plants

Article
Effects of Light Intensity on Growth and Quality of Lettuce and
Spinach Cultivars in a Plant Factory
Chen Miao 1 , Shaojun Yang 2 , Jing Xu 3 , Hong Wang 1 , Yongxue Zhang 1 , Jiawei Cui 1 , Hongmei Zhang 1 ,
Haijun Jin 1 , Panling Lu 1 , Lizhong He 1 , Jizhu Yu 1 , Qiang Zhou 1 and Xiaotao Ding 1, *

1 Shanghai Key Laboratory of Protected Horticultural Technology, Horticulture Research Institute, Shanghai
Academy of Agricultural Sciences, Jinqi Road No. 1000, Fengxian District, Shanghai 201403, China;
[email protected] (C.M.); [email protected] (H.W.); [email protected] (Y.Z.);
[email protected] (J.C.); [email protected] (H.Z.); [email protected] (H.J.);
[email protected] (P.L.); [email protected] (L.H.); [email protected] (J.Y.); [email protected] (Q.Z.)
2 Shanghai Youyou Agricultural Technology Co., Ltd., Yuanqu South Road No. 1000, Chongming District,
Shanghai 202150, China; [email protected]
3 Department of Horticulture, Shanghai Institute of Technology, Haiquan Road No. 100, Fengxian District,
Shanghai 201418, China; [email protected]
* Correspondence: [email protected]

Abstract: The decreased quality of leafy vegetables and tipburn caused by inappropriate light
intensity are serious problems faced in plant factories, greatly reducing the economic benefits. The
purpose of this study was to comprehensively understand the impact of light intensity on the growth
and quality of different crops and to develop precise lighting schemes for specific cultivars. Two
lettuce (Lactuca sativa L.) cultivars—Crunchy and Deangelia—and one spinach (Spinacia oleracea L.)
cultivar—Shawen—were grown in a plant factory using a light-emitting diode (LED) under intensities
of 300, 240, 180, and 120 µmol m−2 s−1 , respectively. Cultivation in a solar greenhouse using only
natural light (NL) served as the control. The plant height, number of leaves, and leaf width exhibited
the highest values under a light intensity of 300 µmol m−2 s−1 for Crunchy. The plant width and leaf
length of Deangelia exhibited the smallest values under a light intensity of 300 µmol m−2 s−1 . The
Citation: Miao, C.; Yang, S.; Xu, J.; fresh weight of shoot and root, soluble sugar, soluble protein, and ascorbic acid contents in the three
Wang, H.; Zhang, Y.; Cui, J.; Zhang, cultivars increased with the increasing light intensity. However, tipburn was observed in Crunchy
H.; Jin, H.; Lu, P.; He, L.; et al. Effects under 300 µmol m−2 s−1 light intensity, and in Deangelia under both 300 and 240 µmol m−2 s−1
of Light Intensity on Growth and
light intensities. Shawen spinach exhibited leaf curling under all four light intensities. The light
Quality of Lettuce and Spinach
intensities of 240 and 180 µmol m−2 s−1 were observed to be the most optimum for Crunchy and
Cultivars in a Plant Factory. Plants
Deangelia (semi-heading lettuce variety), respectively, which would exhibit relative balance growth
2023, 12, 3337. https://doi.org/
and morphogenesis. The lack of healthy leaves in Shawen spinach under all light intensities indicated
10.3390/plants12183337
the need to comprehensively optimize cultivation for Shawen in plant factories to achieve successful
Academic Editors: Hail Rihan, cultivation. The results indicated that light intensity is an important factor and should be optimized
Mick Fuller and Gianluca Caruso
for specific crop species and cultivars to achieve healthy growth in plant factories.
Received: 17 July 2023
Revised: 8 September 2023 Keywords: indoor cultivation; LED light intensity; plant development; photosynthesis; nutrient
Accepted: 14 September 2023 content; tipburn
Published: 21 September 2023

1. Introduction
Copyright: © 2023 by the authors.
A plant factory is an environment-controlled plant production facility [1,2]. Based
Licensee MDPI, Basel, Switzerland.
This article is an open access article
on the lighting systems, plant factories can be divided into three main types: (A) relying
distributed under the terms and
exclusively on artificial light (AL), (B) relying exclusively on natural light (NL), and (C)
conditions of the Creative Commons relying on a combination of NL and AL [3]. Type A involves a nearly fully enclosed
Attribution (CC BY) license (https:// building that uses various sensors and control software to precisely manage the growth
creativecommons.org/licenses/by/ environment of plants. This is commonly used for the commercial production of leafy
4.0/). greens and fruiting vegetables such as lettuce and tomatoes. Types B and C are advanced

Plants 2023, 12, 3337. https://doi.org/10.3390/plants12183337 https://www.mdpi.com/journal/plants

Plants 2023, 12, 3337 2 of 18

greenhouses that are not fully enclosed systems. The main difference between them is that
in the case of a lack of sunlight, such as during the rainy season or winter, type C uses
artificial lighting to prevent growth delays, yield reductions, and quality compromises [3].
Leafy vegetables are considered the most suitable crops for cultivation in plant factories
due to their high yield and quality characteristics [4]. The AL source commonly used in
plant factories is a light-emitting diode (LED), which has many advantages, including low
energy consumption, long lifespan, and narrow spectral range [5].
Light plays a crucial role in the growth and development of plants, providing both
energy and signal [6,7]. Light quality, intensity, and photoperiod are three important factors
that affect plant growth [8]. Light intensity is an important factor that affects crop growth
and quality [9]. It affects the morphology, photosynthetic characteristics, nutrient content,
and antioxidant capacity of crops [10–12], which ultimately affect yield and quality. In
addition, different species and cultivars respond differently to the same light intensity [13].
That is, for specific species or cultivars, precise lighting schemes are required to effectively
improve plant yield and quality. Furthermore, such precise control of light intensity can
reduce electricity consumption.
Light intensity influences the content of soluble sugar, soluble protein, and ascorbic
acid. One crucial aspect of vegetable quality is taste, which is largely determined by the
contents of soluble sugars and soluble protein [14–16]. These substances are responsible for
providing characteristic flavor and texture to vegetables, and their accumulation within
plants is highly dependent on light intensity [17]. A previous study demonstrated that light
intensity plays a significant role in regulating the contents of glucose, fructose, ascorbic
acid, and soluble sugar [18–20]. Moreover, exposure to appropriate light intensity during
storage after harvesting is reported to be beneficial for the preservation of nutrients [21].
Given the importance of vegetable quality, suitable light intensity is a critical factor in
promoting the accumulation of key nutrients and enhancing overall plant quality.
Inappropriate light intensity can create adverse conditions for plant growth. Photosyn-
thesis is driven by the consumption of light energy. It is widely recognized that as the light
intensity increases, the net photosynthetic rate (Pn) increases [22]. However, inappropriate
light intensity may limit the carbon assimilation process and reduce Pn [23]. Therefore,
plants do not use all the absorbed light for photosynthesis. When the absorbed light energy
exceeds the photosynthetic capacity, plants release some of the energy in the form of heat.
Moreover, excessive light intensity may damage plants [24,25], which is reflected by certain
indicators such as tipburn. In addition, the photosynthetic capacity of plants is related to
their variety-specific characteristics imparted by the differences in plant architecture, the
leaf pigment pool, and stomata traits of different cultivars [25,26]. For example, the Pn of
green lettuce is usually higher than that of red lettuce [27].
Different crops and cultivars have varying abilities to adapt to light environments.
When fluorescent lamps were used as the light source, increasing the light intensity from
270 to 570 µmol m−2 s−1 significantly increased the fresh weight, dry weight, leaf thickness,
and other indicators of Lollo Rosso lettuce. However, such effects were not observed when
LED lights were used as the light source [28]. The adaptability of green and red lettuce to
blue light differs among varieties. For example, the rosette fresh weight of red lettuce is
19–25% higher than that of green lettuce under blue light treatment [27].
Currently, one of the major challenges in the production of leafy vegetables in plant
factories is setting up appropriate light intensities for different crops or varieties. This is
because most studies focus on the effects of different light intensities on the same crop, and
research on the response and performance of different crops and varieties to various light
intensities is limited. Additionally, the response of different plant architectures to light
environments needs to be studied. Therefore, we have set up four light-intensity gradients
and selected one spinach (Spinacia oleracea L.) cultivar and two lettuce (Lactuca sativa L.)
cultivars with different morphological characteristics to validate our hypothesis that higher
light intensity promotes faster growth and a higher quality of lettuce and spinach, leading
to increased yields. However, when light intensity exceeds a certain threshold, plants may
Plants 2023, 12, 3337 3 of 18

exhibit stress responses. In this study, we comprehensively analyzed the impact of light
intensity on the growth and development of these crops and cultivars, which will help in
optimizing the delivery of high-quality products by designing an efficient lighting scheme.

2. Results
2.1. Plant Height, Plant Width, Number of Leaves, Leaf Length, Leaf Width, and Fresh Weight of
Shoot and Root
Various growth parameters of the three cultivars were assessed under LED light
intensities of 300, 240, 180, and 120 µmol m−2 s−1 (indicated by LI300, LI240, LI180,
and LI120, respectively). Compared with NL (a solar greenhouse with natural light),
cultivation under LED (a closed-type plant factory with LED light) clearly increased the
plant height of Crunchy and Deangelia (Figures 1 and 2). Under LED treatments, the plant
height of the three cultivars exhibited different growth trends over time. For Crunchy,
the maximum and minimum plant height was observed under LI120 and LI240 on the
17th day, respectively, and the plant height increased with increasing light intensity on
the 28th day (Figures 1A and 2A). For Deangelia, the plant height growth rate was higher
under LI300 than under other LED light intensities from the 17th to 26th day. However,
its plant height under the four LED light intensities was almost the same on the 28th day
(Figures 1C and 2B). For Shawen, on the 28th day, the plant height under LI300 and LI120
was similar and higher than those under LI240 and LI180, and the plant height was the
shortest under NL (Figures 1E and 2C). These results indicated that plant heights of different
species and cultivars exhibited different responses to light intensity.

Figure 1. Lettuce cultivars Crunchy (A,B) and Deangelia (C,D) and spinach cultivar Shawen
(E,F) grown in a solar greenhouse with natural light (NL) and a closed-type plant factory with
light intensities of 300, 240, 180, and 120 µmol m−2 s−1 (indicated by LI300, LI240, LI180, and
LI120, respectively).

Figure 2. Plant height of two lettuce cultivars Crunchy (A) and Deangelia (B) and one spinach cultivar
Shawen (C) grown in a solar greenhouse (NL) and a closed-type plant factory with light intensities of
300, 240, 180, and 120 µmol m−2 s−1 (indicated by LI300, LI240, LI180, and LI120, respectively).
Plants 2023, 12, 3337 4 of 18

Different LED light intensities exhibited different effects on the plant width of lettuce
and spinach (Figures 1 and 3). For Crunchy, the plant width rapidly increased under all LED
treatments from the 7th to the 17th day, after which the growth rate slowed down. The plant
width under LED was slightly larger than that under NL, and no considerable difference
existed among different LED light intensities (Figures 1B and 3A). For Deangelia, the plant
width rapidly increased from the 7th to the 17th day under LED and NL, followed by a
slower increase. On the 17th day, a larger plant width was exhibited under NL than under
four LED light intensities. Among the LED light intensities, LI120 and LI300 exhibited the
largest and smallest plant widths on the 28th day, respectively (Figures 1D and 3B). For
Shawen, the plant width rapidly increased from the 7th to the 24th day, after which the
growth rate slowed down; the plant width under LED was clearly higher than that under
NL (Figures 1F and 3C). The rapid growth period of plant width was longer for Shawen
than for both lettuce cultivars, which may be attributed to interspecific differences.

Figure 3. Plant width of two lettuce cultivars Crunchy (A) and Deangelia (B) and one spinach cultivar
Shawen (C) grown in a solar greenhouse (NL) and a closed-type plant factory with light intensities of
300, 240, 180, and 120 µmol m−2 s−1 (indicated by LI300, LI240, LI180, and LI120, respectively).

The number of leaves under LED treatments was higher than that under NL (Figure 4).
Interestingly, both Crunchy and Shawen demonstrated an increase in the number of leaves
with increasing light intensity, indicating a positive correlation between light intensity and
leaf development (Figure 4A,C). However, Deangelia exhibited some anomalies under high
light intensity. Specifically, the inner leaves could not expand under LI300 and LI240 on
the 22nd day, as it entered the heading stage (Figure 1D). Therefore, we stopped counting
the number of leaves from the 22nd day. Nevertheless, under LI180 and LI120, Deangelia
exhibited normal plant morphology. The number of leaves was higher under LI180 than
under LI120 (Figure 4B). Importantly, the leaves of Crunchy exhibited tipburn under LI300
(Figure 5A), whereas Deangelia exhibited tipburn and leaf shrinkage under LI300 and LI240
(Figure 5B,C).
For both lettuce cultivars, leaf length decreased (Figure 6A,C) but leaf width increased
with increasing light intensity (Figure 6B,D). However, for Shawen, both leaf length and
width were positively correlated with light intensity (Figure 6E,F). Additionally, under
LED treatments, leaf curling occurred in Shawen on the 19th day (Figures 1E and 5D); this
led to a decrease in leaf width, which was smaller than that under NL (Figure 6F).
Plants 2023, 12, 3337 5 of 18

Figure 4. Number of leaves of two lettuce cultivars Crunchy (A) and Deangelia (B) and one spinach cul-
tivar Shawen (C) grown in a solar greenhouse (NL) and a closed-type plant factory with light intensities
of 300, 240, 180, and 120 µmol m−2 s−1 (indicated by LI300, LI240, LI180, and LI120, respectively).

Figure 5. Tipburn in Crunchy under light intensity of 300 µmol m−2 s−1 (A) and in Deangelia under
light intensities of 300 and (B) and 240 (C) µmol m−2 s−1 . Red arrow points to the exact location of
the tipburn. Leaf curling in Shawen (D).
Plants 2023, 12, 3337 6 of 18

Figure 6. Leaf length and leaf width of two lettuce cultivars Crunchy (A,B) and Deangelia (C,D) and
one spinach cultivar Shawen (E,F) grown in a solar greenhouse (NL) and a closed-type plant factory
with light intensities of 300, 240, 180, and 120 µmol m−2 s−1 (indicated by LI300, LI240, LI180, and
LI120, respectively).

The fresh weight of shoot and root increased with the increasing LED light intensity
(Figure 7). The fresh weight of Crunchy was significantly different under four LED light
intensities (Figure 7A,B). The maximum fresh weight of the shoot and root was observed at
LI300, exhibiting a 306.9% and 77.8% increase compared with NL, respectively. However,
no significant differences were observed between LI180 and LI120 in terms of the fresh
weight of the shoots of Deangelia (Figure 7C) and Shawen (Figure 7E); the fresh weight of
the shoots was significantly higher under LI300 in Deangelia and Shawen, exhibiting an
increase of 174.3% (Figure 7C) and 54.2% (Figure 7E) compared with NL, respectively. For
the two lettuce cultivars, the fresh weight of the shoots under LED was significantly higher
than under NL (Figure 7A,C). The fresh weight of the shoot of Shawen was significantly
higher than NL only under LI300 and LI240 (Figure 7E). The effect of different LED light
intensities on the fresh weight of the root was comparable with the variation observed in
the fresh weight of the shoot.
Plants 2023, 12, 3337 7 of 18

Figure 7. Fresh weight of shoot and root of two lettuce cultivars Crunchy (A,B) and Deangelia
(C,D) and one spinach cultivar Shawen (E,F) grown in a solar greenhouse (NL) and a closed-type
plant factory with light intensities of 300, 240, 180, and 120 µmol m−2 s−1 (indicated by LI300, LI240,
LI180, and LI120, respectively). Bars with different letters within each panel indicate significant
differences at p < 0.05 according to Duncan’s test.

2.2. Leaf Photosynthetic Net Rate and Chlorophyll Fluorescence

For lettuce cultivars, leaf Pn increased with increasing light intensity (Table 1). For
Deangelia, the Pn, intercellular CO2 concentration (Ci), stomatal conductivity (Gs), and
transpiration (Tr) were significantly high under LED cultivation compared with NL. For
Crunchy and Shawen, only when light intensity was higher than LI120, Pn was significantly
higher than that under NL. The maximum Pn was observed at LI300 for lettuce cultivars.
For Shawen, no significant difference was observed in Pn under LI300, LI240 and LI180.
Notably, the water use efficiency (WUE) of all three cultivars was significantly higher under
NL than under LED treatments.

Table 1. Photosynthetic parameters of two lettuce cultivars Crunchy and Deangelia and one spinach
cultivar Shawen grown in a solar greenhouse (NL) and a closed-type plant factory with light intensi-
ties of 300, 240, 180, and 120 µmol m−2 s−1 (indicated by LI300, LI240, LI180, and LI120, respectively).

Pn (µmol Ci (µmol Gs (mmol Tr (mmol WUE (mmol

Treatments
CO2 ·m−2 ·s−1 ) CO2 ·mol−1 ) H2 O·m−2 ·s−1 ) H2 O·m−2 ·s−1 ) CO2 ·mol−1 ·H2 O)
Crunchy NL 7.8 ± 0.9 d 144.7 ± 6.7 b 47.0 ± 4.0 e 0.9 ± 0.1 c 8.7 ± 0.2 a
LI300 21.3 ± 0.4 a 356.7 ± 2.5 a 436.3 ± 20.1 a 3.6 ± 0.1 a 5.9 ± 0 b
LI240 18.4 ± 0.4 b 363.3 ± 4.2 a 353.3 ± 24.5 b 3.3 ± 0.1 a 5.6 ± 0.1 b
LI180 11.5 ± 0.1 c 340 ± 14.1 a 144.3 ± 18.2 c 2.0 ± 0.2 b 5.9 ± 0.5 b
LI120 7.8 ± 1.0 d 345 ± 33.8 a 99.0 ± 37.3 d 1.5 ± 0.6 b 5.4 ± 1.3 b
Plants 2023, 12, 3337 8 of 18

Table 1. Cont.

Pn (µmol Ci (µmol Gs (mmol Tr (mmol WUE (mmol

Treatments
CO2 ·m−2 ·s−1 ) CO2 ·mol−1 ) H2 O·m−2 ·s−1 ) H2 O·m−2 ·s−1 ) CO2 ·mol−1 ·H2 O)
Deangelia NL 7.3 ± 1.5 d 167.0 ± 3.0 c 47.0 ± 8.9 d 0.8 ± 0.1 c 9.1 ± 0.2 a
LI300 23.0 ± 2.6 a 342.7 ± 19.9 b 395.3 ± 18.6 a 3.8 ± 0.6 b 6.1 ± 0.6 b
LI240 20.5 ± 0.6 ab 338.3 ± 47.4 b 375.0 ± 9.8 ab 4.0 ± 1.1 b 5.3 ± 1.4 bc
LI180 18.6 ± 0.6 b 407.0 ± 1.0 a 350.0 ± 29.8 b 5.3 ± 0.2 a 3.5 ± 0 d
LI120 12.9 ± 1.2 c 376.0 ± 2.6 ab 301.3 ± 29.8 c 3.0 ± 0.1 b 4.3 ± 0.2 cd
Shawen NL 10.0 ± 1.0 b 124.7 ± 4.0 c 50.0 ± 11.5 d 0.79 ± 0.2 c 11.5 ± 0.2 a
LI300 13.6 ± 1.1 a 420.3 ± 9.5 ab 387.3 ± 21.5 a 5.3 ± 0.1 a 2.6 ± 0.2 c
LI240 14.7 ± 1.9 a 416.3 ± 7.0 ab 345.3 ± 25.5 ab 5.4 ± 0.1 a 2.7 ± 0.3 c
LI180 16.0 ± 0.7 a 441.7 ± 7.1 a 331.7 ± 19.8 b 5.5 ± 0.2 a 2.9 ± 0.1 bc
LI120 10.1 ± 1.3 b 394.3 ± 34.1 b 243.3 ± 34.6 c 2.8 ± 0.7 b 3.8 ± 1.2 b
Different letters indicate significant differences (Duncan’s test at p < 0.05).

Fv/Fm values are widely used to study plant stress response, photosynthetic damage,
and ecosystem productivity. The photosynthetic performance index (PI) is commonly used
to study the response of plants to growth conditions in the environment, as it can indicate
the health status of plant leaves and their photosynthetic efficiency. Under LED treatments,
Crunchy and Deangelia exhibited no difference in Fv/Fm over time (Figure 8A,C). For
both Crunchy and Deangelia, the PI value exhibited an upward trend over time, exhibiting
the order of LI300 > LI240 > LI180 > LI120 on the 28th day (Figure 8B,D). In contrast,
both the Fv/Fm and PI values of Shawen decreased over time under LI300 and LI240,
whereas relatively stable trends or slow increases were displayed under LI180 and LI120
(Figure 8E,F).

Figure 8. Fv/Fm and PI of two lettuce cultivars Crunchy (A,B) and Deangelia (C,D) and spinach cultivar
Shawen (E,F) grown in a solar greenhouse (NL) and a closed-type plant factory with light intensities of
300, 240, 180, and 120 µmol m−2 s−1 (indicated by LI300, LI240, LI180, and LI120, respectively).
Plants 2023, 12, 3337 9 of 18

2.3. Soluble Sugar, Soluble Protein, and Ascorbic Acid Contents

The soluble sugar content positively correlated with light intensity for all three cul-
tivars (Figure 9). Under LED treatments, all three cultivars exhibited the highest and
lowest soluble sugar contents at LI300 and LI120, respectively, with significant differences
between the two levels (Figure 9A–C). Compared with NL, both lettuce cultivars exhibited
significantly higher soluble sugar content under all LED treatments (Figure 9A,B); however,
Shawen exhibited higher soluble sugar content only under LI300, with a significantly lower
content observed at LI120 (Figure 9C).

Figure 9. Soluble sugar content of two lettuce cultivars Crunchy (A) and Deangelia (B) and one
spinach cultivar Shawen (C) grown in a solar greenhouse (NL) and a closed-type plant factory with
light intensities of 300, 240, 180, and 120 µmol m−2 s−1 (indicated by LI300, LI240, LI180, and LI120,
respectively). Bars with different letters within each panel indicate significant differences at p < 0.05
according to Duncan’s test.

Figure 10 indicates the varietal specificity in terms of soluble protein content at differ-
ent light intensities. Among all LED treatments, the soluble protein content was similar
under LI300, LI240, and LI180 for Crunchy, and it was significantly higher than that under
LI120 (Figure 10A). For Deangelia and Shawen, soluble protein content increased with
increasing light intensity, with significantly higher content observed at LI300 than at LI180
and LI120 (Figure 10B,C). Compared with NL, all three cultivars exhibited significantly
higher soluble protein content under LED treatments (Figure 10A–C).
Ascorbic acid contents varied at different LED light intensities in the three cultivars
(Figure 11). Crunchy exhibited significantly higher ascorbic acid content at LI300 than
at LI240, LI180, and LI120, with increases of 157.9%, 191.8%, and 197.9%, respectively
(Figure 11A). Deangelia exhibited significantly higher ascorbic acid content at LI300 among
all LED treatments, whereas no significant difference was observed between LI240 and
LI180 in terms of ascorbic acid content (Figure 11B). The pattern of variation in ascorbic
acid content was similar in Shawen and Deangelia, with significantly higher content under
LI240 and LI180 than under LI120 (Figure 11C). Compared with NL, both lettuce cultivars
exhibited significantly higher ascorbic acid content under all LED treatments (Figure 11A,B);
however, Shawen exhibited significantly higher ascorbic acid content under light intensities
higher than LI180 (Figure 11C).
Plants 2023, 12, 3337 10 of 18

Figure 10. Soluble protein content of two lettuce cultivars Crunchy (A) and Deangelia (B) and one
spinach cultivar Shawen (C) grown in a solar greenhouse (NL) and a closed-type plant factory with
light intensities of 300, 240, 180, and 120 µmol m−2 s−1 (indicated by LI300, LI240, LI180, and LI120,
respectively). Bars with different letters within each panel indicate significant differences at p < 0.05
according to Duncan’s test.

Figure 11. Ascorbic acid content of two lettuce cultivars Crunchy (A) and Deangelia (B) and one
spinach cultivar Shawen (C) grown in a solar greenhouse (NL) and a closed-type plant factory with
light intensities of 300, 240, 180, and 120 µmol m−2 s−1 (indicated by LI300, LI240, LI180, and LI120,
respectively). Bars with different letters within each panel indicate significant differences at p < 0.05
according to Duncan’s test.

3. Discussion
The parameters of light have complex effects on photosynthesis and the quality and
yield of plants [25,29]. This emphasizes the importance of optimizing lighting in plant
cultivation. This is particularly important for plants grown under AL, such as lettuce,
where the regulation of light parameters is crucial. In this study, increasing the light
Plants 2023, 12, 3337 11 of 18

intensity positively affected the plant height and leaf numbers in two lettuce cultivars and
one spinach cultivar (Figures 2 and 4), which is consistent with previous studies [30,31].
Interestingly, the plant width of Crunchy was not affected by varying light intensities,
whereas that of Deangelia decreased as light intensity increased (Figure 3A). However, the
plant width of Shawen positively correlated with light intensity (Figure 3C). These results
highlighted the influence of species-specific traits, such as morphological and physiological
characteristics (e.g., plant structure and stomatal features) [27,32], making certain cultivars
more suitable or less suitable for specific environments. Deangelia is a semi-heading lettuce
variety, and the leaves of this cultivar exhibited leaf shrinkage under LI300 and LI240
(Figure 5), which resulted in a decrease in plant width (Figure 3B). This symptom may be
due to excessive light intensity, as well as species-specific traits.
In addition, tipburn was observed in Crunchy and Deangelia (Figure 5). Tipburn not
only reduces the quality and yield of the products but also decreases the marketable rate.
Tipburn is a serious problem in the indoor cultivation of leafy vegetables [33]. Previous
studies reported that the main reason for tipburn is calcium deficiency at the leaf tips, caused
by rapid growth under high light intensity [34–36]. Plants exhibit higher photosynthetic
rates under higher light intensities (Table 1), resulting in faster growth. However, the
rapidly growing inner leaves were unable to absorb sufficient calcium and translocate it
to the leaf tips, which led to tipburn [35]. Furthermore, during the rapid growth period
of plants, more calcium is transferred to the outer leaves rather than the inner enclosed
leaves [30]. Therefore, tipburn often occurs in the inner leaves. It is possible to alleviate
tipburn by spraying calcium salt solution on the inner leaves [30]. Further comparing
Crunchy and Deangelia, Crunchy exhibited a non-heading trait and Deangelia is a semi-
heading lettuce. Both have completely different plant structures. Therefore, when Deangelia
enters the heading stage, its inner leaves cannot expand fully. This may be the reason why
the incidence of tipburn was higher in Deangelia than in Crunchy.
To address the issue of tipburn, we note that breeding is more important and efficient
than modifying cultivation methods. Ryder and Waycott [37] and SW Jang [38] developed
the tipburn-resistant lettuce varieties Tiber and Sambokhacheong, respectively. However,
breeding tipburn-resistant varieties specifically for plant factories is extremely difficult. As
mentioned earlier, tipburn in lettuce is caused by rapid growth, and plant factories and
farmers want to cultivate lettuce that grows quickly to achieve better economic benefits [39].
This contradiction poses a great challenge for breeding. Plant factories can create a stable
environment throughout the year, which speeds up the breeding process. Moreover, with
in-depth research into the relationship between tipburn and genome [40], Japanese breeders
have successfully bred tipburn-resistant lettuce varieties for plant factories. Increasing the
number of tipburn-resistant lettuce varieties can promote large-scale lettuce production in
plant factories. Our results (Figures 4 and 6) confirmed that lower light intensities led to an
elongated leaf blade and a reduced leaf number in the lettuce [41,42]. In contrast, Shawen
displayed maximum and minimum leaf length and width at LI300 and LI120, respectively
(Figure 6). This is because of the species-specific traits of the response to light intensity.
Leaf structure reflects how environmental factors influence plants or how plants adapt
to the changing environment [43]. In this study, after the 19th day of cultivation under
LED treatments, significant leaf curling was observed in Shawen (Figure 1E,F). This was
considerably different from its morphology under NL. This indicated that the environment
in the plant factory in this experiment was not suitable for the growth of Shawen. In
previous studies, the relative humidity (RH) in indoor cultivation experiments on spinach
fluctuated between 45% and 80% [19,44]. However, in this study, the RH in the plant factory
was 60–90%. Therefore, RH may be an important environmental factor affecting the leaf
curling in Shawen. Therefore, appropriately reducing the RH in the plant factory may be
an effective method to alleviate or eliminate leaf curling in Shawen. However, this should
be further validated in future studies.
To achieve optimal yields and quality of different crop cultivars, it is essential to
develop appropriate lighting schemes that cater to the unique demands of individual
Plants 2023, 12, 3337 12 of 18

plants, thereby enhancing their productivity while reducing energy consumption. Our
results (Figure 7) confirmed that low light intensity reduced the fresh weight of the shoot
and root [42,45,46]. Under low light conditions, leaf chlorophyll content, Gs, and the
activity of photosynthesis-related enzymes decrease [47,48]. Because of this, plants cannot
perform adequate photosynthesis [49,50], which prevents organic matter accumulation
and ultimately reduces both yield and quality. Importantly, in this study, the two lettuce
cultivars displayed a higher fresh weight of shoot and root under LED treatments than
under NL (Figure 7). This could be attributed to the higher emphasis on providing an
optimal light scheme (light intensity and photoperiod) and temperature conditions in plant
factories than in greenhouses during the winter season (Table 2), resulting in better plant
yield and growth.
The magnitude of Pn can be used to assess the suitability of different light intensities
for plants. It is generally acknowledged that low light conditions can inhibit photosynthesis
to a certain extent [51]. Previous studies have reported that light intensity can affect Pn by
altering the levels of enzymes involved in the carbon reduction cycle, electron transport
components, and light-harvesting pigments [52–54]. In this study, high light intensity sig-
nificantly enhanced the Pn, compared with low light intensity in lettuce cultivars (Table 1).
This is consistent with a previous study by Fan [45]. Leaves are regarded as the primary
organ for photosynthesis and transpiration. Under low light intensity, lettuce exhibited
decreased leaf width (Figure 6). This, along with thinner leaf blades [55,56], may ultimately
lead to a reduction in chloroplast number [57] and a subsequent decline in enzyme activity
during the carbon cycle [52]. This may explain the decrease in Pn under low light inten-
sity observed in this study (Table 1). Furthermore, the Pn of the three cultivars grown in
the plant factory was higher or significantly higher than that of the plants grown in the
greenhouse (Table 1). This may be due to a lower temperature [58,59], a shorter photope-
riod [60], and a lower light intensity [22,31] in greenhouses than in plant factories in winter
(January and February). These results further highlighted the superiority and necessity of
the production of plants in plant factories during the winter season.
Agricultural products are dynamic composites of their physicochemical properties
and evolving consumer perception, and they encompass organoleptic, nutritional, and
bioactive components [61]. In all three cultivars, soluble sugar, soluble protein, and ascor-
bic acid contents were higher (significantly higher in Deangelia and Shawen, Shawen,
and all three cultivars, respectively) under LI300 than under other LED light intensities
(Figures 9–11). These results confirmed that plants exhibit higher Pn under higher light
intensity [62], which is beneficial for the accumulation of nutrients. This is consistent with
previous studies [63,64]. In addition, soluble sugar, soluble protein, and ascorbic acid
contents of the three cultivars were higher or significantly higher under LED than under
NL. This is because the light, temperature, and RH can be reasonably regulated under
LED treatments [65], which is more conducive to photosynthesis and more suitable for the
accumulation of nutrients in plants. Importantly, all these results indicated the advantages
of using plant factories over solar greenhouses in terms of plant production in winter.

4. Materials and Methods

4.1. Materials and Treatment Conditions
The experiment was conducted in a fully enclosed plant factory and an advanced solar
greenhouse at Youyou Agricultural Technology Co., Ltd., Chongming District, Shanghai,
China. The experimental materials were two lettuce cultivars (Crunchy and Deangelia)
with different plant architectures and leaf shapes and one spinach cultivar (Shawen). On
23 December 2022, the seeds of the three cultivars were sown in coconut fiber blocks
(10 cm length × 10 cm width × 7 cm height; Van der Knaap, Kwintsheul, the Netherlands)
and germinated in a solar greenhouse until the two-leaf stage. On 3 January 2023, some of
the seedlings were transferred to the fully enclosed plant factory for treatment with different
LED light intensities. The remaining plants continued growing in the solar greenhouse
with ambient average temperature and light intensity (Table 2). The temperature in the
Plants 2023, 12, 3337 13 of 18

plant factory was maintained at 22 ± 2 ◦ C, RH was 60–90%, and CO2 concentration was
approximately 400 µmol mol−1 . During the experiment, all plants were irrigated with
nutrient solution (pH 5.8–6.5 and EC 1.7 mS cm−1 ) every 2–3 days.
A specially designed plant supplement lighting system was used as a light source in
the plant factory (Figure 12A). The system consisted of 4 layers, with 12 LEDs (Shanghai
Sansi Electronic Engineering Co., Ltd., Shanghai, China) installed in each layer. The LED
light tubes had a length of 1214 mm and a diameter of 30 mm. The peak wavelength for red
and blue light was 666 nm and 452 nm, respectively, and the ratio of red (R) to blue (B) was 4
to 1. By integrating an advanced computer-based control system (Figure 12B), light intensity,
light quality, and photoperiod could be precisely regulated. This enabled the control and
manipulation of these parameters, ensuring optimal conditions for experimental purposes.

Figure 12. (A) The supplemental LED lighting system for plants. (B) Computer-based control system
(C) The illumination spectrum used for LED treatments.

The experiment consisted of five treatments, as follows. Treatment 1 involved cul-

tivation in a solar greenhouse using only NL with an average day/night temperature
of 19.8/15.7 ◦ C, average light intensity of 97 µmol m−2 s−1 , and average photoperiod
(day/night) of approximately 10 h/14 h (Table 2). Treatments 2 to 5, respectively, involved
exposure to LED light intensities of 300, 240, 180, and 120 µmol m−2 s−1 (indicated by
LI300, LI240, LI180, and LI120, respectively). The light intensity at the plant canopy was
measured using the Lighting Passport instrument (Model No. ALP-01, Shanghai Hesh-
eng Instrument Technology Co., Ltd., Shanghai, China) in conjunction with the Spectrum
Genius APP (Asensetek Canada Inc., Quebec, Canada). Without intervention, the light
intensity received by the plant changes as the plant grows taller; therefore, the computer-
based LED plant supplementary lighting control system was adjusted to match the desired
experimental light intensity after the measurement of growth parameters. The photoperiod
was set to 16 h. A detailed illustration of the spectra for each LED treatment is presented in
Figure 12C. Each light treatment contained 24 plant replicates in 3 plots, with 8 plants in
each plot.
Plants 2023, 12, 3337 14 of 18

Table 2. Plant growth conditions under LED treatments.

Temperature (◦ C) Light Intensity Photoperiod

Treatment
(Day/Night) (µmol m−2 s−1 ) (Day/Night)
NL 19.8/15.7 97 10 h/14 h
LI300 300 16 h/8 h
LI240 240 16 h/8 h
22 ± 2
LI180 180 16 h/8 h
LI120 120 16 h/8 h

4.2. Sample Collection

All plants were harvested on 3 February 2023. The fresh weight of each plant was
measured immediately after harvest. During sample collection, five individual plants of
each species were randomly selected as replicates for each treatment. Further, five plants
were immediately homogenized using a blender, transferred to test tubes, frozen in liquid
nitrogen, and stored at −40 ◦ C for further analysis.

4.3. Assessment of Various Growth Parameters

4.3.1. Plant Growth
Stem diameter, leaf length, and leaf width were measured using a vernier caliper.
Plant width was measured using a ruler on the 7th, 11th, 17th, 19th, 22nd, 24th, 26th, and
28th days after the start of treatment. Leaf number was measured at the same time. Leaf
length and width were measured using the largest fully expanded leaf, whereas plant
width was measured at the widest point of the plant. The measurements of plant height
were initiated from the 17th day. Plant height was determined from the base of the stem to
the highest point on the plant using a ruler. Five biological replicates were measured for
each treatment.

4.3.2. Leaf Photosynthetic Rate and Chlorophyll Fluorescence

Photosynthetic parameters were determined using a CIRAS-3 portable photosynthesis
system (PP Systems, Amesbury, MA, USA) on the 28th day. Prior to the measurements,
samples were allowed to acclimatize to the measurement conditions for at least 30 min.
Leaves were placed in a 4.5 cm2 conditioning chamber, where a light source provided a
constant photon flux density of 1000 µmol m−2 s−1 , to determine the Pn, Ci, Gs, Tr, and
WUE. The values were recorded once the numerical fluctuations of the parameters were
attenuated or reached a steady state. The photosynthetic parameters were calculated and
presented as mean ± standard deviation (SD). Five biological replicates were measured
for each treatment. The Fv/Fm and PI of leaves were determined using a Pocket Plant
efficiency analyzer (PEA) (Hansatech Instruments Ltd., King’s Lynn, UK) from the 17th
to 28th days. Prior to the measurements, leaves were adapted to the dark with a leaf clip
holder for 30 min to achieve stable fluorescence emission. Five biological replicates were
measured for each treatment.

4.3.3. Soluble Sugar Content

Soluble sugar content was measured according to the method described previously [66].
A homogenized fresh leaf sample (0.5 g) was placed in a glass tube and supplemented
with an appropriate amount of distilled water. The mixture was subsequently boiled for
30 min in a boiling water bath. The supernatant was filtered, and its volume was made up
to 50 mL. The filtrate (0.5 mL) was mixed with 1.5 mL anthrone–ethyl acetate (analytical
reagent (AR); Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) solution and 1.5 mL
distilled water. The mixture was treated with 5 mL sulfuric acid (AR, Sinopharm Chemical
Reagent Co., Ltd., Shanghai, China) and heated for precisely 1 min in a boiling water bath,
which resulted in the formation of a green solution. Once the solution cooled down to
room temperature, the absorbance of the solution was measured at 630 nm using a UV-Vis
Plants 2023, 12, 3337 15 of 18

spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Three biological replicates were

measured for each treatment.

4.3.4. Soluble Protein Content

Soluble protein content was determined using the Coomassie brilliant blue G-250
method [66]. A homogenized fresh leaf sample (0.5 g) was mixed with 1.25 mL distilled
water and centrifuged at 4 ◦ C and 12,000× g for 20 min. The supernatant with the extracted
proteins was collected. The supernatant (0.5 mL) was mixed with 5 mL Coomassie brilliant
blue G-250 (BioReagent; SIGMA-ALDRICH Co., Saint Louis, MO, USA) solution and
incubated for 2 min. Further, its absorbance was measured at 595 nm using a UV-Vis
spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Three biological replicates were
measured for each treatment.

4.3.5. Ascorbic Acid Content

Ascorbic acid content was determined using the acid–base titration method [66]. A
homogenized fresh sample (5 g) was mixed with 20 g/L oxalic acid (guaranteed reagent
(GR); Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) solution until a volume of
50 mL was obtained. An appropriate amount of activated carbon was added to the mixture,
and the mixture was filtered. The filtrate (10 mL) was transferred to a flask and titrated
using a standardized 2,6-dichloroindophenol sodium salt (BioReagent, SIGMA-ALDRICH
Co., Saint Louis, MO, USA) solution. The endpoint was reached when the solution turned
light red and the color persisted for a duration of at least 15 s. Three biological replicates
were measured for each treatment.

4.4. Data Analysis

All treatments were measured at least three times to ensure the accuracy of the data.
Mean values and standard errors were calculated using Microsoft Excel version Mondo
2016 (Microsoft, Redmond, WA, USA). Duncan’s test was performed to determine the
significance of mean differences (p < 0.05) using SPSS software version 22.0 (IBM Corpora-
tion, New York, NY, USA). The figures were drawn using Origin version 2022 (OriginLab
Corporation, Northampton, MA, USA).

5. Conclusions
The photosynthetic rate, leaf width, fresh weight, soluble sugar, soluble protein, and
ascorbic acid contents of both lettuce cultivars increased with the increasing LED light
intensity. However, Crunchy (exhibiting the non-heading trait) developed tipburn when
exposed to the light intensity of 300 µmol m−2 s−1 , whereas tipburn and leaf shrinkage
were observed in Deangelia (semi-heading lettuce cultivar) under both 240 µmol m−2 s−1
and 300 µmol m−2 s−1 . The spinach cultivar, Shawen, exhibited leaf curling under all
LED light intensities, impeding normal growth. These results indicated that although
higher light intensity is beneficial for increasing yield and quality, the light intensity
should be regulated as per the specific variety. Therefore, the optimum light intensities for
Crunchy and Deangelia were 240 and 180 µmol m−2 s−1 , respectively. However, under the
experimental conditions in this study, Shawen spinach was observed to be not suitable for
cultivation in a plant factory, Further studies are required to optimize the light conditions
for the successful cultivation of this variety. Crunchy and Deangelia are two types of
lettuce with different leaf shapes and plant architectures, and non-heading lettuce has
higher adaptability to light intensity than semi-heading lettuce in plant factories. Crunchy
is more suitable for cultivation and production in plant factories. We believe that this study
provides additional insights into the selection of leafy vegetable varieties and lighting
strategies in plant factories for their optimal production.
Plants 2023, 12, 3337 16 of 18

Author Contributions: Conceptualization, X.D. and S.Y.; methodology, C.M. and X.D.; validation,
C.M., H.W. and Y.Z.; formal analysis, P.L. and J.X.; investigation, C.M., J.X., J.C., H.Z. and L.H.;
resources, H.J. and Q.Z.; data curation, C.M. and J.X.; writing—original draft preparation, C.M. and
X.D.; writing—review and editing, X.D.; supervision, J.Y.; project administration, C.M. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by Shanghai Chongming District Ecological Agriculture Science
and Technology Innovation Center (grant no. 2021CNKC-05-02) and the Excellent Team Program of
the Shanghai Academy of Agricultural Sciences (grant no. 2022-022).
Data Availability Statement: The data presented in this study are available upon request from the
corresponding author.
Acknowledgments: The authors would like to thank all the reviewers who participated in the review.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.

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