The selection of solvent is as crucial stage when antioxidants extractions are concerned [23]. In order to evaluate the better solvent for the extraction of murici antioxidants, in terms of TPC and antioxidant activity (DPPH), a preliminary experiment was performed using ethanol, acetone, methanol, and water.

With the polarity change of the solvent, the amount of dissolved antioxidant compound varied. Solvents with low viscosity have low density and high diffusivity, which allows them to easily diffuse into the pores of vegetable materials, and consequently, extract the bioactive compounds [24]. As the result of that, the antioxidant activity of the extract also tends to increase, as observed in this study with the acetone. Hence, this solvent was deemed the better option in this research.

These results match with the results previously described where the mixture water:acetone is the more efficient at the extraction of phenolic antioxidants of oat grains [25] and Etlingera elatior Jack flowers [24], respectively. In addition, this mixture is also recommend as the best system for the extraction of polyphenols [26, 27].

In this study, RSM was employed to determine the optimum conditions for the extraction of antioxidant from fruits of Byrsonima crassifolia (L) Kunth (murici). In order to optimize the extraction parameters (extraction time, temperature, acetone concentration, and solid-liquid ratio), a five-level, four-factor experimental design

Optimization of the Extraction of Polyphenols and Antioxidant Capacity… DOI: http://dx.doi.org/10.5772/intechopen.83457

\*Does not correspond to order of processing. TPC—total phenolic content (mg GAE/100 mg), DPPH radical scavenging activity (IC50—μg/mL), S/S—solid-solvent ratio (mg/mL),T—temperature, and Acet.—acetone in water.

#### Table 2.

2.4 Determination of total phenolic content (TPC)

Plant Physiological Aspects of Phenolic Compounds

performed in three replicates.

50% of DPPH).

the model.

[26, 27].

24

2.6 Statistical analysis

3. Results and discussion

2.5 DPPH radical scavenging activity

The total phenolic compound (TPC) measurements in extracts were adapted from Singleton and Rossi [21] using gallic acid as a standard. Absorbance was measured at 750 nm. Results were expressed as mg equivalent gallic acid (GAE) per 100 mg of murici's dry weight (DW) (mg GAE/100 mg DW). All experiments were

The extract (20 μL) was incubated with 150 μmol/L DPPH (200 μL) in 95% ethanol. Then, it was mixed and kept at room temperature in the dark for 30 min. Finally, the absorbance was measured at 515 nm using a microplate reader. The blank of each concentration was performed with ethanol instead of DPPH solution. The standard curve was a logarithm between 2 and 25 mg/mL gallic acid [22]. The % inhibition was calculated as described by the equation below. The antioxidant activity was expressed as IC50 (required extract concentration to reduce

Statistical analysis was performed using the Statistica Software v.11 and fitted to a second-order polynomial regression model. An analysis of variance (ANOVA) with 95% confidence level was carried. The coefficient of determination (R2

used to estimate the fitness of the polynomial equation to the responses. The experimental and predicted values were compared in order to determine the validity of

The selection of solvent is as crucial stage when antioxidants extractions are concerned [23]. In order to evaluate the better solvent for the extraction of murici antioxidants, in terms of TPC and antioxidant activity (DPPH), a preliminary experiment was performed using ethanol, acetone, methanol, and water.

With the polarity change of the solvent, the amount of dissolved antioxidant compound varied. Solvents with low viscosity have low density and high diffusivity, which allows them to easily diffuse into the pores of vegetable materials, and consequently, extract the bioactive compounds [24]. As the result of that, the antioxidant activity of the extract also tends to increase, as observed in this study with the acetone. Hence, this solvent was deemed the better option in this research. These results match with the results previously described where the mixture water:acetone is the more efficient at the extraction of phenolic antioxidants of oat grains [25] and Etlingera elatior Jack flowers [24], respectively. In addition, this mixture is also recommend as the best system for the extraction of polyphenols

In this study, RSM was employed to determine the optimum conditions for the extraction of antioxidant from fruits of Byrsonima crassifolia (L) Kunth (murici). In order to optimize the extraction parameters (extraction time, temperature, acetone concentration, and solid-liquid ratio), a five-level, four-factor experimental design

Absorbance Control � 100 (1)

) was

% Inhibition ¼ Absorbance Control � Absorbance Sample

Response sheet for RSM experimental design with process variables and experimental results for the preparation of fruits of Byrsonima crassifolia (L.) Kunth.

The values of the TPC ranged from 13.21 to 27.80 mg GAE/100 mg, while the values of the IC 50 ranged from 128.68 to 218.58 μg/mL. The significance and model adequacy were tested by ANOVA, and it revealed that the model was highly significant (p < 0.05) for TPC and DPPH. The lack of fit of each model was not significant (p > 0.05), indicating that the developed model adequately explains the relationship between the independent variables and responses (Tables 3 and 4).

The linear and quadratic effects of the acetone concentration, temperature, and time extraction demonstrated significant effects on TPC (p < 0.01), as well as the

Table 3.

ANOVA for the regression of the second-degree polynomial for the TPC response.

#### Table 4.

ANOVA for the regression of the second-degree polynomial for the DPPH response.

interactions' temperature/time and time/acetone % (p < 0.05). In the case of DPPH, the linear and quadratic effects of temperature, time, and acetone % were significant (p < 0.05). The solid-liquid, in the studied range, did not interfere in the two responses (p > 0.05). Conversely, in the study of [28], the solid-to-solvent ratio (10–30 g/mL) showed a significant effect on the antioxidant compound extraction from germinated chickpea. Probably, an increase in the solvent to sample ratio helps the solute dissolve in solvent and hence the phenolic compounds extraction rate increased with decreasing of solid-to-liquid ratio [29]. However, the extraction rate trend stabilizes when the extraction rate reached a certain value [30]. In this study, we tested low solid-to-solvent ratios (20–140 mg/mL), which seems to have already provided maximum dissolution of the antioxidant compounds.

Analysis of the model clearly shows that temperature and acetone were the most significant factors. The acetone and temperature had a negative effect (coefficient = 4.98 and 3.46, respectively), indicating that a reduction in the temperature and acetone favored the recovery of TPC in the extract. A decrease in the acetone concentration enhanced the solvent polarity, which helps TPC dissolve in solvent [29]. Likewise, the acetone and temperature had a positive effect on DPPH-IC50 (coefficient = 19.35 and 26.12, respectively). This means that the extraction of antioxidants was less favorable at high temperature and acetone because a higher IC50 value corresponds to weaker antioxidant activity of tested extract. These results are similar to those found for [31] for mulberry (Morus nigra) pulp. On the other hand, high ethanol concentration and high temperature were the most effective factors for increasing TPC and antioxidative activities in mango peels [30].

presented in murici [31, 15]. Similarly, the rise in acetone concentration, time, and the temperature decreased the antioxidant activity of murici after DPPH scavenging measurements. This result is in agreement with the findings to Lycium ruthenicum Murr. fruit [29] where lower solvent concentration was found to be conducive to

Response surface plots for the effect of independent variables (stepping time, temperature, and acetone %) on total phenolic content (TPC) and total DPPH radical scavenging activity (IC50 - μg/mL) of fruits from

Optimization of the Extraction of Polyphenols and Antioxidant Capacity…

DOI: http://dx.doi.org/10.5772/intechopen.83457

The determined coefficient for TPC showed a good regression value (R2 = 0.84), and the relationship between the TPC and the extraction parameters of the acetone concentration, the temperature, and the extraction time. By applying multiple regression analysis on the experimental data, the model for the response variable could be expressed by the following quadratic polynomial equation in the form of

total antioxidant activity.

Byrsonima crassifolia (L.) Kunth.

Figure 1.

27

As shown in Figure 1, the TPC increases rapidly as a result of acetone concentration, time and the temperature increase in the range of 10 and 45%, 40–50 min and 10 and 30°C, respectively. However, beyond acetone concentration of 50%, 55 min, and 35°C, the TPC decreases. Generally, temperature ranging from 60 to 70°C might increase the TPC yield due to higher solubility and diffusion in the solvent [30]. However, these temperatures are not recommended for polyphenol extraction from murici. It might be possible that higher temperatures for extended extraction caused the degradation of the some thermosensitive phenolic compounds

Figure 1.

interactions' temperature/time and time/acetone % (p < 0.05). In the case of DPPH, the linear and quadratic effects of temperature, time, and acetone % were significant (p < 0.05). The solid-liquid, in the studied range, did not interfere in the two responses (p > 0.05). Conversely, in the study of [28], the solid-to-solvent ratio (10–30 g/mL) showed a significant effect on the antioxidant compound extraction from germinated chickpea. Probably, an increase in the solvent to sample ratio helps the solute dissolve in solvent and hence the phenolic compounds extraction rate increased with decreasing of solid-to-liquid ratio [29]. However, the extraction rate trend stabilizes when the extraction rate reached a certain value [30]. In this study, we tested low solid-to-solvent ratios (20–140 mg/mL), which seems to have

Source Sum of squares Degrees of freedom Fvalue R2 Regression 385.75 6 18.42 0.8466

Source Sum of squares Degrees of freedom Fvalue R2 Regression 12693.05 5 15.85 0.7906

Lack of fit 69.46 18 17.54

Lack of fit 3325.45 19 9.43

ANOVA for the regression of the second-degree polynomial for the DPPH response.

ANOVA for the regression of the second-degree polynomial for the TPC response.

Residues 69.9 20

Plant Physiological Aspects of Phenolic Compounds

Pure error 0.44 2 Total 455.65 26

Residues 3362.56 21

Pure error 37.11 2 Total 16055.61 26

Table 3.

Table 4.

26

already provided maximum dissolution of the antioxidant compounds.

significant factors. The acetone and temperature had a negative effect (coefficient = 4.98 and 3.46, respectively), indicating that a reduction in the temperature and acetone favored the recovery of TPC in the extract. A decrease in the acetone concentration enhanced the solvent polarity, which helps TPC dissolve in solvent [29]. Likewise, the acetone and temperature had a positive effect on DPPH-IC50 (coefficient = 19.35 and 26.12, respectively). This means that the extraction of antioxidants was less favorable at high temperature and acetone because a higher IC50 value corresponds to weaker antioxidant activity of tested extract. These results are similar to those found for [31] for mulberry (Morus nigra) pulp. On the other hand, high ethanol concentration and high temperature were the most effective factors for increasing TPC and antioxidative activities in mango peels [30]. As shown in Figure 1, the TPC increases rapidly as a result of acetone concentration, time and the temperature increase in the range of 10 and 45%, 40–50 min and 10 and 30°C, respectively. However, beyond acetone concentration of 50%, 55 min, and 35°C, the TPC decreases. Generally, temperature ranging from 60 to 70°C might increase the TPC yield due to higher solubility and diffusion in the solvent [30]. However, these temperatures are not recommended for polyphenol extraction from murici. It might be possible that higher temperatures for extended extraction caused the degradation of the some thermosensitive phenolic compounds

Analysis of the model clearly shows that temperature and acetone were the most

Response surface plots for the effect of independent variables (stepping time, temperature, and acetone %) on total phenolic content (TPC) and total DPPH radical scavenging activity (IC50 - μg/mL) of fruits from Byrsonima crassifolia (L.) Kunth.

presented in murici [31, 15]. Similarly, the rise in acetone concentration, time, and the temperature decreased the antioxidant activity of murici after DPPH scavenging measurements. This result is in agreement with the findings to Lycium ruthenicum Murr. fruit [29] where lower solvent concentration was found to be conducive to total antioxidant activity.

The determined coefficient for TPC showed a good regression value (R2 = 0.84), and the relationship between the TPC and the extraction parameters of the acetone concentration, the temperature, and the extraction time. By applying multiple regression analysis on the experimental data, the model for the response variable could be expressed by the following quadratic polynomial equation in the form of

Table 5.

Predicted and experimental values under optimum conditions based on the multiple responses of TPC and DPPH.

coded values: ŷ = 26.42–1.40X2–0.95X2 2 –1.27X3–0.86X3 2 –1.87X4–4.23X4 2 , where X2 = temperature, X3 = time, and X4 = acetone %.

The determined coefficient for DPPH also showed a reasonable regression value (R2 = 0.84). The model for the response variable could be expressed by the following quadratic polynomial equation in the form of coded values: ŷ = 141.9 + 10.77X2 + 7.88X2 2 + 7.29X3 + 7.32X4 + 23.35X4 2 , where X2 = temperature, X3 = time, and X4 = acetone %.

Based on the experimental results and statistical analysis, numerical optimizations were performed in order to determine the optimum level of independent variables. In our work, the optimum conditions for TPC compounds were as follows: extraction solvent using 44% acetone in water, an extraction temperature of 29°C, and an extraction time of 51 minutes. The optimum conditions for antioxidant activity (DPPH) were as follows: extraction solvent with 45% acetone in water, an extraction temperature of 40°C, and an extraction time of 53 minutes. These results suggest that the optimum parameters of extraction of murici's antioxidants occur with moderate percentages of acetone in water. The optimum temperature for antioxidants extraction evaluated by DPPH was in the middle range, and it was slightly higher for total phenolic compounds.

In order to verify the suitability of the response surface methodology model for quantitative predictions, experiments on estimated optimal conditions were performed. Table 5 shows the predicted and experimental results for the variables selected. The predicted results matched well with the experimental results obtained at optimal extraction conditions proving the validity of the model to describe the process. These findings justified the selection of the RSM design, which was demonstrated to be accurate and reliable for predicting the TPC and antioxidant capacity of extracts from murici.

Author details

(UFRPE), Brazil

29

Mariana Séfora Bezerra Sousa1

and Diego de Souza Buarque2

provided the original work is properly cited.

1 Food Department, Federal Institute of Piauí (IFPI), Brazil

Optimization of the Extraction of Polyphenols and Antioxidant Capacity…

DOI: http://dx.doi.org/10.5772/intechopen.83457

\*Address all correspondence to<EMAIL_ADDRESS>
\*, Jovan Marques Lara Júnior1

2 Academic Unit of Serra Talhada (UAST), Rural Federal University of Pernambuco

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

#### 4. Conclusion

In this study, the RSM was successfully used to determine optimal levels of experimental parameters for the extraction of murici antioxidants. It is possible to verify that temperature, time, and acetone concentration negatively interfere in the extraction of polyphenols and in the antioxidant activity of fruits of Byrsonima crassifolia (L.) Kunth. The efficiency of the extraction can significantly be improved by using the novel extraction techniques, which may contribute to large-scale industrial applications and future pharmacological research.

#### Conflict of interest

All other authors report no potential conflict of interests.

Optimization of the Extraction of Polyphenols and Antioxidant Capacity… DOI: http://dx.doi.org/10.5772/intechopen.83457

#### Author details

coded values: ŷ = 26.42–1.40X2–0.95X2

\*Mean standard deviation (n = 3 replicates).

Plant Physiological Aspects of Phenolic Compounds

Table 5.

DPPH.

ŷ = 141.9 + 10.77X2 + 7.88X2

ity of extracts from murici.

4. Conclusion

Conflict of interest

28

ture, X3 = time, and X4 = acetone %.

slightly higher for total phenolic compounds.

X2 = temperature, X3 = time, and X4 = acetone %.

ing quadratic polynomial equation in the form of coded values:

2

Predicted and experimental values under optimum conditions based on the multiple responses of TPC and

Predicted value 27.61 137.65 Experimental value 28.04 1.14\* 139.00 9.89\*

The determined coefficient for DPPH also showed a reasonable regression value (R2 = 0.84). The model for the response variable could be expressed by the follow-

2 + 7.29X3 + 7.32X4 + 23.35X4

Based on the experimental results and statistical analysis, numerical optimizations were performed in order to determine the optimum level of independent variables. In our work, the optimum conditions for TPC compounds were as follows: extraction solvent using 44% acetone in water, an extraction temperature of 29°C, and an extraction time of 51 minutes. The optimum conditions for antioxidant activity (DPPH) were as follows: extraction solvent with 45% acetone in water, an extraction temperature of 40°C, and an extraction time of 53 minutes. These results suggest that the optimum parameters of extraction of murici's antioxidants occur with moderate percentages of acetone in water. The optimum temperature for antioxidants extraction evaluated by DPPH was in the middle range, and it was

In order to verify the suitability of the response surface methodology model for

In this study, the RSM was successfully used to determine optimal levels of experimental parameters for the extraction of murici antioxidants. It is possible to verify that temperature, time, and acetone concentration negatively interfere in the extraction of polyphenols and in the antioxidant activity of fruits of Byrsonima crassifolia (L.) Kunth. The efficiency of the extraction can significantly be improved by using the novel extraction techniques, which may contribute to large-scale

industrial applications and future pharmacological research.

All other authors report no potential conflict of interests.

quantitative predictions, experiments on estimated optimal conditions were performed. Table 5 shows the predicted and experimental results for the variables selected. The predicted results matched well with the experimental results obtained at optimal extraction conditions proving the validity of the model to describe the process. These findings justified the selection of the RSM design, which was demonstrated to be accurate and reliable for predicting the TPC and antioxidant capac-

–1.27X3–0.86X3

2

TPC (mg GAE/100 mg) DPPH (IC50 - μg/mL)

2

–1.87X4–4.23X4

2 , where

, where X2 = tempera-

Mariana Séfora Bezerra Sousa1 \*, Jovan Marques Lara Júnior1 and Diego de Souza Buarque2

1 Food Department, Federal Institute of Piauí (IFPI), Brazil

2 Academic Unit of Serra Talhada (UAST), Rural Federal University of Pernambuco (UFRPE), Brazil

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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F, Borges SV, Carneiro JDDS.

Medicine. 2013;19(1):26-36

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[3] Silva MR et al. Caracterização química de frutos nativos do cerrado. Ciência Rural. 2008;38(6):1790-1793

[4] Barreto GPM, Benassi MT,

Mercadante AZ. Bioactive compounds from several tropical fruits and correlation by multivariate analysis to free radical scavenger activity. Journal of the Brazilian Chemical Society. 2009; 20(10):1856-1861. DOI: 10.1590/ S0103-50532009001000013

[5] Godon A, Jungfer E, da Silva BA, Maia JGS, Marx F. Phenolic constituents

and antioxidant capacity of four underutilized fruits from the Amazon region. Journal of Agricultural and Food Chemistry. 2011;59(14):7688-7699.

[6] Mariutti LRB, Rodrigues E, Mercadante AZ. Carotenoids from Byrsonima crassifolia: Identification, quantification and in vitro scavenging capacity against peroxyl radicals. Journal of Food Composition and Analysis. 2013;31:155-160. DOI: 10.1016/j.jfca.2013.05.005

[7] Mariutti LR, Rodrigues E, Chisté RC, Fernandes E, Mercadante AZ. The Amazonian fruit Byrsonima crassifolia effectively scavenges reactive oxygen and nitrogen species and protects human erythrocytes against oxidative damage. Food Research International. 2014;64:618-625. DOI: 10.1016/j.

DOI: 10.1021/jf201039r

foodres.2014.07.032

30

[15] Tomšik A, Pavlić B, Vladić J, Ramić M, Brindza VS. Optimization of ultrasound-assisted extraction of bioactive compounds from wild garlic (Allium ursinum L.). Ultrasonics Sonochemistry. 2016;29:502-511. DOI: 10.1016/j.ultsonch.2015.11.005

[16] Angelo PM, Jorge N. Compostos Fenólicos em Alimentos: Uma breve revisão. Revista do Instituto Adolfo Lutz. 2007;66(1):232-240

[17] Tsao R. Chemistry and biochemistry of dietary polyphenols. Nutrients. 2010; 2:1231-1246. DOI: 10.3390/nu2121231

[18] Andreo D, Jorge N. Antioxidantes naturais: Técnicas de extração. Boletim CEEPA. 2006;24(2):319-336. DOI: 10.5380/cep.v24i2.7489

[19] Moure A, Cruz JM, Franco D, Domınguez JM, Sineiro J, Domınguez H, et al. Natural antioxidants from residual sources. Food Chemistry. 2001;72: 145-171. DOI: 10.1016/S0308-8146(00) 00223-5

[20] Rodrigues MI, Iemma AF. Planejamento de Experimentos e Otimização de Processos: Uma estratégia sequencial de planejamentos. 1st ed. São Paulo: Campinas; 2005. 325p

[21] Singleton VL, Rossi JA Jr. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology and Viticulture. 1965;16:144-158

[22] Brand-Williams W, Cuvelier M, Berset C. Use of a free radical method to evaluate antioxidant activity. Lebensmittel-Wissenschaft und Technologie. 1995;28(1):25-30. DOI: 10.1016/S0023-6438(95)80008-5

[23] Ali A, Lim XY, Chong CH, Mah SH, Chua BL. Optimization of ultrasoundassisted extraction of natural antioxidants from Piper betle using response surface methodology. LWT - Food Science and Technology. 2018; 89:681-688. DOI: 10.1016/j.lwt.2017. 11.033

[24] Wijekoon MMJO, Bhat R, Karim AA. Effect of extraction solvents on the phenolic compounds and antioxidant activities of bunga kantan (Etlingera elatiorJack.) inflorescence. Journal of Food Composition and Analysis. 2011; 24(4–5):615-619. DOI: 10.1016/j.jfca. 2010.09.018

[25] Zhou K, Yu L. Effects of extraction solvent on wheat bran antioxidant activity estimation. LWT - Food Science and Technology. 2004;37(7):717-721. DOI: 10.1016/j.lwt.2004.02.008

[26] Sulaiman SF, Sajak AAB, Ooi KL, Seow EM. Effect of solvents in extracting polyphenols and antioxidants of selected raw vegetables. Journal of Food Composition and Analysis. 2011; 24(4–5):506-515. DOI: 10.1016/j. jfca.2011.01.020

[27] Michiels JA, Kevers C, Pincemail J, Defraigne JO, Dommes J. Extraction conditions can greatly influence antioxidant capacity assays in plant food matrices. Food Chemistry. 2012;130(4): 986-993. DOI: 10.1016/j. foodchem.2011.07.117

[28] Hayta M, Isçimen EM. Optimization of ultrasound-assisted antioxidant compounds extraction from germinated chickpea using response surface methodology. LWT - Food Science and Technology. 2017;77:208-216. DOI: 10.1016/j.lwt.2016.11.037

[29] Chen S, Zeng Z, Hu N, Bai B, Wang H, Suo Y. Simultaneous optimization of the ultrasound-assisted extraction for phenolic compounds content and antioxidant activity of Lycium ruthenicum Murr. Fruit using response surface methodology. Food Chemistry.

2018;242:1-8. DOI: 10.1016/j. foodchem.2017.08.105

[30] Sai-Ut S, Benjakul S, Kraithong S, Rawdkuen S. Optimization of antioxidants and tyrosinase inhibitory activity in mango peels using response surface methodology. LWT - Food Science and Technology. 2015;64: 742-749. DOI: 10.1016/j.lwt.2015.06.003

[31] Espada-Bellido E, Ferreiro-González M, Carrera C, Palma M, Barroso CG, Barbero GF. Optimization of the ultrasound-assisted extraction of anthocyanins and total phenolic compounds in mulberry (Morus nigra) pulp. Food Chemistry. 2017;219:23-32. DOI: 10.1016/j.foodchem.2016.09.122

**33**

Section 2

Biosynthetic Routes of

Phenolic Compounds

Section 2

## Biosynthetic Routes of Phenolic Compounds

2018;242:1-8. DOI: 10.1016/j. foodchem.2017.08.105

Rawdkuen S. Optimization of

32

[30] Sai-Ut S, Benjakul S, Kraithong S,

Plant Physiological Aspects of Phenolic Compounds

antioxidants and tyrosinase inhibitory activity in mango peels using response surface methodology. LWT - Food Science and Technology. 2015;64: 742-749. DOI: 10.1016/j.lwt.2015.06.003

[31] Espada-Bellido E, Ferreiro-González M, Carrera C, Palma M, Barroso CG, Barbero GF. Optimization of the ultrasound-assisted extraction of anthocyanins and total phenolic compounds in mulberry (Morus nigra) pulp. Food Chemistry. 2017;219:23-32. DOI: 10.1016/j.foodchem.2016.09.122

**35**

**Chapter 3**

**Abstract**

Compounds

pounds in plants are discussed.

phenylpropanoid metabolism

**1. Introduction**

Shikimic Acid Pathway in

*Norma Francenia Santos-Sánchez, Raúl Salas-Coronado,*

*Beatriz Hernández-Carlos and Claudia Villanueva-Cañongo*

Phenolic compounds are secondary metabolites found most abundantly in plants.  The expression of phenolic compounds is promoted by biotic and abiotic stresses (e.g., herbivores, pathogens, unfavorable temperature and pH, saline stress, heavy metal stress, and UVB and UVA radiation). These compounds are formed via the shikimate pathway in higher plants and microorganisms. The enzymes responsible for the regulation of phenolic metabolism are known, and shikimic acid is a central metabolite. The shikimate pathway consists of seven reaction steps, beginning with an aldol-type condensation of phosphoenolpyruvic acid (PEP) from the glycolytic pathway, and D-erythrose-4-phosphate, from the pentose phosphate cycle, to produce 3-deoxy-D-*arabino*-heptulosonic acid 7-phosphate (DAHP). A key branchpoint compound is chorismic acid, the final product of the shikimate pathway. The shikimate pathway is described in this chapter, as well as factors that induce the synthesis of phenolic compounds in plants. Some representative examples that show the effect of biotic and abiotic stress on the production of phenolic com-

**Keywords:** shikimate pathway, phenolic compounds, biosynthetic routes,

The secondary metabolism is a biosynthetic source of several interesting compounds useful to chemical, food, agronomic, cosmetics, and pharmaceutical industries. The secondary pathways are not necessary for the survival of individual cells but benefit the plant as a whole [1]. Another general characteristic of secondary metabolism is that found in a specific organism, or groups of organisms, and is an expression of the individuality of species [2]. The secondary metabolism provides chemical diversity to organic molecules with low molecular weight that are related by the respective pathways; such organic molecules are called secondary metabolites. The secondary metabolites are often less than 1% of the total carbon in plant molecules [3]. These organic molecules isolated from terrestrial plants are the most studied, and their syntheses have an important role in the protection against

Biosynthesis of Phenolic

#### **Chapter 3**

## Shikimic Acid Pathway in Biosynthesis of Phenolic Compounds

*Norma Francenia Santos-Sánchez, Raúl Salas-Coronado, Beatriz Hernández-Carlos and Claudia Villanueva-Cañongo*

#### **Abstract**

Phenolic compounds are secondary metabolites found most abundantly in plants.  The expression of phenolic compounds is promoted by biotic and abiotic stresses (e.g., herbivores, pathogens, unfavorable temperature and pH, saline stress, heavy metal stress, and UVB and UVA radiation). These compounds are formed via the shikimate pathway in higher plants and microorganisms. The enzymes responsible for the regulation of phenolic metabolism are known, and shikimic acid is a central metabolite. The shikimate pathway consists of seven reaction steps, beginning with an aldol-type condensation of phosphoenolpyruvic acid (PEP) from the glycolytic pathway, and D-erythrose-4-phosphate, from the pentose phosphate cycle, to produce 3-deoxy-D-*arabino*-heptulosonic acid 7-phosphate (DAHP). A key branchpoint compound is chorismic acid, the final product of the shikimate pathway. The shikimate pathway is described in this chapter, as well as factors that induce the synthesis of phenolic compounds in plants. Some representative examples that show the effect of biotic and abiotic stress on the production of phenolic compounds in plants are discussed.

**Keywords:** shikimate pathway, phenolic compounds, biosynthetic routes, phenylpropanoid metabolism

#### **1. Introduction**

The secondary metabolism is a biosynthetic source of several interesting compounds useful to chemical, food, agronomic, cosmetics, and pharmaceutical industries. The secondary pathways are not necessary for the survival of individual cells but benefit the plant as a whole [1]. Another general characteristic of secondary metabolism is that found in a specific organism, or groups of organisms, and is an expression of the individuality of species [2]. The secondary metabolism provides chemical diversity to organic molecules with low molecular weight that are related by the respective pathways; such organic molecules are called secondary metabolites. The secondary metabolites are often less than 1% of the total carbon in plant molecules [3]. These organic molecules isolated from terrestrial plants are the most studied, and their syntheses have an important role in the protection against

**Figure 1.**

*Phenolic compound biosynthesis promoted by biotic and abiotic stresses (e.g., herbivores, pathogens, unfavorable temperature and pH, saline stress, CO2, O3, heavy metal stress, and UVB and UVA radiation).*

pathogens, unfavorable temperature and pH, saline stress, heavy metal stress, and UVB and UVA radiation [3]. Secondary metabolism reflects plant environments more closely than primary metabolism [4]. There are three principal kinds of secondary metabolites biosynthesized by plants: phenolic compounds, terpenoids/ isoprenoids, and alkaloids and glucosinolates (nitrogen- or sulfur-containing molecules, respectively) [5]. Phenolic compounds are biosynthesized by the shikimate pathway and are abundant in plants. The shikimate pathway, in plants, is localized in the chloroplast. These aromatic molecules have important roles, as pigments, antioxidants, signaling agents, electron transport, communication, the structural element lignan, and as a defense mechanism [6], **Figure 1**. The seven steps of the shikimate pathway and the metabolites for branch point are described in this chapter, as factors that induce the synthesis of phenolic compounds in plants. Some representative examples that show the effect of biotic and abiotic stress on the production of phenolic compounds in plants are discussed.

#### **2. The shikimate pathway**

The shikimate biosynthesis pathway provides precursors for aromatic molecules in bacteria, fungi, apicomplexan, and plants, but not in animals [2, 7]. Shikimic acid is named after the highly toxic Japanese *shikimi* (*Illicium anisatum*) flower from which it was first isolated [8]. This biochemical pathway is a major link between primary and secondary metabolism in higher plants [6]. In microorganisms, the shikimate pathway produces aromatic amino acids L-phenylalanine (L-Phe), L-tyrosine (L-Tyr), and L-tryptophan (L-Trp), molecular building blocks for protein biosynthesis [9]. But in plants, these aromatic amino acids are not only crucial components of protein biosynthesis; they also serve as precursors for diverse secondary metabolites that are important for plant growth [10]. These secondary metabolites are called

**37**

**Figure 2.**

*diverse phenolic compounds.*

synthase (CS; EC <IP_ADDRESS>) [13], **Table 1**.

*Shikimic Acid Pathway in Biosynthesis of Phenolic Compounds*

phenolic compounds and are synthesized when needed by the plant [11]. These molecules play an important role in the adaptation of plants to their ecosystem, and their study advances biochemical techniques and molecular biology [3, Bourgaud]. The principal aromatic phenolic compounds synthesized from L-Phe and L-Tyr are cinnamic acids and esters, coumarins, phenylpropenes, chromones (C6-C3), stilbenes,

*The shikimic and chorismic acids are the common precursors for the synthesis of L-Phe, L-Tyr, and L-Trp and*

anthraquinones (C6-C2-C6), chalcones, flavonoids, isoflavonoids, neoflavonoids (C6-C3-C6), and their dimers and trimers, respectively (C6-C3-C6)2,3, lignans, neolignans (C6-C3)2, lignans (C6-C3)n, aromatic polyketides, or diphenylheptanoids (C6-C7-C6) [12]. L-Trp is a precursor of alkaloids in the secondary metabolism [2]. Additionally, diverse hydroxybenzoic acids and aromatic aldehydes (C6-C1) are biosynthesized via branch points in the shikimate pathway, **Figure 2**. Phenolic compounds biosynthesized from the shikimate pathway have structural versatility. The shikimate pathway consists of seven sequential enzymatic steps and begins with an aldol-type condensation of two phosphorylated active compounds, the phosphoenolpyruvic acid (PEP), from the glycolytic pathway, and the carbohydrate D-erythrose-4-phosphate, from the pentose phosphate cycle, to give 3-deoxy-D*arabino*-heptulosonic acid 7-phosphate (DAHP), **Figure 3**. The seven enzymes that catalyze the pathway are known: 3-deoxy-D-*arabino*-heptulosonate-7-phosphate synthase (DAHPS; EC <IP_ADDRESS>, now EC <IP_ADDRESS>), 3-dehydroquinate synthase (DHQS; EC <IP_ADDRESS>), 3-dehydroquinate dehydratase/shikimate dehydrogenase (DHQ/SDH; EC <IP_ADDRESS>/EC <IP_ADDRESS>), shikimate kinase (SK; EC <IP_ADDRESS>), 5 enolpyruvylshikimate 3-phosphate synthase (EPSPS; EC <IP_ADDRESS>), and chorismate

The shikimate pathway has special characteristics that are present only in bacteria, fungi, and plants. The absence of the pathway in all other organisms provides the enzymes catalyzing these reactions with potentially useful targets for the development of antibacterial agents and herbicides. For example, 5-*enol*pyruvylshikimate 3-phosphate synthase (EPSP-synthase) catalyzes the transfer of the enolpyruvyl

(carboxyvinyl) moiety from PEP to shikimic acid 3-phosphate (S3P) [6].

*DOI: http://dx.doi.org/10.5772/intechopen.83815*

*Shikimic Acid Pathway in Biosynthesis of Phenolic Compounds DOI: http://dx.doi.org/10.5772/intechopen.83815*

#### **Figure 2.**

*Plant Physiological Aspects of Phenolic Compounds*

pathogens, unfavorable temperature and pH, saline stress, heavy metal stress, and UVB and UVA radiation [3]. Secondary metabolism reflects plant environments more closely than primary metabolism [4]. There are three principal kinds of secondary metabolites biosynthesized by plants: phenolic compounds, terpenoids/ isoprenoids, and alkaloids and glucosinolates (nitrogen- or sulfur-containing molecules, respectively) [5]. Phenolic compounds are biosynthesized by the shikimate pathway and are abundant in plants. The shikimate pathway, in plants, is localized in the chloroplast. These aromatic molecules have important roles, as pigments, antioxidants, signaling agents, electron transport, communication, the structural element lignan, and as a defense mechanism [6], **Figure 1**. The seven steps of the shikimate pathway and the metabolites for branch point are described in this chapter, as factors that induce the synthesis of phenolic compounds in plants. Some representative examples that show the effect of biotic and abiotic stress on the

*temperature and pH, saline stress, CO2, O3, heavy metal stress, and UVB and UVA radiation).*

*Phenolic compound biosynthesis promoted by biotic and abiotic stresses (e.g., herbivores, pathogens, unfavorable*

The shikimate biosynthesis pathway provides precursors for aromatic molecules in bacteria, fungi, apicomplexan, and plants, but not in animals [2, 7]. Shikimic acid is named after the highly toxic Japanese *shikimi* (*Illicium anisatum*) flower from which it was first isolated [8]. This biochemical pathway is a major link between primary and secondary metabolism in higher plants [6]. In microorganisms, the shikimate pathway produces aromatic amino acids L-phenylalanine (L-Phe), L-tyrosine (L-Tyr), and L-tryptophan (L-Trp), molecular building blocks for protein biosynthesis [9]. But in plants, these aromatic amino acids are not only crucial components of protein biosynthesis; they also serve as precursors for diverse secondary metabolites that are important for plant growth [10]. These secondary metabolites are called

production of phenolic compounds in plants are discussed.

**2. The shikimate pathway**

**36**

**Figure 1.**

*The shikimic and chorismic acids are the common precursors for the synthesis of L-Phe, L-Tyr, and L-Trp and diverse phenolic compounds.*

phenolic compounds and are synthesized when needed by the plant [11]. These molecules play an important role in the adaptation of plants to their ecosystem, and their study advances biochemical techniques and molecular biology [3, Bourgaud]. The principal aromatic phenolic compounds synthesized from L-Phe and L-Tyr are cinnamic acids and esters, coumarins, phenylpropenes, chromones (C6-C3), stilbenes, anthraquinones (C6-C2-C6), chalcones, flavonoids, isoflavonoids, neoflavonoids (C6-C3-C6), and their dimers and trimers, respectively (C6-C3-C6)2,3, lignans, neolignans (C6-C3)2, lignans (C6-C3)n, aromatic polyketides, or diphenylheptanoids (C6-C7-C6) [12]. L-Trp is a precursor of alkaloids in the secondary metabolism [2]. Additionally, diverse hydroxybenzoic acids and aromatic aldehydes (C6-C1) are biosynthesized via branch points in the shikimate pathway, **Figure 2**. Phenolic compounds biosynthesized from the shikimate pathway have structural versatility.

The shikimate pathway consists of seven sequential enzymatic steps and begins with an aldol-type condensation of two phosphorylated active compounds, the phosphoenolpyruvic acid (PEP), from the glycolytic pathway, and the carbohydrate D-erythrose-4-phosphate, from the pentose phosphate cycle, to give 3-deoxy-D*arabino*-heptulosonic acid 7-phosphate (DAHP), **Figure 3**. The seven enzymes that catalyze the pathway are known: 3-deoxy-D-*arabino*-heptulosonate-7-phosphate synthase (DAHPS; EC <IP_ADDRESS>, now EC <IP_ADDRESS>), 3-dehydroquinate synthase (DHQS; EC <IP_ADDRESS>), 3-dehydroquinate dehydratase/shikimate dehydrogenase (DHQ/SDH; EC <IP_ADDRESS>/EC <IP_ADDRESS>), shikimate kinase (SK; EC <IP_ADDRESS>), 5 enolpyruvylshikimate 3-phosphate synthase (EPSPS; EC <IP_ADDRESS>), and chorismate synthase (CS; EC <IP_ADDRESS>) [13], **Table 1**.

The shikimate pathway has special characteristics that are present only in bacteria, fungi, and plants. The absence of the pathway in all other organisms provides the enzymes catalyzing these reactions with potentially useful targets for the development of antibacterial agents and herbicides. For example, 5-*enol*pyruvylshikimate 3-phosphate synthase (EPSP-synthase) catalyzes the transfer of the enolpyruvyl (carboxyvinyl) moiety from PEP to shikimic acid 3-phosphate (S3P) [6].

#### **Figure 3.** *Shikimate pathway.*

In the second reaction step, DAHP loses phosphate (Pi); the enolic-type product is cyclized through a second aldol-type reaction to produce 3-dehydroquinic acid (DHQ ). The 3-dehydroquinate synthase (DHQS) catalyzes this cyclization in the shikimate pathway. The DHQ dehydrates to produce 3-dehydroshikimic acid (DHS) (3-dehydroquinate dehydratase); this compound has a conjugated double carboncarbon, **Figure 3**. The protocatechuic and the gallic acids (C6-C1) are produced by branch-point reactions from DHS [2]. The fourth step in the pathway is a reduction reaction of DHS with reduced nicotinamide adenine dinucleotide phosphate (NADPH), **Figure 3**. The fifth section of the pathway is the activation of shikimic acid with adenosine triphosphate (ATP) (shikimate kinase, SK) to make shikimic acid 3-phosphate (S3P). The sixth chemical reaction is the addition of PEP to S3P to generate 5-*enol*pyruvylshikimic acid 3-phosphate; the enzyme that catalyzes this reaction step, 5-e*nol*pyruvylshikimate 3-phosphate synthase (EPSPS), has been extensively studied. The reason for this interest is because glyphosate [*N*-(phosphonomethyl)

**39**

*Shikimic Acid Pathway in Biosynthesis of Phenolic Compounds*

(PEP), erythrose-4-phosphate

heptulosonic acid 7-phosphate (DAHP)

(DHQ )

(DHS), NADPH + H+

3-phosphate (S3P), PEP

3-phosphate (EPSP)

5 Shikimic acid, ATP Shikimate kinase enzyme

**Reaction step Substrate Enzyme/cofactor Product**

3-Deoxy-D-*arabino*heptulosonate-7-phosphate synthase (DAHPS; EC <IP_ADDRESS>, now EC <IP_ADDRESS>)/ Co2+, Mg2+ or Mn2+ [15]

3-Dehydroquinate synthase DHQS (EC. <IP_ADDRESS>)/Co2+, NAD+

3-Dehydroquinate dehydratase (DHQ dehydratase EC <IP_ADDRESS>) [15]

Shikimate dehydrogenase (SDH; EC <IP_ADDRESS>) [18–21]

(SK; EC <IP_ADDRESS>)

5-*Enol*pyruvylshikimate 3-phosphate synthase, also called aroA enzyme (EPSPS; EC <IP_ADDRESS>) [25]

Chorismate synthase (CS; EC <IP_ADDRESS>)/FMNH2 [2, 19, 30, 31]

*, oxidized nicotinamide adenine dinucleotide; NADPH, reduced nicotinamide adenine*

[15, 16]

3-Deoxy-D-*arabino*heptulosonic acid 7-phosphate (DAHP), Pi

3-Dehydroquinic acid (DHQ ), Pi

3-Dehydroshikimic acid (DHS), H2O

Shikimic acid, NADP+

Shikimic acid 3-phosphate (S3P), ADP

5-*Enol*pyruvylshikimate 3-phosphate (EPSP), Pi

Chorismic acid, Pi

glycine] is a powerful inhibitor of EPSPS [2], so glyphosate has been used as a broadspectrum systemic herbicide. It is an organophosphorus molecule, phosphonic acid, and glycine derivative that has a similar molecular structure to PEP, **Figure 4**.

*PEP and glyphosate (powerful inhibitor of the 5-enolpyruvylshikimate 3-phosphate synthase, EPSPS).*

The last reaction step of the shikimate pathway is the production of chorismic acid from catalytic action on the chorismate synthase (CS). This reaction is a 1, 4-*trans* elimination of Pi, to yield the conjugated molecule, chorismic acid, **Figure 3**.

**2.1 Synthesis of 3-deoxy-D-***arabino***-heptulosonic acid 7-phosphate (DAHP)**

The first reaction of the shikimate pathway is an aldol-type condensation of PEP and carbohydrate erythrose-4-P, to give 3-deoxy-D-*arabino*-heptulosonic acid

*DOI: http://dx.doi.org/10.5772/intechopen.83815*

1 Phosphoenolpyruvate

2 3-Deoxy-D-*arabino*-

3 3-Dehydroquinic acid

4 3-Dehydroshikimic acid

6 Shikimic acid

7 5-*Enol*pyruvylshikimate

*dinucleotide phosphate; FMNH2, reduced flavin mononucleotide.*

*Substrates, enzymes, and products of the shikimate pathway.*

*Pi, phosphate; NAD+*

**Table 1.**

**Figure 4.**

*Shikimic Acid Pathway in Biosynthesis of Phenolic Compounds DOI: http://dx.doi.org/10.5772/intechopen.83815*

*Pi, phosphate; NAD+ , oxidized nicotinamide adenine dinucleotide; NADPH, reduced nicotinamide adenine dinucleotide phosphate; FMNH2, reduced flavin mononucleotide.*

#### **Table 1.**

*Plant Physiological Aspects of Phenolic Compounds*

In the second reaction step, DAHP loses phosphate (Pi); the enolic-type product is cyclized through a second aldol-type reaction to produce 3-dehydroquinic acid (DHQ ). The 3-dehydroquinate synthase (DHQS) catalyzes this cyclization in the shikimate pathway. The DHQ dehydrates to produce 3-dehydroshikimic acid (DHS) (3-dehydroquinate dehydratase); this compound has a conjugated double carboncarbon, **Figure 3**. The protocatechuic and the gallic acids (C6-C1) are produced by branch-point reactions from DHS [2]. The fourth step in the pathway is a reduction reaction of DHS with reduced nicotinamide adenine dinucleotide phosphate (NADPH), **Figure 3**. The fifth section of the pathway is the activation of shikimic acid with adenosine triphosphate (ATP) (shikimate kinase, SK) to make shikimic acid 3-phosphate (S3P). The sixth chemical reaction is the addition of PEP to S3P to generate 5-*enol*pyruvylshikimic acid 3-phosphate; the enzyme that catalyzes this reaction step, 5-e*nol*pyruvylshikimate 3-phosphate synthase (EPSPS), has been extensively studied. The reason for this interest is because glyphosate [*N*-(phosphonomethyl)

**38**

**Figure 3.** *Shikimate pathway.* *Substrates, enzymes, and products of the shikimate pathway.*

**Figure 4.** *PEP and glyphosate (powerful inhibitor of the 5-enolpyruvylshikimate 3-phosphate synthase, EPSPS).*

glycine] is a powerful inhibitor of EPSPS [2], so glyphosate has been used as a broadspectrum systemic herbicide. It is an organophosphorus molecule, phosphonic acid, and glycine derivative that has a similar molecular structure to PEP, **Figure 4**.

The last reaction step of the shikimate pathway is the production of chorismic acid from catalytic action on the chorismate synthase (CS). This reaction is a 1, 4-*trans* elimination of Pi, to yield the conjugated molecule, chorismic acid, **Figure 3**.

#### **2.1 Synthesis of 3-deoxy-D-***arabino***-heptulosonic acid 7-phosphate (DAHP)**

The first reaction of the shikimate pathway is an aldol-type condensation of PEP and carbohydrate erythrose-4-P, to give 3-deoxy-D-*arabino*-heptulosonic acid

**Figure 5.**

*Stereochemistry of the condensation reaction of (Z)-[3-3 H]PEP and D-erythrose-4-phosphate by DAHP synthase [*14*].*

7-phosphate (DAHP), **Figures 3** and **5**. A new stereogenic center is generated in the condensation product DAHP catalyzed by the 3-deoxy-D-*arabino*-heptulosonate-7-phosphate synthase enzyme (DAHPS; EC <IP_ADDRESS>, now EC <IP_ADDRESS>). Results of enzymatic kinetic and labeled PEP with tritium (*Z*)-[3-3 H] PEP suggest that the nucleophilic attack of PEP is from the *Si* face of PEP to the *Re* face of the carbonyl group of D-erythrose-4-P, **Figure 5** [14]. Two isoenzymes of DAHPS have been found for the catalysis of this first reaction step. One isozyme needs only Mn2+, and the other, either Co2+, Mg2+, or Mn2+ for the catalysis [15].

#### **2.2 Synthesis of 3-dehydroquinic acid (DHQ )**

The second reaction of the shikimate pathway is an intramolecular aldol-type reaction cyclization, where the enol (C6-C7) of DAHP nucleophilically attacks the carbonyl group (C2), to produce a six-member cycle, the 3-dehydroquinic acid (DHQ ), **Figures 3** and 6. The enzyme that catalyzes this reaction, 3-dehydroquinate synthase DHQS (EC. <IP_ADDRESS>), is a carbon-oxygen lyase enzyme that requires Co2+ and bound oxidized nicotinamide adenine dinucleotide (NAD+ ) as cofactors [15, 16]. The Co2+ is essential for the catalytic activity of DHQS. Bender et al. [16] found that DHQS, from *Escherichia coli*, is a monomeric metalloenzyme that contains tightly bound Co2+, and DHQS is deactivated with ethylenediaminetetraacetic acid (EDTA). The presence of the substrate (DAHP) blocks the inactivation by EDTA. The NAD+ cofactor dissociates form the DHQS enzyme rapidly in the presence of DAHP [16]. The reaction mechanism of the enzyme-catalyzed conversion of DAHP to DHQ involves five transformations from the DAHP hemiketal form, a pyranose: (1) oxidation of the hydroxyl at C5 adjacent to the lost proton that requires NAD+ (NAD+ need never dissociate from the active site), (2) the elimination of Pi of C7 to make the α,βunsaturated ketone, (3) the reduction of C5 with NADH + H+ , (4) the ring opening of the enol to yield an enolate, and (5) the intramolecular aldol-like reaction to produce DHQ. All five-reaction steps occur through the function of DHQS, **Figure 6**.

The reduction reaction of DHQ leads to quinic acid at this branch point in the shikimate pathway. Quinic acid is a secondary metabolite that is free, forming esters or as part of alkaloids such as quinine. Quinic acid is found in high quantities in mature kiwi fruit (*Actinidia chinensis* and other species of *Actinidia*) and is a distinguishing characteristic of fresh kiwi fruit [7]. Also, the quinic acid is abundant in roasted coffee [17].

#### **2.3 Synthesis of 3-dehydroshikimic acid (DHS) and shikimic acid**

The third and fourth reaction steps of the shikimate pathway are catalyzed by a bifunctional enzyme: 3-dehydroquinate dehydratase/shikimate dehydrogenase

**41**

**Figure 6.**

*synthase DHQS (EC. <IP_ADDRESS>) [16].*

*Shikimic Acid Pathway in Biosynthesis of Phenolic Compounds*

(DHQ dehydratase/SDH; EC <IP_ADDRESS>/EC <IP_ADDRESS>). The DHQ dehydratase enzyme is a hydro-lyase kind, and the SDH is an oxidoreductase enzyme. The DHQ dehydratase, in the third reaction step, converts DHQ into 3-dehydroshikimic acid (DHS) by eliminating water, and this reaction is reversible, **Figure 7**. The DHS is converted to shikimic acid in the fourth reaction step, by the reduction of the carbonyl group at C-5 by the catalytic action of SDH with NADPH, **Figure 3**. The biosynthesis of DHS is a branch point to shikimic acid and to the catabolic quinate pathway. If the DHS dehydrates, it produces protocatechuic acid (C6-C1) or gallic acid, **Figure 3**. Gallic

*Reaction mechanism of DAHP (hemiketal form) to 3-dehydroquinic acid (DHQ ) by 3-dehydroquinate*

Two structurally different kinds of 3-dehydroquinate dehydratase are known: type I (not heat-stable) and type II (heat-stable). Type I enzyme is present in bacteria and higher plants, and type II is found in fungi, which have both types of enzymes [18, 19]. The catalytic mechanism of the type I DHQ dehydratase has been detected by electrospray MS [20]. This catalytic mechanism involves the amino acid residue Lys-241 that forms a Schiff base with the substrate and product, **Figure 7** [21]. The fourth step is the reduction of DHS with NADPH that enantioselectively reduces the carbonyl of the ketone group of DHS to produce shikimic acid (shiki-

Sigh and Christendat [22] reported the crystal structure of DHQ dehydratase/ SDH from the plant genus *Arabidopsis*. The crystal structure has the shikimate bound at the SDH and the tartrate molecule at the DHQ dehydratase. The studies show that Asp 423 and Lys 385 are key catalytic amino acids and Ser 336 is a key-binding group.

The shikimate kinase enzyme (SK; EC <IP_ADDRESS>) catalyzes the phosphorylation of the shikimic acid, the fifth chemical reaction of the shikimate pathway, and the products are shikimic acid 3-phosphate (S3P) and ADP, **Figures 3** and **8**.

acid (C6-C1) is a hydroxybenzoic acid that is a component of tannins [2].

mate dehydrogenase, SDH), **Figure 3**.

**2.4 Synthesis of shikimic acid 3-phosphate (S3P)**

*DOI: http://dx.doi.org/10.5772/intechopen.83815*

*Shikimic Acid Pathway in Biosynthesis of Phenolic Compounds DOI: http://dx.doi.org/10.5772/intechopen.83815*

#### **Figure 6.**

*Plant Physiological Aspects of Phenolic Compounds*

*Stereochemistry of the condensation reaction of (Z)-[3-3*

**Figure 5.**

*synthase [*14*].*

7-phosphate (DAHP), **Figures 3** and **5**. A new stereogenic center is generated in the condensation product DAHP catalyzed by the 3-deoxy-D-*arabino*-heptulosonate-7-phosphate synthase enzyme (DAHPS; EC <IP_ADDRESS>, now EC <IP_ADDRESS>). Results of

nucleophilic attack of PEP is from the *Si* face of PEP to the *Re* face of the carbonyl group of D-erythrose-4-P, **Figure 5** [14]. Two isoenzymes of DAHPS have been found for the catalysis of this first reaction step. One isozyme needs only Mn2+, and

The second reaction of the shikimate pathway is an intramolecular aldol-type reaction cyclization, where the enol (C6-C7) of DAHP nucleophilically attacks the carbonyl group (C2), to produce a six-member cycle, the 3-dehydroquinic acid (DHQ ), **Figures 3** and 6. The enzyme that catalyzes this reaction, 3-dehydroquinate synthase DHQS (EC. <IP_ADDRESS>), is a carbon-oxygen lyase enzyme that requires Co2+

The Co2+ is essential for the catalytic activity of DHQS. Bender et al. [16] found that DHQS, from *Escherichia coli*, is a monomeric metalloenzyme that contains tightly bound Co2+, and DHQS is deactivated with ethylenediaminetetraacetic acid (EDTA). The presence of the substrate (DAHP) blocks the inactivation by EDTA. The NAD+ cofactor dissociates form the DHQS enzyme rapidly in the presence of DAHP [16]. The reaction mechanism of the enzyme-catalyzed conversion of DAHP to DHQ involves five transformations from the DAHP hemiketal form, a pyranose: (1) oxida-

never dissociate from the active site), (2) the elimination of Pi of C7 to make the α,β-

the enol to yield an enolate, and (5) the intramolecular aldol-like reaction to produce

The reduction reaction of DHQ leads to quinic acid at this branch point in the shikimate pathway. Quinic acid is a secondary metabolite that is free, forming esters or as part of alkaloids such as quinine. Quinic acid is found in high quantities in mature kiwi fruit (*Actinidia chinensis* and other species of *Actinidia*) and is a distinguishing characteristic of fresh kiwi fruit [7]. Also, the quinic acid is abundant in

The third and fourth reaction steps of the shikimate pathway are catalyzed by a bifunctional enzyme: 3-dehydroquinate dehydratase/shikimate dehydrogenase

H] PEP suggest that the

*H]PEP and D-erythrose-4-phosphate by DAHP*

) as cofactors [15, 16].

(NAD+

, (4) the ring opening of

need

enzymatic kinetic and labeled PEP with tritium (*Z*)-[3-3

the other, either Co2+, Mg2+, or Mn2+ for the catalysis [15].

and bound oxidized nicotinamide adenine dinucleotide (NAD+

tion of the hydroxyl at C5 adjacent to the lost proton that requires NAD+

**2.3 Synthesis of 3-dehydroshikimic acid (DHS) and shikimic acid**

DHQ. All five-reaction steps occur through the function of DHQS, **Figure 6**.

unsaturated ketone, (3) the reduction of C5 with NADH + H+

**2.2 Synthesis of 3-dehydroquinic acid (DHQ )**

**40**

roasted coffee [17].

*Reaction mechanism of DAHP (hemiketal form) to 3-dehydroquinic acid (DHQ ) by 3-dehydroquinate synthase DHQS (EC. <IP_ADDRESS>) [16].*

(DHQ dehydratase/SDH; EC <IP_ADDRESS>/EC <IP_ADDRESS>). The DHQ dehydratase enzyme is a hydro-lyase kind, and the SDH is an oxidoreductase enzyme. The DHQ dehydratase, in the third reaction step, converts DHQ into 3-dehydroshikimic acid (DHS) by eliminating water, and this reaction is reversible, **Figure 7**. The DHS is converted to shikimic acid in the fourth reaction step, by the reduction of the carbonyl group at C-5 by the catalytic action of SDH with NADPH, **Figure 3**. The biosynthesis of DHS is a branch point to shikimic acid and to the catabolic quinate pathway. If the DHS dehydrates, it produces protocatechuic acid (C6-C1) or gallic acid, **Figure 3**. Gallic acid (C6-C1) is a hydroxybenzoic acid that is a component of tannins [2].

Two structurally different kinds of 3-dehydroquinate dehydratase are known: type I (not heat-stable) and type II (heat-stable). Type I enzyme is present in bacteria and higher plants, and type II is found in fungi, which have both types of enzymes [18, 19]. The catalytic mechanism of the type I DHQ dehydratase has been detected by electrospray MS [20]. This catalytic mechanism involves the amino acid residue Lys-241 that forms a Schiff base with the substrate and product, **Figure 7** [21]. The fourth step is the reduction of DHS with NADPH that enantioselectively reduces the carbonyl of the ketone group of DHS to produce shikimic acid (shikimate dehydrogenase, SDH), **Figure 3**.

Sigh and Christendat [22] reported the crystal structure of DHQ dehydratase/ SDH from the plant genus *Arabidopsis*. The crystal structure has the shikimate bound at the SDH and the tartrate molecule at the DHQ dehydratase. The studies show that Asp 423 and Lys 385 are key catalytic amino acids and Ser 336 is a key-binding group.

#### **2.4 Synthesis of shikimic acid 3-phosphate (S3P)**

The shikimate kinase enzyme (SK; EC <IP_ADDRESS>) catalyzes the phosphorylation of the shikimic acid, the fifth chemical reaction of the shikimate pathway, and the products are shikimic acid 3-phosphate (S3P) and ADP, **Figures 3** and **8**.

#### **Figure 7.** *Reaction mechanism to produce 3-dehydroshikimic acid (DHS) by type I DHQ dehydratase enzyme [*21*].*

**Figure 8.** *Phosphorylation of shikimic acid with ATP.*

Shikimic acid is phosphorylated with ATP in the 5-hydroxyl group of shikimic acid. SK is an essential enzyme in several bacterial pathogens and is not present in the human cell; therefore the SK enzyme has been classified as a protein target for drug design, especially for chemotherapeutic development of antitubercular drugs [23, 24].

#### **2.5 Synthesis of 5-***enol***pyruvylshikimate 3-phosphate (EPSP)**

The 5-*enol*pyruvylshikimate 3-phosphate synthase, also called aroA enzyme (EPSPS; EC <IP_ADDRESS>), catalyzes the condensation of PEP to the 5-hydroxyl group of S3P in the sixth reaction of the shikimate pathway to form 5-*enol*pyruvylshikimate 3-phosphate (EPSP). The reaction mechanism involves the protonation of PEP to subsequent nucleophilic attack of the hydroxyl at C-5 of S3P to form an intermediate that loses Pi to form EPSP, **Figure 9** [25].

EPSPS is the most studied enzyme of the shikimate pathway because it plays a crucial role in the penultimate step. If this enzyme is inhibited, there is an

**43**

**Figure 10.**

*Reaction of mechanism to yield chorismic acid by chorismate synthase [*30*].*

*Shikimic Acid Pathway in Biosynthesis of Phenolic Compounds*

accumulation of shikimic acid [26], and the synthesis of aromatic amino acid is disabled, leading to the death of the plant [27]. Therefore, EPSPS is used as a target for pesticides, like glyphosate, **Figure 4**, the active ingredient in the herbicides RoundUp™, Monsanto Chemical Co., and Touchdown™, Syngenta. Glyphosate (*N*-(phosphonomethyl)glycine) inhibits EPSPS and is a potent nonselective herbicide that mimics the carbocation of PEP and binds EPEPS competitively [28]. Because the glyphosate is nonselective and kills food crops, there is interest in finding glyphosate-tolerant genes for genetically modified crops [29]. Two types of EPSPS enzymes have been identified: type I EPSPS (sensitive to glyphosate) identified mostly in plants and bacteria and type II EPSPS (nonsensitive to glyphosate and has a high affinity for PEP), found in

*Reaction mechanism of the condensation of S3P with PEP by EPSPS (EC <IP_ADDRESS>) to form EPSP [25].*

The seventh and last reaction step of the shikimate pathway is the 1,4-*trans* elimination of the Pi group at C-3 from EPSPS to synthetize chorismic acid. This last step is catalyzed by chorismate synthase (CS; EC <IP_ADDRESS>) that needs reduced flavin mononucleotide (FMNH2) as a cofactor that is not consumed [2, 19]. The FMNH2 transfers an electron to the substrate reversibly [30]. Spectroscopic

*DOI: http://dx.doi.org/10.5772/intechopen.83815*

some bacteria [27].

**Figure 9.**

**2.6 Synthesis of chorismic acid**

*Shikimic Acid Pathway in Biosynthesis of Phenolic Compounds DOI: http://dx.doi.org/10.5772/intechopen.83815*

**Figure 9.**

*Plant Physiological Aspects of Phenolic Compounds*

Shikimic acid is phosphorylated with ATP in the 5-hydroxyl group of shikimic acid. SK is an essential enzyme in several bacterial pathogens and is not present in the human cell; therefore the SK enzyme has been classified as a protein target for drug design, especially for chemotherapeutic development of antitubercular

*Reaction mechanism to produce 3-dehydroshikimic acid (DHS) by type I DHQ dehydratase enzyme [*21*].*

The 5-*enol*pyruvylshikimate 3-phosphate synthase, also called aroA enzyme (EPSPS; EC <IP_ADDRESS>), catalyzes the condensation of PEP to the 5-hydroxyl group of S3P in the sixth reaction of the shikimate pathway to form 5-*enol*pyruvylshikimate 3-phosphate (EPSP). The reaction mechanism involves the protonation of PEP to subsequent nucleophilic attack of the hydroxyl at C-5 of S3P to form an intermedi-

EPSPS is the most studied enzyme of the shikimate pathway because it plays a crucial role in the penultimate step. If this enzyme is inhibited, there is an

**2.5 Synthesis of 5-***enol***pyruvylshikimate 3-phosphate (EPSP)**

ate that loses Pi to form EPSP, **Figure 9** [25].

**42**

drugs [23, 24].

*Phosphorylation of shikimic acid with ATP.*

**Figure 8.**

**Figure 7.**

*Reaction mechanism of the condensation of S3P with PEP by EPSPS (EC <IP_ADDRESS>) to form EPSP [25].*

accumulation of shikimic acid [26], and the synthesis of aromatic amino acid is disabled, leading to the death of the plant [27]. Therefore, EPSPS is used as a target for pesticides, like glyphosate, **Figure 4**, the active ingredient in the herbicides RoundUp™, Monsanto Chemical Co., and Touchdown™, Syngenta. Glyphosate (*N*-(phosphonomethyl)glycine) inhibits EPSPS and is a potent nonselective herbicide that mimics the carbocation of PEP and binds EPEPS competitively [28]. Because the glyphosate is nonselective and kills food crops, there is interest in finding glyphosate-tolerant genes for genetically modified crops [29]. Two types of EPSPS enzymes have been identified: type I EPSPS (sensitive to glyphosate) identified mostly in plants and bacteria and type II EPSPS (nonsensitive to glyphosate and has a high affinity for PEP), found in some bacteria [27].

#### **2.6 Synthesis of chorismic acid**

The seventh and last reaction step of the shikimate pathway is the 1,4-*trans* elimination of the Pi group at C-3 from EPSPS to synthetize chorismic acid. This last step is catalyzed by chorismate synthase (CS; EC <IP_ADDRESS>) that needs reduced flavin mononucleotide (FMNH2) as a cofactor that is not consumed [2, 19]. The FMNH2 transfers an electron to the substrate reversibly [30]. Spectroscopic

**Figure 10.** *Reaction of mechanism to yield chorismic acid by chorismate synthase [*30*].*

techniques and kinetic isotope effect studies suggest that a radical intermediate in a non-concerted mechanism is developed [30, 31], **Figure 10**. Chorismic acid, the final molecule of the shikimate pathway, is a key branch point to post-chorismic acid pathways, to obtain L-Phe, L-Tyr, and L-Trp, **Figure 2**. L-Phe is the substrate to phenylpropanoid and flavonoid pathways [13].

#### **3. Factors that induce the synthesis of phenolic compounds in plants**

The expression of phenolic compounds is promoted by biotic and abiotic stresses (e.g., herbivores, pathogens, unfavorable temperature and pH, saline stress, heavy metal stress, and UVB and UVA radiation). UV radiation is divided into UVC (≤280 nm), UVB (280–320 nm), and UVA (300–400 nm). UVA and UVB radiation are transmitted through the atmosphere; all UVC and some UVB radiation (highly energetic) are absorbed by the Earth's ozone layer. This accumulation is explained by the increase in enzymatic activity of the phenylalanine ammonia-lyase and chalcone synthase enzymes, among others [12]. Studies have been done about the increase of phenolic compounds, such as anthocyanins, in plants when they are exposed to UVB radiation [13]. Another study demonstrates that UVB exposure enhances anthocyanin biosynthesis in "Cripps pink" apples (*Malus* x *domestica* Borkh.) but not in "Forelle" pears (*Pyrus communis* L.) [32]. This effect may be due to UV radiation exposure and the cultivar of the plants studied. It is known that if plants are under stress, they accumulate phenolic compounds.

The increase in phenolic compounds in blueberry (*Vaccinium corymbosum*) plantlets cultivated in vitro exposed to aluminum (Al) and cadmium (Cd) has also been studied. These heavy metals cause high toxicity in plants, because they increase the oxidative stress by the production of reactive oxygen species (ROS). The authors of the study suggest that the phenolic compounds, specifically chlorogenic and ellagic acids, **Figure 11**, reduce the ROS in blueberry plants [33].

An interesting study was carried out in 2011 by Mody et al., where they studied the effect of the resistance response of apple tree seedlings (*Malus* x *domestica*) to a leaf-chewing insect (*Spodoptera littoralis*) [34]. The authors found a significant herbivore preference for undamaged plants (induced resistance) was first observed 3 days after herbivore damage in the most apical leaf. Also, the results showed higher concentrations of the flavonoid phlorizin, **Figure 12**, in damaged plants than undamaged plants. This indicates that insect preference for undamaged apple plants may be linked to phlorizin, which is the main secondary metabolite of the phenolic type in apple leaves.

**45**

*Shikimic Acid Pathway in Biosynthesis of Phenolic Compounds*

Knowledge of the biosynthetic pathway of shikimic acid leads to understanding the reaction mechanisms of enzymes and thus discovering antimicrobials, pesticides, and antifungals. Studies with isotopic labeling of substrates, the use of X-ray diffraction, nuclear magnetic resonance (NMR), mass spectrometry (ES), biotechnology, as well as organic synthesis have contributed to explaining the shikimate pathway. Although the seven steps of the biosynthetic pathway are elucidated, these metabolites are the precursors of phenolic compounds, more complex molecules that are necessary for the adaptation of plants to the environment. So, the shikimate pathway is the basis for the subsequent biosynthesis of phenolic compounds. There is scientific interest in continuing to investigate the biosynthesis of phenolic compounds from several points of view: pharmaceuticals, agronomy, chemical and food industries, genetics, and

The authors thank Carol Ann Hayenga for her English assistance in the prepara-

The authors have no conflict of interest to declare and are responsible for the

This chapter does not contain any studies with human participants or animals

tion of this manuscript. The Technological University of the Mixteca provided

*DOI: http://dx.doi.org/10.5772/intechopen.83815*

**4. Conclusions**

*Chemical structure of phlorizin (C6-C3).*

**Figure 12.**

health.

support.

**Acknowledgements**

**Conflict of interest**

**Ethical approval**

content and writing of the manuscript.

performed by any of the authors.

**Figure 11.** *Chemical structure of chlorogenic (C6-C3) and ellagic (C6-C1) acids.*

*Shikimic Acid Pathway in Biosynthesis of Phenolic Compounds DOI: http://dx.doi.org/10.5772/intechopen.83815*

**Figure 12.** *Chemical structure of phlorizin (C6-C3).*

#### **4. Conclusions**

*Plant Physiological Aspects of Phenolic Compounds*

techniques and kinetic isotope effect studies suggest that a radical intermediate in a non-concerted mechanism is developed [30, 31], **Figure 10**. Chorismic acid, the final molecule of the shikimate pathway, is a key branch point to post-chorismic acid pathways, to obtain L-Phe, L-Tyr, and L-Trp, **Figure 2**. L-Phe is the

**3. Factors that induce the synthesis of phenolic compounds in plants**

The expression of phenolic compounds is promoted by biotic and abiotic stresses (e.g., herbivores, pathogens, unfavorable temperature and pH, saline stress, heavy metal stress, and UVB and UVA radiation). UV radiation is divided into UVC (≤280 nm), UVB (280–320 nm), and UVA (300–400 nm). UVA and UVB radiation are transmitted through the atmosphere; all UVC and some UVB radiation (highly energetic) are absorbed by the Earth's ozone layer. This accumulation is explained by the increase in enzymatic activity of the phenylalanine ammonia-lyase and chalcone synthase enzymes, among others [12]. Studies have been done about the increase of phenolic compounds, such as anthocyanins, in plants when they are exposed to UVB radiation [13]. Another study demonstrates that UVB exposure enhances anthocyanin biosynthesis in "Cripps pink" apples (*Malus* x *domestica* Borkh.) but not in "Forelle" pears (*Pyrus communis* L.) [32]. This effect may be due to UV radiation exposure and the cultivar of the plants studied. It is known that if plants are under stress, they accumulate phenolic

The increase in phenolic compounds in blueberry (*Vaccinium corymbosum*) plantlets cultivated in vitro exposed to aluminum (Al) and cadmium (Cd) has also been studied. These heavy metals cause high toxicity in plants, because they increase the oxidative stress by the production of reactive oxygen species (ROS). The authors of the study suggest that the phenolic compounds, specifically chlorogenic and ellagic acids, **Figure 11**, reduce the ROS in blueberry

An interesting study was carried out in 2011 by Mody et al., where they studied the effect of the resistance response of apple tree seedlings (*Malus* x *domestica*) to a leaf-chewing insect (*Spodoptera littoralis*) [34]. The authors found a significant herbivore preference for undamaged plants (induced resistance) was first observed 3 days after herbivore damage in the most apical leaf. Also, the results showed higher concentrations of the flavonoid phlorizin, **Figure 12**, in damaged plants than undamaged plants. This indicates that insect preference for undamaged apple plants may be linked to phlorizin, which is the main secondary metabolite of the phenolic

substrate to phenylpropanoid and flavonoid pathways [13].

**44**

**Figure 11.**

compounds.

plants [33].

type in apple leaves.

*Chemical structure of chlorogenic (C6-C3) and ellagic (C6-C1) acids.*

Knowledge of the biosynthetic pathway of shikimic acid leads to understanding the reaction mechanisms of enzymes and thus discovering antimicrobials, pesticides, and antifungals. Studies with isotopic labeling of substrates, the use of X-ray diffraction, nuclear magnetic resonance (NMR), mass spectrometry (ES), biotechnology, as well as organic synthesis have contributed to explaining the shikimate pathway. Although the seven steps of the biosynthetic pathway are elucidated, these metabolites are the precursors of phenolic compounds, more complex molecules that are necessary for the adaptation of plants to the environment. So, the shikimate pathway is the basis for the subsequent biosynthesis of phenolic compounds. There is scientific interest in continuing to investigate the biosynthesis of phenolic compounds from several points of view: pharmaceuticals, agronomy, chemical and food industries, genetics, and health.

#### **Acknowledgements**

The authors thank Carol Ann Hayenga for her English assistance in the preparation of this manuscript. The Technological University of the Mixteca provided support.

#### **Conflict of interest**

The authors have no conflict of interest to declare and are responsible for the content and writing of the manuscript.

#### **Ethical approval**

This chapter does not contain any studies with human participants or animals performed by any of the authors.

*Plant Physiological Aspects of Phenolic Compounds*

### **Author details**

Norma Francenia Santos-Sánchez\*, Raúl Salas-Coronado, Beatriz Hernández-Carlos and Claudia Villanueva-Cañongo Institute of Agroindustry, Technological University of the Mixteca, Huajuapan de León, Oaxaca, Mexico

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**47**

*Shikimic Acid Pathway in Biosynthesis of Phenolic Compounds*

2012;**30**:1425-1431. DOI: 10.1016/j.

[9] Weaver LM, Herrmann KM. Dynamic of the shikimate pathway. Trends in Plant Science. 1997;**9**:346-351

[10] Tzin V, Galili G. Amino acids biosynthesis pathways in plants. Molecular Plant. 2010;**3**:956-972. DOI:

[11] Dixon RA, Strack D. Phytochemistry meets genome analysis, and beyond. Phytochemistry. 2003;**62**:815-816

[12] Cheynier V, Comte G, Davies KM, Lattanzio V, Martens S. Plant phenolics: Recent advances on their biosynthesis, genetics and ecophysiology. Plant Physiology and Biochemistry. 2013;**72**: 1-20. DOI: 10.1016/j.plaphy.2013.05.009

[13] Zhang Z-Z, Li X-X, Chu Y-N, Zhang M-X, Wen Y-Q, Duan C-Q, et al. Three types of ultraviolet irradiation differentially promote expression of shikimate pathway genes and production of anthocyanins in grape berries. Plant Physiology and Biochemistry. 2012;**57**:74-83. DOI: 10.1016/j.plaphy.2012.05.005

[14] Floss HG, Onderka DK, Carroll M. Stereochemistry of the 3-deoxy-Darabino-heptulosonate 7-phosphate synthetase reaction and the chorismite synthetase reaction. The Journal of Biological Chemistry. 1972;**247**:736-744

[15] Schmid J, Amrhein N. Molecular organization of the shikimate pathway in higher plants. Phytochemistry.

[16] Bender SL, Mehdi S, Knowles JR. Dehydroquinate synthase: The role of divalent metal cations and of nicotinamide adenine dinucleotide

in catalysis. Biochemistry.

1989;**28**:7555-7560

1995;**39**:737-749

biotechadv.2012.03.001

10.1093/mp/ssq048

*DOI: http://dx.doi.org/10.5772/intechopen.83815*

[1] Adams ZP, Ehlting J, Edwards R. The regulatory role of shikimate in plant phenylalanine metabolism. Journal of Theoretical Biology. 2019;**462**:158-170.

DOI: 10.1016/j.jtbi.2018.11.005

10.1002/9780470742761

perspective. Plant Science. 2001;**161**:839-851. DOI: 10.1016/

10.1016/j.indcrop.2018.07046

copbio.2010.11.004

1999;**207**:325-334

10.1016/j.abb.2013.07.022

[5] Aharoni A, Galili G. Metabolic engineering of the plant primarysecondary metabolism interface. Current Opinion in Biotechnology. 2011;**22**:239-244. DOI: 10.1016/j.

[6] Macheroux P, Schmid J, Amrhein N, Schaller A. A unique reaction in a common pathway: Mechanism and function of chorismate synthase in the shikimate pathway. Planta.

[7] Mittelstädt G, Negron L, Schofiel LR, Marsh K, Parker EJ. Biochemical and structural characterisation of dehydroquinate synthase from the New Zealand kiwifruit *Actinidia chinensis*. Archives of Biochemistry and Biophysics. 2013;**537**:185-191. DOI:

[8] Ghosh S, Chisti Y, Banerjee UC. Production of shikimic acid. Biotechnology Advances.

S0168-9452(01)00490-3

[2] Dewick PM. Medicinal Natural Products: A Biosynthetic Approach. 3rd ed. United Kingdom: John Wiley and Sons Ltd.; 2009. p. 539. DOI:

[3] Bourgaud F, Gravot A, Milesi S, Gontier E. Production of plant secondary metabolites: A historical

[4] Yang D, Huang Z, Jin W, Xia P, Jia Q, Yang Z, et al. DNA methylation: A new regulator of phenolic acids biosynthesis in *Salvia miltiorrhiza*. Industrial Crops and Products. 2018;**124**:402-411. DOI:

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*Shikimic Acid Pathway in Biosynthesis of Phenolic Compounds DOI: http://dx.doi.org/10.5772/intechopen.83815*

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*Plant Physiological Aspects of Phenolic Compounds*

**46**

**Author details**

León, Oaxaca, Mexico

provided the original work is properly cited.

Norma Francenia Santos-Sánchez\*, Raúl Salas-Coronado, Beatriz Hernández-Carlos and Claudia Villanueva-Cañongo

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Institute of Agroindustry, Technological University of the Mixteca, Huajuapan de

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[2] Dewick PM. Medicinal Natural Products: A Biosynthetic Approach. 3rd ed. United Kingdom: John Wiley and Sons Ltd.; 2009. p. 539. DOI: 10.1002/9780470742761

[3] Bourgaud F, Gravot A, Milesi S, Gontier E. Production of plant secondary metabolites: A historical perspective. Plant Science. 2001;**161**:839-851. DOI: 10.1016/ S0168-9452(01)00490-3

[4] Yang D, Huang Z, Jin W, Xia P, Jia Q, Yang Z, et al. DNA methylation: A new regulator of phenolic acids biosynthesis in *Salvia miltiorrhiza*. Industrial Crops and Products. 2018;**124**:402-411. DOI: 10.1016/j.indcrop.2018.07046

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[7] Mittelstädt G, Negron L, Schofiel LR, Marsh K, Parker EJ. Biochemical and structural characterisation of dehydroquinate synthase from the New Zealand kiwifruit *Actinidia chinensis*. Archives of Biochemistry and Biophysics. 2013;**537**:185-191. DOI: 10.1016/j.abb.2013.07.022

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2012;**30**:1425-1431. DOI: 10.1016/j. biotechadv.2012.03.001

[9] Weaver LM, Herrmann KM. Dynamic of the shikimate pathway. Trends in Plant Science. 1997;**9**:346-351

[10] Tzin V, Galili G. Amino acids biosynthesis pathways in plants. Molecular Plant. 2010;**3**:956-972. DOI: 10.1093/mp/ssq048

[11] Dixon RA, Strack D. Phytochemistry meets genome analysis, and beyond. Phytochemistry. 2003;**62**:815-816

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[13] Zhang Z-Z, Li X-X, Chu Y-N, Zhang M-X, Wen Y-Q, Duan C-Q, et al. Three types of ultraviolet irradiation differentially promote expression of shikimate pathway genes and production of anthocyanins in grape berries. Plant Physiology and Biochemistry. 2012;**57**:74-83. DOI: 10.1016/j.plaphy.2012.05.005

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[15] Schmid J, Amrhein N. Molecular organization of the shikimate pathway in higher plants. Phytochemistry. 1995;**39**:737-749

[16] Bender SL, Mehdi S, Knowles JR. Dehydroquinate synthase: The role of divalent metal cations and of nicotinamide adenine dinucleotide in catalysis. Biochemistry. 1989;**28**:7555-7560

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[22] Sigh SA, Christendat D. Structure of *Arabidopsis* dehydroquinate dehydratase-shikimate dehydrogenase and implications for metabolic channeling in the shikimate pathway. Biochemistry. 2006;**45**:7787-7796. DOI: 10.1021/bi060366+

[23] Coracini JD, de Azevedo WF Jr. Shikimate kinase, a protein target for drug design. Current Medicinal Chemistry. 2014;**21**:592-604. DOI: 10.2174/09298673113206660299

[24] Blanco B, Prado V, Lence E, Otero JM, García-Doval C, van Raaij MJ, et al. *Mycobacterium tuberculosis* shikimate kinase inhibitors: Design and simulation studies of the catalytic turnover. Journal of the American Chemical Society.

2013;**135**:12366-12376. DOI: 10.1021/ ja405853p

[25] Lewis J, Johnson KA, Anderson KS. The catalytic mechanism of EPSP synthase revisited. Biochemistry. 1999:7372-7379

[26] Maroli A, Nandula V, Duke S, Tharayil N. Stable isotope resolved metabolomics reveals the role of anabolic and catabolic processes in glyphosate-induced amino acid accumulation in *Amaranthus palmeri* biotypes. Journal of Agricultural and Food Chemistry. 2016;**64**:7040-7048. DOI: 10.1021/acs.jafc.6b02196

[27] Cao G, Liu Y, Zhang S, Yang X, Chen R, Zhang Y, et al. A novel 5-enolpyruvylshikimate-3-phosphate synthase shows high glyphosate tolerance in *Escherichia coli* and *Tobacco Plants*. PLoS One. 2012;**7**:e38718. DOI: 10.1371/journal.pone.0038718

[28] Yi S-y, Cui Y, Zhao Y, Z-d L, Y-j L, Zhou F. A novel naturally occurring class I 5-enolpyruvyl shikimate-3-phosphate synthase from *Janibacter* sp. confers high glyphosate tolerance to rice. Scientific Reports. 2016;**6**:1904

[29] Liu F, Cao Y. Expression of a bacterial aroA gene confers tolerance to glyphosate in tobacco plants. Turkish Journal of Biology. 2018;**42**:187-194. DOI: 10.3906/biy-1712-56

[30] Bornemann S, Theoclitou M-E, Brune M, Webb MR, Thorneley RNF, Abell C. A secondary β deuterium kinetic isotope effect in the chorismate synthase reaction. Bioorganic Chemistry. 2000;**28**:191-204. DOI: 10.1006/bioo.2000.1174

[31] Osborne A, Thorneley RNF, Abell C, Bornemann S. Studies with substrate and cofactor analogues provide evidence for radical mechanism in the chorismate synthase reaction.

**49**

*Shikimic Acid Pathway in Biosynthesis of Phenolic Compounds*

*DOI: http://dx.doi.org/10.5772/intechopen.83815*

The Journal of Biological Chemistry.

[32] Marais E, Jacobs G, Holcroft DM. Postharvest irradiation enhances anthocyanin synthesis in apples but nor in pears. HortScience. 2001;**36**:738-740

[33] Manquián-Cerda K, Cruces E, Escudey M, Zúñiga G, Calderón R. Interactive effects of aluminum and cadmium on phenolic compounds, antioxidant enzyme activity and oxidative stress in blueberry

(*Vaccinium corymbosum* L.) plantlets cultivated in vitro. Ecotoxicology and Environmental Safety. 2018;**150**:320-326.

DOI: 10.1016/j.ecoenv.2017.12.050

[34] Gutbrod B, Mody K, Wittwer R, Dorn S. Within-plant distribution of induced resistance in apple seedlings: Rapid acropetal and delayed basipetal responses. Planta. 2011;**233**:1199-1207

2000;**275**:35825-35830

*Shikimic Acid Pathway in Biosynthesis of Phenolic Compounds DOI: http://dx.doi.org/10.5772/intechopen.83815*

The Journal of Biological Chemistry. 2000;**275**:35825-35830

*Plant Physiological Aspects of Phenolic Compounds*

2013;**135**:12366-12376. DOI: 10.1021/

[25] Lewis J, Johnson KA, Anderson KS. The catalytic mechanism of EPSP synthase revisited. Biochemistry.

[26] Maroli A, Nandula V, Duke S, Tharayil N. Stable isotope resolved metabolomics reveals the role of anabolic and catabolic processes in glyphosate-induced amino acid accumulation in *Amaranthus palmeri* biotypes. Journal of Agricultural and Food Chemistry. 2016;**64**:7040-7048.

DOI: 10.1021/acs.jafc.6b02196

10.1371/journal.pone.0038718

2016;**6**:1904

[28] Yi S-y, Cui Y, Zhao Y, Z-d L, Y-j L, Zhou F. A novel naturally occurring class I 5-enolpyruvyl shikimate-3-phosphate synthase from *Janibacter* sp. confers high glyphosate tolerance to rice. Scientific Reports.

[29] Liu F, Cao Y. Expression of a bacterial aroA gene confers tolerance to glyphosate in tobacco plants. Turkish Journal of Biology. 2018;**42**:187-194.

[30] Bornemann S, Theoclitou M-E, Brune M, Webb MR, Thorneley RNF, Abell C. A secondary β deuterium kinetic isotope effect in the chorismate

synthase reaction. Bioorganic Chemistry. 2000;**28**:191-204. DOI:

[31] Osborne A, Thorneley RNF, Abell C, Bornemann S. Studies with substrate and cofactor analogues provide evidence for radical mechanism in the chorismate synthase reaction.

10.1006/bioo.2000.1174

DOI: 10.3906/biy-1712-56

[27] Cao G, Liu Y, Zhang S, Yang X, Chen R, Zhang Y, et al. A novel 5-enolpyruvylshikimate-3-phosphate synthase shows high glyphosate

tolerance in *Escherichia coli* and *Tobacco Plants*. PLoS One. 2012;**7**:e38718. DOI:

ja405853p

1999:7372-7379

[17] Scholz BM, Maier G. Isomers of quinic acid and quinide in roasted coffee. Zeitschrift für Lebensmittel-Untersuchung und -Forschung.

[18] Harris JM, Gonzalez-Bello C, Kleanthous C, Hawkins A, Coggins J, Abell C. Evidence from kinetic isotope studies for an enolate intermediate in the mechanism of type II

[19] Hermann KM. The shikimate pathway: Early steps in the biosynthesis of aromatic compounds. The Plant Cell.

dehydroquinases. Biochemical Journal.

[20] Shneier A, Kleanthous C, Deka R, Coggins JR, Abel C. Observation of an imine intermediate on dehydroquinase by electrospray mass spectrometry. Journal of the American Chemical Society. 1991;**113**:9416-9418

[21] Sigh SA, Christendat D. The DHQdehydroshikimate-SDH-shikimate-NADP(H) complex: Insights into metabolite transfer in the shikimate pathway. Crystal Growth & Design.

[22] Sigh SA, Christendat D. Structure of *Arabidopsis* dehydroquinate

dehydratase-shikimate dehydrogenase

[23] Coracini JD, de Azevedo WF Jr. Shikimate kinase, a protein target for drug design. Current Medicinal Chemistry. 2014;**21**:592-604. DOI: 10.2174/09298673113206660299

[24] Blanco B, Prado V, Lence E, Otero JM, García-Doval C, van Raaij MJ, et al. *Mycobacterium tuberculosis* shikimate kinase inhibitors: Design and simulation studies of the catalytic turnover. Journal of the American Chemical Society.

and implications for metabolic channeling in the shikimate pathway. Biochemistry. 2006;**45**:7787-7796. DOI:

1990;**190**:132-134

1999;**319**:333-336

1995;**7**:907-919

2007;**7**:2153-2160

10.1021/bi060366+

**48**

[32] Marais E, Jacobs G, Holcroft DM. Postharvest irradiation enhances anthocyanin synthesis in apples but nor in pears. HortScience. 2001;**36**:738-740

[33] Manquián-Cerda K, Cruces E, Escudey M, Zúñiga G, Calderón R. Interactive effects of aluminum and cadmium on phenolic compounds, antioxidant enzyme activity and oxidative stress in blueberry (*Vaccinium corymbosum* L.) plantlets cultivated in vitro. Ecotoxicology and Environmental Safety. 2018;**150**:320-326. DOI: 10.1016/j.ecoenv.2017.12.050

[34] Gutbrod B, Mody K, Wittwer R, Dorn S. Within-plant distribution of induced resistance in apple seedlings: Rapid acropetal and delayed basipetal responses. Planta. 2011;**233**:1199-1207

**51**

**Chapter 4**

**Abstract**

**1. Introduction**

**1.1 Definition of miRNA**

apoptosis, and tumorigenesis.

MicroRNAs Associated with

*González-Márquez Marco Antonio, Rocha Oscar,*

*Cerna-Pantoja Diego and Andrés Cruz-Hernández*

and their use in the strategies to manipulate a controlled manipulation.

**Keywords:** alkaloids, flavonoids, flavonols, gene expression, isoprenoids

mRNAs are noncoding single-stranded RNA molecules that range from a length of 18 to 28 nucleotides. These molecules play an important role in posttranscriptional regulation by the inhibition of the expression of target genes by binding to mRNA [1]. Eukaryotic organisms, such as plants and animals, and some viruses express this type of molecules [2, 3]. Lin-4 is the first miRNA that was identified in *Caenorhabditis elegans*, although later in the same organism were found 22-nt lin-4 and 21-nt let-7; currently, more than 18,226 miRNAs are reported [4]. Although miRNA:target-gene interactions are widely conserved, this process is limited between kingdoms [5]. miRNAs are distributed in genome as single or clusters expressed as polycistronic units and share function relationships [6]. In plants, most miRNAs are encoded by their own primary transcript; few examples of miRNA cluster are reported (i.e., miR395). Introns are the main hotspots for their origination [7]. Some of the mechanisms where they act are the development of time and host-pathogen interactions, as well as cell differentiation, proliferation,

*Aguilar-Ruiz Carlos Agustín,*

Secondary Metabolites Production

MicroRNAs (miRNAs) are noncoding RNAs that play an important role in the regulation of the genetic expression in animals and plants by targeting mRNAs for cleavage or translational repression. Several miRNAs regulate the plant development, the metabolism, and the responses to biotic and abiotic stresses. Characterization of an miRNA has helped to show its role in fine tuning the mechanisms of posttranscriptional gene regulation. Although there is a lot of information related to miRNA regulation of some processes, the role of miRNA involved in the regulation of biosynthesis of secondary plant product is still poorly understood. In this chapter, we summarize the identification and characterization of miRNAs that participate in the regulation of the biosynthesis of secondary metabolites in plants

*Vargas-Hernández Marcela, Vázquez-Marrufo Gerardo,*

#### **Chapter 4**

### MicroRNAs Associated with Secondary Metabolites Production

*Vargas-Hernández Marcela, Vázquez-Marrufo Gerardo, Aguilar-Ruiz Carlos Agustín, González-Márquez Marco Antonio, Rocha Oscar, Cerna-Pantoja Diego and Andrés Cruz-Hernández*

### **Abstract**

MicroRNAs (miRNAs) are noncoding RNAs that play an important role in the regulation of the genetic expression in animals and plants by targeting mRNAs for cleavage or translational repression. Several miRNAs regulate the plant development, the metabolism, and the responses to biotic and abiotic stresses. Characterization of an miRNA has helped to show its role in fine tuning the mechanisms of posttranscriptional gene regulation. Although there is a lot of information related to miRNA regulation of some processes, the role of miRNA involved in the regulation of biosynthesis of secondary plant product is still poorly understood. In this chapter, we summarize the identification and characterization of miRNAs that participate in the regulation of the biosynthesis of secondary metabolites in plants and their use in the strategies to manipulate a controlled manipulation.

**Keywords:** alkaloids, flavonoids, flavonols, gene expression, isoprenoids

#### **1. Introduction**

#### **1.1 Definition of miRNA**

mRNAs are noncoding single-stranded RNA molecules that range from a length of 18 to 28 nucleotides. These molecules play an important role in posttranscriptional regulation by the inhibition of the expression of target genes by binding to mRNA [1]. Eukaryotic organisms, such as plants and animals, and some viruses express this type of molecules [2, 3]. Lin-4 is the first miRNA that was identified in *Caenorhabditis elegans*, although later in the same organism were found 22-nt lin-4 and 21-nt let-7; currently, more than 18,226 miRNAs are reported [4]. Although miRNA:target-gene interactions are widely conserved, this process is limited between kingdoms [5]. miRNAs are distributed in genome as single or clusters expressed as polycistronic units and share function relationships [6]. In plants, most miRNAs are encoded by their own primary transcript; few examples of miRNA cluster are reported (i.e., miR395). Introns are the main hotspots for their origination [7]. Some of the mechanisms where they act are the development of time and host-pathogen interactions, as well as cell differentiation, proliferation, apoptosis, and tumorigenesis.

#### **1.2 Mechanism of action of miRNAs**

The biogenesis of miRNAs in plants and animals presents some differences. In both plants and animals, the precursors of miRNAs are polyadenylated caps and RNAs, and transcribed as for any coding RNA by RNA polymerase II (RNAPII) [5, 8]. However, in the plants, the primary transcript (pri-miRNA) that gives rise to the miRNA is produced by the nuclear RNAase dicer-like 1 (DCL1) and its accessory proteins SERRATE (SE) and hyponastic leaves (HYL1) [8]. Also, Drosha gene is absent in plants [5].

In *Arabidopsis thaliana*, DCL1 and hyponastic leaves 1 (HYL1) cleave the primiRNA in the nucleus of the cell, which gives rise to the precursor-miRNA (premiRNA) dsRNA. Subsequently, there is another cleavage by the action of DCL1 and HYL1 to release the miRNA; the two nucleotide 3′ overhangs are methylated by the action of the sRNA-specific methyltransferase HUA enhancer1 (HEN1). When the mature single-stranded miRNA is found in the cytoplasm, it is loaded onto AGO1 that is present in RNA-induced silencing complexes (RISC), repressing the expression by mRNA cleavage [9]. 3′ untranslated region (3′ UTR) is the union region of the miRNAs to its target mRNAs which allows it to be repressed [10].

The expression of miRNAs is regulated by transcription factors. Negative on TATA less 2 (NOT2) promotes the transcription of protein miRNA genes and facilitates efficient DCL1 recruitment in miRNA biogenesis [11]. Cell division cycle 5 (CDC5) acts as a positive transcription factor associating with miRNA genes [12]. Pleiotropic regulatory locus 1 (PRL1) has the ability to bind to DCL1 and primiRNAs. The miRNA duplex is transported to the cytoplasm by nuclear export factor *Drosophila* Exportin-5 ortholog HASTY (HST).

The miRNA target genes can be a single member of a gene family or regulate a multiple family members. Thus, multiple miRNA genes could be targeting a single member, with tissues and stage specificity, and/or a single miRNA gene could be regulating multiple family members. The spatial and temporal expression and abundance of mature miRNAs are tightly regulated; they vary greatly among different miRNAs; and the abundance also varies depending on the tissue types or developmental stages [13].

#### **1.3 Regulatory processes involving miRNAs**

In the cytoplasm of cells, the miRNAs regulate the expression of genes at the posttranscriptional level via mRNA degradation and/or translational repression [14]. Unlike animals, in plants, there is a perfect complementarity between miRNA and target mRNA [14]. To carry out the silencing, a ribonucleoprotein RNA-induced silencing complex (RISC) is formed [15]. AGO1, AGO2, AGO4, AGO7, and AGO10 slicer activity has been reported, even though AGO1 is associated with most miRNAs [16]. AGO1-catalyzed RNA cleavage (slicing) represses miRNA targets [17].

#### **2. miRNAs and secondary metabolism in plants**

In plants, miRNAs control the expression of genes that encode transcription factors, stress response proteins, and others, which have an impact on biological processes. The miRNAs regulate the biological processes in the plans such as maintenance of genome integrity, primary and secondary metabolism, development, signal transduction, signaling pathways, homeostasis, innate immunity, and adaptive responses to biotic and abiotic stress [18]. Secondary metabolites are a group

**53**

*MicroRNAs Associated with Secondary Metabolites Production*

of phytochemicals that regulate various processes related to the interaction of the plant with its environment [19]. These compounds include terpenoids, alkaloids, phenolics, glycosides, tannins, and saponins, and defend plants from several biotic an abiotic stressors [20]. Even though these types of compounds are synthesized by plants to help in self-defense, they have diverse industrial uses such as insecticides, dyes, flavoring compounds, and nutraceuticals having a positive effect on human health. Commercial importance has resulted in a great interest in studying possibilities of enhancing its production [21]. It is known that miRNAs control several biological processes at the posttranscriptional level. Currently, some studies reveal the role that miRNAs have in the regulation of secondary metabolic pathways [20]. Therefore, the production of compounds derived from secondary metabolism can be managed through the miRNAs. Since they are positively or negatively regulated, the production of desired metabolites can be induced, the production of toxic

metabolites can be limited, and new metabolites can be produced [22].

Computational analysis carried out in two transcriptomes of Swertia resulted in the identification of miRNAs associated to secondary metabolites biosynthesis; miR-156a, miR-166a, miR-166b, miR-168, miR-11071, and miR-11320 targeting metabolic enzymes, such as aspartate aminotransferase, ribulose-phosphate 3-epimerase, acetyl-CoA acetyltransferase, phosphoglycerate mutase, and premnaspirodiene oxygenase, also include a gene encoding a homeobox-leucine zipper protein (HD-ZIP) with a possible association in secondary metabolites biosynthesis in *Swertia chirayita* [23]. Elicited or infected plants induce change in gene expression and production of defensive metabolites and these might be regulated by miRNAs. *Solanum tuberosum* L. under light stimulus found light-responsive miRNAs that are important regulators in alkaloid metabolism, UMP salvage, lipid biosynthesis, and cellulose catabolism [24]. Cadmium stress in oilseed rape (*Brassica napus* L.) reported miRNAs involved in the regulations of TFs, biotic stress defense, ion homeostasis, and secondary metabolism synthesis [25]. *Nicotiana tabacum* plants infected with tobacco mosaic virus (TMV), at the early stage of infection (5 dpi), show a cluster of miRNAs with down-accumulation, while most of the miRNAs were upregulated at 15 and 22 dpi, including both

Flavonoids are secondary metabolites that possess a polyphenolic structure. Those compounds consist of hydroxylated phenolic substances having a benzo--pyrone structure and derived of phenylpropanoid pathway [27]. Within the subgroups of the flavonoids are flavones, flavonols, flavanones, flavanonols, flavanols or catechins, anthocyanins, and chalcones [28]. For plants, this type of compounds is synthesized as a result of the interaction with the environment, other plants, and microorganisms. They have diverse biological functions as anti-oxidative, anti-inflammatory, antimutagenic, and anti-carcinogenic properties, which are structure dependent [28]. The above makes flavonoids a compound with nutraceutical, pharmaceutical, medicinal, and cosmetic applications [28]. The production of secondary metabolites is found in cases regulated by the miRNAs (**Table 1**). Little is known about the miRNAs involved in flavonoid biosynthesis. In *Helianthus*, 323,318 ESTs were computationally screened for the miRNAs identification of them, and a miR911 family was found related to the biosynthesis of tocopherols. Gou et al. [51] demonstrate that accumulation of anthocyanins in the stems of *A. thaliana* is under the regulation of miR156 targeted squamosa promoter binding protein-like (SPL) genes. High miR156 activity promotes accumulation of anthocyanins and activity-induced of flavonols. This study also demonstrates that SPL9 negatively regulates anthocyanin accumulation through

*DOI: http://dx.doi.org/10.5772/intechopen.83804*

miRNAs and miRNA targets [26].

**2.1 Flavonoids**

#### *MicroRNAs Associated with Secondary Metabolites Production DOI: http://dx.doi.org/10.5772/intechopen.83804*

*Plant Physiological Aspects of Phenolic Compounds*

The biogenesis of miRNAs in plants and animals presents some differences. In both plants and animals, the precursors of miRNAs are polyadenylated caps and RNAs, and transcribed as for any coding RNA by RNA polymerase II (RNAPII) [5, 8]. However, in the plants, the primary transcript (pri-miRNA) that gives rise to the miRNA is produced by the nuclear RNAase dicer-like 1 (DCL1) and its accessory proteins SERRATE (SE) and hyponastic leaves (HYL1) [8]. Also, Drosha gene is

In *Arabidopsis thaliana*, DCL1 and hyponastic leaves 1 (HYL1) cleave the primiRNA in the nucleus of the cell, which gives rise to the precursor-miRNA (premiRNA) dsRNA. Subsequently, there is another cleavage by the action of DCL1 and HYL1 to release the miRNA; the two nucleotide 3′ overhangs are methylated by the action of the sRNA-specific methyltransferase HUA enhancer1 (HEN1). When the mature single-stranded miRNA is found in the cytoplasm, it is loaded onto AGO1 that is present in RNA-induced silencing complexes (RISC), repressing the expression by mRNA cleavage [9]. 3′ untranslated region (3′ UTR) is the union region of

The expression of miRNAs is regulated by transcription factors. Negative on TATA less 2 (NOT2) promotes the transcription of protein miRNA genes and facilitates efficient DCL1 recruitment in miRNA biogenesis [11]. Cell division cycle 5 (CDC5) acts as a positive transcription factor associating with miRNA genes [12]. Pleiotropic regulatory locus 1 (PRL1) has the ability to bind to DCL1 and primiRNAs. The miRNA duplex is transported to the cytoplasm by nuclear export

The miRNA target genes can be a single member of a gene family or regulate a multiple family members. Thus, multiple miRNA genes could be targeting a single member, with tissues and stage specificity, and/or a single miRNA gene could be regulating multiple family members. The spatial and temporal expression and abundance of mature miRNAs are tightly regulated; they vary greatly among different miRNAs; and the abundance also varies depending on the tissue types or

In the cytoplasm of cells, the miRNAs regulate the expression of genes at the posttranscriptional level via mRNA degradation and/or translational repression [14]. Unlike animals, in plants, there is a perfect complementarity between miRNA and target mRNA [14]. To carry out the silencing, a ribonucleoprotein RNA-induced silencing complex (RISC) is formed [15]. AGO1, AGO2, AGO4, AGO7, and AGO10 slicer activity has been reported, even though AGO1 is associated with most miRNAs [16]. AGO1-catalyzed RNA cleavage (slicing) represses

In plants, miRNAs control the expression of genes that encode transcription factors, stress response proteins, and others, which have an impact on biological processes. The miRNAs regulate the biological processes in the plans such as maintenance of genome integrity, primary and secondary metabolism, development, signal transduction, signaling pathways, homeostasis, innate immunity, and adaptive responses to biotic and abiotic stress [18]. Secondary metabolites are a group

the miRNAs to its target mRNAs which allows it to be repressed [10].

factor *Drosophila* Exportin-5 ortholog HASTY (HST).

**1.3 Regulatory processes involving miRNAs**

**2. miRNAs and secondary metabolism in plants**

**1.2 Mechanism of action of miRNAs**

absent in plants [5].

developmental stages [13].

miRNA targets [17].

**52**

of phytochemicals that regulate various processes related to the interaction of the plant with its environment [19]. These compounds include terpenoids, alkaloids, phenolics, glycosides, tannins, and saponins, and defend plants from several biotic an abiotic stressors [20]. Even though these types of compounds are synthesized by plants to help in self-defense, they have diverse industrial uses such as insecticides, dyes, flavoring compounds, and nutraceuticals having a positive effect on human health. Commercial importance has resulted in a great interest in studying possibilities of enhancing its production [21]. It is known that miRNAs control several biological processes at the posttranscriptional level. Currently, some studies reveal the role that miRNAs have in the regulation of secondary metabolic pathways [20]. Therefore, the production of compounds derived from secondary metabolism can be managed through the miRNAs. Since they are positively or negatively regulated, the production of desired metabolites can be induced, the production of toxic metabolites can be limited, and new metabolites can be produced [22].

Computational analysis carried out in two transcriptomes of Swertia resulted in the identification of miRNAs associated to secondary metabolites biosynthesis; miR-156a, miR-166a, miR-166b, miR-168, miR-11071, and miR-11320 targeting metabolic enzymes, such as aspartate aminotransferase, ribulose-phosphate 3-epimerase, acetyl-CoA acetyltransferase, phosphoglycerate mutase, and premnaspirodiene oxygenase, also include a gene encoding a homeobox-leucine zipper protein (HD-ZIP) with a possible association in secondary metabolites biosynthesis in *Swertia chirayita* [23]. Elicited or infected plants induce change in gene expression and production of defensive metabolites and these might be regulated by miRNAs. *Solanum tuberosum* L. under light stimulus found light-responsive miRNAs that are important regulators in alkaloid metabolism, UMP salvage, lipid biosynthesis, and cellulose catabolism [24]. Cadmium stress in oilseed rape (*Brassica napus* L.) reported miRNAs involved in the regulations of TFs, biotic stress defense, ion homeostasis, and secondary metabolism synthesis [25]. *Nicotiana tabacum* plants infected with tobacco mosaic virus (TMV), at the early stage of infection (5 dpi), show a cluster of miRNAs with down-accumulation, while most of the miRNAs were upregulated at 15 and 22 dpi, including both miRNAs and miRNA targets [26].

#### **2.1 Flavonoids**

Flavonoids are secondary metabolites that possess a polyphenolic structure. Those compounds consist of hydroxylated phenolic substances having a benzo--pyrone structure and derived of phenylpropanoid pathway [27]. Within the subgroups of the flavonoids are flavones, flavonols, flavanones, flavanonols, flavanols or catechins, anthocyanins, and chalcones [28]. For plants, this type of compounds is synthesized as a result of the interaction with the environment, other plants, and microorganisms. They have diverse biological functions as anti-oxidative, anti-inflammatory, antimutagenic, and anti-carcinogenic properties, which are structure dependent [28]. The above makes flavonoids a compound with nutraceutical, pharmaceutical, medicinal, and cosmetic applications [28]. The production of secondary metabolites is found in cases regulated by the miRNAs (**Table 1**). Little is known about the miRNAs involved in flavonoid biosynthesis. In *Helianthus*, 323,318 ESTs were computationally screened for the miRNAs identification of them, and a miR911 family was found related to the biosynthesis of tocopherols. Gou et al. [51] demonstrate that accumulation of anthocyanins in the stems of *A. thaliana* is under the regulation of miR156 targeted squamosa promoter binding protein-like (SPL) genes. High miR156 activity promotes accumulation of anthocyanins and activity-induced of flavonols. This study also demonstrates that SPL9 negatively regulates anthocyanin accumulation through

**55**

**Plant species** Korean ginseng

miR854b and miR854c

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), farnesyl

diphosphate synthase (FPS), geranyl-diphosphate synthase, squalene

synthase, and squalene epoxidase (SE)

MYB transcription factor, geranyl diphosphate synthase, GCPE protein,

Primary and secondary metabolism,

[41]

Isoprenoid/terpenoid biosynthesis

iridoid production in higher plants

Terpenoid biosynthesis

Mono-, sesqui-, di-, and tri-terpenoids

biosynthesis

Terpene biosynthesis

[43]

UDP-glucose iridoid glucosyltransferase

Nontranscriptional factor proteins, such as DXS, HMGR, IDS, and IDI,

R-linalool synthase, gibberellin 3-oxidase, ent-kaurene synthase, squalene

epoxidase, beta-amyrin synthase, and germacrene A oxidase

essential to produce IPP and DMAPP

(*Panax ginseng*

Meyer)

*C. roseus* *Xanthium*

miR7539, miR5021, and

*strumarium* L.

*Xanthium*

miR7540, miR5183,

miR6449, miR5255,

miR5491, and miR6435

*strumarium* L.

*Ferula gummosa*

miR2919, miR5251,

SPL7, SPL11, and ATHB13 TFs

miR838, miR5021, and

miR5658

*Pogostemon cablin*

*A. thaliana* *Mentha* spp.

*Others*

*S. chirayita*

miR-168, miR-11320, miR-166a, miR-11071, miR-156a

Acetyl-CoA acetyltransferase (AACT), aspartate aminotransferase (PHAT),

premnaspirodiene oxygenase (PSO), ribulose-phosphate 3-epimerase (RPE),

phosphoglycerate mutase (PGM), and a gene encoding homeobox-leucine

zipper protein (HD-ZIP)

Family of small molecules of methyltransferases

Auxin receptors (TIR1, AFB2 and AFB3)

Secondary metabolism

Increase of glucosinolate and decrease

of camalexin

[45]

[46]

and miR-166b

*A. thaliana* *A. thaliana*

miR393

miR163

miR156, miR414, and

Basic helix-loop-helix (bHLH) geranyl di-phosphate synthase subunit alpha-

like protein (NACA), respectively

miR5021

miR156

miRNA156

Squamosa promoter binding protein-like (SPL)

SPL transcription factor

Sesquiterpene biosynthesis

Modulate sesquiterpene synthase gene

TPS21 responsible for the biosynthesis

of (E)-β-caryophyllene

Terpenoid backbone biosynthesis, sesquiterpenoid and triterpenoid

[38]

biosynthesis

Secondary metabolites biosynthesis

[23]

[44]

[44]

miR1134

mir-5021

**miRNA**

**Target**

**Function**

**References**

[40]

*MicroRNAs Associated with Secondary Metabolites Production*

[42]

*DOI: http://dx.doi.org/10.5772/intechopen.83804*

[42]

#### *Plant Physiological Aspects of Phenolic Compounds*

#### *MicroRNAs Associated with Secondary Metabolites Production DOI: http://dx.doi.org/10.5772/intechopen.83804*

*Plant Physiological Aspects of Phenolic Compounds*

**54**

**Plant species**

*Flavonoids*

Sunflower *A. thaliana* *Diospyros kaki* *Lonicera japonica*

miRNAs (U436803,

U977315, U805963,

U3938865 and U4351355)

*A. thaliana* *Halostachys*

*caspica*

*Podophyllum*

miR1873/miR5532

*hexandrum*

*Alkaloids*

Opium poppy

pso-miR13, pso-miR2161,

7-O-methyltransferase, *S*-adenosyl-l-methionine:3′-hydroxy-

methylcoclaurine 4′-O-methyltransferase 2/ 4′-O-methyltransferase2

(4-OMT)/FAD-binding and BBE domain-containing protein, also known as

reticuline oxidase-like protein

QPT1, QPT2, CYP82E4, and PMT2

Taxane 13α hydroxylase and taxane 2α-O-benzoyltransferase

Targets kaempferol 3-O-beta-D-galactosyltransferase

Basic helix-loop-helix (bHLH)

3-Deoxy-7-phosphoheptulonate synthase (DAHP synthase)

Terpenoid biosynthesis ultimately

[39]

affecting the production of picroside-I

Paclitaxel biosynthetic genes

Flavonol glycosides

Flavone/flavonol biosynthesis

[38]

[36]

[37]

Nicotine biosynthesis and catabolism

[35]

*N*-

Benzylisoquinoline alkaloids

[34]

and pso-miR408

(*Papaver*

*somniferum)*

Tobacco *T. baccata* *R. serpentina*

*Mentha* spp.

*Terpenoids*

*P. kurroa*

iRNA-4995

miR156

miR396b

miRX17, miRX27, miRX20,

and miRX19

miR164 and miR171

MicroRNA858a

miR6194 and miR5655

R2R3-MYB transcription factors

Flavanone 3-hydroxylase

Dihydroflavonol 4-reductase C/-hydroxyisoflavanone dehydratase

miR395p-3p and miR858b

bHLH and MYB, respectively

R2R3-MYB transcription factors

miR156

miR2911

Gamma-tocopherol methyl transferase

SPL transcription factor

**miRNA**

**Target**

**Function** Tocopherols biosynthesis

Accumulation of anthocyanins,

whereas reduced miR156 activity

results in high levels of flavonols

Proanthocyanidin biosynthesis

Flavonoid biosynthesis

Flavonoid biosynthesis

Flavonols, anthocyanidins

proanthocyanidins synthesis

Flavonoid/isoflavonoid biosynthesis

[33]

[31]

[32]

[30]

[2, 3]

[29]

[51]

**References**

**Table 1.**

**57**

biosynthesis [38].

**2.3 Terpenoids**

**2.2 Alkaloids**

*MicroRNAs Associated with Secondary Metabolites Production*

soflavanone dehydratase is related to isoflavonoid biosynthesis [33].

Alkaloids are naturally compounds that have one or more of their nitrogen atoms. Alkaloids are classified into different groups: indole, piperidine, tropane, purine, pyrrolizidine, imidazole, quinolizidine, isoquinoline, and pyrrolidine alkaloids [52]. Because of their toxicity, alkaloids act as defense compounds against diverse pathogens or herbivores. Understanding the regulation of alkaloid biosynthesis is crucial for its production. Target transcript identification analyses in Opium poppy (*Papaver somniferum*) revealed that pso-miR13, pso-miR2161, and pso-miR408 (**Table 1**) might be involved in BIA biosynthesis. pso-miR13 might cleave 7-OMT transcript, involved in the conversion of S-reticuline to morphinan alkaloids. 4-OMT is the target of pso-miR216 and mediates the production of S-reticuline that is also an intermediate molecule in BIA biosynthesis. On the other hand, pso-miR408 possibly targets mRNA from reticuline oxidase-like protein in charge of the conversion of S-reticuline to (S)-scoulerine in the BIA pathway [34]. Studies in tobacco (*Nicotiana tabacum*), identified four unique tobacco-specific miRNAs miRX17, miRX27, miRX20, and miRX19 that were predicted to target key genes of the nicotine biosynthesis and catabolism pathways, QPT1, QPT2, CYP82E4, and PMT2 genes, respectively [35]. In *Taxus baccata*, two paclitaxel biosynthetic genes, taxane 13α hydroxylase and taxane 2α-O-benzoyltransferase, are the cleavage targets of miR164 and miR171, respectively [36]. *In silico* analysis reveals that miR396b in *Rauwolfia serpentina* targets kaempferol 3-O-beta-D-galactosyltransferase whose activity as transferase activity, transferring hexosyl groups is essential for formation of flavonol glycosides [37]. A computational approach in *Mentha* spp., revealed that miR156, miR414, and miR5021 are essential for regulation of essential oil biosynthesis.

miR156 participates in flavone, flavonol biosynthesis, and terpenoid backbone

Plant terpenoids secondary metabolites are synthesized from C5 precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). They are

destabilization of a MYB-bHLH-WD40 transcriptional activation complex. *Diospyros kaki* fruits collected at two examined stages (15 and 20 WAF) showed differential expression of the mRNAs, indicating that these miRNAs might regulate PA synthesis during development, and some of them are miR858 and miR156, which regulate PA synthesis. miR858 positively regulates the genes responsible for the production of PA, while miR156 does so in a negative way. miR395 is another miRNA that has an influence on PA biosynthesis [30]. Some miRNAs (U436803, U977315, U805963, U3938865, and U4351355) regulate fatty acid and flavonoid biosynthesis in *Lonicera japonica* [2, 3]. The characterization in *A. thaliana* shows that miR858a targets MYB transcription factors that are involved in flavonoid biosynthesis, growth, and development. Over-expression of miR858a downregulates several MYB transcription factors, and the higher expression of MYBs in MIM858 lines leads to the redirection of the metabolic flux toward the synthesis of flavonoids [31]. Yang et al. [32] indicate that salt stress conditions regulate miRNAs; some salt stress-related biological pathways includes calcium signaling pathway, MAPK signaling pathway, plant hormone signal transduction, and flavonoid biosynthesis [32]. Himalayan mayapple (*Podophyllum hexandrum*), miR1438 target caffeoyl-CoA O-methyltransferase and is related to phenylalanine metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis, stilbenoid, diarylheptanoid, and gingerol biosynthesis. miR1873 targets dihydroflavonol 4-reductase C related to flavonoid biosynthesis. miR5532 2-hydroxyi-

*DOI: http://dx.doi.org/10.5772/intechopen.83804*

*miRNA related to secondary metabolite production.* destabilization of a MYB-bHLH-WD40 transcriptional activation complex. *Diospyros kaki* fruits collected at two examined stages (15 and 20 WAF) showed differential expression of the mRNAs, indicating that these miRNAs might regulate PA synthesis during development, and some of them are miR858 and miR156, which regulate PA synthesis. miR858 positively regulates the genes responsible for the production of PA, while miR156 does so in a negative way. miR395 is another miRNA that has an influence on PA biosynthesis [30]. Some miRNAs (U436803, U977315, U805963, U3938865, and U4351355) regulate fatty acid and flavonoid biosynthesis in *Lonicera japonica* [2, 3]. The characterization in *A. thaliana* shows that miR858a targets MYB transcription factors that are involved in flavonoid biosynthesis, growth, and development. Over-expression of miR858a downregulates several MYB transcription factors, and the higher expression of MYBs in MIM858 lines leads to the redirection of the metabolic flux toward the synthesis of flavonoids [31]. Yang et al. [32] indicate that salt stress conditions regulate miRNAs; some salt stress-related biological pathways includes calcium signaling pathway, MAPK signaling pathway, plant hormone signal transduction, and flavonoid biosynthesis [32]. Himalayan mayapple (*Podophyllum hexandrum*), miR1438 target caffeoyl-CoA O-methyltransferase and is related to phenylalanine metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis, stilbenoid, diarylheptanoid, and gingerol biosynthesis. miR1873 targets dihydroflavonol 4-reductase C related to flavonoid biosynthesis. miR5532 2-hydroxyisoflavanone dehydratase is related to isoflavonoid biosynthesis [33].

#### **2.2 Alkaloids**

*Plant Physiological Aspects of Phenolic Compounds*

[49]

[50]

**56**

**Plant species**

Potato *S. rebaudiana*

*Arabidopsis*

miR826 and miR5090

AOP2

*thaliana*

*A. thaliana* *Salvia miltiorrhiza*

**Table 1.**

*miRNA related to secondary metabolite production.*

miR5072

miR826

Alkenyl hydroxyalkyl producing 2

Acetyl-CoA C-acetyl transferase

miRstv\_7

mirn79

**miRNA**

**Target** AP2/ERF transcription factor

and kaurene oxidase (KO)

UDP-glycosyltransferase 76G1 (ugt76g1), kaurenoic acid hydroxylase (KAH),

**Function** JA-responsive secondary metabolites

Steviol glycoside biosynthesis

Glucosinolate biosynthesis

Glucosinolate synthesis

Tanshinones biosynthesis

[48]

[47]

[24]

**References**