can increase leaf photosynthesis. Plant evolved under sunlight for hundreds of millions of years, and it seems likely that the relatively low absorptance of green light contributes to the overall photosynthetic efficiency of plants ( Nishio, 2000).

Conclusion

There was an interactive effect of light spectrum and PPFD on leaf photosynthesis. Under low PPFD, QY inc was lowest under green and highest under red light. The low QY inc under green light at low PPFD was due to low absorptance. In contrast, at high PPFD, green and red light achieved similar QY inc, higher than that of blue light. The strong absorption of blue light by chlorophyll creates a large light gradient from the top to the bottom of leaves. The large amount of excitation energy near the adaxial side of a leaf results in upregulation of nonphotochemical quenching, while chloroplasts near the bottom of a leaf receive little excitation energy under blue light. The more uniform distribution of green light absorption within leaves reduces the need for nonphotochemical quenching near the top of the leaf, while providing more excitation energy to cells near the bottom of the leaf. We also found that the interactive effect of light spectrum and PPFD on photosynthesis was a result of the light-dependent reactions; gross assimilation and J were strongly correlated. We detected no synergistic or antagonistic interactions between blue, green, and red light.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Author Contributions

JL and MI designed the experiment, discussed the data, and revised the manuscript. JL performed the experiment, analyzed data, and prepared the first draft. Both authors contributed to the article and approved the submitted version.

Funding

This study was funded by the USDA-NIFA-SCRI award number 2018-51181-28365, project Lighting Approaches to Maximize Profits.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2021.619987/full#supplementary-material

Supplementary Figure 1 | (Related to Figure 6) Quantum yield of CO ₂ assimilation of "Green Towers" lettuce as a function of incident ( QY inc) (A,C,E,G) and absorbed PPFD ( QY abs) (B,D,F,H) under nine light spectra (see Table 1). Error bars represent standard deviation ( n = 9).

Supplementary Figure 2 | (Related to Figure 7) Differential quantum yield of CO ₂ assimilation ( differential QY) of "Green Towers" lettuce under nine light spectra as a function of the PPFD. Inserts show differential QY at PPFDs of 1,000-1,300 μmol⋅m ⁻² s ⁻¹s to better show differences at high PPFD (note the different y-axis scale). The composition of the nine light spectra is shown in Table 1. The light spectra in the graphs are (A) 100B, 100G and 100R; (B) 100B, 80B20G, 20B80G and 100G; (C) 100G, 80G20R, 20G80R and 100R; and (D) 20B80R, 16B20G64R and 100G.

Supplementary Figure 3 | (Related to Figure 6) The correlation between electron transport ( J) and maximum Rubisco carboxylation rate ( V c,max) of "Green Towers" lettuce estimated from curves under PPFD (1000 μmol m ⁻² s ⁻¹) under nine light spectra ( p < 0.001).

Supplementary Figure 4 | (Related to Figure 6) The comparison between QY inc before (A) and after (B) correcting for light-suppression of respiration under blue, green, and red LED light. Note that the initial increase in QY inc became more pronounced after correction of light suppressed respiration.

Supplementary Figure 5 | The comparison between QY abs before (A) and after (B) correcting for alternative electron sinks under blue, green, and red LED light. Assuming a simplified electron sink that diverts energy of 15 μmol m ⁻² s ⁻¹ of absorbed photons (an arbitrary value used for illustrative purposes only) away from the Calvin cycle under all PPFDs, the corrected QY abs was calculated based on remaining photons available to support Calvin cycle processes (B). Note that the pattern of QY inc after correcting of alternative electron sink (B) is similar to quantum yield of PSII measured by chlorophyll fluorescence by Weaver and van Iersel (2019).

Supplementary Table 1 | Dark respiration rate (R d), maximum quantum yield of CO ₂ assimilation (QY m,inc) and maximum gross assimilation rate (A g,max) of "Green towers" lettuce derived from the light response curves for nine different spectra using Eq. 1. The light response curves are shown in Figure 3. *See light composition of nine lights presented here in Table 1.

Abbreviations

PPFD, photosynthetic photon flux density; RuBP, ribulose 1,5-bisphosphate; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; VPD, vapor pressure deficit; FWHM, full width at half maximum; A n, net CO ₂ assimilation rate; R d, dark respiration rate; QY m,inc, maximum quantum yield of CO ₂ assimilation; A g,max, light-saturated gross assimilation rate; QY m,abs, maximum quantum yield of CO ₂ assimilation on absorbed light base; QY inc, quantum yield of CO ₂ assimilation based on incident PPFD; A g, gross CO ₂ assimilation rate; QY abs, quantum yield of CO ₂ assimilation on absorbed light base; QY, quantum yield of CO ₂ assimilation; A/Ci curve, assimilation - internal leaf CO ₂ response curve; RACiR, rapid response technique; V c,max, maximum rate of Rubisco carboxylation; J, rate of electron transport; CA1P, 2-carboxy- D-arabinitol-1-phosphate; NPQ, non-photochemical quenching.

References

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# data[].attributes.attachment_link.title
Photosynthetic Physiology of Blue, Green, and Red Light: Light Intensity Effects and Underlying Mechanisms

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Red and blue light are traditionally believed to have a higher quantum yield of CO2 assimilation (QY, moles of CO2 assimilated per mole of photons) than green light, because green light is absorbed less efficiently. However, because of its lower absorptance, green light can penetrate deeper and excite chlorophyll deeper in leaves.

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by , , and *, *

Key Laboratory of Mariculture (Ministry of Education), Fisheries College, Ocean University of China, Qingdao 266003, China

Authors to whom correspondence should be addressed.

1. Introduction

Light is one of the most significant abiotic factors that modulate the diverse physiological processes in freshwater and marine microalgae [ 1]. One feature of light is that the light wavelength can play a significant role in their growth and development [ 2, 3]. For instance, blue light promoted the growth of Tisochrysis lutea, Chlamydomonas reinhardtii, and Dunaliella sp. by regulating their physiological metabolisms or increasing DNA or RNA synthesis for cell division [ 4, 5, 6]. Blue light may also generally enhance the photosynthetic activities of various microalgal species by activating their photosynthetic electron transfer chain reactions, especially the activity of ribulose-1,5-diphosphate carboxylase [ 7, 8]. Additionally, it grew fastest for Haematococcus lacustris (Girod-Chantrans) Rostafinski under red light because the energy of red photons might be the most suitable to meet its photosynthesis requirements [ 9]. However, the amino acids of Amphora sp. under yellow light may be mainly used for the synthesis of pigments rather than for cell proliferation, thereby resulting in the lowest cell concentration [ 10]. Therefore, the selection of appropriate light wavelength is very important to optimize the biomass production of microalgae and support their high-density cultures.

Numerous studies have clarified that light wavelength can greatly impact the pigment content of microalgae. Zhao et al. [ 11] indicated that blue light-stimulated lutein synthesis in Chlamydomonas sp. by regulating its protein synthesis and related enzyme activities. For Chlorella sp., both red light and blue light could increase its lutein content, and maximal lutein production was found under red light [ 12]. The astaxanthin content of H. lacustris under blue light was two to eight times higher than under red or white light [ 13]. Mixing red and blue light could also significantly promote the astaxanthin accumulation of H. lacustris [ 14, 15]. Therefore, the manipulation of light wavelength may be an effective way to improve the content of economically valuable pigments. Similar to lutein and astaxanthin, fucoxanthin is also a commercial carotenoid that has received widespread attention. Due to its function as an antioxidant, anti-cancer, and anti-obesity, fucoxanthin has been used as a nutritional supplement in foods, nutraceuticals, and pharmaceuticals, and has been verified to be safe for human consumption [ 16, 17, 18]. Light conditions have also been verified to play a crucial role in improving the fucoxanthin content in microalgae. For Odontella aurita, Isochrysis zhangjiangensis and Stephanocyclus cryptica (Reimann, Levin & Guillard) Kulikovskiy, Genkal & Kociolek, low light intensity accelerated their fucoxanthin accumulation by inducing light-harvesting mechanisms and by modulating the diadinoxanthin cycle [ 19, 20, 21]. However, little information is currently available on the impact of light wavelength on fucoxanthin accumulation in microalgae and its potential application.

Emiliania huxleyi is one of the most abundant marine coccolithophores and is widely distributed from polar to tropical waters worldwide [ 22, 23, 24]. This species plays a significant role in the carbon cycle of seawater by fixing carbon from photosynthesis and calcification [ 25, 26, 27]. E. huxleyi is deemed to be an important raw material in biomolecules such as alkenones, neutral and polar lipids, and polyunsaturated fatty acids, which are widely applied in the food and pharmaceutical industries [ 28, 29, 30]. It is also an ideal candidate for fucoxanthin production because of its richness in pigments and rapid proliferation [ 22, 31]. Given the commercial and ecological significance of this species, a great number of studies exploring the influences of various environmental factors on its physiological and biochemical characteristics have been performed [ 32, 33, 34, 35]. However, the effects of light wavelength on this species, particularly its growth and pigment synthesis, have rarely been investigated.

In this study, we examined the effects of light wavelength on the biomass (cell density), growth (cell diameter and dry weight), net photosynthetic rate, and pigment contents (chlorophyll a, fucoxanthin, and lutein) of E. huxleyi. The effects of the change to light wavelength on its cell density and fucoxanthin content were also evaluated. Our findings will provide important information to improve fucoxanthin production by light wavelength regulation.

2. Materials and Methods

2.1. Microalgal Strain and Experimental Preparation

The strains of naked cells Emiliania huxleyi (Lohmann) W.W.Hay & H.Mohler (LAMB244) used in this study were obtained from the microalgae culture collection at the Laboratory of Applied Microalgal Biology, Ocean University of China. The stock was grown in 3000 mL Erlenmeyer flasks containing 2500 mL aseptic IMK liquid medium [ 36] under the conditions of 20 ± 0.5 °C, 100 µmol photons m −2 s −1 and a 12:12 h light/dark cycle. In subsequent experiments, E. huxleyi was maintained in its exponential growth phase by transferring it to fresh aseptic IMK mediums every week. The IMK liquid medium contained 200 mg L −1 NaNO ₃, 1.4 mg L −1 Na ₂HPO ₄, 5 mg L −1 K ₂HPO ₄, 2.68 mg L −1 NH ₄Cl, 0.52 mg L −1 Fe-EDTA, 0.0332 mg L −1 Mn-EDTA, 37.2 mg L −1 Na ₂-EDTA, 0.023 mg L −1 ZnSO ₄·7H ₂O, 0.014 mg L −1 CoSO ₄·7H ₂O, 0.0073 mg L −1 Na ₂MoO ₄·4H ₂O, 0.0025 mg L −1 CuSO ₄·5H ₂O, 0.0018 mg L −1 MnCl ₂·4H ₂O, 0.2 mg L −1 Thiamin-HCl, 0.0015 mg L −1 Biotin, and 0.0015 mg L −1 Vitamin B12.

2.2. Single Light Wavelength Experiment

A culture experiment was carried out for 9 days under five single light wavelengths (LS-DD34, LEESA, Guangzhou, China): red light (620-630 nm), blue light (400-470 nm), green light (510-515 nm), yellow light (590-595 nm), and white light. There was a total of five experimental treatments, and each treatment was performed for nine replicates. Each of the three replicates was used for the determination of growth parameters, photosynthesis, and pigments. This experiment used 45 Erlenmeyer flasks (500 mL), with each flask containing 450 mL of fresh aseptic IMK medium. An initial cell density of 1.5 × 10 ⁴ cells mL −1 was set for each replicate. During the experiment, a temperature of 20 ± 0.5 °C, an irradiance of 100 µmol photons m −2 s −1, and a 12:12 h light/dark cycle were held constant. The light intensity was determined using a Quantitherm Light Meter (QRT1, Hansatech Instruments, UK). These flasks were manually shaken several times per day.

During this experiment, the cell density of E. huxleyi in each replicate was determined using a phytoplankton counting chamber (XKJ-01B, Xiamen Kexi Instrument Company, China) every 3 days. To immobilize these cells, a 1 mL suspension from each replicate was transferred to centrifuge tubes (1.5 mL), to which 10 µL Lugol's iodine was added. These samples were homogenized, and a 0.1 mL sample was transferred to the phytoplankton counting chamber using a pipette. Sixty fields of view were randomly selected, and cell counting was performed under an inverted light microscope (IX73, Olympus, Japan) at ×400 magnification. The cell density (N; cell mL −1) was calculated using the following Equation (1) [ 37]:

N = Cs · V · Pn/(Fs · Fn · U),

where Cs is the area of the total grid on the chamber, V is the volume of the total sample, Pn is the number of counted cells, Fs is the area of the measured grid, Fn is the number of measured grids, and U is the volume of the chamber.

After 9 days, the diameters of 120 cells from each replicate were measured under a light microscope (CX31, Olympus, Japan) at ×1000 magnification after calibration.

The algal cells were used to measure the dry weight after the culture experiment. A 200 mL suspension from each replicate was collected and centrifuged at 7000 rpm for 5 min. The deposits were adequately rinsed and centrifuged again in the same way. The pellets were then flash-frozen in liquid nitrogen and lyophilized for 24 h. Finally, dry deposits were weighed.

After 9 days, the net photosynthetic rate was determined using a Fiber-Optic Oxygen Meter (FSO2-C4, PyroScience GmbH, Germany). A 350 mL E. huxleyi suspension from each replicate was moved to the oxygen electrode cuvette, and the cell density was adjusted to approximately 1.0 × 10 ⁵ cells mL −1. The suspension was then magnetically stirred to ensure the even diffusion of oxygen. The temperature and irradiance levels were the same as for the above-mentioned experiment. The oxygen increase in the suspension was regarded as the net photosynthetic oxygen evolution. Prior to conducting measurements, these cells were allowed to acclimate to the conditions in the cuvette for 5 min. The oxygen concentration was recorded every 1 min for 15 min. The net photosynthetic rate was normalized to µmol O ₂ × 10 ⁵ cell −1 mL min −1.

To obtain the Chl a measurement, a 5 mL suspension was collected from each replicate and centrifuged at 7000 rpm for 5 min. An amount of 3 mL precool methanol (100%) was added to the deposits, which were then resuspended for 2 h under low temperature and dark conditions. Extracts were centrifuged at 7000 rpm for 5 min. The absorbance was determined to be 665 and 750 nm using a spectrophotometer (U-2900, HITACHI, Japan). The concentration of Chl a (mg L −1) was calculated using the following Equation (2) [ 38]:

Chl a (mg L −1) = 13.3 × (OD ₆₆₅ − OD ₇₅₀) × V m/V t,

where 13.3 is the extinction coefficient for Chl a in 100% methanol, OD ₆₆₅ and OD ₇₅₀ are the absorptions of extracts at 665 nm and 750 nm, respectively, V m is the volume of the measured suspension, and V t is the volume of the total suspension.

The extraction and measurements of lutein and fucoxanthin were carried out according to [ 39]. Specifically, a 200 mL suspension from each replicate was collected and centrifuged at 7000 rpm for 5 min. The deposits were adequately rinsed and centrifuged again in the same way. The deposits were then flash-frozen in liquid nitrogen and lyophilized for 24 h. Next, 5 mL of a precooled mixture of methanol and acetone (1:1, v/v) was added to a 20 mg freeze-dried sample and mixed under a low temperature and dark condition for 2 h. After 5 min centrifugation at 3000 rpm, the supernatant was filtered using 0.22 µm polycarbonate filters for high-performance liquid chromatography (HPLC) analysis. The contents of filtered pigments were determined using an Agilent 1200 HPLC (Agilent Technologies, CA, USA) equipped with an Rx-C18 analytical column (4.6 mm × 250 mm; Agilent Technologies, USA). The standard commercial lutein and fucoxanthin used for quantification were from Sigma-Aldrich (USA). Processing of the mobile phase, which was composed of I (water/methanol/acetonitrile, 3:6:11, v/v/v), II (methanol/acetonitrile, 3:17, v/v), III (water/methanol/acetonitrile/ethyl acetate, 3:3:7:7, v/v/v/v), and IV (methanol/ethyl acetate, 3:7, v/v), was performed as follows: 0-15 min, linear gradient from I to II; 15-17 min, linear gradient from II to III; and 17-40 min, linear gradient from III to IV. The flow rate was 0.75 mL min −1, and the column temperature was 50 °C. The pigments were detected using their absorbance at 443 nm.

2.3. Light Wavelength Change Experiment

To investigate the impact of light wavelength change on E. huxleyi, it was cultured under blue light for 3 days, and then changed to red light for 6 days. The cell density and fucoxanthin content of E. huxleyi cultured under these conditions were compared with those cultured under three single light wavelengths of white, blue, and red light for 9 days. Cell density and fucoxanthin content were determined in the same way as described above. A total of 12 Erlenmeyer flasks (500 mL) were prepared. Each flask contained 450 mL of fresh aseptic IMK medium. An initial cell density of 1.5 × 10 ⁴ cells mL −1 was set for each replicate. During the experiment, a temperature of 20 ± 0.5 °C, an irradiance of 100 µmol photons m −2 s −1, and a 12:12 h light/dark cycle were maintained. These flasks were manually shaken several times per day.

2.4. Statistical Analysis

The significant effects of light wavelength condition on cell density, cell diameter, dry weight, net photosynthetic rate, and pigment contents were analyzed using a one-way analysis of variance. Before analyses, the data variances were subjected to the homogeneity test. A Duncan multiple range test was applied when significant differences were found between means. Differences were significant at a probability of 5% (p < 0.05). Data analysis was conducted using SPSS statistical software (version 26.0).

3. Results

3.1. Single Light Wavelength Experiment

A culture experiment was performed for 9 days under five single light wavelengths. During this experiment, the cell density of E. hurleyi in each replicate was determined every 3 days. After the culture, the growth (cell diameter and dry weight), net photosynthetic rate, and pigment contents (chlorophyll a, fucoxanthin, and lutein) of E. huxleyi were measured.

3.1.1. Cell Density

The cell densities of E. huxleyi were significantly different between light wavelengths on the 3rd, 6th, and 9th day (one-way ANOVA, the 3rd day: df = 4, F = 747.3789, p < 0.001; the 6th day: df = 4, F = 321.786, p < 0.001; the 9th day: df = 4, F = 242.214, p < 0.001; Figure 1). On the 3rd day, the cell density under blue light was significantly greater than under the other light wavelengths. The cell density under red light was significantly lower than under white light and green light. On the 6th day, the cell density under blue light was significantly greater than under the other light wavelengths. The cell density under red light was significantly greater than under white light and green light. On the 9th day, the cell density under white light was significantly greater than under the other light wavelengths. The cell density under green light was significantly lower than under red light and blue light. On each day, the cell density under yellow light was significantly lower than under the other light wavelengths.

3.1.2. Growth

The cell diameters of E. huxleyi also differed significantly between light wavelengths (one-way ANOVA, df = 4, F = 78.288, p < 0.001; Figure 2). The cell diameter of E. huxleyi was significantly greater under red light than under the other light wavelengths. Additionally, the cell diameters under white light and green light were significantly greater than under blue light and yellow light. The cell diameter under yellow light was significantly lower than under blue light.

The dry weights of E. huxleyi were significantly different between light wavelengths (df = 4, F = 3172.595, p < 0.001; Figure 2). After 9 days, the dry weight under red light reached the maximum value of 864.20 ± 7.16 mg L −1, which was significantly greater than under the other light wavelengths. The dry weights under blue, green, and yellow lights were significantly lower than under white light.

Our data showed that the cell diameter and the dry weight of E. huxleyi were significantly greater under red light than under the other light wavelengths, indicating that red light could significantly contribute to the cell growth of E. huxleyi. However, yellow light exhibited the greatest inhibition to the cell diameter and the dry weight of E. huxleyi, implying that this species might not effectively harvest yellow light.

3.1.3. Net Photosynthetic Rate

The net photosynthetic rate of E. huxleyi was significantly affected by light wavelength (df = 4, F = 87.631, p < 0.001; Figure 3). The net photosynthetic rate showed its maximum value under red light. The net photosynthetic rates under red light and blue light were significantly greater than under the other light wavelengths. The net photosynthetic rates under green light and yellow light were significantly lower than under white light. The net photosynthetic rate under yellow light was significantly lower than under the other light wavelengths.

3.1.4. Pigment Contents

The Chl a content was significantly affected by light wavelength (one-way ANOVA, df = 4, F = 364.175, p < 0.001; Figure 4). The Chl a content under blue light was significantly greater than under the other light wavelengths. The Chl a content under red, green, and yellow light was significantly lower than under white light. The Chl a content under yellow light was significantly lower than under the other light wavelengths.

The fucoxanthin content differed significantly between light wavelengths (df = 4, F = 679.564, p < 0.001; Figure 4). The fucoxanthin content under red light was significantly greater than under the other light wavelengths. The fucoxanthin content under blue, green, and yellow lights was significantly lower than under white light. The fucoxanthin content under blue light and yellow light was significantly lower than under the other light wavelengths.

There were significant differences in lutein content between light wavelengths (df = 4, F = 548.092, p < 0.001; Figure 4). The lutein content showed a significant continuous decrease in the following order: white, red, blue, green, and yellow light.

3.2. Light Wavelength Change Experiment

In the single light wavelength experiment, blue light could effectively enhance the cell proliferation of E. hurleyi, and red light had a stimulating effect on its cell growth and fucoxanthin synthesis. Given the positive role of the two light wavelengths, a light wavelength change experiment was performed. During this experiment, the cell density of E. hurleyi in each replicate was determined every 3 days. After the experiment, the fucoxanthin content of E. huxleyi was measured.

3.2.1. Cell Density

The cell density of E. huxleyi was significantly different between light wavelength conditions on the 3rd, 6 th, and 9th day (one-way ANOVA, the 3rd day: df = 3, F = 732.291, p < 0.001; the 6th day: df = 3, F = 179.763, p < 0.001; the 9th day: df = 3, F = 46.004, p < 0.001; Figure 5). On the 3rd and 6th days, the cell density under blue light and the light wavelength change were significantly greater than under white light and red light. On the 3rd day, the cell density under red light was significantly lower than under white light. On the 9th day, the cell density under light wavelength change reached the maximum value of 0.2830 ± 0.0065 × 10 ⁵ cell mL −1, which was significantly greater than under the other single light wavelengths. The cell density under white light was significantly greater than under red light and blue light.

3.2.2. Fucoxanthin Content

The fucoxanthin content differed significantly between light wavelength conditions (one-way ANOVA, df = 3, F = 613.777, p < 0.001; Figure 6). After 9 days, the fucoxanthin content under light wavelength change reached the maximum value of 0.2717 ± 0.0113 mg L −1, which was significantly greater than that of the other single light wavelengths. The fucoxanthin content under red light was significantly greater than under white light and blue light. In addition, the fucoxanthin content under blue light was significantly lower than under the other light wavelength conditions.

4. Discussion

Our study showed that the cell density of E. huxleyi on the third and sixth day was significantly greater under blue light than under the other light wavelengths, indicating that blue light could stimulate its cell proliferation. Similar findings have been documented for Tetraselmis sp. and Nannochloropsis sp. Specifically, it was shown that the cell density of these species was the highest under blue light, probably due to the promotion of gene transcription that was related to photosynthetic enzymes and carbohydrate degrading enzymes and the regulation of activated enzymes [ 40]. Additionally, the maximum growth rate of Rhodomonas sp. was detected under blue light because its phycoerythrin and photosynthetic pigments could effectively absorb blue light to obtain energy for cell division [ 41]. Coincidentally, the Chl a content of E. huxleyi reached the maximum value under blue light. This is basically accordant with the results of previous studies and implies that blue light significantly enhanced the photosynthetic pigment syntheses in microalga species, including Isochrysis galbana, Dunaliella salina, Chaetoceros gracilis, and Nannochloropsis sp. [ 42, 43]. Similar to the above species, blue light is likely to promote the energy absorption of E. huxleyi, which leads to its hypermetabolism and gene expression modulation, thereby stimulating its cell proliferation. Further studies are needed to identify the correlative physiological mechanisms of this finding.

As we know, carotenoids play a critical role in the photosynthetic system of microalgae, such as by protecting the photosynthetic apparatus against photooxidative damage, and are greatly affected by light wavelength [ 44, 45]. Red light has been proven to significantly enhance the total carotenoid content of Dunaliella sp. and Cyanobium sp., probably due to the fact that red light induced their photoreceptors to accumulate carotenoids and activated correlative metabolic pathways [ 5, 46, 47]. Amaro et al. [ 48] demonstrated that red light could remarkably increase the β-carotene content of Tetradesmus obliquus (Turpin) M.J.Wynne under low cell density conditions because of its role in enhancing the function of the photosynthetic complex and, as such, avoidance of possible photodamage. Several previous studies have suggested that the change in the contents of different pigments, especially fucoxanthin and 19′-hexanoyloxyfucoxanthin, enhanced the photoacclimation capacities of microalgae including E. huxleyi, and that the light wavelength had a significant effect on the balance between the content of fucoxanthin and 19′-hexanoyloxyfucoxanthin [ 49, 50]. In addition, Garrido et al. [ 49] have suggested that E. huxleyi had a higher content of fucoxanthin under red light in order to maintain its high photosynthetic performance. Similarly, the fucoxanthin content of E. huxleyi was significantly greater under red light than under the other light wavelengths, suggesting that red light is optimal for fucoxanthin production by this species. As it is a type of light stressor, red light could inhibit the biomass growth of E. huxleyi; however, it may motivate the specific photoprotective mechanisms of this species to eliminate this photodamage, thereby resulting in the enhancement of fucoxanthin accumulation. In addition, compared with the inhibition of cell proliferation by red light, our results showed that the cell diameter, dry weight, and net photosynthetic rate of E. huxleyi reached the maximum value under red light, which indicates that red light could significantly contribute to the cell growth of E. huxleyi. Similarly, the dry weight of Chlorella vulgaris reached the maximum value under red light because it could enhance the activity of photosystem II, thereby promoting microalgal growth [ 51].

The dry weight and the net photosynthesis rate of E. huxleyi under green light were significantly lower than under white light, which indicates that green light is not appropriate for the cultivation of E. huxleyi. This is coincident with the previous finding that the dry weight of microalgal species, for instance, Microchloropsis salina (D.J.Hibbard) M.W.Fawley, I.Jameson & K.P.Fawley and Gloeothece membranacea, decreased significantly under green light, which may be due to the reflection of green light by these species that led to insufficient light energy and growth inhibition [ 22, 52]. In addition, the Chl a content of E. huxleyi under green light was significantly lower than under white light. This provides additional support for the possible interference of green light on its photosynthetic activity. Therefore, we suggest that E. huxleyi may not be able to effectively utilize green light through relevant pathways to generate the photosynthetic energy that contributes to its growth. Moreover, compared with the other light wavelengths, all the measured physiological parameters of E. huxleyi were the lowest under yellow light, which implies that this species might not harvest yellow light. Similar to our results, yellow light exhibited the greatest inhibition of the cell density and growth rate of Coscinodiscus granii of all light wavelength settings [ 53]. However, the physiological mechanism of the negative effect of yellow light on microalgae is still unclear.

The combined application of various light wavelengths may strongly impact microalgae biomass by activating the correlative photoreceptors and promoting availability of energy [ 54]. For example, a ratio of blue-to-red light of 50:50 was optimal for increasing the cell density of I. galbana and Phaeodactylum tricornutum. This combination could induce and enhance the activation of photosystems, thereby producing more photosynthetic energy for cell division [ 55, 56]. In addition, this light combination can significantly impact biomolecule synthesis. The combination of blue and red light could promote the lipid synthesis of microalgae such as C. vulgaris, T. obliquus, Diacronema lutheri, and Arthrospira platensis [ 57, 58]. The conversion from red to blue light promoted the synthesis of lipids in P. tricornutum, while the conversion from blue to red light increased the content of carbohydrates [ 59]. In our study, on each experimental day, E. huxleyi had significantly greater cell density under blue light followed by red light compared with white light, while its cell density was significantly lower under blue light than under white light on the ninth day. These results suggest that the conversion from blue to red light can more effectively promote the sustainable cell proliferation of E. huxleyi compared with single blue light. Similarly, Granata et al. [ 60] demonstrated that the combination of blue and red light could remarkably increase the biomass of E. huxleyi, probably due to the Emerson effect for algae, which promotes the photosynthetic rate. As it is a physiological basis for the biomass enhancement of this alga, we speculate that the application of combined light with different spectra is likely to separately stimulate the two photosystems to generate sufficient energy. Furthermore, microalgal species can respond to light wavelength pressure by modulating the syntheses of relevant antioxidant pigments. Compared with single blue or red light, the mixed light composed of both induced greatly higher lutein content in D. salina [ 61]. The combination of blue and red light provided moderate external stress that stimulated lutein synthesis, thereby avoiding irreversible damage to this species [ 61]. Similarly, our results showed that the fucoxanthin content under blue light followed by red light was significantly higher than under the other single light wavelengths. The fucoxanthin synthesis in diatoms has been shown to be primarily supported by the photosynthetic energy from absorbed blue light and red light [ 62]. Hence, the change in light wavelength from blue to red light is considered to be an effective technical measure to enhance the biomass and fucoxanthin production of E. huxleyi.

5. Conclusions

In conclusion, we have clarified that light wavelength is a very significant variable that affects the growth, photosynthetic activity, and pigment synthesis of E. huxleyi. Blue light significantly stimulated the cell proliferation of E. huxleyi, while red light could promote its cell growth and fucoxanthin synthesis. Compared with all single light wavelength regimes, the combination of blue and red light significantly increased the cell density and fucoxanthin content of E. huxleyi. Our findings are likely to be highly valuable in the cultivation management and commercial exploitation of this productive species. Because of our limited data, further studies are needed to determine the correlative physiological and molecular mechanisms.

Author Contributions

Conceptualization and methodology, Q.W. and X.G.; writing-original draft preparation, J.Z.; writing-review and check, X.G.; project administration and funding acquisition, X.G.; investigation, formal analysis, and data curation, F.L. and J.Z.; validation and resources, Q.G. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by Fundamental Research Funds for the Central Universities (No. 202262002) and the Young Talent Program from the Ocean University of China (No. 202212015).

Institutional Review Board Statement

Informed Consent Statement

Data Availability Statement

Our data are available from the corresponding author upon reasonable request.

Acknowledgments

We sincerely thank Baohua Zhu from the Ocean University of China for his assistance with experimental methods. We sincerely thank Guangce Wang from the Institute of Oceanology, Chinese Academy of Sciences, for helping with the analysis of lutein and fucoxanthin concentrations.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 4. Content of Chl a, fucoxanthin and lutein of E. huxleyi cultured under different light wavelengths (white, red, blue, green, and yellow light) on the 9th day. Data are presented as mean ± SE (n = 3). Lowercase letters indicate significant differences between light wavelengths at p < 0.05.

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AMA Style

Zhang J, Liu F, Wang Q, Gong Q, Gao X. Effect of Light Wavelength on Biomass, Growth, Photosynthesis and Pigment Content of Emiliania huxleyi (Isochrysidales, Cocco-Lithophyceae). Journal of Marine Science and Engineering. 2023; 11(2):456. https://doi.org/10.3390/jmse11020456

Chicago/Turabian Style

Zhang, Jing, Falong Liu, Qiaohan Wang, Qingli Gong, and Xu Gao. 2023. "Effect of Light Wavelength on Biomass, Growth, Photosynthesis and Pigment Content of Emiliania huxleyi (Isochrysidales, Cocco-Lithophyceae)" Journal of Marine Science and Engineering 11, no. 2: 456. https://doi.org/10.3390/jmse11020456

# data[].attributes.attachment_link.title
Effect of Light Wavelength on Biomass, Growth, Photosynthesis and Pigment Content of Emiliania huxleyi (Isochrysidales, Cocco-Lithophyceae)

# data[].attributes.attachment_link.description
Light wavelength is a critical abiotic factor in modulating the development and pigment accumulation of microalgae. In the present study, we investigated the influences of white, red, blue, yellow, and green light on biomass (cell density), growth (cell diameter and dry weight), net photosynthetic rate, and pigment contents (chlorophyll a, fucoxanthin, and lutein) of the coccolithophore Emiliania huxleyi.