agronomy

Article
Ultraviolet-to-Blue Light Conversion Film Affects Both Leaf
Photosynthetic Traits and Fruit Bioactive Compound
Accumulation in Fragaria × ananassa
Hafsa El Horri 1 , Maria Vitiello 2 , Costanza Ceccanti 1,3, *, Ermes Lo Piccolo 4, *, Giulia Lauria 1 ,
Marinella De Leo 2,3,5 , Alessandra Braca 2,3,5 , Luca Incrocci 1 , Lucia Guidi 1,3 , Rossano Massai 1,3 ,
Damiano Remorini 1,3 and Marco Landi 1,3

1 Department of Agriculture, Food and Environment, University of Pisa, 56124 Pisa, Italy;
[email protected] (H.E.H.); [email protected] (G.L.); [email protected] (L.I.);
[email protected] (L.G.); [email protected] (R.M.); [email protected] (D.R.);
[email protected] (M.L.)
2 Department of Pharmacy, University of Pisa, 56126 Pisa, Italy; [email protected] (M.V.);
[email protected] (M.D.L.); [email protected] (A.B.)
3 Interdepartmental Research Center Nutrafood “Nutraceuticals and Food for Health”, University of Pisa,
56124 Pisa, Italy
4 Department of Agriculture, Food, Environment and Forestry, University of Florence, 50144 Florence, Italy
5 CISUP, Centre for Instrumentation Sharing, University of Pisa, 56126 Pisa, Italy
* Correspondence: [email protected] (C.C.); [email protected] (E.L.P.)

Abstract: The influence of light downconversion films (red, pink and blue films) on leaf physiological
features and fruit bioactive compound accumulation was studied in strawberry plants. Red, pink and
blue films were able to convert light less utilised by plants into more efficient light wavebands with
various possibilities depending on the film characteristics (blue film shifts UV into blue radiation; red
film shifts green into red radiation, pink film shifts UV and green into blue and red radiation but to a
Citation: El Horri, H.; Vitiello, M.; lesser extent than red film). Indeed, by enhancing the quality of light available for photosynthesis, the
Ceccanti, C.; Lo Piccolo, E.; Lauria, G.; utilization of these films holds the potential to improve agricultural productivity and sustainability.
De Leo, M.; Braca, A.; Incrocci, L.; All of these light downconversion films resulted in higher plant fresh weight compared to a control
Guidi, L.; Massai, R.; et al.
colourless (Cnt) film, with plants grown under blue film (UV-to-blue light conversion) showing the
Ultraviolet-to-Blue Light Conversion
most impressive results in terms of total leaf area (+25%), leaf thickness (+10%) and leaf mass per
Film Affects Both Leaf Photosynthetic
area (+15%). Simultaneously, during the flowering stage, plants under blue film had a higher net
Traits and Fruit Bioactive Compound
photosynthetic rate due to the increase in photosynthetically efficient wavelengths. Moreover, blue
Accumulation in Fragaria × ananassa.
Agronomy 2024, 14, 1491. https://
film resulted in the highest total phenolic (+40% and +28% than red and pink films, respectively)
doi.org/10.3390/agronomy14071491 and flavonoid content (+54%, +84%, +70% than Cnt, red and pink films, respectively) in fruit, with
specific effects on targeted phenols, i.e., quercetin, ellagic acid and its glycoside, ellagitannins, and
Academic Editors: Mengyao Li and
procyanidins. In conclusion, the use of the UV-to-blue conversion light film tested herein represents
Ya Luo
an innovative solution to increase strawberry yield and promote fruit nutraceutical features, playing
Received: 28 May 2024 a pivotal role in ensuring food quality and security and sustainable agricultural practices.
Revised: 2 July 2024
Accepted: 5 July 2024 Keywords: light downconversion film; light spectrum; phenolic compound; photosynthesis; shifting
Published: 9 July 2024 light film; strawberry

Copyright: © 2024 by the authors.

1. Introduction
Licensee MDPI, Basel, Switzerland.
This article is an open access article Balancing crop productivity with market demands is a daily challenge for most farm-
distributed under the terms and ers. Simultaneously implementing sustainable practices and reducing energy consumption
conditions of the Creative Commons adds an extra layer of complexity. The production system is constantly evolving to align
Attribution (CC BY) license (https:// with the market in response to the high demand for fresh fruit. This evolution is facil-
creativecommons.org/licenses/by/ itated by integrating new technologies to enhance fruit quality and yield [1–3]. In this
4.0/). context, as light is one of the main drivers that affect plant physiology, researchers have

Agronomy 2024, 14, 1491. https://doi.org/10.3390/agronomy14071491 https://www.mdpi.com/journal/agronomy

Agronomy 2024, 14, 1491 2 of 20

continuously attempted to increase available light as well as to manipulate the spectral

composition of light to possibly use light as an “eustress” able to modulate the accumula-
tion of targeted bioactive compounds in fruit [4–6]. Notably, it has been observed that red
and blue lights have pronounced effects on leaf photosynthetic traits when administered
as monochromatic light (actually as a narrow-band spectrum) or as supplemental light
in greenhouses, as reviewed by Landi et al. [4]. For instance, supplementing red light
during daylight hours has been shown to enhance plant productivity in strawberries [6,7].
Red light supplementation can also influence the secondary metabolisms, leading, for
example, to increased starch and sucrose levels as observed in tomatoes and higher levels
of anthocyanins as detected in strawberry fruits [6,8–10]. It has long been known that
supplementation with blue light commonly leads to an increase in secondary metabolite
concentrations in a range of plant species, including green and red sweet basil, red lettuce,
tomatoes, and strawberries [6,11,12]. However, it should be noted that high intensities of
blue light supplementation (e.g., 250 µmol m−2 s−1 ) could be detrimental to plant and fruit
productivity, leading to a decline in physiological performance [6,13].
In horticulture, covering materials are highly used due to their versatility. The most
used covering technologies enable shade adjustment by modulating light intensity or
selecting specific wavebands. These techniques optimize the light environment to promote
crop growth and development [14–16]. Nowadays, researchers are testing a new technology
for covering films that are able to manipulate the spectral distribution of transmitted solar
radiation, known as light downconversion films [17–20]. Light downconversion films, also
referred as to fluorescent or light conversion films, can convert light radiation less usable
by chlorophyll, such as UV (280–380 nm) and green radiation (510–575 nm), into light
radiation that is more efficient to kick off photosynthesis, such as blue and red light [20].
Furthermore, the specific effects of the conversion films depend on the composition and
structure of the fluorescent dye blends within the plastic polymer matrix [18].
The alteration by light downconversion films of the sunlight spectrum reaching the
plant, consisting of an increase in red and blue light wavebands, has positively affected
crop production without compromising fruit qualitative characteristics [21]. This effect was
observed in early potato crops (+12% yield), melons, and watermelons (+10% yield) [21].
Furthermore, when subjected to a green-to-red light conversion film, ‘Buttercrunch’ let-
tuce plants had a significant increase on leaf area, leading to a 21.7% increase in total
aboveground fresh weight [22]. In the case of Solanum lycopersicum var. Beefsteak, a light
conversion film focused solely on the red light peak at 600 nm resulted in a 23% increase in
light efficiency measured as crop production per unit of photosynthetically active radiation
light (PAR), leading to a 10% faster vegetative growth rate and a 36% reduction in total
tomato waste [23]. Furthermore, variations in light spectra can impact plant quality by
influencing the accumulation of bioactive compounds [22–24]. For instance, Cozzolino
et al. [24] reported increased chlorophyll and vitamin C contents in Valerianella locusta L.
plants cultivated under blue light-diffusing films. Moreover, Shen et al. [22] reported an
increase in vitamin C content in lettuce grown under a green-to-red light-converting film.
Strawberry fruits have significant economic importance in various climate zones
worldwide [25,26]. In an experiment conducted by Peng et al. [27], strawberry plants
grown under various light selective films differed significantly in leaf area and shoot
biomass. The films that transmitted a higher proportion of red light led to the best results,
including the highest anthocyanin fruit yield.
To the best of our knowledge, little information is present in the literature regarding
the use of different light downconversion films on the physiological features of strawberry
plants and, especially, on fruit nutraceutical properties. Hemming et al. [28] found a
weak relationship between vegetative growth and fruit yield among plants grown under
different light downconversion films. Only the fluorescent film in the blue spectrum showed
promising effects on fruit production. Differently, a work conducted by Kang et al. [3]
suggests that green-to-red spectrum downconversion films boost the electron flow, leading
to an enhanced photosynthetic capacity and superior fruit nutraceutical value in strawberry.
Agronomy 2024, 14, 1491 3 of 20

Assuming that little and inconsistent information is available about the effects of light
downconversion films on strawberry plants, the present research aimed to investigate the
effects of three distinct light downconversion films (UV-to-blue, UV/green-to-blue/red,
and green-to-red light shifting features) on the physiological features of strawberry leaves,
as well as their impact on fruit secondary metabolites accumulation. Our hypothesis is
based on the assumption that the selective enrichment with red and blue light may enhance
the photosynthetic efficiency of strawberry leaves and, at the same time, stimulate the
production of specific secondary metabolites in the fruit.

2. Materials and Methods

2.1. Plant Material and Growing Conditions
Commercial plantlets of Fragaria × ananassa cv. “Aromas” were cultivated under
four tunnels (6 m × 4.15 m × 1.9 m) with two open ends located at the facilities of the
Department of Agriculture, Food and Environment (DAFE), University of Pisa (43.7041371◦
N, 10.4270071◦ E). The transplantation took place on the 31 of August 2022; 5 plants were
put in commercial substrate bags (X-BAG P30, Virgoplant Italia srl, Fombio, LO, Italy) with
coco-fibre and perlite (60:40; v/v) under each tunnel, and the experiment lasted 3 months.
The automated drip irrigation was scheduled 3 times per day with a nutrient solution
containing the following nutrient concentrations: 11.0 mM NO3 − , 0.5 mM NH4 + , 1.6 mM
H2 PO4 − , 5.7 mM K+ , 5.0 mM Ca2+ , 1.7 mM Mg2+ , 2.5 mM Na+ , 2.9 mM SO4 2− , 2.8 Cl− ,
18.0 mM Fe2+ , 19.0 mM B, 0.8 mM Cu2+ , 8.0 mM Zn2+ , 15.0 mM Mn2+ , and 0.5 mM MoO4 2−
Water chemical adjustments were carried out periodically to maintain 1.9 mS cm−1 for
electrical conductivity (EC) and 5.5 for pH values.
A total of 60 strawberry plants were split into 3 treatments (20 plants per treatment),
involving the utilization of 150 µm thick polyethylene films manufactured and provided
by CASCADE SAS (Clamart, France) along with 20 plants subjected to a control colourless
polyethylene film (Cnt; without the fluorescent dye formulation). The treatments were: blue
film (blue; shift of UV into blue radiation), red film (red; shift of green into red radiation),
light red film (pink; shift of UV and green into blue and red radiation, respectively but
to a lesser extent than the red film). A spectroradiometer (SpectraPen, Photon Systems
Instruments, Drásov, Czech Republic) was used to measure the light spectra distribution
under each light downconversion film compared to the control film.
On the field, non-destructive analyses were focused on leaf physiological traits through
gas exchanges and chlorophyll a (chl a) fluorescence measurements. Leaf physiological
analysis started after 25 days of plant acclimation under the agricultural films (T1; Figure 1);
thereafter, the same analyses were carried out around every 20 days for a total of 3 times
(T2, T3 and T4; Figure 1). Ambient daily minimum and maximum temperatures during the
trial period are reported in Figure 1.
Strawberry fruits were harvested at the commercial stage, ensuring that fruit from
different treatments had visually reached the desired ripening level of 75–90% maximum
redness. Fruit harvest started at the end of October and was prolonged until the end of
November. For biochemical investigations on the fruit, fruit samples were stored at −80 ◦ C
using liquid nitrogen and then cryogenically ground for homogenous replicates.

2.2. Plant and Leaf Morphological Parameters

At the end of the experiment, five plants were randomly selected from each treatment
for morphological measurements [plant fresh and dry weight, leaf thickness, leaf area and
leaf mass per area (LMA)]. Plant fresh weight (n = 5) was obtained by weighing the whole
aerial part of the plant composed by leaf, stem, and collar. Then, the aerial part samples
were put in a ventilated oven (Memmert GmbH Co., KG Universal Oven UN30, Schwabach,
Germany) at 80 ◦ C on the first day, followed by 105 ◦ C on the second day until a constant
weight was reached for the determination of the dry weight. Leaf thickness and foliar
area measurements (n = 20) (4 leaves for each biological replicate) were performed using
High Precision Digital Thickness Gauge and ImageJ software (version 1.52t). LMA was
 Agronomy 2024, 14, 1491 4 of 20

Agronomy 2024, 14, 1491 4 of 21

evaluated utilising one leaf per biological replicate (n = 5), calculated as the ratio of leaf dry
weight to leaf area, and expressed as g m−2 .

Stage
40
Vegetative Flowering Fructification

T1 T2 T3 T4
30
Temperature (°C)

20

10

T-Max
T-Min
0
2 2 22 22 22 2 2 2 2 2 2 2 2 2 2 2
02 02 20 20 20 02 02 02 02 02 02 02 02 02 02 02
-2 -2 p- p- p- -2 -2 -2 -2 -2 -2 -2 -2 -2 -2 -2
ep ep ct ct ct ct ct ct v v v v v
S S Se Se Se 1-
O
7-
O -O -O -O -O No -N
o
-N
o
-N
o
-N
o
1- 7- 13
-
19
-
25
-
13 19 25 31 6- 12 18 24 30

Date
Figure 1. Ambient daily minimum and maximum temperatures during the trial period with cor-
Figure 1. Ambient
responding daily
dates minimum and maximum
of non-destructive temperatures
data analysis times (T1:during the trial
vegetative period
stage; with corre-
T2: flowering stage;
sponding dates of non-destructive
T3: fruit stage; T4: fruit stage). data analysis times (T1: vegetative stage; T2: flowering stage; T3:
fruit stage; T4: fruit stage).
2.3. Gas Exchange and Chl a Fluorescence Analysis
Strawberry
Gas exchangefruits measurements
were harvested(nat=the 10) commercial
were performed stage,using
ensuring that fruit
a portable fromgas
infrared
different treatments had visually reached the desired ripening level
analyser LI-6800 system (Li-Cor, Lincoln, NE, USA) on randomly selected fully expanded of 75–90% maximum
redness.
leavesFruit
fromharvest started
11:00 a.m. at the
to 1:00 end at
p.m. ofaOctober and wasofprolonged
light intensity 1500 µmoluntil the end
photons m−of2 s−1 .
November.
Using theFor CObiochemical
2 mixer, the investigations
CO2 concentration on the fruit,the
inside fruit samples
leaf chamber were
wasstored at −80
set at 400 µmol
mol−1liquid
°C using , and nitrogen
the flow andratethen 500 µmol s−1ground
wascryogenically . Oncefor thehomogenous
steady statereplicates.
was reached, the
following parameters were recorded: net photosynthetic rate (Pn ), stomatal conductance
2.2.(g
Plant and Leaf Morphological
s ), intercellular Parameters
CO2 concentration (Ci ), and apparent carboxylation efficiency (Pn /Ci ).
Chlend
At the a fluorescence parameters
of the experiment, were measured
five plants were randomly (n = 6)selected
on 20 min from dark-adapted
each treatment leaves
for using a portable
morphological fluorometer [plant
measurements (Plantfresh
Efficiency
and dry Analyzer–Handy PEA, Hansatech
weight, leaf thickness, leaf area and Ltd.,
Norfolk, UK). Samples were flashed for 1 s with a saturated (2700 µmol m −2 s−1 ) red light
leaf mass per area (LMA)]. Plant fresh weight (n = 5) was obtained by weighing the whole
emitting
aerial part ofdiodes (LED)
the plant light pulse
composed by(650
leaf,nm).
stem,Plant
andphotosynthetic
collar. Then, the performance
aerial partwas assessed
samples
using the maximum quantum efficiency of Photosystem
were put in a ventilated oven (Memmert GmbH Co., KG Universal Oven UN30, II (PSII), i.e., Fv /F m Schwa- Fv
, where
represents the difference between the maximal (F ) and minimal
bach, Germany) at 80 °C on the first day, followedmby 105 °C on the0 second day until (F ) fluorescence of a dark-
a
adapted
constant sample,
weight was Freached
m is the for
maximum Chl a fluorescence
the determination of the dryrecorded
weight.afterLeaf athickness
saturating andlight
pulse,
foliar areaand F0 is the basal
measurements (n fluorescence
= 20) (4 leaves before a saturation
for each pulse,
biological extrapolated
replicate) from the line
were performed
of best fit determined through the initial data point recorded
using High Precision Digital Thickness Gauge and ImageJ software (version 1.52t). LMA at the onset of illumination.
was evaluated utilising one leaf per biological replicate (n = 5), calculated as the ratio of
2.4. Phenolic Extraction for Spectrophotometric Assay
leaf dry weight to leaf area, and expressed as g m−2. ◦
Homogenous fruit samples (stored at −80 C) of about 0.1 g fresh material were
combined withand
2.3. Gas Exchange 1 mL
Chlofa aFluorescence
solution of Analysis
methanol diluted to 80% (v/v). The homogenates were
◦
sonicated at 4 C with a sonicator (Digital ultrasonic Cleaner, DU-45, Argo Lab, Modena,
Gas exchange measurements (n = 10) were performed using a portable infrared gas
Italy) for 30 min and then centrifuged at 4 ◦ C with a laboratory centrifuge (MPW 260R,
analyser
MWPLI-6800 system (Li-Cor,
Med. instruments, Lincoln,
Warsaw, NE, at
Poland) USA) on rpm
10,000 randomly
for 15 selected
min. Thefully expanded
obtained extracts
leaves from 11:00 a.m. to
◦ 1:00 p.m. at
were stored at −20 C until the analysis.a light intensity of 1500 μmol photons m −2 s−1. Using

the CO2 mixer, the CO2 concentration inside the leaf chamber was set at 400 μmol mol−1,
and the flow rate was 500 μmol s−1. Once the steady state was reached, the following pa-
rameters were recorded: net photosynthetic rate (Pn), stomatal conductance (gs), intercel-
lular CO2 concentration (Ci), and apparent carboxylation efficiency (Pn/Ci).
Agronomy 2024, 14, 1491 5 of 20

2.5. Total Phenolic Content (TPC)

The procedure presented by Dewanto et al. [29] for TPC was followed with some
reagent volume modifications. Basically, 62.5 µL extract sample was mixed with 62.5 µL
Folin–Ciocalteu reagent and 250 µL distilled water and kept standing for 6 min at room tem-
perature. Then, 625 mL of Na2 CO3 7% (w/v) aqueous solution was added and the mixtures
were incubated for 90 min in the dark at room temperature. The absorbance at 760 nm was
measured with a spectrophotometer (Ultrospec 2100 Pro, GE Healthcare Ltd., Chalfont, Buck-
inghamshire, UK) against a blank solution (free of fruit sample). All of the obtained results
(n = 5) were compared with a gallic acid standard curve (y = 0.002x + 0.0008; R2 = 0.9934) and
were expressed as mg gallic acid equivalents per g fresh weight (FW; mg GAE g−1 FW).

2.6. Total Flavonoid Content (TFC)

TFC was determined following the procedure described by Silva et al. [30]. An aliquot
(100 µL) of fruit extract, 30 µL of NaNO2 5% (w/v) aqueous solution and 400 µL distilled
water were mixed. After 5 min, 30 µL of AlCl3 10% (w/v) aqueous solution was added
for the aluminium–flavonoid complexes formation. After 6 min, 200 µL of NaOH 1 M
and 240 µL of distilled water were added to the mixture. The absorbance at 510 nm was
spectrophotometrically measured against a blank solution for each extract replicate (which
contained all of the reagents except the AlCl3 10% (w/v) aqueous solution, which was
replaced with distilled water). All of the obtained results (n = 5) were compared with
a catechin standard curve (y = 0.0005x + 0.0465; R2 = 0.9956) and were expressed as mg
catechin equivalents per g FW (mg CAE g−1 FW).

2.7. Total Ascorbic Acid Content

Total ascorbic acid content was measured following the procedure described by
Kampfenkel et al. [31]. An amount (0.2 g) of homogenous fresh fruit sample powder
stored at −80 ◦ C was homogenized with 6% (w/v) trichloroacetic acetic acid (TCA) aqueous
solution and centrifuged at 10,000 rpm for 10 min at 4 ◦ C. From each replicate supernatant,
50 µL of extract was combined with 50 µL of 10 mM (w/v) dithiothreitol and 100 µL of
0.2 M (w/v) phosphate buffer Na-P (pH 7.4) and then the mixture was placed in a water
bath at 42 ◦ C. After 15 min incubation, 50 µL 0.5% (w/v) N-ethylmaleimide, 250 µL 10%
(w/v) TCA, 200 µL 42% (w/v) H3 PO4 , 200 µL 4% (w/v) 2,2’-dipyridil and 100 µL 3% (w/v)
FeCl3 were added to the mixture, which was incubated again at 42 ◦ C for 40 min. The
blank solution was prepared by replacing the extract with 6% TCA (w/v). The adsorption of
the final solution was measured spectrophotometrically at 525 nm and all of the obtained
results (n = 5) were expressed as mg ascorbic acid per g FW (mg ASA g−1 FW) using an
ascorbic acid standard calibration curve (y = 0.0147x − 0.0042; R2 = 0.9954).

2.8. Total Anthocyanin Content (TAC)

TAC was determined following the differential pH method [32]. For the sample
extraction, an amount (0.2 g) of fresh fruit sample (stored at −80 ◦ C) was homogenized
with 1% (v/v) acidified methanol and centrifuged at 10,000 rpm for 15 min at 4 ◦ C. For
the TAC determination, 100 µL of fruit extract was diluted in 900 µL of either 0.4 M (w/v)
sodium acetate buffer (pH 4.5) or 0.025 M (w/v) potassium chloride (pH 1), depending on
the pH to test. After 15 min at room temperature, the absorbance at 530 and 700 nm was
spectrophotometrically registered for both pH dilutions and the final absorbance (Af ), and
TAC was calculated as follows:

Af = (A530 − A700 )pH1.0 − (A530 − A700 )pH4.5

A f × MW Pg3glu × D f
TA =
E × 1 × Vext × Wsample
Agronomy 2024, 14, 1491 6 of 20

where MW is the molecular weight of pelargonidin 3-O-glucoside, Df is the dilution factor,

1 is the path length (in cm), ε is the molar extinction coefficient (in L mol−1 cm−1 ), Vext is
the volume extract and Wsample is the weight of the sample (in g).
All obtained results (n = 5) were expressed as mg pelargonidin 3-O-glucoside equiva-
lent on g FW (mg Pg-3-O-glu g−1 FW).

2.9. Antioxidant Activity (AA) Assays

The AA was measured using both the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free
radical scavenging assay [33] and the 2,29-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)
(ABTS) test [34].
For the DPPH assay, after the preparation of the methanolic solution of 3.12 × 10−5 M
DPPH (w/v), 10 µL of fruit extract was mixed with 990 µL of DPPH methanolic solution.
The absorbance was spectrophotometrically determined at 515 nm against a blank solution
(free of extract).
For the ABTS assay, a buffer solution (5 mM w/v) was prepared from Na2 PO4 and
Na2 H2 PO4 to reach pH = 7.4. Firstly, 0.0192 g of 7 mM (w/v) ABTS and 0.0034 g of 2.5 mM
(w/v) K2 S2 O8 solution were solubilized in 5 mL of buffer solution and the mixture was
stored in a dark environment. For the analysis, 50 µL of fruit extract was mixed with 950 µL
of ABTS solution and the kinetics of the reaction were spectrophotometrically followed for
90 s at 734 nm.
The obtained results (n = 5) from both antioxidant activity assays were expressed as
mg Trolox equivalents per g FW (mg TE g−1 FW).

2.10. Chemical Fingerprint by UHPLC-DAD-HR-ESI-MS Analysis

Each strawberry fruit sample was subjected to ultrasound-assisted extraction utilizing
an ethanol solution 80% (v/v) for the extraction of phenolic compounds (solid:liquid ratio
of 1:3 g mL−1 ) and a mixture of 2% HCl in methanol (solid:liquid ratio of 1:6 g mL−1 ) for
anthocyanin recovery. The defrosted strawberry samples (1.5 g each) were placed in the
corresponding amount of solvent and treated with ultrasound at 20 ◦ C for 15 min. After
centrifugation for 5 min at 3500 rpm, the supernatants were directly analysed in triplicate
using ultra-high performance liquid chromatography (UHPLC) coupled to a diode array
detector (DAD) and a high-resolution mass spectrometer (HR-MS).
The LC-MS system employed a Vanquish Flex Binary pump coupled with DAD and a
HR-MS Q Exactive Plus Orbitrap-based FT-MS with an electrospray ionization (ESI) source
and Xcalibur 4.1 software (Thermo Fisher Scientific Inc., Bremen, Germany). A volume
of 2 µL of each phenolic extract and of 5 µL of anthocyanin extract were injected into the
LC-MS system. All of the analyses were performed on a C-18 Kinetex® Biphenyl column
(100 × 2.1 mm, 2.6 µm particle size) provided with a Security GuardTM Ultra Cartridge
(Phenomenex, Bologna, Italy), eluting with a mixture of HCOOH in H2 O 0.1% (v/v) for
solvent A and HCOOH in methanol 0.1% (v/v) for solvent B at a flow rate of 0.5 mL min−1 ,
using a splitting system of 1:1 to MS and DAD. For phenols, a linear gradient was applied,
increasing from 5 to 80% of solvent B over a period of 32 min. In the case of anthocyanins,
the solvent gradient was 10 to 35% of solvent B in 5 min. The column and autosampler
temperatures were maintained at 35 and 4 ◦ C, respectively. Data from the DAD were
registered within the range of 200–600 nm. Selected channels at 254, 280, and 325 nm
were utilized according to the characteristic absorbances of phenolic compounds, while
for anthocyanins, the absorbance was targeted at 515 nm. The ESI interface was used in
negative ion mode to analyse phenols and in positive ion mode for anthocyanins. The HR-
MS spectra were acquired in a m/z scan range of 170–1200 for phenols and m/z 250–1200 for
anthocyanins, operating in full (resolution 70,000 and maximum injection time of 220 ms)
and data dependent-MS/MS (resolution 17,500 and maximum injection time of 60 ms). The
ionization parameters were optimized as reported in Cioni et al. [35].
Agronomy 2024, 14, 1491 7 of 20

2.11. Statistical Analysis

After checking the normality of distribution (Shapiro–Wilk test, 95% confidence inter-
val), one-way ANOVA was performed on the plant morphological and fruit nutraceutical
compounds data using the type of film as the factor of variation. A two-way ANOVA
was carried out on the physiological data (gas exchange and chlorophyll fluorescence
parameters) using the type of film and time as factors of variation. Significant differences
among treatments were determined by the LSD Fisher post hoc test (p ≤ 0.05). GraphPad
software (v.10, GraphPad, La Jolla, CA, USA) was used for the statistical analysis.

3. Results
3.1. Light-Converting Properties of Polyethene Films
The blue film significantly increased the intensity of the blue light spectrum, which
falls in the range of 420–480 nm, by a noticeable 8.4% when compared to the Cnt film
(Figure 2). On the other hand, the red film primarily boosted the red light spectrum,
encompassing wavelengths from 600–700 nm, showing an increase of 12.6% in comparison
to the Cnt film. Interestingly, the pink film demonstrated enhancements in both the red
and blue light spectra, registering increases of 4 and 5%, respectively, over the Cnt film.
However, despite these differences in specific spectra, it is important to note that the PAR,
which spans from 400–700 nm, remained consistent across all of the treatments, hovering
Agronomy 2024, 14, 1491
around approximately 1200 µmol m−2 s−1 on a full sunny day. Furthermore, through 8 of 21
a
comparison with PAR natural sunlight, all of the films exhibited an irradiance that fell short
of the sun’s irradiance, which measured at around 1500 µmol m−2 s−1 (Figure 2).

Cnt Blue Red Pink Sun

8
Irradiance (mmol m– 2 s–1)

0
380 420 460 500 540 580 620 660 700 740 780
Wavelength (nm)
Effect of
Figure2.2.Effect
Figure of control
control (Cnt),
(Cnt), blue,
blue,red,
red,and
andpink
pinklight downconversion
light films
downconversion used
films in the
used experiment
in the experi-
on the
ment ontransmitted wavelengths
the transmitted of sunlight
wavelengths inside
of sunlight the tunnel.
inside the tunnel.

3.2. Plant Morphological Parameters

3.2. Plant Morphological Parameters
The plants cultivated under the light downconversion films experienced a notable
The plants cultivated under the light downconversion films experienced a notable
increase in both fresh and dry weights, showing an uplift of +40 and +34%, respectively,
increase in both fresh and dry weights, showing an uplift of +40 and +34%, respectively,
when compared to those grown under the Cnt film, as evidenced by the data presented in
when compared to those grown under the Cnt film, as evidenced by the data presented in
Table 1. Strawberry plants under blue and red films showed a greater leaf area (+25 and
Table 1. Strawberry plants under blue and red films showed a greater leaf area (+25 and
31%, respectively) than those under Cnt film (Table 1). Delving deeper into the values, the
31%, respectively) than those under Cnt film (Table 1). Delving deeper into the values, the
blue film exhibited particularly impressive results. The plants grown under the blue film
blue film exhibited particularly impressive results. The plants grown under the blue film
had higher values of leaf thickness and LMA (10 and 15%, respectively) when compared to
had higher values of leaf thickness and LMA (10 and 15%, respectively) when compared
the plants grown under the Cnt film.
to the plants grown under the Cnt film.

Table 1. Plant fresh and dry weight, leaf area, leaf thickness, and leaf mass area (LMA) of Fragaria ×
ananassa plants grown under control (Cnt; no fluorescence), blue, red, and pink light downconver-
sion films.

Variable 1 Cnt Blue Red Pink

Plant fresh weight (g plant−1) 71.98 ± 23.17b 100.10 ± 11.99a 105.60 ± 6.04a 96.67 ± 8.61a
Agronomy 2024, 14, 1491 8 of 20

Table 1. Plant fresh and dry weight, leaf area, leaf thickness, and leaf mass area (LMA) of Fra-
garia × ananassa plants grown under control (Cnt; no fluorescence), blue, red, and pink light
downconversion films.

Variable 1 Cnt Blue Red Pink

Plant fresh weight (g plant−1 ) 71.98 ± 23.17b 100.10 ± 11.99a 105.60 ± 6.04a 96.67 ± 8.61a
Plant dry weight (g plant−1 ) 16.50 ± 4.43b 22.08 ± 2.52a 21.97 ± 2.62a 22.30 ± 3.02a
Leaf area (cm2 ) 50.87 ± 10.69b 63.40 ± 13.19a 66.52 ± 11.93a 53.00 ± 9.810b
Leaf thickness (µm) 247.2 ± 26.8b 271.7 ± 35.80a 233.5 ± 23.00b 242.9 ± 29.70b
LMA (g m−2 ) 70.09 ± 3.19b 80.56 ± 5.06a 63.39 ± 5.74b 69.98 ± 10.39b
1 Each value is the mean ± SD of 5 replicates for plant fresh, dry weight and leaf mass area measurements,
mean ± SD of 20 replicates for leaf morphological parameters (leaf area and leaf thickness). Means were subjected
to one-way ANOVA with type of film as the source of variations. Means with different letters are significantly
different at p ≤ 0.05 for Fisher’s least significant difference post hoc test.

3.3. Gas Exchange and Chl a Fluorescence Analysis

The analysis of gas exchanges during different phenological stages indicated that
plants under the blue film had superior net CO2 assimilation performances than Cnt, with
an average increase of 16% throughout all stages. The blue film promoted an enhancement
of Pn , closely followed by the red film, which reported a 15% average increase. The pink
Agronomy 2024, 14, 1491
film demonstrated a comparable Pn improvement (+16%; Figure 3a) only9 during
of 21
the final
two stages (T3 and T4).

Figure3. 3.
Figure NetNet photosynthesis
photosynthesis (Pn ;stomatal
(Pn; (a)), (a)), stomatal conductance
conductance (gs ; (b)), intercellular
(gs; (b)), intercellular CO2 concentration
CO2 concentration
(Ci; i(c)),
(C ; (c)), apparent
apparent carboxylation
carboxylation efficiency
efficiency (Pn/Ci;(P n /C
(d)) i ; (d))
and and photosystem
photosystem II maximum II photochemical
maximum photochemical
efficiency
efficiency (F(F
v/Fm; (e)) of Fragaria × ananassa plants grown under control (Cnt; no fluorescence), blue,
v /Fm ; (e)) of Fragaria × ananassa plants grown under control (Cnt; no fluorescence), blue,
red, and pink light downconversion films, measured at vegetative (T1), flowering (T2), fructification
(T3 and T4) stages. Means were subjected to two-way ANOVA with the type of film and sampling
time as the source of variations. Means with different letters are significantly different at p ≤ 0.05
with Fisher’s least significant difference post hoc test. When the F ratio of the interaction between the
variability factors was not significant, the letters indicate statistically significant differences between
means over time.

During T3, all treatments exhibited the lowest Pn values, but the differences reported
above were maintained. The gs declined in accordance with the plant phenological stage.
From T2 onwards, the highest gs value was observed in plants under the blue film (Figure
Agronomy 2024, 14, 1491 9 of 20

red, and pink light downconversion films, measured at vegetative (T1), flowering (T2), fructification
(T3 and T4) stages. Means were subjected to two-way ANOVA with the type of film and sampling
time as the source of variations. Means with different letters are significantly different at p ≤ 0.05
with Fisher’s least significant difference post hoc test. When the F ratio of the interaction between the
variability factors was not significant, the letters indicate statistically significant differences between
means over time.

During T3, all treatments exhibited the lowest Pn values, but the differences reported
above were maintained. The gs declined in accordance with the plant phenological
stage. From T2 onwards, the highest gs value was observed in plants under the blue
film (Figure 3b). At T3, plants under the blue and red films displayed statistically signifi-
cant differences compared to those under the Cnt film, with improvements of 45 and 27%,
respectively (Figure 3b). At T4, all of the light downconversion films showed higher gs
values than the Cnt film (+54, +34 and +29% for blue, red and pink compared to the Cnt
film, respectively; Figure 3b). At T1, the highest Ci values were observed in plants under
the pink film, showing a 3% increase compared to the Cnt, whereas the blue film led to a
3% reduction (Figure 3c). At T2, both the red and blue films showed approximately 3%
lower Ci values compared to the Cnt. However, by T4, the blue film led to the highest Ci
values, with a 7% increase relative to the Cnt. The Pn /Ci patterns mirrored the Pn trends.
Throughout the experiment, plants under the blue and red films consistently recorded
higher Pn /Ci values than those under the Cnt film, averaging increases of 15 and 16%,
respectively (Figure 3d). The lowest Pn /Ci values were observed across all treatments at
T3. Starting from T3 to T4, plants grown under the pink film again showed higher values
of Pn /Ci than the Cnt plants (+16% on average).
At the beginning of the experiment, during the vegetative stage (T1), strawberry leaves
under all films showed the highest Fv /Fm (Figure 3e) when compared with the other growth
stages (flowering and fructification). During the flowering (T2) and the fructification (T3)
stages, leaves from all treatments showed a significant decrease of Fv /Fm , with a subsequent
increase at the end of the fructification stage, independent of the treatment.

3.4. Fruit Nutraceutical Features

The fruit from plants grown under the blue film showed the highest content in TPC
(+38% than the Cnt fruit; Figure 4a), even though no significant differences were reported
between fruits harvested under blue and Cnt film. Moreover, fruits from plants grown
under Cnt film yielded similar TPC to those grown under the red and pink films.
The TFC was significantly higher in fruits under the blue film (+127% than the Cnt fruit)
than in those under the Cnt film or the rest of the light downconversion films (Figure 4b).
Fruits under the red film had lower TFC in comparison with those under the Cnt film but
similar to those harvested under the pink film. For TAC, the pink film fruits exhibited
the lowest value in comparison to the rest of the films (−43% compared to the Cnt fruit;
Figure 4c). However, the light downconversion films did not positively affect the total
ascorbic acid content of strawberry fruit (−36, −21 and −36% for fruit grown under blue,
red, and pink films, respectively, compared with the Cnt fruit; Figure 4d). No significant
differences were observed in the antioxidant activity between the DPPH and ABTS assays
(Figure 4e,f).
The chemical composition of strawberry fruit subjected to light downconversion films
was elucidated through the application of the highly sensitive UHPLC-DAD-HR-ESI-MS
technique. The LC-MS obtained chromatograms for phenolic and anthocyanin profiles are
reported in Figures 5 and 6, respectively.
 3.4. Fruit Nutraceutical Features
The fruit from plants grown under the blue film showed the highest content in TPC
(+38% than the Cnt fruit; Figure 4a), even though no significant differences were reported
Agronomy 2024, 14, 1491 between fruits harvested under blue and Cnt film. Moreover, fruits from plants grown 10 of 20
under Cnt film yielded similar TPC to those grown under the red and pink films.

10 0.5 0.5
(a) (b) (c)

(mg Pg-3-O-glu. eq. g FW)

a
8 0.4 a 0.4

–1
(mg CAE g FW)
(mg GAE g FW)
ab b 0.3 0.3

–1
6

–1
b

TAC
TFC
a

TPC
a
4 0.2 0.2 a
b
bc b
2 0.1 c 0.1

0 0.0 0.0
Cnt Blue Red Pink Cnt Blue Red Pink Cnt Blue Red Pink

0.5 5 5
(d) (e) (f)
0.4 4 4
Total ascorbic acid
(mg ASA g FW)

(mg TE g FW)

(mg TE g FW)
0.3
–1

3 3

DPPH
–1

–1
ABTS
a
0.2 b
c bc
2 2

0.1 1 1

0.0
0 0
Cnt Blue Red Pink
Cnt Blue Red Pink Cnt Blue Red Pink

Figure 4. Total phenolic (a), flavonoid (b), anthocyanin (c), ascorbic acid (d) content and antioxidant
Figure 4. Total phenolic (a), flavonoid (b), anthocyanin (c), ascorbic acid (d) content and antioxidant
activity
activity detected
detected with DPPH
with DPPH (e) and (e)
ABTSand(f)ABTS
assays (f) Fragaria
assays ×ofananassa
of Fragaria × from
fruit ananassa
plantsfruit from plants grown
grown
under control (Cnt; no fluorescence), blue, red and pink light downconversion films.
under control (Cnt; no fluorescence), blue, red and pink light downconversion films. Means wereMeans were
subjected to one-way ANOVA with the type of film as the source of variation. Means (n = 5 ± SD)
subjected to one-way ANOVA with the type of film as the source of variation. Means (n = 5 ± SD)
with different letters are significantly different at p ≤ 0.05 for Fisher’s least significant difference post
Agronomy 2024, 14, 1491 hoc test. different letters are significantly different at p ≤ 0.05 for Fisher’s least significant
with 11 of 21 difference post
Agronomy 2024, 14, 1491 hoc test. 11 of 21
The TFC was significantly higher in fruits under the blue film (+127% than the Cnt
fruit) than in those under the Cnt film or the rest of the light downconversion films (Figure
4b). Fruits under the red film had lower TFC in comparison with those under the Cnt film
but similar to those harvested under the pink film. For TAC, the pink film fruits exhibited
the lowest value in comparison to the rest of the films (−43% compared to the Cnt fruit;
Figure 4c). However, the light downconversion films did not positively affect the total
ascorbic acid content of strawberry fruit (−36, −21 and −36% for fruit grown under blue,
red, and pink films, respectively, compared with the Cnt fruit; Figure 4d). No significant
differences were observed in the antioxidant activity between the DPPH and ABTS assays
(Figure 4e,f).
The chemical composition of strawberry fruit subjected to light downconversion
films was elucidated through the application of the highly sensitive UHPLC-DAD-HR-
ESI-MS technique. The LC-MS obtained chromatograms for phenolic and anthocyanin
profiles are reported in Figure 5 and Figure 6, respectively.

Figure 5.5.Phenolic
Figure Phenolic UHPLC-DAD-HR-ESI-MS
UHPLC-DAD-HR-ESI-MS profile recorded
profileinrecorded
negative ion mode of Fragaria
in negative ×
ion mode of Fragaria
Figure
ananassa5.fruit
Phenolic
fromUHPLC-DAD-HR-ESI-MS
plants grown under controlprofile recorded in negative ion red
mode of pink
Fragaria ×
× ananassa fruit from plants grown under(Cnt; no fluorescence),
control blue,
(Cnt; no fluorescence), and light
blue, red and pink light
ananassa fruit from
downconversion plants
films. Peakgrown under
data are control
shown (Cnt;
in Table 2. no fluorescence), blue, red and pink light
downconversion
downconversion films. films.
PeakPeak data
data are are shown
shown in Tablein
2. Table 2.

Figure 6. Anthocyanin UHPLC-DAD-HR-ESI-MS profile recorded in positive ion mode of Fragaria

Figure
Figure 6.6.Anthocyanin
× ananassa fruit UHPLC-DAD-HR-ESI-MS
from plants
Anthocyanin grown under control profile
UHPLC-DAD-HR-ESI-MS recorded
(Cnt; no in positive
fluorescence),
profile blue,ion
recorded mode
inred and of Fragaria
pink
positive light
ion mode of Fragaria
×downconversion
ananassa fruit from
films.plants
Peak grown
data areunder
showncontrol (Cnt;
in Table 3. no fluorescence), blue, red and pink light
× ananassa fruit from plants grown under control (Cnt; no fluorescence), blue, red and pink light
downconversion films. Peak data are shown in Table 3.
downconversion
All phenolic and films. Peak datacompounds
anthocyanin are shown in Table
were 3.
tentatively identified by comparison
All elution
of their phenolic and anthocyanin
order, compounds
molecular formula, were full
and both tentatively identified bymass
and fragmentation comparison
spectra
of their 2elution
(Tables and 3)order, molecular
with data formula,
reported and both full
in the literature and fragmentation
[6,35–38]. A mass error mass spectra
< 5 ppm was
(Tables 2 and
considered for3)compound
with data annotation.
reported inAll
theanalysed
literaturestrawberry
[6,35–38]. A mass error
samples < 5 ppm
exhibited was
a highly
considered
comparablefor compound showing
composition, annotation. All analysed
a chemical strawberry
fingerprint thatsamples exhibited
was almost a highly
superimposa-
comparable
ble. In total, composition, showing a chemical
43 phenolic compounds fingerprint
were attributed that was almost
as constituents superimposa-
of strawberry fruit,
ble. In total,
according to 43 phenolic reported
previously compounds were The
studies. attributed as constituents
identified substances of strawberry
belong fruit,
to different
Agronomy 2024, 14, 1491 11 of 20

Table 2. Retention time (tR ) and mass spectral data of phenols tentatively identified in Fragaria × ananassa fruit from plants grown under control (Cnt; no fluorescence),
blue, red and pink light downconversion films.

N. 1 tR [M-H]− Product Ions Error ppm Formula Compound Peak Area × 106
Cnt Blue Red Pink
Phenols
Hydroxybenzoic acid
2 4.6 299.0779 137.02 +2.21 C13 H16 O8 74.16 ± 13.14ab 78.92 ± 13.71a 49.98 ± 16.17b 72.12 ± 9.44ab
hexoside
447.1506 401.14, 269.10,
12 7.2 ([M+HCOO]− ) 161.04, 113.01, −0.44 C18 H26 O10 Icariside F2 48.17 ± 12.20 55.72 ± 14.15 50.59 ± 10.09 39.41 ± 20.70
101.02
271.06, 164.01, Hydroxybenzoic acid
23 11.0 317.1030 137.02 +0.18 C17 H18 O6 57.30 ± 5.06b 86.83 ± 17.51a 73.93 ± 10.10ab 94.80 ± 14.89a
derivative
Flavonoids
14 8.4 431.0982 269.04 +0.39 C21 H20 O10 Apigenin glucoside 744.32 ± 185.72 764.70 ± 20.33 745.19 ± 131.46 562.73 ± 92.57
287.04, 269.04, Dihydrokaempferol
19 10.2 449.1089 243.06 −0.09 C21 H22 O11 340.26 ± 102.53 364.15 ± 15.29 364.61 ± 80.15 274.11 ± 55.75
glucoside
448.82, 287.06, Dihydrokaempferol
21 10.5 595.1692 269.04 +0.60 C27 H32 O15 21.80 ± 5.75 21.24 ± 3.61 18.56 ± 4.52 14.36 ± 2.23
rhamnosylglucoside
24 11.4 463.0519 300.99 +0.19 C21 H18 O13 Quercetin 3-O-glucoside 18.99 ± 6.68b 28.86 ± 6.23a 14.29 ± 2.03b 14.24 ± 2.70b
28 14.4 477.0673 301.03 +2.34 C21 H20 O11 Quercetin 3-O-glucuronide 32.43 ± 4.26 47.49 ± 9.13 34.75 ± 11.90 44.38 ± 16.47
29 15.4 447.0932 285.04 −0.20 C21 H18 O12 Kaempferol 3-O-glucoside 45.07 ± 19.12 33.53 ± 10.49 26.30 ± 1.29 28.47 ± 2.60
30 15.9 461.0725 285.04 −0.11 C23 H22 O12 Kaempferol 3-O-glucuronide 80.63 ± 15.60 76.93 ± 8.92 60.86 ± 15.86 68.08 ± 9.98
31 16.7 489.1038 327.05, 285.04, −0.10 C24 H22 O14 9.86 ± 1.41 10.02 ± 3.17 7.56 ± 1.24 7.31 ± 1.77
284.03, 255.03 Kaempferol acetyl hexoside
Kaempferol
32 16.8 533.0937 285.04 +0.04 C30 H26 O13 21.60 ± 3.22ab 23.63 ± 3.73a 18.00 ± 3.31ab 16.18 ± 3.19b
O-malonyl-O-hexoside
Kaempferol 3-O-(6-O-p-
33 19.4 593.1301 447.09, 285.04 +0.07 C21 H20 O10 44.66 ± 29.45 34.53 ± 28.03 24.12 ± 8.70 42.62 ± 12.96
coumaroylglucoside)
Ellagitannins
1 3.5 783.0695 300.99 +1.08 C34 H24 O22 Pedunculagin 12.85 ± 4.02b 19.55 ± 2.41a 10.15 ± 1.53b 12.74 ± 3.06b
7 6.0 633.0735 633.07, 300.99 +0.25 C27 H22 O18 Strictinin 24.85 ± 8.03b 38.14 ± 9.41a 20.39 ± 4.30b 21.54 ± 4.09b
467.0359 633.07, 391.03, Galloyl-diHHDP-glucose
15 9.3 ([M-H]2− ) 300.99 +0.96 C41 H28 O26 51.16 ± 2.81ab 64.79 ± 28.00a 35.29 ± 8.77b 30.38 ± 7.98b
(casuarictin)
466.0264 −0.51 31.20 ± 12.03a
22 10.6 ([M-H]2− ) 466.03, 300.99 C41 H26 O26 Castalgin 16.66 ± 3.22b 15.92 ± 1.74b 18.14 ± 1.49b
1567.14, 935.07, Digalloyl-tetraHHDP-
934.0717 −0.07 28.02 ± 3.43a
25 11.9 ([M-H]2− ) 783.06, 633.07, C82 H54 O52 diglucose/Sanguin H-6 0.31 ± 0.16b 2.64 ± 0.88b 27.89 ± 10.99b
300.99 isomer
Ellagic acid conjugates
26 13.9 447.0566 300.99, 299.99 −0.67 C20 H16 O12 Ellagic acid deoxyhexose 163.72 ± 43.63b 263.61 ± 21.79a 149.52 ± 1.18b 154.12 ± 8.41b
27 14.0 300.9988 257.00 −0.63 C14 H6 O8 Ellagic acid 83.07 ± 16.34b 126.32 ± 12.16a 76.39 ± 6.47b 80.92 ± 11.86b
Agronomy 2024, 14, 1491 12 of 20

Table 2. Cont.

N. 1 tR [M-H]− Product Ions Error ppm Formula Compound Peak Area × 106
Cnt Blue Red Pink
Cinnamic acid conjugates
187.04, 163.04, p-Coumaric acid glucoside
454.59 ± 46.69a 381.59 ±
5 5.9 325.0929 145.03 +0.03 C15 H18 O8 364.40 ± 56.53ab 83.21ab 325.10 ± 65.62b
(isomer I)
187.04, 163.04, p-Coumaric acid glucoside
10 6.3 325.0929 145.03 +0.03 C15 H18 O8 93.3 ± 24.90ab 130.52 ± 11.49a 126.00 ± 22.09a 73.18 ± 27.26b
(isomer II)
17 9.9 355.1034 309.10, 207.05, +0.38 C15 H18 O7 140.63 ± 10.31 109.04 ± 85.33 142.97 ± 37.28 123.94 ± 21.05
([M+HCOO]− ) 147.04 Cinnamoyl glucose
487.1456 14.92 ± 0.47a 14.50 ± 2.68a 13.38 ± 3.56a
18 10.1 ([M+HCOO]− ) 441.14, 147.04 +0.18 C21 H28 O13 Cinnamoyl xylosylglucose 8.81 ± 0.61b

Catechin and proanthocyanidins

451.10, 425.09, 122.85 ± 10.79a 123.75 ± 16.62a
3 4.8 577.1353 289.07, 125.02 +0.26 C30 H26 O12 Procyanidin dimer (isomer I) 97.31 ± 18.82ab 90.41 ± 11.29b
451.10, 425.09, Procyanidin dimer
4 5.3 577.1353 +0.26 C30 H26 O12 90.55 ± 28.42ab 123.04 ± 2.72a 64.29 ± 1.29b 67.35 ± 21.07b
289.07, 125.02 (isomer II)
5 5.5 289.0718 289.07, 245.08, +0.14 C15 H14 O6 414.32 ± 327.94 649.95 ± 58.15 526.28 ± 18.90 618.72 ± 118.12
109.03 Catechin
435.11, 289.07, Propelargonidin dimer
8 6.2 561.1408 271.06 +1.00 C30 H26 O11 12.27 ± 2.03b 16.59 ± 2.94a 14.43 ± 0.33ab 13.58 ± 2.05ab
(isomer I)
577.14, 407.08, 46.01 ± 2.43a
9 6.3 865.1998 289.07, 125.02 +0.30 C45 H38 O18 Procyanidin trimer 34.23 ± 10.83ab 25.44 ± 3.48ab 22.13 ± 12.73b
435.11, 289.07, Propelargonidin dimer
11 6.9 561.1408 271.06 +1.00 C30 H26 O11 15.04 ± 2.26b 20.68 ± 1.60a 14.33 ± 1.34b 16.78 ± 2.86ab
(isomer II)
720.1595 863.17, 575.11,
12 7.2 ([M-H]2− ) 407.08, 289.07, +0.67 C75 H62 O30 Procyanidin pentamer 11.60 ± 1.42b 27.64 ± 2.36a 12.31 ± 1.33b 15.71 ± 3.48b
125.02
576.1276 407.08, 289.07,
13 7.4 ([M-H]2− ) 125.02 +0.31 C60 H50 O24 Procyanidin tetramer 4.19 ± 0.70b 8.09 ± 0.83a 3.65 ± 0.78b 6.28 ± 2.74ab
577.14, 407.08, Proanthocyanidin C1
16 9.4 865.1998 289.07, 125.02 +0.30 C45 H38 O18 22.16 ± 3.15 25.78 ± 2.21 19.68 ± 4.09 21.62 ± 4.52
(catechin trimer)
864.1915 407.08, 289.07, 2.47 ± 0.44c 11.37 ± 1.99a 1.93 ± 0.34c
20 10.3 ([M-H]2− ) 125.02 +0.91 C90 H74 O36 Procyanidin hexamer 6.13 ± 1.79b
1 Means (n = 3 ± SD) with letters are significantly different after one-way ANOVA followed by Fisher’s LSD post hoc test (p = 0.05) considering the type of film as the source of variability.
Agronomy 2024, 14, 1491 13 of 20

Table 3. Retention time (tR ) and mass spectral data of anthocyanins tentatively identified in Fragaria × ananassa fruit from plants grown under control (Cnt; no
fluorescence), blue, red and pink light downconversion films.

N. 1 tR [M-H]− Product Ions Error ppm Formula Compound Peak Area ×106
Cnt Blue Red Pink
Pelargonidin
1 1.4 579.1496 433.11, 271.06 −0.17 C30 H27 O12 p-coumaroylhexoside 30.32 ± 7.98a 20.91 ± 5.01ab 15.30 ± 0.80b 14.87 ± 3.85b
(isomer I)
1641.74 ± 187.24a 1241.09 ± 684.79 ± 170.04c
2 1.7 433.1130 271.06 +0.18 C21 H21 O10 Pelargonidin 3-O-glucoside 971.00 ± 106.53bc 183.71b
Pelargonidin
3 2.3 519.1133 271.06 −0.04 C24 H23 O13 2.07 ± 0.40a 1.36 ± 0.54a 1.34 ± 0.39ab 0.52 ± 0.42b
malonylglucoside
Pelargonidin
4 2.6 579.1496 433.11, 271.06 −0.17 C30 H27 O12 p-coumaroylhexoside 10.49 ± 6.00 6.56 ± 1.35 4.52 ± 0.26 5.90 ± 1.89
(isomer II)
5 3.0 533.1292 271.06 +0.43 C25 H25 O13 Pelargonidin derivative 2.18 ± 0.1a 1.58 ± 0.65ab 1.55 ± 0.64ab 0.90 ± 0.68b
6 3.2 449.1077 287.05 −0.31 C21 H21 O11 Cyanidin 3-O-glucoside 12.30 ± 0.95a 8.83 ± 1.98ab 10.00 ± 2.92ab 7.85 ± 1.17b
Cyanidin
7 3.6 535.1083 287.05 +0.13 C24 H23 O14 3.71 ± 0.44a 2.70 ± 0.61ab 2.53 ± 0.83b 1.60 ± 0.56b
malonylglucoside
8 4.0 477.1026 287.05 −0.31 C22 H21 O12 Cyanidin derivative 16.56 ± 2.48a 12.00 ± 1.62ab 10.82 ± 4.03b 13.81 ± 2.00ab
9 4.3 549.1241 287.05 +0.40 C25 H25 O14 Cyanidin derivative 5.59 ± 1.35 5.80 ± 0.76 4.62 ± 0.72 4.71 ± 1.17
Cyanidin
10 4.8 595.1444 287.05 −0.37 C30 H27 O13 6.99 ± 2.48 8.12 ± 3.85 15.75 ± 8.82 6.56 ± 2.12
p-coumaroylhexoside
1 Means (n = 3 ± SD) with letters are significantly different after one-way ANOVA followed by Fisher’s LSD post hoc test (p = 0.05) considering the type of film as the source of variability.
Agronomy 2024, 14, 1491 14 of 20

All phenolic and anthocyanin compounds were tentatively identified by comparison

of their elution order, molecular formula, and both full and fragmentation mass spectra
(Tables 2 and 3) with data reported in the literature [6,35–38]. A mass error < 5 ppm was
considered for compound annotation. All analysed strawberry samples exhibited a highly
comparable composition, showing a chemical fingerprint that was almost superimpos-
able. In total, 43 phenolic compounds were attributed as constituents of strawberry fruit,
according to previously reported studies. The identified substances belong to different
classes, such as simple phenols (hydroxybenzoic acid derivatives and icariside F2), cinnamic
acid derivatives (coumaric and cinnamic acid glycosides), flavonoids (mainly flavonols as
kaempferol and quercetin glycosides), anthocyanins (pelargonidin and cyanidin glycosides),
ellagic acid and its glycoside, ellagitannins, and catechin together with proanthocyanidins
(in the form of dimers, trimers, tetramers, and larger molecules). Among the anthocyanins,
pelargonidin hexoside was the most represented compound in all of the samples. Although
the presence of strawberry metabolites was confirmed in all studied samples by LC-MS
metabolomic analysis, variation in their amount was observed among fruit from plants
under different light downconversion films. The change in the concentration of each
metabolite throughout the treatments was estimated by relative quantification integrating
the peak areas in the chromatograms obtained by LC-MS experiments (Tables 2 and 3).
Based on the results, blue, red, and pink downconversion films did not show benefit on an-
thocyanins production compared to the Cnt film. Indeed, a slight but significant decrease in
their level was observed (Table 3), particularly in fruit from plants exposed to the pink film
(isomer I of pelargonidin p-coumaroylhexoside, pelargonidin 3-O-glucoside, pelargonidin
malonylglucoside, pelargonidin derivative, cyanidin 3-O-glucoside, and cyanidin malonyl-
glucoside) and exposed to the red film (isomer I of pelargonidin p-coumaroylhexoside,
pelargonidin 3-O-glucoside, cyanidin malonylglucoside and a cyanidine derivative). On
the contrary, fruit from plants grown under the blue film showed a slight but significant
increase in the content of almost all of the other phenolic compounds (hydroxybenzoic
acid derivative, quercetin 3-O-glucoside, pedunculagin, strictinin, castalgin, digalloyl-
tetraHHDP-diglucose, ellagic acid and ellagic acid deoxyhexose, propelargonidin dimer,
procyanidin pentamer, tetramer and hexamer) compared to fruit from plants under the
Cnt film, even though the use of light downconversion films did not seem to dramatically
promote any class of metabolites or, inside each group, any individual phenolic molecules.
Anyway, the compounds wholly influenced were found to be ellagic acid conjugates and
ellagic tannins. In the plants subjected to red or pink films, no significant differences
were observed for most of the phenolic metabolites content when compared with those
found in the Cnt fruit, except for cinnamoyl xylosylglucose, the content of which was
significantly lower in fruit from plants under the pink film when compared with the Cnt
fruit. Interestingly, the proportion of the main metabolites in the strawberry samples was
not altered by the application of the light downconversion films.

4. Discussion
4.1. Strawberry Plant Growth Was Boosted by Light Downconversion Films, Especially by the
UV-to-Blue Shifting One
Light plays a pivotal role in modulating plant developmental processes, strongly
influencing photomorphogenesis and photosynthesis. This modulation arises from a
complex network of photoreceptors such as cryptochromes for blue light and phytochromes
(Pfr) for the red/far-red (R:FR) light ratio, acting as primary molecular channels, translating
light quality to photomorphogenesis signals as reviewed by Landi et al. [4]. There is a
growing interest in the cost-effective manipulation of the solar spectrum transmitted by
plastic film by enhancing the blue and red light, which is able to enhance the ability of
plants to use the film converted light as a “eustress” to induce an accumulation of bioactive
compounds in fruit [7]. Indeed, recent advances have introduced polyethylene films with
solar spectrum downconversion technology for tunnel crop applications. This innovative
technology employs fluorescent agents (dyes, organic and inorganic rare-earth complexes)
Agronomy 2024, 14, 1491 15 of 20

that are adept at converting less photosynthetically active wavelengths, such as green or
UV radiation, to more effective radiation, such as red and blue [3,20,21,23].
In our experiment, strawberry plants exhibited enhanced vegetative growth (both
aerial biomass and leaf area) with all of the light downconversion films tested herein.
Several studies have assessed the effect of downconversion films that promote red light
on plant biomass and nutraceutical characteristics [22,23,39,40]. For instance, Li et al. [39]
demonstrated that light downconversion film, by shifting green–yellow light wavebands
into orange–red light wavebands, significantly enhanced the crop yield of leafy vegetables,
especially in haze-prone regions like North China, by addressing weak light challenges and
boosting photosynthetically active radiation. Similarly, Shen et al. [22] achieved promising
results in enhancing lettuce yield by converting green light into more active red light. Other
reports include those of Hebert et al. [23] and Parrish et al. [40], who utilized quantum
dots in greenhouse films to shift UV/blue photons to red emissions to modify the sunlight
spectrum, resulting in improved plant growth parameters in tomato (+9% of vine growth)
and lettuce plants (+11% of fresh biomass and +13% leaf area).
Several factors could contribute to the enhanced development of plants exposed to
films that convert different light wavelengths into red light. According to our results,
some studies have reported increased photosynthetic rates in various plants, including
strawberries, pepper, and lettuce, when grown under light downconversion films that shift
blue or green wavelengths into red and far-red regions [3,41,42]. However, the pink, red
and blue films used in the present experiment did not influence the activity of PSII since its
photochemical efficiency in light downconversion was not statistically different from those
of plants under Cnt film at each plant stage according to the findings of Khramov et al. [42].
Therefore, the observed variations in carbon assimilation rates are likely attributable to
other intrinsic factors that influence photosynthesis. In our experiment using pink and
red films, the observed increased plant biomass could not just be related to the higher
percentage of available red light for photosynthesis. It is conceivable that a higher R:FR
light ratio increased the active form of Pfr due to the greater quantity of red light emitted
by these two films compared to the Cnt film. Elevated Pfr levels can lead to increased CO2
assimilation rates, promoting plant growth and resilience [42,43] and explaining the less
pronounced decrease in gas exchange parameters such as Pn and Pn /Ci rates during colder
periods at both fruiting stages.
Unfortunately, there is a lack of research into the impacts of UV-to-blue light con-
version films on plant physiology and development, with only a few reports [28,44]. In
these cases, it was observed that blue film can exert a stimulating growth effect on crop
species. For example, Hemming et al. [28] reported an increase in fruit production by
11% in strawberry plants grown under UV-to-blue light downconversion film, while
Guerrero-Gonzalez et al. [44] reported increased growth rate indices of about 125% in
seedlings of Ipomea compared to controls. In our experiment, the increase in the blue light
fluence under the blue film improved plant photosynthetic processes, as underscored by the
very elevated photosynthetic rate values with respect to controls in all of the analysed plant
stages. This increase in photosynthetic performances resulted in increased LMA since more
C was potentially available for the vegetative structures with respect to other films [45].
Notably, a higher blue light fraction can induce the development of sun-adapted leaves
with high LMA, thickness and photosynthetic capacity [46,47]. The elevated photosynthetic
rates in plants grown under the blue film were attributed to both leaf biochemistry, as
indicated by higher Pn /Ci values, and enhanced stomatal conductance, which remained
higher compared to the Cnt film at certain plant stages (T3 and T4). Indeed, it is worth
noting that Pfr can also respond in plants subjected to blue light wavelengths, even though
in these plants, the conversion to Pfr is less effective than in plants subjected to red light [4].
Additionally, in cucumber seedling leaves grown under pure blue light, Su et al. [48]
observed an increase in the Rubisco biosynthesis and related gene expression that, together
with a higher stomatal conductance, improved the photosynthetic process compared to
other monochromatic lights. The higher stomatal conductance exhibited by plants grown
Agronomy 2024, 14, 1491 16 of 20

under the blue film (compared to the others) was more evident during the fruiting stage,
coinciding with a significant temperature drop. This phenomenon was directly linked to
the properties of the additional blue light in stimulating stomatal opening, mediated by the
blue light absorbing carotenoid zeaxanthin [4].
Although research on plant physiological responses to blue film remains limited, blue
light exposure has shown remarkable effects on plant physiology, including increased
photosynthetic efficiency and positive influences on leaf morphology. These findings
emphasize the significance of light spectrum manipulation as a promising approach to
optimizing plant growth and resilience across different agricultural contexts. Our observa-
tions are in agreement with the work of Hemming et al. [28], where fluorescent pigments
in films emitting blue spectrum fluorescence have potential to positively affect the growth
and development of strawberry plants.

4.2. UV-to-Blue Downconversion Film Enhances the Accumulation of Polyphenols in

Strawberry Fruit
The current literature lacks any information concerning the accumulation of secondary
metabolites in fruit from crops cultivated under the influence of light downconversion films.
This conspicuous absence of data underscores the need for clarification and investigation in
this specific domain. To clarify how light downconversion films affect the accumulation of
secondary metabolites in fruit, findings related to the use of LED monochromatic lights, an
aspect that has been extensively studied [4,49,50], will be considered to discuss our results.
Anthocyanins are of pivotal importance in strawberries because, besides their powerful
antioxidant prerogatives [51], they are responsible for the bright fruit colour, thereby driving
consumer selection of the fruit [52]. It is reported in the literature that short and high-
energetic wavelengths, such as UV and blue, are the most efficient for stimulating the
increase in fruit anthocyanin contents [53,54]. However, there is also evidence that longer
wavelengths, such as red, can enhance the intensity of fruit redness [6,53]. Indeed, in
a previous work with monochromatic supplemental LED lighting, red light has proven
to be the most efficient in improving the accumulation of anthocyanins in strawberry
fruit [6]. The possible discrepancies between our results and others from studies using LED
monochromatic light can probably be attributed to the different growing conditions and
then to the different light quality and quantity.
In the present study, blue film induced a higher TPC and TFC in strawberry fruit than
Cnt, red and pink films. Indeed, blue film was the most promising light downconversion
film for increasing the phenolic compounds in strawberry fruit when compared with Cnt
film, with a specific effect on hydroxycinnamic acids (hydroxybenzoic acid derivative),
flavonoids (quercetin 3-O-glucoside), and most of the ellagitannins, ellagic acid conjugates
and condensed tannins identified in the present experiment. In particular, the contents of
quercetin 3-O-glucoside and ellagic acid conjugates increased by more than 50% in fruit
grown under blue film compared to Cnt fruit.
Ellagic acid and quercetin 3-O-glucoside are powerful antioxidant molecules with
many biological effects, including antioxidant, anti-inflammatory, antidiabetic, cardiopro-
tective, and neuroprotective effects [55,56], so their increase in fruit grown under the blue
film is interesting for human health. To the best of our knowledge, no research has been
conducted on the effect of blue light on ellagic acid content. However, it was reported
for Triticum aestivum [57], Camellia sinensis [58] and Pisum sativum [59] that there was an
increase in gallic acid content, a precursor of ellagic acid, in plants grown under blue
light. Furthermore, the observed increase in flavonoids, and especially quercetin 3-O-
glucoside, can be due to the ability of the blue light waveband to increase the activity of
shikimate dehydrogenase and the transcript levels of genes responsible for phenylalanine
ammonia-lyase (PAL) and flavonoid-3′ -hydroxylase (F3’H) [60,61].
While blue light enrichment has been shown to positively impact the accumulation of
TPC and TFC by stimulating the phenylpropanoid metabolism, it is important to note that
the use of all light downconversion films led to reduced levels of ascorbic acid (vitamin C)
Agronomy 2024, 14, 1491 17 of 20

in fruit. Despite the decrease in ascorbic acid, the antioxidant activity of the strawberries
grown under all of the light downconversion films remained comparable to that of Cnt
fruit, suggesting the compensatory effect of other ROS-scavenging molecules differentially
stimulated by downconversion films. Ascorbic acid plays a key role in plants, acting mainly
as an antioxidant in the response and adaptation to environmental stressors such as UV
radiation [62]. Previous studies have shown that exposure to UV radiation promoted
the increase in ascorbic acid content in vegetative and fruit tissues [63–65]. Conversely,
UV depletion resulted in reduced levels of ascorbic acid [66]. Therefore, we suggest that
the observed reductions in the ascorbic acid could be due to the higher limitation of UV
light transmission (Figure 2) to plants grown under light downconversion films compared
to Cnt.

5. Conclusions
Light downconversion films are promising for improving protected cultivation. The
common theme throughout this study is the potential to modify light conditions to optimize
plant photosynthesis and growth and, at the same time, to enhance the ability of plants to
use the converted light as a “eustress” to induce an accumulation of antioxidant and healthy
compounds in fruit. In this regard, plants developed under a blue film (UV-to-blue light
conversion) showed the highest vegetative growth, with an enhanced leaf area, thickness
and mass area and an elevated net photosynthetic rate associated with higher apparent
carboxylation efficiency values. At the same time, the use of the blue film induced an
increase in total phenolic and flavonoid contents in fruit and promoted the stimulation
of targeted health-related polyphenols. Therefore, the use of a pre-harvest blue film
resulted in an efficient solution to enhance the nutraceutical properties of fruit from tunnel
crops, maintaining their productivity and physiological plant status. These innovative
downconversion films, which combine traditional agricultural practices with cutting-edge
technologies, might play a vital role in ensuring food quality and security and sustainable
agricultural practices. Further research might focus on a deeper investigation into the
organoleptic quality induced by the use of these films to assess consumer appreciation of
the horticultural products grown under these conditions.

Author Contributions: Methodology, formal analysis, investigation, writing—original draft,

writing—review and editing, H.E.H.; methodology, investigation, formal analysis, writing—original
draft, writing—review and editing, M.V.; conceptualization, methodology, formal analysis, investi-
gation, writing—original draft, writing—review and editing, C.C.; conceptualization, methodology,
investigation, formal analysis, writing—original draft, writing—review and editing, E.L.P.; formal
analysis, investigation, data curation, writing—original draft, G.L.; methodology, formal analysis,
writing—review and editing, M.D.L.; methodology, formal analysis, writing—review and editing,
A.B.; writing—review and editing, L.I.; writing—review and editing, L.G.; writing—review and edit-
ing, R.M.; writing—review and editing, D.R.; conceptualization, project administration, methodology,
resources, writing—review and editing, supervision, M.L. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was carried out within the Agritech National Research Center and received
funding from the European Union Next-Generation EU (PIANO NAZIONALE DI RIPRESA E
RESILIENZA (PNRR)–MISSIONE 4 COMPONENTE 2, INVESTIMENTO 1.4–D.D. 1032 17/06/2022,
CN00000022).
Data Availability Statement: The data are contained within the article.
Acknowledgments: The authors acknowledge CASCADE SAS (Clamart, France), for the supply of
the light converting films.
Conflicts of Interest: The authors declare no conflicts of interest.
Agronomy 2024, 14, 1491 18 of 20

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