Spectral quality as an elicitor of bioactive compound production in Solanum aculeatissimum JACQ cell suspension

Journal Pre-proof Spectral quality as an elicitor of bioactive compound production in Solanum aculeatissimum JACQ cell suspension

Luciana Arantes Dantas, Márcio Rosa, Erika Crispim Resende, Fabiano Guimarães Silva, Paulo Sérgio Pereira, Ana Cristina Lourenço Souza, Fernando Higino de Lima e Silva, Aurélio Rubio Neto PII:

S1011-1344(19)30747-X

DOI:

https://doi.org/10.1016/j.jphotobiol.2020.111819

Reference:

JPB 111819

To appear in:

Journal of Photochemistry & Photobiology, B: Biology

Received date:

11 June 2019

Revised date:

4 February 2020

Accepted date:

8 February 2020

Please cite this article as: L.A. Dantas, M. Rosa, E.C. Resende, et al., Spectral quality as an elicitor of bioactive compound production in Solanum aculeatissimum JACQ cell suspension, Journal of Photochemistry & Photobiology, B: Biology(2020), https://doi.org/ 10.1016/j.jphotobiol.2020.111819

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier.

Journal Pre-proof SPECTRAL QUALITY AS AN ELICITOR OF BIOACTIVE COMPOUND PRODUCTION IN Solanum aculeatissimum JACQ CELL SUSPENSION

Luciana Arantes Dantasa, Márcio Rosaa, Erika Crispim Resendeb, Fabiano Guimarães Silvac, Paulo Sérgio Pereirad, Ana Cristina Lourenço Souzac, Fernando Higino de Lima and Silvac,

Plant Biotechnology, Program in Biotechnology and Biodiversity, Pro-Centro Oeste Network -

ro

a

of

Aurélio Rubio Netoc,*

-p

Federal Institute of Education, Science and Technology Goiano (IF Goiano), Rio Verde, GO, Brazil, [email protected]

re

b

[email protected]

Plant Tissue Culture Lab, IF Goiano, Rio Verde, GO, Brazil, [email protected]

na

c

lP

Department of Biomolecules, IF Goiano, Iporá campus, Iporá, GO, Brazil,

d

Biomolecules and Bioassays Laboratory, IF Goiano, Rio Verde, GO, Brazil,

*

Jo

ur

[email protected]

Corresponding author

Aurélio Rubio Neto E-mail: [email protected] Address: Rodovia Sul Goiana, Km 01, Zona Rural | Rio Verde - GO | CEP: 75.901-970 - Brazil Phone: + 55-64-3620 5617 Fax: +55-64-3620 5640

1

Journal Pre-proof Abstract Solanum aculeatissimum Jacq. is a common plant in much of Brazil. Despite containing metabolites with a wide range of pharmacological applications, there are few tissue culture reports for this plant. The possibility of large-scale in vitro production of this material has significant biotechnological potential. Therefore, the objective of this study was to investigate the effect of light conditions on the growth of cells in suspension, observing the production and

of

yield of biomass and bioactive compounds and the enzymatic behavior. Calli obtained from leaf

ro

segments were cultured in solid medium supplemented with 1 mg L-1 of 2,4-D, 2.5 mg L-1

-p

kinetin, pH 5.7, in the dark. After 110 days of subculture, the calli were transferred to liquid

re

medium. Cells were kept in the dark under agitation at 110 rpm and 25 °C and subcultured every 30 days. After 90 days of culture, 20 mL aliquots of cell suspension were added to flasks

lP

containing approximately 20 mL of medium (1:1) and cultured at different wavelengths (white,

na

green, blue, red, and blue/red) under a photoperiod of 16 h with irradiance of 50 μmol m-2 s-1 ) and in the absence of light. The experiment was performed in a 6 × 6 factorial design (light

ur

condition × culture time). The cell cultures showed viability throughout the entire cycle, and

Jo

chlorogenic and ferulic acids, orientin, quercitrin and, in higher amounts, quercetin, were detected in the first 7 days of culture. There was an increase in superoxide dismutase and catalase and a decrease in ascorbate peroxidase after exposure to different light conditions; for phenylalanine ammonia lyase, no differences were observed. The different light conditions were not sufficient to trigger responses in the concentrations of bioactive compounds, despite the detection of increased levels of the enzymes involved in cellular homeostasis.

Keywords: oxidative stress, phenolic compounds, enzymes

2

Journal Pre-proof

Abbreviations

ROS: Reactive Oxygen Species LED: Light-Emitting Diode MS: Murashige & Skoog

of

FDA: Fluorescein Diacetate

ro

PI: Propidium Iodide

-p

SOD: Superoxide Dismutase

APX: Ascorbate Peroxidase

lP

PAL: Phenylalanine Ammonia Lyase

re

CAT: Catalase

OR: Orientin QTIN: Quercetin

Jo

QTRIN: Quercitrin

ur

na

FA: Ferulic acid

PROT: Total Proteins

3

Journal Pre-proof 1 Introduction Solanum aculeatissimum Jacq. is present in tropical and subtropical regions in much of the world, and South America is the center of diversity and distribution of this species [1]. The Solanum genus has abundant bioactive metabolites in various parts of the plant, such as groups of steroidal alkaloids, pyridines, withanolides, sesquiterpenes and diterpenes, glycoalkaloids and flavonoids, among others, and this species can be a source of numerous compounds used for

of

medicinal purposes with cytotoxic, antifungal and antitumor effects [2,3].

ro

The potential of using cell culture for the production and accumulation of chemical

-p

compounds with the same bioactivity of the mother plant has been known since the emergence of

re

tissue culture techniques [4]. Sustainable cell cultures are a promising way to achieve a continuous, large-scale supply of compounds of interest because they have the flexibility to

lP

produce plant material anywhere in the world, regardless of the geographic or environmental

growth cycles [5,6].

na

conditions, and are free of microbiological and chemical contamination, with rapid and safe

ur

Despite these advantages, there are few reports of commercial plant cell culture

Jo

biofactories due to insufficient information about the metabolic pathways of the compounds of interest. This culture method has the potential to investigate various elicitation processes, avoiding or detecting possible interfering compounds through physiological and biochemical processes, in addition to providing valuable information that enables the screening of metabolically produced compounds [6]. Much of the difficulty related to the use of new lightbased methods is the lack of available literature on the subject, in addition to a lack of information on the types of lamps to be used, such as fluorescent or LED, hindering the reproduction of the method in other species [7].

4

Journal Pre-proof Spectral condition, photon flux and photoperiod are the main triggers for plant growth and development. Light-emitting diode (LED) technology has been recently used in several studies on micropropagation, both in vitro and ex vitro, being considered a great alternative light source, with an efficient emission of a certain radiation [6,8]. The advantages of LED lamps are low power consumption and a longer service life. A large part of the supplied energy is converted as desired, with minimal heat production and high energy efficiency, and they are able

of

to emit light at the desired color without the need to use filters, as is the case in a traditional

ro

fluorescent lamp [8].

-p

By subjecting the plant to severe stress, the cells can accumulate reactive oxygen species

re

(ROS). Exposure to several external biotic or abiotic stress factors triggers the biosynthesis of substances involved in cellular redox homeostasis, such as the enzymes superoxide dismutase,

lP

catalase, ascorbate peroxidase and phenylalanine ammonia lyase [9]. However, when there is

na

excessive production of ROS, the cell function is impaired by the oxidative stress that is generated, which has several adverse effects, such as enzymatic oxidation, membrane lipid

ur

peroxidation and DNA damage [10].

Jo

The objective of this study was to detect and quantify bioactive compounds of biotechnological interest in S. aculeatissimum and to analyze the regulatory behavior of the enzymes associated with oxidative stress that are generated by exposure to different light conditions. If substantial production of bioactive compounds of biological and pharmacological importance are observed, the study will provide relevant information on the biochemical response, in addition to preliminary data on the production yield an industrial scale.

2 Materials and methods

5

Journal Pre-proof The experiments were conducted at the Laboratory of Plant Tissue Culture of the Federal Institute of Education, Science and Technology Goiano (IF Goiano) - Rio Verde Campus, State of Goiás (GO), Brazil. Approximately 200 ripe fruits of S. aculeatissimum were collected from plants located in the municipality of Rio Verde, at coordinates 17º 48’ 343’’ S - 50º 54’ 00’’ W, altitude 616 m. After collection, the fruits were pulped to remove the seeds and subsequently

of

dried at 35 °C in a forced air oven.

-p

ro

2.1 In vitro establishment

re

2.1.1 Seed disinfection and in vitro establishment

The S. aculeatissimum seeds were wrapped in gauze and immersed in running water for

lP

30 minutes, followed by 70% ethanol for 1 minute and then immersed in a sodium hypochlorite

na

(NaClO) solution (20% commercial solution) containing one droplet of polysorbate (Tween®) for 15 minutes. The seeds were then rinsed three times with autoclaved, distilled water in a laminar

ur

flow cabinet to remove the disinfection solution residue.

Jo

Disinfected seeds were aseptically inoculated into test tubes (25 × 150 mm) containing 10 mL of medium Murashige & Skoog (MS) [11] with 50% salt, 30 g L-1 sucrose, 3.5 g L-1 agar and the pH was adjusted to 5.7 ± 0.3. The medium was autoclaved at 121 °C and a pressure of 1.05 kg cm-2 for 20 minutes. The test tubes were sealed with a plastic lid (polypropylene) and kept in a growth room at 25 ± 3 ºC with a relative humidity of 45 ±%. The medium was exchanged every 30 days. The tubes were maintained for a 16 h photoperiod under photosynthetically active radiation of 45-55 μmol m-2 s-1 , provided by fluorescent lamps.

6

Journal Pre-proof 2.1.2 Callus induction Leaf fragments (1 cm2 ) of previously established seedlings were inoculated into glass flasks containing 40 mL of 50% MS medium, 30 g L-1 sucrose and 3.5 g L-1 agar, and the pH adjusted to 5.7 ± 0.3 (Figure 1A and B). A combination of the growth regulators kinetin (KIN) (2.5 mg L-1 ) and 2,4-dichlorophenoxyacetic acid (2,4-D) (2.5 mg L-1 ) was added to the culture medium. The flasks were kept in a room under the same environmental conditions as described

of

above in the absence of light. 12

Branca white

6

4

2

8

6

4

2

0

E

H

-2

-2

400

AZUL blue

450

500

550

600

650

700

750

800

12 300

Comprimento de onda (nm)

8

6

lP

4

2

0

F

-2 12300

350

400

450

500

550

600

650

VermelhaComprimento de onda (nm) red

700

750

800

10

400

450

500

550

600

650

700

750

800

8

6

4

2

0

I

-2

300

350

400

450

500

550

600

650

700

750

800

Comprimento de onda (nm)

Wavelength (nm)

na

10

8

6

4

2

0

-2

300

350

Azul/Verm. blue/red Comprimento de onda (nm)

re

10

350

Irradiancia (Unidade relativa)

300

350

400

450

500

550

600

650

750

800

G

A

B

C

D

Jo

Coprimento de onda (nm) Wavelength (nm)

700

ur

Irradiância (Unidade relativa)

Irradiance Irradiância(relative (Unidade relativa) unit)

0

10

ro

Irradiância (Unidade relativa)

Verde green 8

-p

Irradiancia (Unidade Relativa)

10

Figure 1. S. aculeatissimum Jacq. cell culture under different spectral compositions (green, blue, red, white, blue/red light) with irradiance of 50 μmol m-2 s-1 (AE); leaf segment used for induction of callogenesis (F), callus induced after 30 days (G), friable calli after 90 days of induction (H) and stable cell suspension (I); Bar = 1 cm.

2.1.3 Suspension cell culture

7

Journal Pre-proof To establish the suspension cell culture, friable calli (Figure 1C) were selected and transferred to glass flasks containing 40 mL of liquid medium with their respective salt concentrations and growth regulators in which they were grown. The cultures were placed under agitation (LS -183, SolabT M shaker table) at 100 rpm under the same light and temperature conditions described above. After three months, stable and

of

contamination-free cultures were selected (Figure 1D) to evaluate their kinetics.

-p

ro

2.2 Abiotic elicitation

re

2.2.1 Exposure to different light conditions

To begin the elicitation proces, approximately 20 mL of inoculum and 20 mL of medium

lP

were added, identical to cell subculturing. The cultures were maintained for seven days for

na

adaptation. They were subsequently transferred to environments illuminated with different LED lamps (white light: 300-750 nm, blue: 400-490 nm, green: 490-560 nm, red: 600-700 nm and

ur

blue/red 1:1) using a metal frame attached to the lamps over the shakers (Figure 1E-I), and the

Jo

samples were subjected to a photoperiod of 16 h, with photosynthetically active radiation of 50 μmol m-2 s-1 . The spectral composition was determined using a USB2000 (OceanOptics) spectroradiometer. Evaluations were performed every seven days for 35 days. The control suspensions were cultured in the absence of light under the same environmental conditions as the others, also under agitation.

2.2.2 Biometric evaluations and cell viability monitoring

8

Journal Pre-proof At the end of the elicitor exposure period, the pH and electrical conductivity of the suspensions were measured, and they were then filtered through 0.45 μm filter paper to remove the culture medium. After filtration, the retained cell masses were weighed to determine the fresh weight and then dried in a forced air oven at 35 °C for 24 h and weighed again to obtain the dry mass. For the cell viability analysis, 100 μL of the S. aculeatissimum cell suspension was added

of

to 2 mL microtubes along with 100 μL of trypan blue dye (Sigma-Aldrich, Brazil) at 0.4% (v/v).

ro

For analysis and counting under an optical microscope, 50 μL aliquots of the mixture were

-p

transferred to slides, and cover slips were placed over the samples to trap the suspension. A total

re

of 100 cells were counted per sample to find the equivalent value of cell viability in percentage, and the entire procedure was performed in duplicate. To validate the viability analysis, the dyes

lP

fluorescein diacetate (FDA) and propidium iodide (PI) were used. The cells that emitted green

na

fluorescence were considered viable and those that emitted red fluorescence were considered nonviable. This analysis was performed using a fluorescence microscope (Olympus BX 60), with

ur

filters at excitation/emission wavelengths of 460/510 nm for FDA and 530/615 nm for PI and a

Jo

total magnification of 100× [12].

2.2.3 Phytochemical evaluations The extracts were prepared using 0.1 g of dry mass with 2 mL of HPLC-grade methanol (Neon) in an ultrasound bath for 30 minutes. Filtering was then performed in a cotton membrane filter (Advantec HP020AN - 20 μm). Next, the chromatographic analysis was performed using a Shimadzu HPLC with a SPD-M20A photodiode array detector (λ = 254 nm) and an LC18

9

Journal Pre-proof column (25 cm × 4.6 mm, 5 μm, SupelcosilT M) coupled to a 2 cm LC18 pre-column (Supelguard, Supelco), and the oven was set at 30 ºC (S1.1). The compounds present in the samples were detected by comparison with the peaks of known phenolic and flavonoid standards and were quantified using the equations for the following standards: gallic acid, epicatechin, caffeic acid, chlorogenic acid, ferulic acid, orientin, vitexin, rosmarinic acid, myristicin, isovitexin, hesperidin, rutin, quercetin-3-glucoside,

of

kaempferol-3-galactoside, quercetin, kaempferol-3-glucoside, kaempferol-3-rutinoside,

-p

ro

quercitrin, kaempferol, luteolin and apigenin.

re

2.2.4 Enzymatic evaluations

To determine the activity of the enzymes involved in the cellular detoxification process,

lP

0.3 g of the cell suspension was macerated in a mortar and pestle with liquid nitrogen and

na

contained 2 mL of the following extraction medium: 50 mM potassium phosphate buffer (pH 6.8), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonyl fluoride

ur

(PMSF), and 2% polyvinylpyrrolidone (PVP). The enzyme extract was centrifuged at 12,000 g

Jo

for 15 minutes at 4 °C, and the supernatant was used as the crude extract. Superoxide dismutase (SOD) activity was determined by measuring the enzyme’s ability to photochemically reduce nitro blue tetrazolium (NBT). A unit of SOD is defined as the amount of enzyme required to inhibit 50% of NBT photoreduction (S1.2) [13]. Catalase (CAT) activity was determined by the rate of hydrogen peroxide (H2 O2 ) breakdown at 240 nm for 1 minute at 25 ºC (S1.3). A molar extinction coefficient of 36 M-1 cm-1 was used for calculations of enzyme activity. For ascorbate peroxidase (APX) activity. The APX activity was measured by the oxidation rate of the ascorbic acid at 290 nm for 1 minute at 25 °C.

10

Journal Pre-proof A molar extinction coefficient of 2.8 mM-1 cm-1 was used to calculate the APX activity (S1.4). The methodology proposed by Data et al. [14] with modifications was used to quantify the phenylalanine ammonia lyase (PAL) activity. One unit of activity was defined as 1 μmol of trans-cinnamic acid formed per minute under the assay conditions (S1.5).

2.2.5 Determination of total protein content

of

To determine the total soluble protein content, the methodology proposed by Bradford

ro

[15] was used, with bovine serum albumin (BSA) as the standard curve. The mixture was then

re

-p

read in a spectrophotometer (Shimadzu) at 595 nm (S1.6).

2.3 Statistical analysis

lP

The experimental design was completely randomized in a 6 × 6 factorial arrangement,

na

with six spectral bands (white, green, blue, red, blue/red and dark) and six culture times (0, 7, 14, 21, 28 and 35 days) and 4 replicates (40 mL/flask). The data were statistically evaluated by

ur

analysis of variance with the F test (5%), and the means were analyzed by regression analysis for

Jo

quantitative variables and by Tukey’s test for qualitative variables using SISVAR software [16]. Pearson’s correlation was used to assess relationships between the variables. To obtain the estimates, the software R version 3.5.2 was used, and a heatmap was created using the Corrplot package.

3 Results and discussion

3.1 Biometric evaluations and cell viability monitoring

11

Journal Pre-proof Independent of the light condition applied to the in vitro culture, the suspensions exhibited a linear increase in fresh and dry weight as a function of culture time, reaching 10.48 g and 0.64 g, respectively (Figure 2A and B). At 35 days, a higher dry biomass was observed when cells were cultured under blue/red light, reaching 0.70 g (Figure 3). The increase in biomass production in suspension cultures is promoted by an appropriate balance between the auxin and cytokinin concentrations in cell proliferation, as observed in Linum usitatissimum L. cv. Modran

of

cell cultures, and regardless of the light band evaluated, the cells were able to growth [17].

A 11

0.7

re

0.6

lP

dry weight (g)

9

7

na

fresh weight (g)

10

8

5 0

7

14

ur

6

21

Jo

days of cultivation

28

B

y= 0.00406x + 0.29673 **r2=0.82 y= 0.004114x + 0.281460 **r2=0.58 y= 0.00505x + 0.28097 r2=0.86 y= 0.00512x + 0.27760 **r2=0.68 y= 0.00467x + 0.28964 **r2=0.63 y= 0.00067x2 - 0.01509x + 0.33745 **r2=0.82

-p

y= 0.1426x + 5.3859 **r2=0.93

ro

MF

0.5

0.4

0.3

0.2

35

0

7

14

21

28

35

days of cultivation

Figure 2. Fresh (A) and dry (B) weight of S. aculeatissimum Jacq. cells cultured under green, blue, white, red, blue/red light and darkness. The bars indicate the standard error of the means; significance level: **p<0.01.

12

ro

of

Journal Pre-proof

-p

Figure 3. Dry weight at 35 days of S. aculeatissimum Jacq. cell culture after exposure to green,

re

blue, white, red, and blue/red light. Means were compared using Tukey’s test. The bars indicate

lP

the standard error of the means.

na

Biomass accumulation is due to cell growth, and the inability of cells to use light energy

ur

as a source of energy indicates that at certain periods of culture it is entirely heterotrophic, as observed in cells of Thevetia peruviana (Pers.) K. Schum [18]. Even in this nutritional condition,

Jo

cells exert primary the functions of photosystem II (PSII) that may be important, such as in O 2 production and survival [19].

Similar to our observations, the light condition was shown to influence the biochemical and morphological characteristics of Silybum marianum L. and Fagonia indica L. [19,20]. Some authors have shown that white light leads to a decrease in cell growth due to photochemical alterations of the culture medium [20]. In S. aculeatissimum, however, this decrease was not observed.

13

Journal Pre-proof The pH values varied over time and after exposure to different light conditions (Figure 4A). Regardless of the light condition, a quadratic behavior was observed that explains the variation in the pH of the medium as a function of the culture time. During cell growth, the concentrations of hydrogen ions in the medium change; the pH of the medium decreases during the assimilation of ammonia and increases during the absorption of nitrate [5]. This same result was observed in Ajuga multiflora Bunge cells. According to the authors, the decrease in the

of

initial pH can be attributed to the adaptation process to the growth environment, and its increase

ro

is due to the absorption of ammonium ions [21]. Monitoring the electrical conductivity in all

-p

culture conditions showed a linear decrease over time (Figure 4B), and as biomass increased,

re

there was a decrease in conductivity due to the cell consumption of ions from nutrients in the culture medium, with consumption occurring gradually [21].

2.0

ur

6.0

Jo

pH

6.2

5.8 5.6

electric conductivity (µs cm-1)

y= 0.0018x2 - 0.0655 + 6.2687 **r2=0.73

6.4

Condutividade

B 2.2

na

6.6

lP

pH A

y= 1.9687 - 0.0352x **r2=0.97

1.8 1.6 1.4 1.2 1.0 0.8

5.4

0.6 0

7

14

21

days of cultivation

28

35

0

7

14

21

28

35

days of cultivation

Figure 4. pH (A) and electrical conductivity (B) of the S. aculeatissimum Jacq culture medium. Cell suspension after exposure to different light conditions throughout the 35-day cell cycle. Means were compared using Tukey’s test. The bars indicate the standard error of the means.

14

Journal Pre-proof

The cell viability was above 85% in all of the light conditions, as assessed by both fluorescein diacetate (Figure 5A and B) and trypan blue staining (Figure 5C and D). The cells were predominantly small and isodiametric with meristematic features. They were able to multiply; therefore, they were used for further study. When culturing Byrsonima verbascifolia L. (DC) calli, the evaluation of cell viability and morphology provided important information for

of

the selection of cells with embryogenic competence [12]. Similarly, the S. aculeatissimum cells

Jo

ur

na

lP

re

-p

ro

showed high multiplication capacity and high viability.

Figure 5. Images obtained by epifluorescence microscopy showing the viability of S. aculeatissimum Jacq. cells stained with fluorescein diacetate (FDA). Green fluorescence (A) indicates viable cells. When staining with propidium iodide (PI), red fluorescence (B) indicates nonviable cells. Staining with trypan blue reveals viable cells (white) and nonviable cells (blue) (C). (D) Characteristic cell clusters seen in the culture; v: viable cells; nv: nonviable cells; cc: cell clusters.

15

Journal Pre-proof 3.2 Phytochemical evaluations In the elicitation process proposed in this study, gallic acid, rosmarinic acid, kaempferol, luteolin, apigenin, vitexin, isovitexin, rutin, myricitrin, hesperidin, quercetin-3-glucoside, epicatechin, kaempferol-3-galactoside, kaempferol-3-glucoside and kaempferol-3-rutinoside were not detected in the S. aculeatissimum cell suspension. However, chlorogenic acid, ferulic acid, orientin, quercitrin and quercetin were detected (S2.1, S2.2).

of

There was an effect of time on the concentration and yield of chlorogenic acid, but it was

ro

not significantly influenced by the light conditions (Figure 6A), where differences occurred only

-p

between the length of culturing. On the first day of culture, the concentration of chlorogenic acid was higher (0.177 mg g-1 ), but after 7 days, its concentration decreased by half (0.087 mg g-1 ),

re

and at 14 days, there was a drop of 85% (0.026 mg g-1 ) relative to the initial concentration. At 21

lP

and 28 days, the metabolite was not detected, but during the 35 days, low concentrations were

na

detected when compared to that in the beginning of the culture (0.011 mg g-1 ). Regarding the

Jo

day (0.058 mg/flask).

ur

yield per flask, the same behavior occurred, with the highest concentration occurring on the first

16

Journal Pre-proof B 0.05

0.15

0.15

0.10

0.10

0.05

0.05

0.05

0.04

0.04

0.03

0.03

0.02

0.02

0.01

0.01

0.00

0.00

0.00 14 21 days of cultivation

28

35

0 concentration yield

C

-p

y= 0.0054 - 0.00016x **r2= 0.90 y= 0.001825 - 0.00005316x **r2= 0.88

0.004

0.003

0.003 0.002

na

0.002

0.001

0.000 7

14

21

days of cultivation

28

Jo

0

ur

0.001

35

0.000

21

28

35

days of cultivation

D 0.018 0.016

0.014

0.014

0.012

0.012

0.010

0.010

0.008

0.008

0.006

0.006

0.004

0.004

0.002

0.002

0.000

0.000

re

0.004

-1

0.005

lP

0.005

14

y= 0.01526 - 0.00048x **r2=0.85 y= 0.005171 - 0.000158x **r2= 0.81

0.016

0.006

quecetrin (mg g )

0.006

0.018

0.007

orientin (mg/ flask)

0.007

7

0

7

14

21

28

35

days of cultivation

Figure 6. The concentration and yield of chlorogenic acid (A), ferulic acid (B), orientin (C) and quercitrin (D) in S. aculeatissimum Jacq. cells cultured under an irradiance of 50 μmol m-2 s-1 with different light conditions.

There was a quadratic behavior for the concentration and yield of ferulic acid as a function of the culture time. There was no interaction between the factors. There was no isolated effect of the light condition on the concentration of this compound (Figure 6B). The highest 17

quecetrin (mg/ flask)

7

ro

0

orientin (mg g-1 )

y= 0.00005x2 -0.002x + 0.03986 **r2=0.74 y= 0.00002x2 -0.00096x + 0.01344 **r2=0.66

of

0.00

ferulic acid (mg g-1 )

0.20

y= 0.0003x2 - 0.0149x + 0.1718 **r2= 0.97 y= 0.625701x2 - 0.004856x + 0.05869 **r2= 0.97

chlorogenic acid (mg/ flask)

chlorogenic acid (mg g-1)

0.20

ferulic acid (mg/ flask)

A

Journal Pre-proof concentration was observed after 7 days of culture (0.037 mg g-1 ); after 14 days, its concentration decreased by 87% (0.005 mg g-1 ), and the minimum value found throughout the cycle was 0.001 mg g-1 at 21 days. There was a gradual increase (0.004 mg g-1 ) up to 35 days (0.011 mg g-1 ) after 28 days. Regarding yield, the highest values found were after 7 days (0.013 mg/flask) of culture. The production and yield of orientin showed a decreasing linear trend over time, and as the culture time increased, there was a decrease in these parameters. The light condition did not

of

have an interaction with the culture time to produce any effect (Figure 6C). On the first day, the

ro

detected concentration was 0.005 mg g-1 , and after 21 days, there was a decrease (0.0024 mg g-1 )

-p

of 50% compared to the first day of culture. The same trend was observed for yield, where

re

0.0019 mg/flask was detected on the first day, and a 50% drop in yield was observed, decreasing to 0.0010 mg/ flask.

lP

Regarding quercitrin production, the light condition and its interaction with the culture

na

time did not have any effect, and the maximum concentration found was observed after 7 days of culture (0.015 mg g-1 ), with a linear decrease. The minimum concentration (0.001 mg g-1 ) was

ur

observed after 35 days. Similarly, the yield of this compound peaked after 7 days of culture,

Jo

producing 0.005 mg/ flask (Figure 6D). For quercitrin production, there was a linear decrease (Figure 7A) over the culture time. The highest concentration was found on the first day of culture (0.432 mg g-1 ), and the lowest concentration was 0.209 mg g-1 at 35 days. There was no interaction between the factors.

18

Journal Pre-proof

B

A y= 0.397 - 0.0060x **r2=0.91

0.18

0.45 quercetin (mg/ flask)

0.16

0.40 0.35 0.30 0.25

0.14 0.12 0.10 0.08

0.20

of

0.06

0.02

0.15 0

7

14

21

28

ro

0.04 0

35

14

21

28

35

days of cultivation

-p

days of cultivation

7

re

Figure 7. Concentration (A) and yield (B) of quercetin in S. aculeatissimum Jacq. cells cultured

lP

under an irradiance of 50 μmol m-2 s-1 with different light conditions. The bars indicate the

na

standard error of the means.

There was variation in the yield of quercetin (Figure 7B). The results showed a quadratic

ur

behavior in the treatment with white light, reaching a maximum yield on the first day (0.156 mg/flask) and a minimum concentration (0.062 mg/flask) at 14 days of culture The yield under

Jo

quercetin (mg g-1 )

blue ( y= 0.135108 - 0.002116x **r2= 0.72) blue/red ( y= 0.117325 - 0.001723x *r2= 0.51) white ( y= 0.00020x2 - 0.0015x + 0.15612 **r2=0.59)

0.20

0.50

both blue light and blue/red light behaved linearly, both showing a maximum production (0.135 mg and 0.117 mg/flask) at the beginning of culture and a minimum production (0.061 mg and 0.057 mg/flask) at 35 days. The other light conditions did not differ from each other. The elicitation process with different light conditions over time did not favor the production of quercetin in S. aculeatissimum cells. The effect of different light conditions on the production of secondary antioxidant metabolites was investigated in Prunella vulgaris L. calli, and high levels of phenolic compounds were observed when the cells were grown under blue light [21]. Similar results were 19

Journal Pre-proof observed for Stevia rebaudiana (Bert) calli [7]. However, in the present study with S. aculeatissimum, this behavior was not observed. This confirms a previous report [22] demonstrating that the biochemical and physiological response varies among species. In S. marianum, white and red light were considered to be the best light conditions for the accumulation of flavonoids and phenols. Red light also favors antioxidant activity and superoxide dismutase (SOD) activity, while the dark spectrum favors peroxidase activity [23].

of

When comparing the effect of red and blue light on the biosynthesis of phenolic compounds and

ro

flavonoids between Lactuca sativa L. and Ocimum basilicum L., it was observed that the

-p

biosynthesis of these compounds was species-dependent, i.e., it depends on the genetic makeup

re

of the species [24].

When evaluating the effect of different light conditions in T. peruviana cell culture on the

lP

phenolic content of cells grown in the dark when compared to light, the authors found a greater

na

accumulation of phenolic content and a higher antioxidant activity. In this particular case, light may have a deleterious effect on the production of polyphenols, whereas darkness has a

ur

protective effect [18]. However, it was not possible to confirm this effect in S. aculeatissimum,

Jo

as cells exposed to both dark and light conditions did not show differences in the production of phenolic compounds.

Comparing the phenolic and flavonoid content in the cell suspension with Artemisia absinthium L. calli, there was variation in the production of these compounds according to culture time, with the highest production at 6 days in suspension and after 30 days in the calli [4]. Similarly, in S. aculeatissimum, the highest level of phenolic compounds in the current study was detected at the beginning of culturing and decreased over time. Light was not a determining factor for the production of the phenolic compounds in S.

20

Journal Pre-proof aculeatissimum cells; therefore, there was variation in the concentrations over the culture time. The phytochemical responses to the elicitation process are due to the type of elicitors and the length of culture time [25]. For cell cultures with a low accumulation of secondary compounds, in many cases this may not be due to a lack of key biosynthetic enzymes but rather to inhibitory feedback mechanisms, enzymatic degradation (or lack thereof), or to volatilization from the

of

culture [5].

ro

3.3 Enzymatic evaluations

-p

The results obtained for proteins from the enzymatic extracts of cells grown under

re

different light conditions show a quadratic behavior, with no difference between the light conditions. After 9 days, the amount of protein increased 9.68% relative to the initial amount. At

lP

28 days, there was a decrease of 61% in protein content relative to the maximum peak, and the

na

minimum value was observed after 35 days of culture. In Prunella vulgaris L. callus cultures under different light conditions, the total protein content also did not increase during culture [26],

Jo

under red light [24].

ur

but in the culture of Artemisia absinthium L. calli, the highest total protein content was observed

Regarding SOD activity, there was no difference after exposure to the different light conditions, with a small concentration at the beginning of culturing that gradually increased over time, reaching a maximum at 35 days (Figure 8A). This increase was pronounced under blue light in Oncidium sp. seedlings [27] and in Fagonia indica L. callus cultures when comparing the different light conditions [19]. In Prunella vulgaris L., the maximum SOD activity was observed under yellow light [23], in contrast to S. aculeatissimum cells, where SOD activity did not vary among the different light conditions over the culture time.

21

Journal Pre-proof A 0.020

B y= 0.000001x2 - 0.000005x + 0.000203 **r2=0.86

y= 0.0002x + 0.0099 *r2=0.71

0.0010

0.0008

0.016 0.014

0.0006

0.012 0.0004

0.010 0.008

0.0002

µmol-1 min-1 mg protein

U min-1 mg-1 de protein

0.018

7

14

21

28

35

0

days of cultivation APX (µmol)

0.0000 21

28

35

ro

D 120

100

re

0.18

PAL

-p

y= 0.193959 - 0.002942x **r2=0.82

0.20

80

0.12

^y = y 72.28

na

0.10

60

-1

0.14

-1

lP

0.16

0.06 0

7

14

ur

0.08

21

days of cultivation

28

35

40

20 0

7

14

21

28

35

days of cultivation

Jo

µmol-1 min-1 mg protein

14

days of cultivation

C 0.22

7

nmol min mg protein

0

of

0.006

Figure 8. SOD (A), CAT (B), APX (C) and PAL (D) activity in S. aculeatissimum Jacq. cells cultured under different light conditions. The bars indicate the standard error of the means.

SOD activity acts as part of the defense system of plant cells, providing better tolerance to stress conditions and counteracting the increase in ROS [9]. SOD acts as the first line of cell defense against the oxidation of lipid membranes, whose integrity plays a central role in cell viability and defense against free radicals [28]. We observed that the primary detoxification 22

Journal Pre-proof system of S. aculeatissimum cells was efficient because the cells were growing and viable throughout the culture cycle. Regarding the CAT activity in the cells, there were no differences between the different light conditions, but the CAT activity varied over the culture time (Figure 8B). On the first day, the activity was low (0.0002 μmol-1 min-1 mg protein); after 21 days, the activity doubled (0.0004 μmol-1 min-1 mg protein) and, compared to the first day, there was a four-fold increase

of

(0.0008 μmol-1 min-1 mg protein) at 35 days. Regarding APX activity, there was no effect of light

ro

or its interaction with the culture time, but a gradual decrease in its activity was observed over

-p

time (Figure 8C), showing a decreasing linear trend throughout the whole culture cycle, with the

re

lowest activity (0.0873 μmol-1 min-1 mg protein) at 35 days.

In S. aculeatissimum cells, there was no difference regarding the light conditions, but the

lP

increase of CAT throughout the culture was similar to that of Oncidium sp. seedlings [27] and

na

Eutrema salsugineum (Pall.) calli. Despite the considerable increase in CAT activity, this previous study reports that the increase is likely associated with ROS formation, metabolic

ur

acceleration, senescence, or apoptosis. After the formation of hydrogen peroxide (H2 O2 ) by SOD

Jo

(which occurs either spontaneously or not), the CAT enzyme becomes more active at higher H2 O 2 concentrations, whereas APX has a higher affinity for low concentrations in the conversion of H2 O2 into water and molecular O 2 [29]. The PAL activity results showed no difference after exposure to different light conditions, and there was no interaction with time. During the culture period, the activity of this enzyme was maintained relatively constant, with a mean value of 72.28 nmol-1 min-1 mg of protein (Figure 8D).

23

Journal Pre-proof Under white fluorescent light in Prunus sp., the in vitro culture showed an increased PAL expression and, consequently, an increased production of phenolic compounds and reduced lignification [30]. In S. aculeatissimum, such accumulation of phenolic compounds did not occur even after culturing under different light conditions. However, the variation in the PAL activity observed during the culture cycle suggests that the pathway that regulates this production derives from multiple branches of the biosynthesis and that the response intensity is regulated at the

of

transcriptional level of the genes involved with the cell wall [7]. In Dianthus caryophyllus L.

ro

grown in vitro, red LED light significantly increased the PAL activity, and the lowest activity

-p

levels were found using white fluorescent light [31]. In Picea abies (L.) Karst, the PAL gene is

re

regulated under blue LED light [32].

The findings varied among the investigations due to the different light sources

lP

(fluorescent and LED) used and the variation inherent to the different species. General effects of

na

most abiotic stresses are often dependent on the genotype, organism age, stress intensity, and time of exposure to the stress [22].

ur

LED technology has a high potential because it provides clean and interference- free

Jo

spectra; however, it is not a limiting factor for the physiological and biochemical responses in S. aculeatissimum cell culture because there are numerous intrinsic and extrinsic factors involved in the entire biosynthesis process for possible compounds of biotechnological interest. We emphasize the importance of this study in the elucidation of the physiological and biochemical mechanisms of a hardy species that is underappreciated and rarely studied.

3.4 Correlation

24

Journal Pre-proof The correlation coefficients were estimated to assess the association between the variables analyzed, except for chlorogenic acid and yield, as their values were null (Figure 9). To this end, among the analyzed times, we chose to obtain estimates for the intermediate period of 21 days. This time period was chosen because at the beginning (7 days), there was no elicitation, i.e., the mean values did not vary between the evaluated light conditions, and at the end (35

ur

na

lP

re

-p

ro

of

days), the lowest values were observed.

Jo

Figure 9. Estimates of correlation Heatmap according to Pearson’s correlation coefficient (dry and fresh weight; pH; electrical conductivity; concentrations of ferulic acid, orientin, quercetin, quercitrin, and the enzymes CAT, APX, PAL, and SOD) after 21 days of Solanum aculeatissimum Jacq. cell culture. ** and * are significant at 1 and 5% using the t-test, respectively. DW, dry weight; FW, fresh weight; FA, ferulic acid; OR, orientin; QTIN, quercetin; QTRIN, quercitrin; PROT, total proteins; CAT, catalase; APX, ascorbate peroxidase; PAL, phenylalanine ammonia lyase; SOD, superoxide dismutase; pH, potential of hydrogen; EC, electrical conductivity. ** and * are significant at 1 and 5% by the t-test, respectively.

25

Journal Pre-proof There were positive and significant correlations (p<0.05) between dry weight and fresh weight (0.89) and PAL levels (0.88). The observed correlation between the dry and fresh weight was expected, as plants with greater biomass accumulation tend to have a higher dry biomass. Furthermore, there was a negative correlation between the dry weight and the electrical conductivity of the culture medium (-0.88), demonstrating that the greater the amount of dry biomass, the greater the amount of PAL and the lower the conductivity of the medium. In regard

of

to fresh weight, a negative correlation (p<0.01) was observed with the electrical conductivity of

ro

the medium (-0.93), where a higher fresh weight resulted in a lower conductivity (S3).

-p

There was evidence of a high positive and significant correlation (p<0.01) between CAT and the pH of the medium (0.94), suggesting that the higher the amount of CAT in the cells, the

re

higher the pH of the medium. This increase in pH is due to the transport of NO 3 - through an

lP

active process (symport system) with simultaneous transport of H+ and NO 3 - into the cells,

na

triggering an increase in the potential of hydrogen in the culture medium [33]. However, there is still a lack of data that explain the correlation between the CAT enzyme and the possible increase

ur

in the pH of the cell culture medium.

Jo

A negative and significant (p<0.05) correlation was observed between APX and quercetin (-0.83), where higher amounts of quercetin corresponded to lower amounts of APX. The enzyme APX requires ascorbic acid as a reducing agent. Despite having a high affinity for H2 O2 , APX is involved in the ascorbate-glutathione cycle, where H2 O2 is formed by SOD [34]. This action requires a reducing molecule (cytochrome or thioredoxin) to act as a cofactor for regeneration to remove H2 O 2 [35]. Complementary studies of these cofactors are necessary to determine whether they are actually responsible for the production of quercetin, because the increased quercetin in the experiments with S. aculeatissimum confirms the decrease in APX.

26

Journal Pre-proof Future proteomic, metabolomic and genomic studies may contribute to a better understanding of the biochemical networks responsible for cellular responses to oxidative stress, helping to elucidate the role of ROS in the physiological and biochemical aspects of in vitro cell culture.

Conclusion

of

Regardless of the light condition, S. aculeatissimum cell cultures showed viability

ro

throughout the cycle, and metabolites with biotechnological applications were detected, such as

-p

chlorogenic and ferulic acids, orientin, quercitrin and, in greater amounts, quercetin, especially

re

until up to 7 days of culture.

There was an increase in SOD and CAT activity and a decrease in APX activity

lP

regardless of the light condition throughout the entire in vitro culture; however, there was no

na

difference in PAL. It was evident that the repair mechanism was efficient because the cells continued to grow and remained viable.

ur

The different light conditions were not sufficient to trigger responses in the

enzymes.

Jo

concentrations of the bioactive compounds despite the increased detection of antioxidant

Acknowledgements We thank the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES), the National Council for Scientific and Technological Development (CNPq), the

27

Journal Pre-proof Goiás Research Foundation (FAPEG), the Pro-Centro Oeste Network and the Federal Institute of Education, Science and Technology Goiano (IF Goiano) for their support.

Declarations of interest The authors declare that they have no conflict of interest.

J.B. Harborne, H. Baxter, Phytochemical Dictionary. A Handbook of Bioactive

ro

[1]

of

References

L.A. Dantas, A.M. Melo, P. Pereira, L. Souza, S. Filho, F.G. Silva, Histochemical

re

[2]

-p

Compounds from Plants, Taylor and Francis, London, Washington DC, 1993.

screening of leaves compared to in situ and in vitro calluses of Solanum aculeatissimum

F.C.L. Pinto, D.E.A. Uchoa, E.R. Silveira, O.D.L. Pessoa, R. Braz-Filho, F.M. Silva,

na

[3]

lP

Jacq, J. Agric. Sci. 9 (2017) 80.

P.N.E.T. Theodoro, L.S. Espíndola, Glicoalcaloides antifúngicos, flavonoides e outros

M. Ali, B.H. Abbasi, H. Ihsan ul, Production of commercially important secondary

Jo

[4]

ur

constituintes químicos de Solanum asperum, Quím. Nov. 34 (2011) 284-288.

metabolites and antioxidant activity in cell suspension cultures of Artemisia absinthium L, Ind. Crop. Prod. 49 (2013) 400-406. [5]

S.R. Rao, G.A. Ravishankar, Plant cell cultures: chemical factories of secondary metabolites, Biotechnol. Adv. 20 (2002) 101-153.

[6]

K. Ramirez-Estrada, H. Vidal-Limon, D. Hidalgo, E. Moyano, M. Golenioswki, R.M. Cusido, J. Palazon, Elicitation, an effective strategy for the biotechnological production

28

Journal Pre-proof of bioactive high-added value compounds in plant cell factories, Molecules 21 (2016) 182. [7]

N. Ahmad, A. Rab, N. Ahmad, Light-induced biochemical variations in secondary metabolite production and antioxidant activity in callus cultures of Stevia rebaudiana (Bert), J. Photochem. Photobiol. B 154 (2016) 51-56.

[8]

S.D. Gupta, A. Karmakar, Machine vision based evaluation of impact of light emitting

of

diodes (LEDs) on shoot regeneration and the effect of spectral quality on phenolic

ro

content and antioxidant capacity in Swertia chirata, J. Photochem. Photobiol. B 174

G. Agati, E. Azzarello, S. Pollastri, M. Tattini, Flavonoids as antioxidants in plants:

re

[9]

-p

(2017) 162-172.

location and functional significance, Plant Sci. 196 (2012) 67-76. F. Martelli, F.M.F. Nunes, Radicais livres: em busca do equilíbrio, Ciên. Cult. 66 (2014)

lP

[10]

[11]

na

54-57.

T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tabacco

R.B. Chiavegatto, A.H.F. Castro, M.G. Marçal, M.S. Pádua, E. Alves, V.H. Techio, Cell

Jo

[12]

ur

tissue culture, Physiol. Plant. 15 (1962) 473-497.

viability, mitotic index and callus morphology of Byrsonima verbascifolia (Malpighiaceae), Tropic. Plant Biol. 8 (2015) 87-97. [13]

C. Beauchamp, I. Fridovich, Superoxide dismutase: improved assays and an assay applicable to acrylamide gels, Anal. Biochem. 44 (1971) 276-287.

[14]

E.S. Data, M.A. Quevedo, L.A. Gloria, Pruning techniques affecting the root quality of cassava at harvest and subsequent storage, in: I. Uritani, E.D. Reyes (Eds.) Tropical Root Crops: Postharvest Physiology and Processing, JSSP, Tokyo, 1984, pp. 127-143.

29

Journal Pre-proof [15]

M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248-254.

[16]

D.F. Ferreira, Sisvar: a computer statistical analysis system., Ciênc. Agrotéc. 35 (2011) 1039-1042.

[17]

A. Seta-Koselska, E. Skórzyńska-Polit, Optimization of in vitro culture conditions for

of

obtaining flax (Linum usitatissimum L. cv. Modran) cell suspension culture, Bio.

J.P. Arias, K. Zapata, B. Rojano, M. Arias, Effect of light wavelength on cell growth,

-p

[18]

ro

Technol. 98 (2018) 183-188.

re

content of phenolic compounds and antioxidant activity in cell suspension cultures of Thevetia peruviana, J. Photochem. Photobiol. B 163 (2016) 87-91. P.P. Pashkovskiy, T.N. Soshinkova, D.V. Korolkova, A.V. Kartashov, I.E. Zlobin, V.Y.

lP

[19]

na

Lyubimov, V.D. Kreslavski, V.V. Kuznetsov, The effect of light quality on the pro/antioxidant balance, activity of photosystem II, and expression of light-dependent genes

T. Khan, B.H. Abbasi, M.A. Khan, The interplay between light, plant growth regulators

Jo

[20]

ur

in Eutrema salsugineum callus cells, Photosynth. Res. 136 (2018) 199-214.

and elicitors on growth and secondary metabolism in cell cultures of Fagonia indica, J. Photochem. Photobiol. B 185 (2018) 153-160. [21]

Y.C. Wang, J.J. Zhao, D.F. Chi, beta-Ecdysterone accumulation and regulation in Ajuga multiflora Bunge suspension culture, 3 Biotech. 8 (2018) 87.

[22]

U. Tariq, M. Ali, B.H. Abbasi, Morphogenic and biochemical variations under different spectral lights in callus cultures of Artemisia absinthium L, J. Photochem. Photobiol. B 130 (2014) 264-271.

30

Journal Pre-proof [23]

M. Younas, S. Drouet, M. Nadeem, N. Giglioli-Guivarc'h, C. Hano, B.H. Abbasi, Differential accumulation of silymarin induced by exposure of Silybum marianum L. callus cultures to several spectres of monochromatic lights, J. Photochem. Photobiol. B Biol. 184 (2018) 61-70.

[24]

K. Taulavuori, V. Hyöky, J. Oksanen, E. Taulavuori, R. Julkunen-Tiitto, Species-specific differences in synthesis of flavonoids and phenolic acids under increasing periods of

D. Mahendran, P.B.K. Kishor, S. Sreeramanan, P. Venkatachalam, Enhanced

ro

[25]

of

enhanced blue light, Environ. Exp. Bot. 121 (2016) 145-150.

-p

biosynthesis of colchicine and thiocolchicoside contents in cell suspension cultures of

re

Gloriosa superba L. exposed to ethylene inhibitor and elicitors, Ind. Crops Prod. 120 (2018) 123-130.

H. Fazal, B.H. Abbasi, N. Ahmad, S.S. Ali, F. Akbar, F. Kanwal, Correlation of different

lP

[26]

na

spectral lights with biomass accumulation and production of antioxidant secondary metabolites in callus cultures of medicinally important Prunella vulgaris L, J.

L. Mengxi, X. Zhigang, Y. Yang, F. Yijie, Effects of different spectral lights on

Jo

[27]

ur

Photochem. Photobiol. B Biol. 159 (2016) 1-7.

Oncidium PLBs induction, proliferation, and plant regeneration, Plant Cell Tissue Org. Cult. 106 (2011) 1-10. [28]

H. Aldesuquy, H.A. Elshafii, H. Hanem, Exogenous silicon ameliorate alkalinity stress in sorghum (Sorghum bicolor L.) plants by up-regulating the membrane characteristics and antioxidant capacity defense, Adv. Agric. Technol. Plant Sci. 1 (2018) 180002.

[29]

I. Heiser, W.F. Osswald, Formação e função das espécies reativas de oxigênio nas interações planta-patógeno, in: S.F. Pascholati, B. Leite, J.R. Stangarlin, P. Cia (Eds.)

31

Journal Pre-proof Interação Planta-Patógeno – Fisiologia, Bioquímica e Biologia Molecular, FEALQ, Piracicaba, 2008, pp. 249-283. [30]

A. Pina, P. Errea, Differential induction of phenylalanine ammonia- lyase gene expression in response to in vitro callus unions of Prunus spp, J. Plant Physiol. 165 (2008) 705-714.

[31]

A. Manivannan, P. Soundararajan, Y.G. Park, H. Wei, S.H. Kim, B.R. Jeong, Blue and red light-emitting diodes improve the growth and physiology of in vitro-grown carnations

F. OuYang, J. Wang, Y. Li, Effects of cutting size and exogenous hormone treatment on

ro

[32]

of

‘green beauty’ and ‘purple beauty’, Horticult. Environ. Biotechnol. 58 (2017) 12-20.

-p

rooting of shoot cuttings in Norway spruce [Picea abies (L.) Karst.], New For. 46 (2015)

[33]

re

91-105.

G. Rocha, L.M. Ferreira, O.C.H. Tavares, A.M. Santos, S.R. Souza, Cinética de absorção

lP

de nitrogênio e acúmulo de frações solúveis nitrogenadas e açúcares em girassol,

[34]

na

Pesquisa Agropecu. Tropic. 44 (2014) 381-390. P. Sharma, A.B. Jha, R.S. Dubey, M. Passarakli, Reactive oxygen species, oxidative

ur

damage, and antioxidative defense mechanism in plants under stressful conditions, J.

[35]

Jo

Botan. 2012 (2012) 217037.

A. Mhamdi, G. Noctor, A. Baker, Plant catalases: peroxisomal redox guardians, Arch. Biochem. Biophys. 525 (2012) 181-194.

32

Journal Pre-proof Highlights

All light conditions resulted in increased biomass and cell viability.



The light condition was not a determining factor of phenolic compound production.



Bioactive compounds were detected in higher quantities at seven days of culture.



The redox process repair mechanism was efficient in the cell cultures.

Jo

ur

na

lP

re

-p

ro

of



33

Journal Pre-proof Authors contributions Fabiano and Paulo was responsible for funding acquisition; Luciana, Márcio and Ana did the original draft; Writing – review & editing and performed the experiments; Luciana, Erika and Paulo did Supervision; Validation and performed the chromatographic analyzes; Luciana and Márcio performed enzymatic analyzes; Luciana, Aurélio, Fabiano and Fernando contributed to Project administration; Resources; Software; Supervision of the research and

Jo

ur

na

lP

re

-p

ro

of

to the writing of the paper.

34

Journal Pre-proof Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Jo

ur

na

lP

re

-p

ro

of

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

35