Keywords:

Phytonutrients

Fragaria x ananassa

Rubus adenotrichos

Antioxidants

HPTLC

Biokinetic mechnisms of anthocyanins in red fruits produced in the state of Michoacan, Mexico

Mecanismos biocinéticos de antocianinas en frutos rojos producidos en el estado de Michoacán,

México

Mecanismos biocineticos das antocianinas em frutos vermelhos produzidos no estado de Michoacan,

Mexico

Jesús Di Carlo Quiroz Velásquez

Cristian Lizarazo Ortega

Jesús Gerardo García Olivares

Jorge Alberto Torres Ortega

Israel García León

Jose Luis Hernández Mendoza

Rev. Fac. Agron. (LUZ). 2023, 40(3): e234029

ISSN 2477-9407

DOI: https://doi.org/10.47280/RevFacAgron(LUZ).v40.n3.07

Food Technology

Associate editor: Dra. Gretty R. Ettiene Rojas

University of Zulia, Faculty of Agronomy

Bolivarian Republic of Venezuela

Instituto Politécnico Nacional – Centro de Biotecnología

Genómica. Laboratorio de Biotecnología Experimental.

Ciudad Reynosa Tamaulipas, México.

Instituto Politécnico Nacional – Centro de Biotecnología

Genómica. Laboratorio de Biotecnología Vegetal. Ciudad

Reynosa Tamaulipas, México.

Instituto Politécnico Nacional – Centro de Biotecnología

Genómica. Laboratorio de Biotecnología Ambiental. Ciudad

Reynosa Tamaulipas, México.

Received:

15-05-2023

Accepted: 18-07-2023

Published: 28-08-2023

Abstract

Berry fruits are a rich source of phytonutrients, especially phenolic

compounds such as avonoids, which have antioxidant properties. Among

these fruits, the most cultivated and consumed are those of the genus

Fragaria (Strawberries) and Rubus (Raspberries, blackberries, dewberries),

which have been widely studied for their benecial eects on human and

animal health. One of the most important bioactive compounds of these

fruits are anthocyanins, which have shown potential benets for health by

their antimicrobial, anti-inammatory and anticancer activity. Therefore, the

study of anthocyanins is of great pharmaceutical and nutraceutical interest.

The objective of this research is to analyze the biokinetic mechanisms of

anthocyanins in Rubus adenotrichos and Fragaria x ananassa produced

in the state of Michoacán, Mexico. For this purpose, research strategies

that included the extraction and quantication of anthocyanins, as well

as bioinformatic tools to understand their biosynthetic pathway in the

mentioned fruits were used. The use of informatic platforms allowed to

identify the regulatory genes and enzymes involved in the biosynthesis of

anthocyanins in R. adenotrichos and F. x ananassa, nding that most are

common, with some specic dierences, and that there are only a few

exceptions, such as the enzymes catechol-O-methyltransferase (OMT),

UDP-glucosyltransferase (UGT) and beta-glucuronidase (GUSB), which

only occur in Rubus adenotrichos and not in Fragaria x ananassa.

This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.

Rev. Fac. Agron. (LUZ). 2023, 40(3): e234029. July-September. ISSN 2477-9407.2-7 |

Resumen

Los frutos del bosque o berries son una fuente rica de

tonutrientes, especialmente de compuestos fenólicos como los

avonoides, que tienen propiedades antioxidantes. Entre estos frutos,

los más cultivados y consumidos son los del género Fragaria (Fresas)

y Rubus (Frambuesas, moras, zarzamoras), que han sido ampliamente

estudiados por sus efectos benécos para la salud humana y animal. Uno

de los compuestos bioactivos más importantes de estos frutos son las

antocianinas, que han demostrado potenciales benecios para la salud

por su actividad antimicrobiana, antiinamatoria y anticancerígena.

Por ello, el estudio de las antocianinas es de gran interés farmacéutico

y nutraceútico. El objetivo de esta investigación es analizar los

mecanismos biocinéticos de las antocianinas en Rubus adenotrichos y

Fragaria x ananassa producidos en el estado de Michoacán, México.

Para ello, se utilizaron estrategias de investigación que incluyeron la

extracción y cuanticación de antocianinas, así como herramientas

bioinformáticas para comprender su vía biosintética en los frutos

mencionados. El uso de las plataformas informáticas permitió

identicar los genes reguladores y las enzimas que intervienen en

la biosíntesis de antocianinas en R. adenotrichos y F. x ananassa,

encontrando que la mayoría son comunes, con algunas diferencias

especícas, y que solo hay unas pocas excepciones, como las enzimas

catecol-O-metiltransferasa (OMT), UDP-glucosiltransferasa (UGT)

y beta-glucuronidasa (GUSB), que solo se presentan en Rubus

adenotrichos y no en Fragaria x ananassa.

Palabras clave: tonutrientes, Fragaria x ananassa, Rubus

adenotrichos, antioxidantes, HPTLC.

Resumo

Os frutos silvestres ou berries são uma fonte rica de tonutrientes,

especialmente de compostos fenólicos como os avonoides, que têm

propriedades antioxidantes. Entre estes frutos, os mais cultivados

e consumidos são os do gênero Fragaria (morangos) e Rubus

(framboesas, amoras, amora-preta), que têm sido amplamente

estudados por seus efeitos benécos para a saúde humana e animal.

Um dos compostos bioativos mais importantes destes frutos são as

antocianinas, que têm demonstrado potenciais benefícios para a saúde

pela sua atividade antimicrobiana, anti-inamatória e anticancerígena.

Por isso, o estudo das antocianinas é de grande interesse farmacêutico

e nutracêutico. O objetivo desta pesquisa é analisar os mecanismos

biocinéticos das antocianinas em Rubus adenotrichos e Fragaria x

ananassa produzidos no estado de Michoacán, México. Para isso,

foram utilizadas estratégias de pesquisa que incluíram a extração e

quanticação de antocianinas, bem como ferramentas bioinformáticas

para compreender sua via biossintética nos frutos mencionados.

O uso das plataformas informáticas permitiu identicar os genes

reguladores e as enzimas envolvidas na biossíntese de antocianinas

em R. adenotrichos e F. x ananassa, encontrando que a maioria são

comuns, com algumas diferenças especícas, e que há apenas algumas

exceções, como as enzimas catecol-O-metiltransferase (OMT), UDP-

glucosiltransferase (UGT) e beta-glucuronidase (GUSB), que só

ocorrem em Rubus adenotrichos e não em Fragaria x ananassa.

Palavras chave: tonutrientes, Fragaria x ananassa, Rubus

adenotrichos, antioxidantes, HPTLC.

Introduction

Berries are crops that have great agricultural potential, due to

their protability, being an activity with high labor requirements,

versatility in production for consumption and wide export possibilities

(Lagunes-Fortiz et al., 2020). Mexico has a cultivated area of berries

and has high productive potential in the state of Michoacán, Mexico.

(SAGARPA, 2017) in addition to exporting about 41 % of the national

production of berries to countries such as: Netherlands, United

States of America and Canada (Mexicana et al., 2019) Strawberries

(Fragaria x ananassa), belonging to the Rosaceae family, contain

particularly abundant secondary metabolites. These metabolites have

been of great interest in the investigation of phenolic compounds

such as avonols, anthocyanins, proanthocyanidins, phenolic acids,

ellagitannins, and galiolglucoses (Haugeneder et al., 2018). The

conjugated bonds of anthocyanins result in owers and fruits with

purple, blue, and red coloration (Salinas Moreno et al., 2013).

Since anthocyanins are polar in nature they can be easily dissolved

in dierent solvents such as methanol, ethanol, water and acetone.

High performance thin layer chromatography (HPTLC) analytical

techniques are widely used for the quantication of anthocyanins.

The objective of this research is to analyze the biokinetic mechanisms

of anthocyanins in Rubus adenotrichos and Fragaria x ananassa

produced in the state of Michoacán, Mexico.

Materials and methods

Comparative study of metabolic pathways

A study focused on the anthocyanin biosynthetic pathway in Rubus

adenotrichos and Fragaria x ananassa was carried out by developing

the metabolic pathway of anthocyanin synthesis and breakdown in

the genus Rubus based on data recorded in NCBI, GDR, KEGG and

BlastKOALA,

Anthocyanin extraction and HPTLC analysis

Multiple lots of “Sayulita” strawberries and “Tupy” blackberries

were acquired from the agricultural regions of the state of Michoacán.

The strawberries were obtained from “Cerrito de Cotijarán” and the

blackberries were obtained from the municipality of “Los Reyes

de Salgado”, both samples in a ripe, fresh, and rm consistency.

The batches of each species were mixed with a high-performance

dispersion instrument to acquire more representative samples. For

anthocyanin extraction, the procedure described by Brito et al. (2014)

was used, which consisted of taking three (3) g of sample and 15 mL

of acidied ethanol (Ethanol and 1N HCl; 85:15 v/v) and macerated

in a mortar. Subsequently, the solutions were vortexed vigorously and

the pH was adjusted to 1 with hydrochloric acid.

The solutions were then shaken (EBERBACH

reciprocating

shaker) at 120 rpm for 16 h at room temperature. After this time, the

solutions were centrifuged at 3,000 rpm for 30 min, the supernatant

was recovered, and made up to 25 mL with acidied ethanol. Samples

were stored at -20 °C until use. Subsequently, 1 mg of the standard

anthocyanins pelargonidin-3-glucoside and cyanidin-3-glucoside in

chloride salt form (Sigma-Aldrich®, USA) was dissolved in 10 mL

of acidied methanol respectively (0.5% HCl) (0.1 mg.mL

-1

). After

sample preparation, the samples were analyzed by HPTLC. Silica

gel plates 60, 10 x 20 cm, with uorescence indicator F254 (Merck

Switzerland) were used. A 25 µL syringe was used for sample

application. Sample application was performed automatically using

the Automatic Sampler 4 (ATS4, Camag

) at a distance of 8 mm from

This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.

Quiroz et al. Rev. Fac. Agron. (LUZ). 2023 40(3): e234029

3-7 |

the bottom edge of the plate. The left - right distance (edges) for a plate

with 14 applications was 48 mm. The distance between applications

was 8 mm. The samples were applied in the form of 6 mm strips. A

sample volume of 8 µL of Rubus adenotrichos and 15 µL of Fragaria

x ananassa was applied. After sample application, the plates were

automatically developed in the Automatic Development Chamber 2

(ADC 2, Camag

), with the following conditions: equilibration time

ve (5) min, temperature 20 ± 2 °C, relative humidity was adjusted to

33 ± 2 % (MgCl

, 260 g per 100 H

O). The humidity at the beginning

of the analysis was 77 % and at the end was 67 %. The plate was

developed in 10 mL of mobile phase (ethyl acetate/acetic acid/acetic

acid/formic acid/water (AE/AA/AF/A), ratio 100:11:11:11:27, v/v/v/

v/v). The migration distance was 70 mm and the migration time was

30 min. After development, the plate was dried for ve (5) min, in the

same chamber. The developed plates were removed from the chamber

and evaluated with transmitted white light, UV light 366 nm and 254

nm using the TLC Visualizer (Camag

). The data were processed with

Camag

visionCATS software. After chromatographic separation, the

plate was heated in the TLC Plate Heater III (Camag

) at 100 °C for

ve (5) min. Derivatization with a 1 % solution of Natural Products

was carried out to reveal phenolic and avonoid compounds (1 g of

diphenylboryloxyethanolamine diluted in 100 mL of methanol) in the

TLC Immersion Device III (Camag

) at a vertical speed of 3 cm.s

-1

The immersion time was three (3) seconds. After derivatization, the

plate was dried with cold air for ve (5) min.

Linearity and quantication

The verication of the normal distribution of the results was

evaluated with the linearity through the relationship between the

concentration of cyanidin-3-glucoside, pelargonidin-3-glucoside

and the absorbance of the HPTLC detector. The calibration line was

performed for the cyanidin-3-glucoside and pelargonidin-3-glucoside

compounds that were analyzed and, thus, to know the extent of the

total variability of the response that could be explained by the linear

regression model.

The quantication of the concentration of the compounds was

determined through a working factor obtained by dividing the

concentration of the working standard (mg.L

-1

) by the absorbance

milli-units of the area in the standard. Subsequently, the sample

values were obtained and the respective factor of the standard was

multiplied by the area of the test sample.

Identication of genes of anthocyanin biosynthetic enzymes

Genomic DNA (gDNA) was extracted from the samples using

the specialized Wizard Genomic DNA Purication Kit Technical

Manual - Promega, following the manufacturer’s instructions. The

oligonucleotides described by Chen et al. (2012) were used (table 1).

PCR reactions were performed on a Veriti thermal cycler from Applied

Biosystems, with the following program: an initial denaturation cycle

at 95 ºC for 3 min, 25 denaturation cycles at 95 ºC for 30 s, alignment

at 55 ºC for 30 s and extension at 72 ºC for 30 s, and a nal extension

cycle at 72 ºC for 3 min and 34 s.

Results and discussion

Comparative study of metabolic pathways

Tables 2a and 2b show the results of the enzymes involved in

anthocyanin biosynthesis in Rubus adenotrichos and Fragaria x

ananassa as a comparative control method.

The development of anthocyanins in ripening berries involves

the coordinated expression of genes encoding a number of enzymes

involved in phenylpropanoid synthesis. Recent research has focused

on the development of heat maps depicting the transcriptomic proles

of the key avonoid pathway and anthocyanin biosynthetic enzymes,

modifying enzymes, and their respective transcriptional regulatory

genes (Garcia-Seco et al., 2015; Thole et al., 2019). Based on the

comparative analyses performed, it was observed that the biosynthetic

enzymes are: phenylalanine ammonia lyase (PAL), anthocyanidin

reductase (ANR), chalcone synthase (CHS), anthocyanidin synthase

(ANS), chalcone isomerase (CHI), dihydroavonol 4-reductase

(DFR), avonone 3-hydroxylase (F3H), avonone synthase (FNS)

and avonol synthase (FLS). Similarly, it was recorded that the main

pathway modifying enzymes are uridine diphosphate-glucose (UDP),

avonoid-O-glucosyl transferase (UFGT) and beta-glucuronidase

(GUSB); while the transcriptional regulatory genes are MYB, bHLH

and WDR (Jaakola, 2013).

Table 1. List of oligonucleotides for amplication of genes of interest in Rubus adenotrichos (R.a) and Fragaria x ananassa (F.a),

describing the size of the expected products.

Gene Description Amplied Sequence (5’-3’) Amplicon reported

pRuCHS

F CAG TGA CAC CCA CCT TGA CAGT 58

R TGC TGC ACC ATC ACC GAAT

pRuANS

F GGC CTC GGG AAA AAT TCA AG 56

R GCC CGG AAG CAT TGT TTG

pRuDFR

F ACA GTT CGA AGG CTG GTG TTT AC 63

R CTT CTG GTG CTC TTC GAC ATA CA

pRuGT

F GGA GCT GAA GAA AAG ACT CCA GAA 63

R GCC CGG AAG CAT TGT TTG

pRuANR

F TCT CTG ATG GCT GGT GCT AGT C 60

R CGT GGC GAG GCC AAT ACT

pRuLAR

F GCA TCC TTC CGA GGT TGT TC 62

R TGA CAG TGC CAT CAC CGT AGA

pβ-actina

F TGA CAA TGG GAC TGG AAT GGT 57

R GCC CTG GGA GCA TCA TCA

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Rev. Fac. Agron. (LUZ). 2023, 40(3): e234029. July-September. ISSN 2477-9407.4-7 |

Taking into consideration the name of the enzyme present in

the metabolic pathway, its respective enzyme identication code

was searched in KEGG and recorded in the comparative table. In

the process, to achieve the acquisition of the participating enzyme

code, BlastKOALA Rubus chingii was searched. Based on the

results acquired from the platform, each enzyme that was available

was identied and corroborated, and it was compared whether it

was also present in Fragaria x ananassa. Subsequently, the enzyme

identication code KEGG was introduced in the NCBI platform for

the GenBank code registry.

HPTLC analysis

In the samples of Rubus adenotrichos and Fragaria x ananassa,

it was observed that both fruits had abundant concentrations of the

main anthocyanin by which they are recognized. The chromatograms

obtained from the comparison of fruit samples with the standard

samples of cyanidin-3-glucoside and pelargonidin-3-glucoside

are shown below, the results are shown in gures 1 and 2 and also

visualizing the retention factor (Rf) of the compound pelargonidin-

3-glucoside (Pg3G), which was 0.54, while for cyanidin-3-glucoside

(Cy3G) it was 0.43 (gures 1 and 2, respectively).

Table 2a. Comparison of enzymes involved in anthocyanin biosynthesis for Rubus adenotrichos and Fragaria x ananassa.

KEGG Gene Bank Enzyme Biosynthesis pathway Rubus adenotrichos Fragaria x ananassa

EC 4.3.1.24 AAF40223,1 Phenylalanine ammonia lyase (PAL) phenylpropanoid O O

EC:1.3.1.77 AMP19723,1 Anthocyanidin reductase (ANR) Flavonoid O O

EC:1.14.204 AQP31154,1 Anthocyanidin synthase (ANS) Flavonoid O O

EC:2.3.1.74 AEQ61979,1 Chalcone synthase (CHS) Flavonoid O O

EC:5.5.1.6 S14705 Chalcone isomerase (CHI) Flavonoid O O

EC:1.1.1.219 AXK92787,1 Dihydroavonol 4-reductase (DFR) Flavonoid O O

EC 1.14.20.5 CAP09052,1 Flavonova synthase (FNS) Flavonoid X O

EC 1.14.11.9 ABX74780,1 Flavonone 3 - hydroxylase (F3H) Flavonoid O O

Note: In the comparative table, the symbol “O” represents that the genus carries the enzyme, while “X” represents that at least one of the fruits does not have the enzyme

or it has not been reported.

The comparative table was elaborated based on the data registered in “G.D.R”, “K.E.G.G” and “BlastKOALA”, for the verication of the reported ndings.

Table 2b. Continued comparative of enzymes involved in anthocyanin biosynthesis for the genus Rubus adenotrichos and Fragaria x

ananassa.

KEGG Gene Bank Enzyme Biosynthesis pathway Rubus adenotrichos Fragaria x ananassa

EC 1.14.20.6 AAZ78661, Flavonol Synthase (FLS) Flavonoid O O

EC 2.1.1.6 AEA30241,1 Catechol O - methyltransferase (OMT) Betalains O X

EC 1.17.1.3 AFD54430,1 Leucoanthocyanidin reductase (LAR) Flavonoid O O

EC:1.14.20.4 AAG50980,1 Leucoanthocyanidin deoxygenase (LDOX) Flavonoid X O

EC:2.4.1.271 AWT04749,1 UDP-glycosyl transferase (UGT) Secondary metabolites O X

EC2.4.1.91 AF171901,1 Flavonoid-O-glycosyl transferase (UFGT) Flavone and Favonol O O

EC3.2.2.31 WP000945878,1 Beta-glucuronidase (GUSB) Flavone and Flavonol O X

Note: In the comparative table, the symbol “O” represents that the genus carries the enzyme, while “X” represents that at least one of the fruits does not have the enzyme

or it has not been reported.

The table was prepared based on the data recorded in “G.D.R”, “K.E.G.G” and “BlastKOALA”, for the verication of the reported ndings.

Figure 1. Chromatogram of Fragaria x ananassa. Chromatogram

of strawberry extracts derivatized with Natural

Products, with white light. One plate required to

perform the experiment in triplicate (15 µL). From right

to left, last ve lanes: Pelargonidin-3-glucoside (Rf =

0.54). From left to right, Lanes 1-9: natural strawberry

fruits.

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Figure 2. Chromatogram of Rubus adenotrichos. Chromatogram

of blackberry extracts derivatized with Natural

Products, with white light. Only one plate required to

perform the experiment in triplicate (8 µL). From right

to left, last ve lanes: Cyanidin-3-glucoside (Rf = 0.43).

From left to right, Lanes 1-9: natural blackberry fruits.

The quantication data of Pg3G present in the extracts of Fragaria

x ananassa and Cy3G in Rubus adenotrichos can be visualized in

table 3. The anthocyanin quantication data corresponding to the

concentration in volume, amount applied on the band of the plate and

amount of anthocyanin present in one gram of dry weight are shown

in table 3.

The performance data of the HPTLC method for the determination

of anthocyanins in the extracts are shown in table 4, the method was

validated for instrumental precision, repeatability, specicity and

linearity.

Records described by (Hurtado and Pérez, 2014) mention that the

approximate amount of pelargonidin-3-glucoside that can be found

in dry weight in strawberries is around 726.14 µg.g

-1

. While, for

blackberries the amount of cyanidin-3-glucoside that can be found

in the fruit in dry weight range from 0.30 to 0.40 mg.g

-1

(Bhagwat et

al., 2013).

Table 3. Quantication of Cy3G and Pg3G.

Rubus adenotrichos

Cyanidin-3-glucoside (Samples)

Volume 8 mL Concentration (mg.mL

-1

) ng.Band

-1

mg.g

-1

dry weight

Track 2 12.29 98.35 0.31

Track 3 14.48 115.9 0.36

Track 4 16.61 132.9 0.42

Track 5 19.29 154.4 0.48

Track 6 19.84 158.7 0.5

Track 7 18.76 150.1 0.47

Track 8 17.42 139.4 0.44

Track 9 12.73 101.8 0.32

Mean 16.43 131.44 0.41

Std. Dev. 2.95 23.6 0.07

Rubus adenotrichos

Pelargonidin-3-glucoside (Samples)

Volume 15 mL Concentration (mg.mL

-1

) ng.Band

-1

mg.g

-1

dry weight

Track 2 56.18 842.7 1.40

Track 3 56.02 840.3 1.40

Track 4 60.68 910.2 1.52

Track 5 67.73 1.016 1.69

Track 6 68.86 1.033 1.72

Track 7 67.74 1.016 1.69

Track 8 58.45 876.8 1.46

Mean 62.24 933.57 1.56

Std. Dev. 5.72 85.83 0.14

Note: Anthocyanin quantication data corresponding to the concentration in volume, amount applied on the plate band and amount of anthocyanin present in one gram of

dry weight.

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6-7 |

Table 4. HPTLC Analysis Results.

Genre Compound Rf Equation Regression

Mode

Correlation

coecient ®

Coecient of

Variation

Rubus adenotrichos

Cyanidin-3-glucoside 0.43

Y = 3.485 x 10

-7

Linear 91.383026 % 14.1693 %

Fragaria x ananassa

Pelargonidin-3-glucoside 0.54

Y = [(2.908 x 10

-1

x) / (3.587 x 10

-6

+ x)] – 2.809 x 10

-2

Polynomial 99.527551 % 3.7992 %

Note: Performance data of the HPTLC method for the determination of cyanidin-3-glucoside in Rubus adenotrichos and pelargonidin-3-glucoside in Fragaria x ananassa.

The ve (5) point calibration curve indicates limits of

quantication for each fruit: 1.1 - 5.5 (µg.Band

-1

) for Pg3G in F. x

ananassa and 1.1 - 3.3 (µg.Band

-1

) for Cy3G for R. adenotrichos.

gures 3 and 4 respectively.

Figure 3. Polynomial regression line for Fragaria x ananassa.

Polynomial calibration of the pelargonidin-3-glucoside

standard performed by maximum height in white

light, with a correlation coecient of 99.527551 %. (Y

= [(2.908 x 10

-1

x) / (3.587 x 10

-6

+ x)] - 2.809 x 10

-2

Figure 4. Linear regression line for Rubus adenotrichos. Linear

calibration of the cyanidin-3-glucoside standard

performed by maximum height in white light, with a

correlation coecient of 91.383026 %. (Y = 3.485 x 10

-7

x).

The HPTLC analysis proved to be an excellent technique to

obtain the anthocyanin concentrations in the fruits of R. adenotrichos

and F. x ananassa, based on what is reported in the literature.

Molecular identication of anthocyanins

The enzymes that perform the conversion of phenylalanine to

anthocyanins and proanthocyanidins are: chalcone synthase (CHS),

dihydroavonol reductase (DFR), anthocyanidin synthase (ANS),

glycosyltransferase (GT), leucoanthocyanidin reductase (LAR) and

anthocyanidin reductase (ANR). The rst 3 enzymes CHS, DFR and

ANS allow the generation of former substrates for anthocyanins and

avonols. While the last mentioned enzymes GT, LAR and ANR,

perform glycosylation processes to increase the water stability of

anthocyanins and redirect two distinct pathways for the synthesis of

avonols and proanthocyanidin polymers, respectively (Chen et al.,

2012).

In the PCR analysis of Rubus adenotrichos and Fragaria x

ananassa, the expected products were as described in Table 1, with

a fragment size of less than 100 base pairs, as described in Figure

5. This means that, based on the gDNA extracted and incorporated

with the requested oligonucleotides, both fruits have practically all

the regulatory genes involved in anthocyanin biosynthesis.

Figure 5. Detection of amplied products in R. adenotrichos

and F. x ananassa. Oligonucleotide amplication

using a gDNA sample from R. adenotrichos (A) was

found to be positive in its entirety. Similarly, most of

the oligonucleotides to be examined were shown to be

present in the gDNA samples for F. x ananassa (B), with

the exception of the pRuGT and pβ-actin oligos.

The lack of amplication of pRuGT and pβ-actin genes in gDNA

of F. x ananassa is supported by previous studies carried out in the

Arabidopsis thaliana and Vitis vinifera genera, where it is noted

that the amplication of this gene is observed during the early stage

of the fruit, when it is green, and decreases when the fruit is ripe

(Khater et al., 2012). This background would support the absence

of amplication, because during the experimental process we

worked with ripe fruit and not in the early green stage. There are

several glycosyltransferases (GT) that transfer sugars to a variety of

acceptors ranging from secondary metabolites and hormones to biotic

and abiotic chemicals (Chen et al., 2012).

Conclusions

Through the use of the informatics platforms we were able to

identify the regulatory genes and enzymes involved in anthocyanin

biosynthesis in R. adenotrichos and F. x ananassa, nding that most

are common, with some specic dierences, and that there are only

a few exceptions, such as the enzymes catechol-O-methyltransferase

(OMT), UDP-glucosyltransferase (UGT) and beta-glucuronidase

(GUSB), which are only present in Rubus adenotrichos and not in

Fragaria x ananassa. These results were corroborated by metabolic

and molecular analyses, which allowed us to obtain the anthocyanin

concentrations in the fruits of R. adenotrichos and F. x ananassa, in

agreement with those reported in the literature.

This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.

Quiroz et al. Rev. Fac. Agron. (LUZ). 2023 40(3): e234029

7-7 |

Acknowledgments

To the Centro de Biotecnología Genómica del Instituto Politécnico

Nacional, as well as to the project SIP 20221377, for the funding

granted for this research, the IPN EDI system and to the IBT Jaime

Alberto Morales Baquera, thesis student and BEIFI IPN student.

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