Effects of red and blue light on leaf anatomy, CO2 assimilation, and the photosynthetic electron transport capacity of sweet pepper (Capsicum annuum L.) seedlings

doi:10.21203/rs.2.24179/v1

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Research article

Effects of red and blue light on leaf anatomy, CO2 assimilation, and the photosynthetic electron transport capacity of sweet pepper (Capsicum annuum L.) seedlings

https://doi.org/10.21203/rs.2.24179/v1

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published 06 Jul, 2020

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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 and stomatal traits, 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/m²·s.

Results: The results revealed that seedlings grown under R light had lower biomass accumulation, CO₂ assimilation, and photosystem II (PSII) electron transportation compared to plants grown under the other treatments. These changes are probably due to inactivation of the photosystem (PS). Biomass accumulation and CO₂ assimilation were significantly enriched in B- and RB-grown plants, especially the latter treatment. Their leaves were also thicker, and stomatal conductance, photosynthetic electron transport capacity, and the photosynthetic rate were enhanced. The up-regulation of the expression and activities of Rubisco, fructose-1, 6-bisphosphatase (FBPase), and fructose-1, 6-bisphosphate aldolase (FBA), which are involved in the Calvin cycle and are probably the main enzymatic factors contributing to RuBP (ribulose-1, 5-bisphosphate) synthesis, 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.

Plant Physiology and Morphology

Plant Molecular Biology and Genetics

Sweet pepper (Capsicum annuum L.)

Light quality

Anatomy

Photosynthesis

CO2 assimilation

Light is one of the most important environmental factors affecting plant growth and development [1]. Using light rather than chemicals to control plant architecture could 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. [3–6].

It is known that plants can respond to light intensity, photoperiod, and light quality [7, 8]. Light quality, which refers to the color or wavelength of the light, is a key factor in numerous adaptive responses and development transitions in plants [9–11]. Specific light qualities have precise effects on plants. For example, B and 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 [12, 13]. A few studies have used two wavelengths to examine the effects of light quality on plants. In general, R light plays an important role in controlling the functions of the chloroplast, stem and petiole growth, and the reproductive system [14, 15]. B light affects plant growth, leaf expansion, photomorphogenesis, stomatal opening, photosynthesis, and pigment accumulation [16, 17]. Furthermore, studies have revealed that plants under B light had greater stomatal opening, higher chlorophyll (Chl) a/b ratios, smaller amounts of light harvesting Chl a/b-binding protein in photosystem II (PSII), higher photosynthetic electron-transport activity per unit of Chl content, and higher Rubisco activity levels and expressions of Calvin cycle-related genes than those plants under R light [18, 19].

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 [20]. Various studies have found that mixed R and B light was an effective lighting source that improved plant development, and a suitable proportion of R and B light accelerates photosynthesis and the growth of tomato, cucumber, and sweet pepper, etc. [20–22]. Furthermore, Klein [23] and Naznin [22] 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 [24, 25]. 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 [26]. Mixed R and B light has an apparent influence on the growth and physiology of pepper plants [22, 27, 28]. 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 [29–31]. 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 seedling photosynthesis and growth. The objective of this study was to examine how R and/or B light sources affected pepper seedling photomorphogenesis, photosynthetic characteristics, and the transcriptional and translation levels of key enzymes in the Calvin cycle.

Plant morphology and biomass accumulation under the 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) is shown in Figs. 2 and 3 and the differences were significant. The plant shoot dry weight (DW) under RB significantly increased compared to W (P < 0.05), and it was also higher than those in the other treatments, whereas, R light produced the lowest DWs (Fig. 4A). The root DWs showed similar trends under all the treatments (Fig. 4B).

Leaf anatomy and stomatal traits under the different light treatments

Table 2 and Fig. 5 show that R and B light had a significant effect on the anatomical structure of pepper leaves. Leaf thickness was 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 the RB treatment (P < 0.05). They 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

Primes sequences used for real time RT-PCR assays.
Gene	Forward primer	Reverse primer
actin	5'-TGAAAATCAAGGTGGTGGCG-3'	5'-TCCGGTGAACAATGGAAGGA-3'
FBA	5'-ATGAACCAAGCACCAAACCC-3'	5'-TGTACTTTCCGAGCTGAGCA-3'
FBPase	5'-TCGTCAGCCATTTCTTCCCT-3'	5'-CACCCACATCAACTCCTCCT-3'
GAPDH	5'-GACTACTGTGCACGCAACAA-3'	5'-AGCAGCACCAGTTGAACTTG-3'
TK	5'-CCCAATGTTCTGATGCTCCG-3'	5'-CCACCCTTTGCTGTTCCTTC-3'

Table 2

Effects of different light treatments on leaf anatomy of sweet pepper seedlings at 28 DAT.
Treatments	Leaf thickness (µm)	Palisade mesophyll tissue thickness (µm)	Spongy mesophyll tissue thickness (µm)	Upper epidermis thickness (µm)	Lower epidermis thickness (µm)	Palisade mesophyll tissue/ spongy mesophyll tissue ratio
W	122.54 ± 4.92 b	39.73 ± 2.11 b	67.92 ± 3.02 b	8.35 ± 0.39 b	6.21 ± 0.11 ab	0.58 ± 0.02 ab
R	103.25 ± 3.78 c	30.21 ± 1.32 c	59.03 ± 2.82 c	6.23 ± 0.15 c	5.88 ± 0.19 b	0.51 ± 0.04 b
B	130.22 ± 3.15 b	43.33 ± 1.87 b	73.24 ± 1.45 b	7.96 ± 0.27 b	6.07 ± 0.14 b	0.59 ± 0.02 ab
RB	146.90 ± 5.21 a	50.07 ± 2.56 a	81.02 ± 2.56 a	10.18 ± 0.11 a	6.42 ± 0.12 a	0.62 ± 0.02 a
Note: 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 same as below.

The different light qualities resulted in different stomata traits distributions (Table 3, Fig. 6). The highest stomatal density was observed under RB, which was 398.88 no./mm², followed by W and B light, and the lowest value (233.43 no./mm²) occurred under R. The lengths and widths of the stomata and the stomatal aperture, and the stomatal index under B and RB significantly increased compared to W (P < 0.05), but there was no significant difference between B and RB (P > 0.05). However, they were inhibited under R light. Therefore, B and RB light promoted stomatal opening.

Table 3

Effects of different light treatments on stomatal traits of sweet pepper seedlings at 28 DAT.
Treatments	Stomatal size (µm)				Stomatal density (no./mm²)	Stomatal index (%)
Treatments	Length	Width	Length	Width	Stomatal density (no./mm²)	Stomatal index (%)
W	19.46 ± 0.71 b	14.02 ± 0.61 ab	9.44 ± 0.68 b	3.09 ± 0.12 b	321.19 ± 18.32 b	62.02 ± 9.22 b
R	18.93 ± 0.82 b	13.68 ± 0.29 b	7.90 ± 0.42 c	1.42 ± 0.08 c	233.43 ± 25.67 c	47.35 ± 6.33 c
B	23.69 ± 0.98 a	15.67 ± 0.79 a	13.04 ± 0.72 a	4.32 ± 0.21 a	306.81 ± 27.41 b	76.64 ± 12.11 a
RB	24.16 ± 0.74 a	16.89 ± 0.47 a	11.35 ± 0.87 ab	3.78 ± 0.33 ab	398.88 ± 17.94 a	83.39 ± 8.34 a

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

The net photosynthetic rate (Pn) of the leaves increased as the PPFD (Fig. 7A) and CO₂ concentration (Fig. 7B) rose. Furthermore, this value gradually became stable as the PPFD and CO₂ concentration increased. The highest Pn-PPFD response curve value was detected under RB, followed by B and W, whereas R produced the lowest value. All treatments produced responses to CO₂ concentration. The apparent quantum efficiency (AQY), light saturation point (LSP), light-saturated maximum (Pn_max), carboxylation efficiency (CE), and CO₂ saturation point (CSP) levels, and the maximum RuBP regeneration rate were significantly higher under RB (P < 0.05) than under W, whereas, the light compensation point (LCP) and CO₂ compensation point (CCP) values decreased under this treatment (Table 4 and Table 5).

Table 4

Effects of different light treatments on photosynthetic light-response curve parameters of sweet pepper seedlings at 28 DAT.
Treatments	AQY (µmol/m²·s)	LCP (µmol/m²·s)	LSP (µmol/m²·s)	Pn_max (µmol/m²·s)
W	0.051 ± 0.003 b	26.6 ± 2.34 a	729 ± 38.34 c	13.0 ± 0.55 b
R	0.030 ± 0.002 c	27.2 ± 2.13 a	520 ± 29.56 d	6.1 ± 0.42 c
B	0.050 ± 0.002 b	23.7 ± 1.82 b	924 ± 27.45 b	15.2 ± 0.72 a
RB	0.056 ± 0.001 a	22.8 ± 2.91 b	968 ± 28.17 a	16.3 ± 0.68 a
Note: AQY, apparent quantum efficiency; LCP, light compensation point; LSP, light saturation point; Pn_max, light-saturated maximum.

Table 5

Effects of different light treatments on photosynthetic CO₂-response curve parameters of sweet pepper seedlings at 28 DAT.
Treatments	CE (mol/m²·s¹)	CCP (µmol/m²·s)	CSP (µmol/m²·s)	Maximum RuBP regeneration rate (µmol/m²·s)
W	0.047 ± 0.006 b	81 ± 5.64 b	1087 ± 25.12 c	23.1 ± 3.92 b
R	0.032 ± 0.004 c	92 ± 3.12 a	1213 ± 12.39 b	11.0 ± 1.87 c
B	0.057 ± 0.009 b	57 ± 3.28 c	1040 ± 17.28 d	21.4 ± 1.93 b
RB	0.066 ± 0.003 a	61 ± 6.77 c	1443 ± 21.32 a	39.5 ± 2.67 a
Note: CE, carboxylation efficiency; CCP, CO₂ compensation point; CSP, CO₂ saturation point.

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

The effects of R and B light on the pepper seedling Chl fluorescence parameters are shown in Fig. 8. F_v/F_m, which represents the greatest light conversion efficiency or the maximum quantum yield of PS II, was higher under RB, W, and B than under R, but there were no significant differences among the RB, W, and B treatments (Fig. 8A). 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. 8B). F'_v/F'_m indicates how efficiency the excitation energy is captured by open PSII reaction centers, and it was enhanced in B- and RB-grown seedlings compared to W, but there were no significant differences among these three treatments (P > 0.05) (Fig. 8C). However, seedlings grown under R had significantly lower F'_v/F'_m values than the other treatments (P < 0.05).

The typical polyphasic Chl a fluorescence transient (OJIP) increases at different experimental time points are shown in Figs. 9A-D. In general, the results showed that the W, B, and RB treatments decreased the amplitude of the OJIP curves compared to 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). The JIP-test was applied to the fluorescence induction transients to further investigate the mechanisms underlying the observed changes (Figs. 10A-H). All parameters under the W, R, and B treatments initially increased and then decreased, but the parameters fluctuated under RB. 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 to 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 under the other treatments (P < 0.05).

Calvin cycle enzymes activity under the different light treatments

Rubisco, FBPase, 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 as the duration of different light quality treatments increased (Figs. 11A-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. 11). In contrast, R-grown plants had a significantly lower activity levels (15% less) than W-grown plants.

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. 11B). Activities of this enzyme in plants under B light remained significantly higher than under the 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 under the other treatments during the experimental period. The FBA activities in plants treated with W and R light increased slowly during the experimental period (Fig. 11C), 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. 11D). 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 under the other treatments (Fig. 11E).

Gene expression under the different light treatments

The RT-PCR method was used to analyze the relative expression levels of the FBA, FBPase, GAPDH, and TK genes involved in the Calvin cycle after pepper seedling exposure to the different light qualities for 28 d. Figures 12A-D show 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 the 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.

During light-controlled development, it is generally assumed that the photoreceptors perceive and interpret incident light and transduce signals to modulate light-responsive nuclear genes. Amongst the light spectra, R and B wavelengths are the primary spectral wavelengths and can highly influence plant photosynthesis, physiological metabolism and morphology [32–34]. 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 greater than under the 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 [28]. 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 [35–37]. The pepper seedling biomass significantly increased under RB compared to the other treatments and this was probably due to the enlarged leaf area (LA) [38] 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, the RB treatment greatly increased the PT, SPT, and upper and lower epidermis thickness, which led to thicker leaves, and this was consistent with the results of Arena et al. [39]. 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 [40]. This improved the photosynthetic structure, which should increase the light capture and absorbance capacities, and might contribute to better photosynthetic light acclimation. In addition, leaf thickness plays a key role in determining space availability for chloroplast development [41]. The RB treatment increased leaf thickness, which enhanced the chloroplast ultrastructure [42], and this could be another important reason why RB was able to improve photosynthetic efficiency.

The stomata are important channels that exchange air and water with the outside environment. Light quality affects stomatal development [43], which in turn will affect air conductance via the leaf mesophyll and stomata. Furthermore, the stomatal density and the cumulative effect of the stomatal aperture size influence stomatal conductivity. It is well known that B light has a significant effect on the development of stomata [44, 45]. Nevertheless, unlike previous studies, we found that there were increases in stomatal size, aperture size, and density under RB compared to monochromatic B light, which indicated that the mixed R and B light had a synergistic effect on stomatal conductivity and development. The results suggested that a larger LA and increased leaf and PT cells thickness improved light interception by the pepper seedlings. In addition, higher stomatal density and conductivity increased acquisition efficiency and improved the availability of CO₂ in the mesophyll, which would lead to higher photosynthetic activity levels [46, 24]. This may have led to the significant rise in biomass [47]. The RB light can also increase shoot regeneration and development by stimulating cell division [48]. In contrast, the lower stomatal density under R light could restrain photosynthesis rates by increasing diffusive resistance to CO₂ uptake, which may reduce the activities of the photosynthetic organs [49].

The light- and CO₂-response curves reflect the plant capacity to benefit from increments in light energy and CO₂. This information offers interesting insights into the mechanisms that underlie light capture and CO₂ fixation. In this study, Pn-PPFD under the different light qualities was significantly lower than Pn-CO₂. This might be due to a CO₂ concentration limitation. The AQY and CE values showed the initial slopes of the light- and CO₂-response curves, respectively. They represent the capacity of the plant to capture low light energies and low CO₂ levels. Our results confirmed a previous study [50], 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 indicated that mixed R and B light worked synergistically to increase photosynthetic capacity [51]. 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 significantly decreased under RB, which showed that the treatment improved photosynthetic performance and light energy utilization efficiency.

Light qualities can regulate photosynthesis by affecting the formation of different types of chloroplast proteins and electron transport between light systems [52]. Chl fluorescence can partly reflect the photosynthetic ability of plants [53] and the efficiency of PSII photochemistry (Φ_PSII) can be used to reveal the physiological state of plants [54]. 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 [55]. Additionally, the higher F'_v/F'_m and Φ_PSII levels under the 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.

The J-step, I-step, and IP phases of Chl fluorescence transients are correlated with the redox state of quinone electron acceptor (Q_A), the redox state of plastoquinone, and the redox state of the end acceptors at the PSI electron acceptor side, respectively [56, 57]. 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, which would reduce CO₂ assimilation. Monochromatic B and mixed R and B light induced a decrease in all the OJIP steps during the experimental period compared to the other treatments, which altered both the donor and acceptor sides of PSII and affected electron transport [58]. 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. 10). This indicated that there was less damage to the photochemical and non-photochemical redox reactions and that there was an enhanced capacity for electron transport, which would accelerate ATP synthesis and RuBP regeneration [59].

In C3 plants, the Calvin cycle is the predominant pathway for CO₂ assimilation [60]. 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 [61, 62]. Light is an important environmental signal that triggers gene expression and regulates corresponding enzyme activities in plants during development. A few studies have examined how light regulates the expression and activities of enzymes that are involved in photosynthesis [51, 63]. These previous studies were verified by the present study. The Rubisco activity in B- and RB-treated plants was significantly higher than in the plants treated with the other light wavelengths. This finding suggested that the application of B or RB could increase carbon assimilation and RuBP regeneration in the Calvin cycle. We also found that under R light, the decreased photosynthetic rate was accompanied by reductions in stomatal aperture size, density, and index, Rubisco activity, and the transcriptional levels of most genes involved in the Calvin cycle. This result was consistent with an earlier observation and implied that the inhibition of CO₂ 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 CO₂ assimilation rate in R-grown seedlings compared to the other light treatments [64, 31]. Furthermore, a previous study found that the stomatal factor, which differentially regulates the availability of RuBP and CO₂, might also be involved in the regulation of gene expression because the expression levels of the genes examined were highly correlated with the changes in stomatal conductance [31].

The FBA and FBPase activities directly affect photosynthetic efficiency and carbon accumulation [65]. 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 [66]. In our study, the activities of these enzymes under B and RB and the relative expression of their associated genes, except for TK, were significantly elevated, which promoted RuBP regeneration and increased Pn [65, 66]. Chloroplast GAPDH is a key enzyme involved in the carbon reduction process during photosynthesis [67] and the greater GAPDH expression level under RB light in the present study may be due to the increased demand for carbon flux [68]. Changes in TK activities in all treatments were not consistent with the pattern observed for TK expression, which implied that TK activity is only partially dependent on light quality, but this needs further investigation.

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 stomatal closure, which limited the diffusion of CO₂ to the chloroplast and caused a reduction in CO₂ 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 FBA, 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.

Plant material and climate conditions

The experiment was performed in June, 2018 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. Sweet pepper (Capsicum annuum L. cv. Hongqijian) seeds (Jinan Weili Seeds Co., Ltd., Shandong, China) were immersed in water for 15 min at 55 ^oC and then soaked in cold water (4 ^oC) 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, a total of 480 seedlings were selected, moved into the ACC, and cultured under one of four 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 CO₂ level was the same as the CO₂ level of atmosphere outside. The relative humidity (RH) was maintained at 70 ± 10%, with a 12 h photoperiod and a temperature of 26 ± 1 ^oC during the daytime and 18 ± 1 ^oC 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 with a maximum intensity at 657 nm or mixed R and B light (3:1, RB: 75% R light at a wavelength of 657 nm and 25% B light at a wavelength of 457 nm). There was a multi-wavelength W light treatment as control (Fig. 1). The light intensity, expressed as PPFD at the canopy level, was set at 300 µmol/m²·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 distance between the LEDs and the canopy was approximately 10 cm. The spectral photon flux density distributions (SPDs) of the LEDs were measured using a spectroradiometer (Unispec-SC Spectral Analysis System, PP Systems Inc., Haverhill, MA, USA).

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 and stomatal traits

Leaf anatomy was measured on the fully expanded second leaves from five pepper seedlings at a similar position for each replication each treatment [69] 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).

Stomatal traits were determined using transparent nail polish according to Wu and Zhao [70]. Epidermal impressions were collected from both leaf surfaces, placed on microscope slides, and analyzed using a bright field microscope (JSM-6610LV, JEOL Inc., Tokyo, Japan). Five slides from five plants each replication each treatment were used for the determination. Five fields of view were randomly selected from each slide and the stomatal number was counted to calculate stomatal density, which was the number of stomata per mm² (no./mm²). The stomatal size was determined by randomly selecting 20 stomata from each slide and then measuring their length and width [17]. The stomatal aperture numbers, and width and length were determined according to Chen et al. [71]. The stomatal index was calculated as the number of stomatal apertures / number of stomata × 100.

Photosynthetic light- and CO₂-response curves

The photosynthetic light-response curves and CO₂-response curves were measured on the second fully expanded leaf between 09:00 am and 14:00 pm 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. [51]. The leaf chambers were set to temperature 26 ± 1 ∘C, air relative humidity 65 ± 5%, and flow rate 300 µmol/s. The light-response curves were measured under a graded PPFD series of 1800, 1500, 1200, 1000, 800, 600, 400, 300, 200, 150, 100, 50, 20, and 0 µmol/m²·s. When the CO₂-response curve measurements were taken, the light intensity and CO₂ concentration of the leaf cuvette were set to 1000 µmol/m²·s and 400 µmol/mol, respectively, for 30 min. After it reached a steady state, a CO₂ mixer was used to measure the CO₂-response curves under a graded Ci value series of 400, 300, 200, 100, 50, 100, 200, 300, 400, 600, 800, 1000, 1200, 1500, and 1800 µmol·CO²/mol. It took about 120 to 180 s for the leaf chamber to adjust to its new microclimate for each measurement with one match. Each curve was measured three times and fitted with a non-linear regression equation, as previously reported [72, 73], so that the LCP, LSP, Pn_max, CCP, CSP, and the maximum RuBP regeneration rate. The AQY was the initial slope of the light-response curve and the CE was the initial slope of the CO₂-response curve.

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. The leaves were then illuminated by natural light for 1 h, and the F'_o, F'_m, and F_s values were measured under 800 µmol/m²·s activating light. The saturation pulse intensity and duration were 3000 µmol/m²·s and 0.8 s, respectively. F'_o and F'_m represent the minimum and maximum fluorescence yields of an illuminated leaf, respectively, and were measured using the saturation pulse method. F_s represents the steady state 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.

The OJIP was measured on the second leaves by a plant efficiency analyzer (Handy PEA, Hansatech Instruments Ltd., King’s Lynn, Norfolk, UK). The JIP-test formulae and glossary of terms were calculated according to Strasser [74, 75]. The following derivative parameters were determined according to Lin et al. [59] and Miao et al. [25]: RC/ABS, S_m, DI_o/RC, TR_o/RC, PI_ABS, PI_total, Φ_Ro, and δ_Ro.

Calvin cycle enzymes activity

The second leaves from the top of 15 plants per treatment were sampled at 7, 14, 21, and 28 DAT 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 MgSO₄, 5 mM dithiothreitol (DTT), 1 mM Na₂EDTA, 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 [76].

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 were determined using an ELISA kit (Shanghai Yanji Biological Technology Ltd., Shanghai, China) and the extraction methods used for these enzymes were according to Rao and Terry [77] and Wang et al. [31] with some modification. The frozen leaf samples (0.5 g) were ground to a fine powder in liquid nitrogen with a mortar and pestle, transferred to a centrifuge tube, and then extracted in pre-chilled extraction buffer (5 mL). The enzyme extraction solution was centrifuged at 12,000 × g for 15 min at 4 ^oC. The supernatant was used for the Calvin cycle enzymes activity assay. Subsequently, the activities of the Calvin cycle enzymes were determined using a microplate absorbance reader (Bio-Tek ELX800, Bio-Tek Instruments, Winooski, VT, USA) at an absorbance of 450 nm according to the manufacturer’s instruction.

The protein concentration of each enzyme extraction solution was measured according to Bradford [78]. The results were expressed as U/g of protein.

Gene expression

Total RNA was extracted using Quick RNA Isolation Kit according to the supplier’s instructions (Huayueyang Biotech Co., Ltd., Beijing, China). Reverse transcription was conducted using a ReverTra Ace qPCR RT-Kit (Toyobo Bio-Technology, Co., Ltd., Osaka, Japan). The gene expression analysis was conducted using real-time PCR, with 18S rRNA as an internal control. The thermal cycler program was one initial cycle of 94 °C for 2 min, followed by 40 cycles of 94 °C for 10 s, 60 °C for 20 s, and 72 °C for 30 s. Relative gene expressions were analyzed using the method described in Livak and Schmittgen [79]. The specific gene primers used for real-time PCR analysis of the genes involved in the PS complexes are shown in Table 1.

Data analysis

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

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: CO2 compensation point; CSP: CO2 saturation point; AQY: Apparent quantum efficiency; CE: Carboxylation efficiency; Fv/Fm: Maximum photochemical efficiency of PSII; ΦPSII: 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; ΦRo: Quantum yield for reduction of end electron acceptors at the PSI acceptor side; δRo: 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.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Competing interests

The authors declare that they have no conflict of interest.

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.

Authors’ contributions

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

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.

Authors’ details

^aCollege of Horticultural Science and Engineering, Shandong Agricultural University

^bScientific Observing and Experimental Station of Environment Controlled Agricultural Engineering in Huang-Huai-Hai Region, Ministry of Agriculture

^cShandong Collaborative Innovation Center of Fruit & Vegetable Quality and Efficient Production

^dState Key Laboratory of Crop Biology, Daizong Street 61, Tai’an, Shandong, 271018, China

^eEntomology and Nematology Department, University of Florida, 1881 Natural Area Dr, Gainesville, FL U.S.A

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Effects of red and blue light on leaf anatomy, CO2 assimilation, and the photosynthetic electron transport capacity of sweet pepper (Capsicum annuum L.) seedlings

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Results

Plant morphology and biomass accumulation under the different light treatments

Leaf anatomy and stomatal traits under the different light treatments

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

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

Calvin cycle enzymes activity under the different light treatments

Gene expression under the different light treatments

Discussion

Conclusions

Methods

Plant material and climate conditions

Light treatments

Biomass analysis

Leaf anatomy and stomatal traits

Photosynthetic light- and CO₂-response curves

Chlorophyll fluorescence and chlorophyll fluorescence transients

Calvin cycle enzymes activity

Gene expression

Data analysis

Abbreviations

Declarations

References

Status:

Journal Publication

Version 1

Effects of red and blue light on leaf anatomy, CO2 assimilation, and the photosynthetic electron transport capacity of sweet pepper (Capsicum annuum L.) seedlings

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Results

Plant morphology and biomass accumulation under the different light treatments

Leaf anatomy and stomatal traits under the different light treatments

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

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

Calvin cycle enzymes activity under the different light treatments

Gene expression under the different light treatments

Discussion

Conclusions

Methods

Plant material and climate conditions

Light treatments

Biomass analysis

Leaf anatomy and stomatal traits

Photosynthetic light- and CO2-response curves

Chlorophyll fluorescence and chlorophyll fluorescence transients

Calvin cycle enzymes activity

Gene expression

Data analysis

Abbreviations

Declarations

References

Status:

Journal Publication

Version 1

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

Photosynthetic light- and CO₂-response curves