Responses of the photosynthetic characteristics and chloroplast ultrastructure of Welsh onion (Allium fistulosum L.) to different LED light qualities

Background: The optimized illumination of plants (LEDs) is beneficial to their photosynthetic performance. Because of this, in recent years LEDs have been widely used in horticultural facilities. However, there are significant differences in the responses of different crops to light quality. The influence of light quality on photosynthesis needs to be further explored to provide theoretical guidance for the adjustment of the light environment in industrial crop production. This study tested the effects of different qualities of LED lighting (white, W; blue, B; green, G; yellow, Y; and red, R) with the same photon flux density (300 μmol/m2·s) on the growth and development, photosynthesis, chlorophyll fluorescence characteristics, leaf structure, and chloroplast ultrastructure of Welsh onion (Allium fistulosum L.) plants. Results: The results showed that the plant height, leaf area, and fresh weight of plants in the W and B treatments were significantly higher than those in the other treatments. The photosynthetic pigment content and net photosynthetic rate (Pn) in the W treatment were significantly higher than those in the monochromatic light treatments, while the transpiration rate (E) and stomatal conductance (Gs) were the highest in the B treatment, and the intercellular CO2 concentration (Ci) was the highest in the Y treatment. Among the chlorophyll fluorescence characteristics tested, the non-photochemical quenching coefficient (NPQ) was the highest in the Y treatment, while the maximum photochemical efficiency of photosystem II (PSII) under dark adaptation (Fv/Fm), maximum photochemical efficiency of PSII under light adaptation (Fv'/Fm'), photochemical quenching coefficient (qP), actual photochemical efficiency (ΦPSII), and apparent electron transport rate (ETR) all differed among treatments in the following order: W

> B > R > G > Y. Both leaf structure and chloroplast ultrastructure showed the most complete development in the B treatment. Conclusions: In summary, in addition to W light, B light significantly improved the photosynthetic efficiency of Welsh onion, whereas Y light significantly reduced the photosynthetic efficiency of this plant.

Background
Light is not only the primary source of energy for photosynthesis in plants, but is also an important signal for plant growth and development [1]. Light intensity, light quality, and photoperiod can regulate plant growth and development [2]. Johkan [3] found that the net photosynthetic rate (P n ) in the leaves of Lactuca sativa plants irradiated with green light from a light-emitting diode (LED) at a photosynthetic photon flux (PPF) of 200 μmol/m 2 ·s was significantly higher than that at a PPF of 100 μmol/m 2 ·s, and the Pn of plants irradiated with G510 light (peak wavelength: 510 nm; band width at half peak height: 18 nm) was the highest among all the light sources they tested. Many processes can also be regulated by adjusting the wavelength of light to which plants are exposed, such as seed germination, photomorphogenesis, photosynthesis, carbon and nitrogen metabolism, biomass accumulation, chloroplast ultrastructure, and leaf anatomical structure [4][5][6][7][8][9]. Studies have shown that the regulation of red light and the far-red light ratio can regulate the flowering time of Arabidopsis, and also provided evidence of the existence of light quality pathways regulating plant flowering times [10].
Photosynthesis is an important biological process for maintaining plant life, and has played a very important role in the evolution of the Earth's ecosystems. Increasing rates of photosynthesis is critical to increasing crop yields to meet rising human needs for food [11,12]. Chloroplast development and chlorophyll metabolism are important activities involved in photosynthesis in green plants. Previous studies have demonstrated the existence of chlorophyll synthesis-related enzymes that are key regulators of chloroplast development [13,14]. It was previously found that the photosynthesis rate in cucumber seedlings under white light was significantly higher than that of red, blue, yellow and green light, and the morphology and photosynthetic rate of these plants were significantly different under different monochromatic light treatments [15]. It was also previously shown that RGB light (33% red, 33% green, and 33% blue) and RB light (66% red and 33% blue) reduced the plant height, plant biomass, and leaf area of tomato plants compared to those of plants grown under white light [16]. In a study of the effects of different light quality treatments on tobacco, monochromatic light treatments reduced tobacco growth, Pn, stomatal conductance, intercellular CO 2 , and transpiration rates compared to those of plants grown under white light [8]. In another study of the effects of light quality (white, blue, yellow, and red light) on the growth and photosynthesis of Camptotheca acuminata seedlings, it was found that red light promoted the development of chloroplasts and improved the photosynthetic efficiency of the seedlings of this plant [17].
Ribose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO; EC 4.1.1.39) is a key enzyme in plant photosynthesis that controls both CO 2 and carbon fixation. The Calvin cycle and the photorespiration cycle are shunted by this process, and the relative magnitudes of their activities directly affect the photosynthetic rate [18].
Gao [19]found that when the ratio of red to blue light was 4:1, the ribose-1,5bisphosphate carboxylase (RuBPCase) activity in purple lettuce was significantly higher than that in control plants and those in other light treatments.
Although previous studies have determined the effects of light quality on the growth, physiology, and morphology of various plant species, few studies have reported upon the responses of the growth, photosynthetic characteristics, and flavor of Welsh onion to different LED light qualities. Welsh onion (Allium fistulosum L.) is an important seasoning vegetable. In recent years, the scale of the production of Welsh onion in industrial facilities has been expanding. To create good environmental conditions for the growth of crops in such facilities, previous studies have been conducted on the relationships between light quality and the growth, photosynthetic characteristics, carbon and nitrogen metabolism, and volatile production of other plants. Research by Lin [20]showed that the root fresh weight and dry weight of lettuce treated with combined red-blue-white light (RBW) and fullspectrum light (FL) were higher than those of lettuce treated with RB light. Zhang [21]found that the content of sucrose, fructose, and glucose in peach fruits grown under natural light was higher than that in those grown covered with blue film, red film, green film, and yellow film. In addition, previous studies also examined the responses of plant volatiles to different light qualities. For instance, basil plants grown under light at blue-red-yellow (BRY) or blue-red-green (BRG) wavelengths evaporated higher levels of monoterpenoid volatiles, while basil plants grown under a combined far-infrared-blue-red (far-RBR) treatment evaporated even higher levels of most sesquiterpenoid volatiles [22]. The main flavor of Welsh onion comes from an organic sulfide, which is an important indicator of its nutritional quality [23]. The organic sulfide content of the plant can be expressed as the content of pyruvic acid, its decomposition product [24]. The objective of this study was to test the effects of different LED light qualities on the photosynthetic characteristics and chloroplast ultrastructure of Welsh onion plants.

Response of growth and development of Welsh onion to different LED light qualities
The number of leaves, LA, plant height, cauloid diameter, and cauloid FW of Welsh onion plants were significantly higher after 30 days under W light than under any monochromatic light treatment, and the growth of plants in the B treatment was significantly higher than that under all the other monochromatic light treatments.
The shoot dry matter content of plants in the B treatment were slightly higher than those of plants in the W treatment, indicating that the water content of the Welsh onion was higher in the W treatment. Dickson's Quality Index (DQI) could assess seedling quality and performance attributes [25]. According to the calculation of DQI, we found that the seedlings quality of Welsh onion under the monochromatic light treatments differed in the following order: B > R > G >Y (Fig. 1B, Table 1). The content of chlorophyll a and chlorophyll b in the W treatment were significantly higher than those in all the monochromatic light treatments, and among the monochromatic light treatments these were the highest in the B treatment. The trends in carotenoid content and chlorophyll content were consistent (Table 2).
Chlorophyll b (Chl b) plays an important role in adapting plants to low-light conditions. In low light, plants synthesize more Chl b and increase their chlorophyll a / b ratio, which helps to form a larger light-harvesting system as an adaptation to low-light environments [26].

Responses of chlorophyll fluorescence parameters in Welsh onion to different LED light qualities
The Fv/Fm and Fv'/Fm' of Welsh onion in all light quality treatments except the W treatment were significantly higher than those in the Y treatment, but those in all other combinations of treatments were not significantly different ( Fig. 3A-B, Fig. 4).
The qP was significantly higher in the W treatment than in all of the other treatments. Among the monochromatic light treatments, the qP in the B treatment was the highest, and that in the Y treatment was the lowest (Fig. 3C). The ΦPS II and ETR showed consistent trends among treatments with those in qP, and were both significantly higher in the W treatment than in the monochromatic light treatments, and decreased sequentially in the B, R, G, and Y treatments, respectively ( Fig. 3D-E). The NPQ and qP showed opposite trends, with the NPQ being the highest in the Y treatment, and the lowest in the W treatment (Fig. 3F).
These results indicated that among the monochromatic light treatments, the B treatment could increase the proportion of the reaction centers in PSII opening under light adaptation, enhance PSII reaction center activity, and increase the electron transfer rate, while the Y treatment increased the heat dissipation capacity of Welsh onion plants.

Responses of leaf anatomy and chloroplast ultrastructure in Welsh onion to different LED light qualities
Welsh onion (Allium fistulosum L.) plants have fistular leaves. The fistular lamina of Welsh onion leaves changes from being solid to hollow during development, and the cells around the cavity break up until the remaining 1-2 layers of cells from the palisade show cell wall residues ('arrowheads') [27]. Through the observation of leaf slices after red-green staining, the palisade tissue cells in each layer of the Btreated leaves in this study were found to have the same size and tight arrangement, and the chlorophyll in them was dense, which meant they could use the spaces in the leaf to more efficiently improve the absorption efficiency of light energy, thereby contributing to improved photosynthesis. On the other hand, in the W-, R-, and G-treated Welsh onion leaves, the palisade tissue cells were arranged in a relatively disordered manner, and in the Y treatment their arrangement was loose.
Differences in leaf vascular bundle sizes were found in the following order: W > B > R > G > Y. There were no significant differences in the spongy mesophyll tissue thickness or arrangement among treatments (Fig. 5).
Chloroplasts are the locations where chlorophyll is found, and photosynthesis occurs in plant cells. If chlorophyll synthesis is reduced or blocked, this will result in a change in chloroplast structure [28]. The different light quality treatments greatly affected the development of chloroplasts in the leaves of Welsh onion in this study.
The size and shape of the chloroplasts in the mesophyll cells of Welsh onion were observed by transmission electron microscopy after sampling them on the 30th day of treatment with different LED light qualities. The normal shape of the chloroplast is fusiform or elliptical, and they are arranged along the plasma membrane in Welsh onion cells. In normal chloroplasts, the structure of the granular sheets (grana lamellae) in the chloroplasts is clearly visible and runs parallel to the long axis of the chloroplast, and the thylakoids are closely packed and arranged in a neat and orderly manner; such normal chloroplasts were observed in the W and B treatments herein ( Fig. 6W1-B3). In addition, the thylakoid membranes in B-treated plants' leaf cells grew the most, which means that the B-treated Welsh onion plants had a greater light-capturing ability, and thus an improved energy conversion efficiency of the photosynthetic membrane [29]. However, the chloroplasts of Y-treated Welsh onion leaves became smaller, and the granular lamellae of the thylakoids in the chloroplasts degraded, resulting in them having decreased photosynthetic capacity ( Fig. 6Y1-Y3). Our results indicated that the chloroplasts of cells in leaves treated with B and R light were intact and still contributed well to photosynthesis, while the development of chloroplasts was inhibited in the Y treatment.

Response of RuBPCase activity in Welsh onion to different LED light qualities
RuBPCase is a key enzyme for photosynthesis. Different light qualities had shading actually occurs [33].
In this study, there were significant differences in the growth and development of Welsh onion plants among LED light treatments with different wavelengths (Table   1). We found that the whole-spectrum W-treated Welsh onion plants grew the best, which is consistent with the conclusions of previous studies done on cucumber and Cyclocarya paliurus [15,34]. Among the monochromatic light treatments tested, the plants grown in the B treatment were more compact, whereas those grown in the G and Y treatments were not as compact (Fig. 1B). This may have been due to the different responses of the leaves to different optical signals, which would be consistent with the results of previous studies done on rice and lettuce [35,36].
However, different results were reported for studies done on Camptotheca acuminata Decne. seedlings, wheat, and other crops [6,37], which may have been due to differences in the responses of different species to variation in light quality.
In a previous study [38] of the effects of combined red-blue light on cucumber seedlings, the growth rate and leaf area were reduced as the proportion of blue light in the environment decreased. Plant growth rate was the lowest in that study in the 0% B:100% R treatment, and the fastest in the 100% B:0% R treatment, which may have been related to a variety of morphological and physiological factors [38].
Previous studies have shown that photosynthetic pigments can absorb and transmit light energy, which makes these pigments the material basis for photosynthesis in plants. Light quality affects the synthesis of photosynthetic pigments, which in turn affects the photosynthesis of plants, and thus plays an important role in regulating plant growth and development [39,40]. The pigments found in plant leaves are closely related to their physiological functions. It was previously found that a reduction in CO 2 assimilation occurred of rose under blue light, which was related to the decreased photosynthetic pigment content of the tested plants [41]. Chlorophyll absorbs light energy and transfers it to the chloroplasts for photosynthesis [42]. In our study, chlorophyll content and its ratio were significantly affected by different light qualities ( Table 2). The decrease in total chlorophyll content under monochromatic light indicates that monochromatic light causes damage to photosynthetic pigments. However, compared to R light, B light treatment in this study resulted in a significant increase in leaf chlorophyll content, which was inconsistent with the reports in lettuce and Anoectochilus roxburghii [43,44]. In this study, the P n of Welsh onion seedlings grown under monochromatic light decreased significantly compared to that of seedlings grown under W light, especially under Y and G light ( Fig. 2A). This is consistent with the results of previous studies of cucumber and Acacia mangium seedlings [45,46]. In the present study, the Pn of Welsh onion was significantly associated with its chlorophyll content and RuBisCO content. The transpiration rate (E) and stomatal conductance (G s ) were the highest under B light (Fig. 2B-C), probably because the stomata of the seedlings exposed to blue light developed well, meaning that the stomatal conductance and nitrogen accumulation were high in them compared with those in seedlings raised under other monochromatic light treatments [47].
RuBisCO, acting as RuBPCase, is known to be a key enzyme in the Calvin cycle, the nature of which determines the photosynthetic efficiency and ultimate productivity of photosynthetic organisms. Previous studies have shown that light quality affects RuBPCase activity in algae [48,49]. In our experiments, the higher rate of photosynthesis observed in the B treatment may have been related to the chlorophyll content (Table 2), light energy conversion efficiency (Fig. 3B), and RuBPCase activity of plants in this treatment ( Fig. 7), which were all high and thus would have promoted good Welsh onion growth.
The measurement of the chlorophyll fluorescence of green plants reflects their photosynthetic potential in a complex manner [50]. Green plants absorb light, some of which is used for photosynthesis, while some re-emerges in the form of chlorophyll (Chl) fluorescence, and some is used for heat dissipation [51]. The in the form of superheat [52]. In addition, ΦPSII is often used to indicate the quantum yield of electron transfer in plant photosynthesis, reflecting the actual primary light energy capture rate when the reaction centers are partially closed. In this study, among the monochromatic light treatments, the Fv/Fm, ETR, qP, and ΦPSII were the highest in B-treated Welsh onion plants, while the NPQ was decreased in these (Fig. 3). Therefore, we propose that treatment with B light increased the rate of photosynthesis in Welsh onion, but reduced the heat dissipation ability of PSII in this plant. It was thus shown in this study that B light is beneficial to Welsh onion because it improves the light energy conversion efficiency of this plant and allows it to accumulate more energy for carbon assimilation in the dark reactions (Fig. 3A-B). In a similar study, Phalaenopsis had lower Fv/Fm values in a 0% B:100% R light treatment compared to that in treatments containing more blue light [53].
In the present study, the anatomical structure of the leaves of Welsh onion was significantly changed by treatments with different LED light qualities. In the W treatment, loosely arranged palisade mesophyll cells were observed, and the spaces among the palisade cells were the largest observed (Fig. 5W), which was similar to the findings of a previous study of potato (Solanum tuberosum L.) leaves [54]. Other monochromatic light did not show significant differences in leaf thickness, while on cucumber leaves, red light severely reduced the thickness of the leaf fence and sponge tissue, resulting in thinning of the leaves [55]. Saebø [56]showed that when the in vitro culture of Betula pendula Roth was carried out under light of different quality, the areas of epidermal cells were the largest under blue light irradiation, and the smallest under red light. These comparisons confirm that the response of plant morphological characteristics to changes in light quality is species-specific.
Chloroplasts are the major photosynthesis organelles, which are rich in thylakoid membranes that carry light absorption, transport and transformation in photosynthesis [57]. The light quality greatly influenced the ultrastructure of chloroplast and thylakoid membrane [58,59]. In this study, on the 30 th day of treatment, the number of chloroplasts per cell was the highest in the plants grown under B light, and the number of grana lamellae in each chloroplast was the highest (Fig. 6W1-B3), which was consistent with the results of previous studies done on cucumber leaves [60]. Li [61] also reported that upland cotton (Gossypium hirsutum significantly. Grana has a large diameter and irregular shape, and many prominent thylakoids. In some areas, the thylakoids are disordered [63]. The structure of chloroplasts in leaves of R-treated Welsh onion is not the same. On the contrary, the chloroplasts of R-treated Welsh onion leaves are relatively intact, which may be related to different species.

Conclusions
The growth and development of plants is strongly influenced by the spectrum of light in their growing environment. In this study, the growth, photosynthetic characteristics, and chlorophyll fluorescence characteristics of Welsh onion grown under LED light source of different quality were studied. The purpose of this study was to reveal the effects of light quality on the photosynthesis of Welsh onion, as well as to provide a theoretical basis for the regulation of light environments used in the growth of Welsh onion.
As expected, the growth and morphology of Welsh onion plants were altered by growing them under different light spectra, and different light quality treatments had significant effects on photosynthesis-related processes. In this study, the fullspectrum W treatment was the most beneficial for the growth of Welsh onion.
Among the monochromatic light treatments, the chlorophyll content, Chl a/Chl b ratio, net photosynthetic rate, stomatal conductance, and transpiration rate were the highest in the B treatment, indicating that B light is beneficial to the photosynthesis of Welsh onion. At the same time, it can be seen from the leaf structural changes observed that R light may play an important role in chloroplast development and delaying leaf senescence. However, the Y treatment induced NPQ, affecting plant morphology, destroying leaf tissue and thylakoid membrane structure, reducing the photosynthetic pigment content, and significantly reducing the net rate of photosynthesis. In summary, different monochromatic light qualities were found to each play unique roles in the growth and photosynthesis of Welsh onion.

Materials and treatments
The experiment was carried out in the light quality culture room of the College of The Welsh onion variety tested was 'Yuanzang', which was seeded in 50-hole trays.
Seedlings were watered with 1/2 Hoagland nutrient solution every 3 days after sowing. When the seedling height was about 5 cm, they were thinned so that there

Data analysis
Sampling of plants followed the principle of random sampling in this study. The test data were processed, plotted, and statistically analyzed using Excel 2016 and DPS software, and tested for significant differences (P ≤ 0.05) among treatments using Responses of the photosynthetic parameters of Welsh onion to different LED light qualities, i Chlorophyll fluorescence imaging analysis of Welsh onion leaves under different LED light qu Responses of the leaf anatomy of Welsh onion to different LED light qualities. The fistular lam Figure 6 Responses of chloroplast ultrastructure in Welsh onion leaf cells to different LED light qualiti Responses of the RuBPCase activity in Welsh onion leaves to different LED light qualities. Th

Supplementary Files
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