Phenylalanine promotes phenolic compounds and quality of Qızıl Uzum grape (Vitis vinifera L.) fruit by regulating expression patterns of PAL and CHS genes and modulating the antioxidant system

doi:10.21203/rs.3.rs-1821067/v1

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Phenylalanine promotes phenolic compounds and quality of Qızıl Uzum grape (Vitis vinifera L.) fruit by regulating expression patterns of PAL and CHS genes and modulating the antioxidant system

https://doi.org/10.21203/rs.3.rs-1821067/v1

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With the aim of quality improvement, the effects of foliar application of phenylalanine at 5 concentrations on grape (Vitis vinifera cv. Qızıl Uzum) berry compositions were studied. The studied parameters included antioxidant activity, total phenolics, total flavonoids, total anthocyanin, catalase, phenylalanine ammonia-lyase enzyme activity, and phenolic compounds content based on HPLC analyses (myricetin, quercetin, kaempferol, syringetin, catechin, gallic acid, caffeic acid, p-coumaric acid, resveratrol). Phenylalanine at 1000 μM increased total phenols, flavonoids, anthocyanins, and PAL enzyme activity. In addition, HPLC analysis revealed a significant accumulation of individual phenolic compounds by phenylalanine treatment. The highest values were recorded at 100 and 500 μM phenylalanine treatments. Real-time quantitative RT-PCR results showed that the expression of PAL and CHS genes was induced by phenylalanine. The highest PAL and CHS gene expression was observed at 500 μM and then at 1000 μM phenylalanine.

Antioxidant capacity

PAL and CHS genes

Phenolic compounds

Phenylalanine

Phenylalanine ammonia-lyase

Grape (Vitis vinifera L.) is one of the most important fruit products globally and has a high nutritional value with potent antioxidant and anti-cancer activities. Hence, its antioxidant properties are considered one of the leading indicators of its quality¹. It’s a good source of polyphenols that protect against chronic diseases such as diabetes, cancer, and heart diseases. Recently, much attention has been paid to using natural and safe substances in terms of fruit quality improvement, leading to better marketing of fruits. Studies have shown that amino acids can directly or indirectly affect the physiological activities of plants. In addition, they considered the essential ingredients of protein synthesis.

In current years, phenylalanine application has been particularly noticed for enhancing the nutritional quality of horticultural crops. Phenylalanine, one of the most important amino acids, is an organic molecule that directly or indirectly can influence plant physiological activities associated with the cell’s growth and development². It also plays an essential role in many biochemical processes in the plant via the phenylpropanoid pathway. It is noted that phenylalanine is found as an abundant amino acid in grapes and is described in the first stage of the phenylpropanoid pathway, the precursor of various phenolic compounds³. It is well demonstrated that apart from the fact that phenylalanine incorporation into grape berries influences berry phenolic metabolism, also it is detected as a positive compound for grape and wine aromatic characteristics⁴, respect to these facts, this aromatic amino acid drastically participates in the production of aromatic compounds and particular antioxidant components⁵.

At present, the phenolic composition of grapes has been considered to increase their concentration in fruits because their properties are directly or indirectly related to the quality of grapes and affect their color and astringency. Phenylalanine has been reported to improve the content of phenolic compounds, including anthocyanins and flavonoids in grape fruit⁶.

Phenolic compounds are described as a heterogeneous group of secondary metabolites that significantly affect the organoleptic properties of grapes with their help on color, stability, and mouthfeel, such as astringency or bitterness⁷. These bioactive compounds are of great importance in plant metabolism, exhibiting a variety of biological functions. Interestingly, these bioactive compounds show effective antioxidant activity; it can be attributed to the properties of their radical inhibition and their ability to chelate metals by donating hydrogen, absorbing single oxygen, or acting as a chelator of reducing factors. In addition, the use of phenylalanine has been claimed to increase the antioxidant potential of fruits by regulating phenolic biosynthesis⁸. As reported, phenylalanine-treated grapes showed improvements in antioxidant potential, and phenol synthesis affected antioxidant activity⁹.

Different genes such as chalcone synthase (CHS) and phenylalanine ammonia-lyase (PAL) are involved in the biosynthetic pathway of phenolic compounds whose expression levels vary under different conditions. The CHS gene controls the first step in the flavonoid biosynthesis pathway¹⁰. PAL is a key enzyme in the phenylpropanoid pathway involved in the biosynthesis of phenolics and may also reflect the relationship between the metabolism of secondary metabolite synthesis and the treatments applied. Also, it is considered a regulatory and significant enzyme in the phenylpropanoid pathway of the grape berry. It is a key enzyme between primary and secondary phenylpropanoid metabolism that modulates the synthesis of phenolic compounds¹¹. This pathway catalyzes the kinematic synthesis of cinnamic acid from phenylalanine, the primary mediator in the biosynthesis of phenols and the precursor of various phenylpropanoids. It has been claimed that the use of phenylalanine significantly increased PAL activity by affecting membrane lipid peroxidation and ROS accumulation and the higher content of phenols and flavonoids in plum fruit^8,11. It should be noted that the right time to apply nitrogen in grapes can improve the phenolic composition. The veraison stage is considered to be only the most prosperous period of phenolic compounds synthesis, so it can be suggested that foliar application of phenylalanine during veraison may increase the synthesis of biologically active compounds and improve the antioxidant potential of grapes⁹.

In general, phenylalanine plays a key role in linking primary and secondary metabolism in plants. Phenylalanine is a building block of proteins and a precursor to a number of essential plant compounds involved in growth, reproduction, and defense against various stresses. Phenylalanine also plays an important role in the transfer of synthetic carbon in the process of photosynthesis for the biosynthesis of phenylpropanoids¹². Therefore, foliar application of phenylalanine may be considered a suitable organic matter to improve the quality of Qizil Uzum table grape. Qızıl Uzum, a seeded, red-purple color table grape is considered one of the commercial grape cultivars with good quality maintenance in West Azarbaijan province, Iran. This variety has vigorous growth, regular and high yield with good marketability.

Due to the importance of this grape cultivar concerning its nutritional and economic significance and lack of knowledge about the effect of phenylalanine application on phenolic content and quality of grapes, the present study was performed to evaluate the impact of phenylalanine foliar application on the improvement of phenolic compounds and expression patterns of PAL and CHS genes involved in their biosynthesis in Qızıl Uzum table grape.

Antioxidant activity (AA). The results of determining total antioxidant activity are shown in Fig. 2. According to the results, foliar application of phenylalanine significantly increased the AA content of the berries (p ≤ 0.01). The highest AA based on DPPH was recorded in berries treated with 2000 µM phenylalanine and then 500 µM. No significant difference was observed among other applied concentrations.

Total phenolic content. Phenylalanine significantly affected the phenol content of berries (P ≤ 0.01). The highest total phenolic content was recorded at 1000 µM phenylalanine, while no significant difference was observed between the concentration of 500 µM and 2000 µM and the lowest was observed at the concentration of 100 µM (Fig. 2).

Total flavonoid content. Results of grape flavonoid content were displayed in Fig. 3. According to the results, foliar application of phenylalanine significantly influenced flavonoids content (P ≤ 0.05). The highest flavonoids content was recorded in fruits treated with 1000 µM phenylalanine, although there was no significant difference among all functional concentrations of phenylalanine.

Total anthocyanin contents. The results of anthocyanin determination were shown in Fig. 3. Based on these results, foliar application of phenylalanine significantly increased the anthocyanin content in treated fruits (P ≤ 0.01). Phenylalanine at a concentration of 500 µM resulted in the highest amount of anthocyanin, while there was no significant difference among the other concentrations.

Phenolic compositions by HPLC analysis. Flavonols compounds. The results of individual flavonols for the control and treated grape samples are shown in Table 1. Based on this research, myricetin, quercetin, kaempferol, and syringetin, the major components of flavonols, were found in all samples. These compounds were significantly affected by phenylalanine application (P ≤ 0.01). The highest amount of myricetin was obtained by applying 500 µM phenylalanine. Quercetin, kaempferol, and syringetin showed the highest values at 500 µM phenylalanine application.

Flavanols compounds. The results of the HPLC analysis of flavanol compounds are shown in Table 1. Catechin content was significantly affected by phenylalanine foliar application (P ≤ 0.01). The highest level of flavanols was obtained from 100 µM phenylalanine and then 500 µM.

Non-flavonoid compounds. The results of the HPLC analysis of grape non-flavonoid compounds are shown in Table 1. The present results identified gallic acid, caffeic acid, p-coumaric acid, and resveratrol as the main non256 flavonoid compounds identified in the treated grape samples. The results showed that foliar application of phenylalanine at different concentrations significantly affected non-flavonoid components (P ≤ 0.01). The highest amount of gallic acid was recorded at a concentration of 1000 µM phenylalanine, but no significant difference was observed among all applied concentrations. In addition, caffeic acid showed the highest value using 100 µM and 500 µM phenylalanine. Also, p-coumaric acid reached its maximum rate at 100 and 500 µM phenylalanine concentrations, followed by 2000 µM. In this case, the highest amount of resveratrol was observed due to 500 µM phenylalanine, and no significant difference was observed among other concentrations.

Table 1

Effect of foliar spray with different doses of Phe. (0, 100, 500, 1000, 2000 µM) on phenolic compounds of ‘Qızıl Uzum’ table grape berries (µgg− 1 FW)
Phe.	Myricetin	Quercetin	Kaempferol	Syringetin	Catechin	Gallic acid	Caffeic acid	p-Coumaric acid	Resveratrol
0 µM	1.17^c	9.10^d	2.16^c	12.90^d	12.00^e	2.57^b	1.50^b	0.70^c	0.38^c
100 µM	2.79^a	18.55^b	6.37^a	20.27^a	26.98^a	4.70^a	2.35^a	3.50^a	0.90^b
500 µM	2.93^ab	22.44^a	3.43^b	18.43^b	22.20^b	4.38^a	2.45^a	3.20^a	1.45^a
1000 µM	2.10^ab	14.75^c	2.45^c	15.25^c	19.05^c	4.95^a	1.80^b	2.05^b	0.60^b
2000 µM	1.55^bc	17.60^b	2.20^c	14.27^d	17.30^d	4.80^a	1.65^b	2.65^ab	0.73^b
Means indicated with different letters in a column are significantly different at p ≤ 0.01 according to Duncan’s Multiple Range Tests

PAL activity enzyme. The results of determining PAL activity are shown in Fig. 4. Based on the data, a significant effect of phenylalanine application was observed in all treated samples (P ≤ 0.01). The highest PAL activity was recorded at 500 µM phenylalanine followed by 100 and 1000 µM, although no significant difference was observed among the following concentrations.

CAT enzyme activity. Figure 4 shows the results of CAT enzyme activity. According to the results, 1000 µM phenylalanine had the highest CAT activity, followed by 500 µM, and no significant difference was observed between 100 µM and control. The results showed that foliar application of phenylalanine had a significant effect on CAT activity.

Expression of PAL gene. The relative mRNA expression level of PAL gene was investigated using qRT-PCR at 2 sampling times, 48 and 72 hours after phenylalanine foliar application. PAL gene expression levels were significantly affected by different concentrations of phenylalanine, sampling time, and their interactions at the level of 1% (P ≤ 0.01). The highest PAL transcription accumulation was observed at 500 µM phenylalanine-treated samples, followed by 1000 µM (Fig. 5).

Expression of CHS gene. According to qRT-PCR results, CHS gene expression in phenylalanine-treated samples increased significantly at 48 and 72 h after phenylalanine foliar application compared to the control (Fig. 5). A significant difference (P ≤ 0.01) was observed for CHS gene expression at different levels of phenylalanine concentrations and among sampling times. The phenylalanine concentrations and sampling time interaction were also significant on CHS gene expression level. Over-expression of CHS at 500 µM phenylalanine application was observed at both sampling times, and there was no significant difference among other concentrations of phenylalanine (Fig. 5).

Many antioxidant systems with different chemical and physical properties are involved in plants with free radicals and reactive oxygen species (ROS) scavenging, which exhibit differential inhibitory mechanisms of antioxidant activity. In this regard, phenolic compounds such as flavonoids and anthocyanins as important components of non-enzymatic antioxidant defense systems, as well as catalase (CAT) and superoxide dismutase (SOD) are one of the main types of enzymes in plants that aim to protect plant cells against various biotic and abiotic stresses⁸.

It has been explained that there is a high relationship between total phenolic content and the antioxidant activity of grape berries. There is also a positive relationship between grape flavonoid content and antioxidant activity¹³. It has been shown that the antioxidant activity of phenolic compounds can be affected by their structure, such as the position of hydroxyl groups. It should be noted that each phenolic component has a certain amount of antioxidant activity. Given all these effects, it can be suggested that the total phenol and flavonoid content is a good indicator of the antioxidant activity of grape berries¹³.

According to our results, phenylalanine foliar application increased the antioxidant activity of grapes (Fig. 2). These results are consistent with Cheng et al.⁹ findings on Cabernet Sauvignon grape that reported antioxidant activities promotion with phenylalanine. They claimed that phenylalanine foliar application could significantly improve the antioxidant properties of grape fruits. Based on their study, the increase in antioxidant activity with phenylalanine was associated with high phenol content and total flavonoid content in grapes because the advanced antioxidant activity in Cabernet Sauvignon was obtained by regulating phenolic biosynthesis⁹.

Due to the increase of antioxidant activity through phenylalanine treatment, it was concluded that the increase in ABA content and the related gene expression is closely associated with the increase in antioxidant activity due to phenolic contents promotion, especially anthocyanins and acetylbones. From this perspective, ABA primarily activates the expression of genes involved in synthesizing anthocyanins and stilbenes, followed by the accumulation of secondary metabolites¹⁴. In addition, phenylalanine has been shown to increase DPPH scavenging capacity by producing more NADPH⁹.

Phenolic compounds are considered critical secondary metabolites of grapes due to their essential role in determining the quality of grape berry and its organoleptic properties such as color, taste, astringency, bitterness, and aroma¹⁵. Mainly due to the contribution of these compounds as sources of natural compounds such as anthocyanins, flavanols, stilbenes, and their biological and organoleptic properties, it reveals the critical role of phenolics in the quality of grape and its extract¹⁶. They also act as bioactive compounds in various biochemical and pharmacological activities and show antimicrobial, anticancer, and antioxidant properties¹⁷.

Due to the participation of phenylalanine in the biosynthesis pathway of bioactive compounds, phenylalanine is considered a source of grape phenol-derived compounds, including anthocyanins, hydroxy cinnamates, flavonols, flavan-3-ols, tannins, and stilbenes⁴. All of these compounds are synthesized from phenylalanine via the phenylpropanoid pathway. Phenylalanine, a product of the shikimate pathway, links carbohydrate metabolism with aromatic amino acids and the biosynthesis of secondary metabolites¹⁸.

Similar to our results, Portu et al.⁶ found that phenylalanine and urea application improved polyphenolic compounds and flavanol content compared to the control group. In addition, these results were consistent with the results obtained by Gutiérrez-Gamboa et al.¹⁹, who reported that phenylalanine foliar application could increase the amino acids contents as essential precursors of secondary metabolism in grape, so its application can be suggested that due to its effect on phenylalanine accumulation in grapes, the use of phenylalanine may increase the quantity of phenolics and fatty acids²⁰.

Flavonoids are a subset of phenols that are the most abundant in grapes and play an important role in fruit quality parameters. The polyphenolic substances in grapes are usually divided into flavonoids and non-flavonoids. Flavonoids can be classified as anthocyanins, flavanols, and flavan-3-ols²¹. The most common flavonoids in grapes are known flavonols, such as quercetin, kaempferol, and myricetin. The most flavan-3-ols include catechin, epicatechin, tannins, and anthocyanins and the most abundant non-flavonoids are stilbenes, hydroxycinnamic acids, and benzoic acids. All these compounds are synthesized through the flavonoid metabolism pathway, for which phenylalanine is the most crucial precursor²⁰. Flavonoids, which are abundant in the skin of grape berries, are considered important compounds for the quality of grapes and wine due to anthocyanins and related derivatives. In addition, flavonoids in grapes have been shown to have antioxidant and anti-inflammatory activities and reduce LDL oxidation and oxidative DNA damage⁸. Concerning the contribution of phenylalanine to the synthesis of flavonoids, it can be argued that phenylalanine may improve grape quality because flavonols are involved in copigmentation interactions²². In this regard, higher levels of flavonol components were observed in high-quality grapes and wines, which indicates that the content of flavonols in grapes may be a helpful indicator of grape quality characteristics⁹. The present results confirm the findings of Portu et al.⁶ in the case of grapes, who reported that the phenolic content of grapes increased with foliar application of phenylalanine. Similar results were found with phenylalanine treatment for these parameters³. In addition, these results are parallel to the results obtained by the Aghdam et al.¹¹, which stated that in phenylalanine-treated tomato fruits, the content of phenols and flavonoids is higher than the control samples and leads to reduced chilling injury, membrane lipid peroxidation, and ROS accumulation during cold storage.

As a group of phenolic compounds, anthocyanins are mainly responsible for the red color of grapes and wine, indicating the fruit's high acceptable quality²³. These natural colorants exert antioxidant, antimicrobial, and anticarcinogenic properties by participating in many processes that enhance grape color through copigmentation and the formation of polymeric pigments. In addition, anthocyanins have been shown to have a variety of biological functions in plants, such as UV protection, pathogen attacks, oxidative damage, and free radical scavenging¹⁶.

Grape berry peel produces at least 16 different species of anthocyanins derived from dehydroxylated anthocyanin, cyanidin, or anthocyanidin delphinidin. Moreover, glucosides (3-glc) of delphinidin, cyanidin, petunidin, peonidin, malvidin, and their corresponding acetyl (3-acglc) and trans-p-coumaroyl (3-cmglc) derivatives were identified as the main anthocyanins in grapes. It is noted that the type of anthocyanin in grape varieties affects the color of the grape berries²⁴.

Anthocyanin synthesis and the way vine uses precursors to synthesize phenolic compounds can be influenced by several factors such as genotype, environmental conditions such as temperature, light and vineyard location, nutrient status, vintage and cultural practices⁶. Among all the mentioned factors, especially environmental factors, temperature plays a vital role in the quality and composition of grape anthocyanin¹⁶. It is also noteworthy that temperature is negatively correlated with the transcription of anthocyanin biosynthetic genes²⁵.

Due to the effect of phenylalanine on improving color parameters, it has been reported that phenylalanine can increase the anthocyanin content in grapes and enhance the color intensity⁹. The results of this study seem to be parallel to the reports of Portu et al.⁶ which show that the use of phenylalanine in grapes significantly affects anthocyanin compounds, especially malvidin and peonidin, in comparison with the control. Similar results have been reported by Chassy et al.⁴, which found an increase in anthocyanins in phenylalanine treated red grape berries and improved color parameters because these compounds are considered the main ones responsible for red grape color.

In addition, according to a study by Cheng et al.⁹, anthocyanin accumulation in grapes was significantly improved after foliar application with phenylalanine. They claimed that this could be explained because phenylalanine significantly increased endogenous ABA content and increased expression of related genes (VvPAL, VvCHS, VvF3H, and VvUFGT) in the anthocyanin biosynthetic pathway. Furthermore, these results were consistent with the results reported by Sogvar et al.⁸, that high concentrations of phenols and anthocyanins were observed in plum fruits treated with phenylalanine. They concluded that higher PAL activity in response to phenylalanine application resulted in a more significant accumulation of phenolic compounds, including total flavonoids, phenols, and anthocyanins.

It is worth mentioning that the effect of different treatments on the pathway of anthocyanins, apart from all the mentioned factors, the profile of anthocyanins can be strongly related to grape variety and environmental factors and crop management methods. This is the reason that reflects different results in different experiments^8,20. Also, it has been stated that the accumulation of sugars such as glucose, fructose, and sucrose during the process of plant photosynthesis, seems to play a key role in the expression of several structural genes in the flavonoid pathway and can cause the accumulation of anthocyanins in grape berries²⁶.

Flavonols are a common and dispersed group of plant phenolic compounds and a pervasive class of flavonoids in monomeric, oligomeric, or polymeric forms in all species of Vitis. Flavonols, along with anthocyanins, are important cofactors involved in the copigmentation phenomenon of red wines. They even play a role in stabilizing the red color of anthocyanins. Flavanols, together with anthocyanins, act as light-protective components because they strongly absorb UV-A and UV-B wavelengths. In general, these bioactive compounds are believed to be valuable markers in grape classification because they are responsible for red grapes' bitter and astringent properties¹⁸. Quercetin and myricetin derivatives have been shown to be prominent forms of flavonols that are consistent with several previous studies on Tempranillo grapes^6,20,24.

The profile of flavonols in grapes is highly variable among genotypes and can also be affected by various factors such as environmental parameters¹⁸. Moreover, in red varieties, their profile is closely related to anthocyanins because flavonols, as flavonoid biosynthetic products, share a large part of their biosynthetic pathway with anthocyanins⁶. In addition, the presence of flavonols has often been associated with the quality of grapes and wine, in which case higher levels of flavonol components have been reported in high-quality grapes, indicating that the flavonol content in grapes can reflect the quality of grapes⁹.

According to our results, non-methylated flavonols such as glycosides of kaempferol, quercetin, and myricetin seem to have the most significant effect on phenylalanine (Table 1). Since flavonols, like all phenolic compounds in the phenylpropanoid pathway, are synthesized by converting phenylalanine to 4- coumaroyl-CoA and later to tetrahydroxychalcone, it can be concluded that different classes of flavonoids, including the biosynthesis of flavonols, reflect the key role of phenylalanine in this pathway, which starts by phenylalanine¹⁸.

These results are consistent with Portu et al.⁶ findings on grapes, which reported that phenylalanine foliar application increased flavonol content compared to control. Similar results were observed by Cheng et al.⁹ with phenylalanine on the increase of flavonoids in grapes.

In addition, several previous studies on Tempranillo grape confirm these results^6,20,24. Because phenylalanine participates in copigmentation interactions, it can be concluded that the use of phenylalanine may increase the quality of grapes by affecting the increase of flavonols²².

Flavan-3-ol monomers are a group of phenolics that play an important role in the organoleptic properties of grapes and wine, such as astringency and bitterness, which contribute to color stability, mouthfeel, or wine aging capacity. They are characterized by a high bitter taste and significantly improve the quality and health of grapes and wine. In addition, they are believed to be the main building blocks of proanthocyanidins or dense tannins²⁷.

It has been reported that the use of phenylalanine can significantly affect the accumulation of flavonoids in grapes, and depending on the different applications of nitrogen, the positive effects on the composition of grape flavonoids can vary⁹. In this regard, it has been concluded that phenylalanine may reduce the enzymatic activity of the flavonoid glucosyltransferase and increase the enzymatic activity related to the synthesis of non-anthocyanin flavonoids. Then more phenylalanine may be used to synthesize flavonols and flavonoids²⁸.

According to our study, the content of catechins was significantly affected by foliar application of phenylalanine, which seems to be consistent with the previous results in grapes²¹. These results are consistent with the results previously reported by Portu et al.⁶, who found that in phenylalanine-treated grapes, the content of individual flavanols significantly increased, mainly due to the substantially higher content of procyanidin b1. In addition, similar results were reported in other studies by Cheng et al.⁹ and Portu et al.²⁰. In general, it should be noted that the differences observed in the results of different studies can be related to the differences among grape cultivars and the situation in the region.

Non-flavonoids are considered a group of phenolic acids, including cinnamic hydroxy acids and hydroxybenzoic acids found in grapes. Hydroxycyanic acids are usually esterified with tartaric acid in grapes, so trans-caftaric acid is grapes’ primary derivative²⁹. It has been suggested that hydroxycinnamic acids, as a group of anthocyanin-derived orange-red pigments, play a vital role in the long-term color stability of grapes. In addition, non-flavonoid compounds, apart from being the main copigment and critical constituents of grape color, can increase plant resistance and enhance the health effects of grapes and wine³⁰. The present study identified gallic acid as a significant hydroxybenzoic acid in treated grapes (Table 1). Also, caffeic acid, p-coumaric acid, and resveratrol were predominant hydroxycinnamic acids. All of these compounds were significantly affected by the use of phenylalanine (P ≤ 0.01).

In this study, resveratrol as the main stilbene was influenced considerably by phenylalanine. Stilbenes are believed to be a type of phytoalexin in grape berries that have beneficial effects on the nutritional quality of fruit. Resveratrol is also known for its antioxidant, anti-inflammatory, anti-cancer, and heart-protecting activities in grapes. They are synthesized in grapes as active defense compounds against exogenous attacks⁹.

However, it was found that these results are consistent with the results obtained by Cheng et al.⁹, who reported that phenylalanine significantly improved grape stilbenes content and observed a positive relationship between stilbenes biosynthesis and anthocyanin. Endogenous ABA accumulation may be a possible explanation for the expression of related genes that lead to the development of phenolic compounds accumulation³¹.

PAL is the main enzyme in the phenylpropanoid pathway that converts L-phenylalanine to transcinnamic acid as a primary mediator in the biosynthesis of phenolic compounds. In addition, it is a key enzyme in this pathway between primary and secondary metabolism, which regulates phenylalanine flow to the biosynthesis of secondary metabolites³². This enzyme is activated by various bio- and abiotic stresses that accumulate bioactive compounds such as flavonoids, phenolic acids, stilbenes, and lignins³³. Furthermore, PAL is the first and the most critical enzyme during anthocyanin biosynthesis through the synthesis of cinnamic acid from phenylalanine, a precursor to various phenylpropanoids. Considering all these pathways, it can be concluded that the positive effect of phenylalanine in improving anthocyanin and improving fruit quality can be attributed to the role of phenylalanine on PAL gene expression and activation⁸.

The results seem consistent with Sogvar et al.⁸ results, which showed that in plum fruits, phenylalanine application led to a significant increase in PAL activity during cold storage. According to their study, phenylalanine-treated fruits had a higher content of anthocyanins, flavonoids, and phenols with a higher total antioxidant activity. They related the higher accumulation of phenolic compounds to the higher activity of PAL, and a positive relationship was observed between this enzyme activity, total phenolic contents, and antioxidant activity. Moreover, in parallel with these results, Aghdam et al.¹¹ reported that phenylalanine application significantly improved PAL activity and the contents of phenols and flavonoids, resulting in decreased fruit chilling injury.

The results seem to be consistent with Sogvar et al.⁸ results, which showed that in plum fruit, the use of phenylalanine leads to a significant increase in PAL activity during cold storage. According to their study, fruits treated with phenylalanine had a higher content of anthocyanins, flavonoids, and phenols with higher total antioxidant activity. They linked the higher accumulation of phenolic compounds to higher PAL activity, and a positive relationship was observed between the activity of this enzyme, total phenol content, and antioxidant activity. In addition, in parallel with these results, Aghdam et al.¹¹ reported that the application of phenylalanine significantly improved the activity of PAL and the contents of phenols and flavonoids.

Similar to our results, Sogvar et al.⁸ reported that using phenylalanine in plum fruit improved catalase activity and antioxidant enzymes such as superoxide dismutase and ascorbate peroxidase, thereby reducing the accumulation of hydrogen peroxide. This agrees with Aghdam et al.¹¹ study that observed a significant effect between phenylalanine-treated and control samples.

Multi-gene families encode PAL enzyme in grapevines. It has been reported that PAL, as a key gene, encodes phenylalanine ammonia-lyase, which is responsible for the first step in the phenylpropanoid pathway resulting in the accumulation of phenolic compounds such as phenolic acids and flavonoids³⁴. Moreover, it has been shown that some factors could affect PAL activity mainly by promoting transcription or translation of the PAL gene. In this regard, in grapes, several MYB family proteins have been reported to control various points in the phenylpropanoid pathway³⁴.

Previous studies focused on evaluating grape composition without notifying the expression levels of related genes in the biosynthetic pathway. The present work showed a significant increase in PAL expression under phenylalanine application at two sampling time points, 48 and 72 hours after phenylalanine foliar application in grape berry, especially at 500 µM followed by 1000 µM and it was observed that with an increased amount of phenylalanine concentrations, no more PAL transcription was observed (Fig. 5). It could be concluded that according to the present results, the best range of applied phenylalanine should not necessarily be the maximum rate but that it could also be more active at its optimal rate, as observed at 500 µM followed by 1000 µM.

The present results are similar to those reported by Cheng et al.⁹on Cabernet Sauvignon grape variety. They reported that foliar application of phenylalanine increased the expression levels of key genes of the phenolic synthesis pathway, including PAL, CHS, F3H, UFGT, and STS, from veraison to harvest.

Overall, in the biosynthetic pathway of flavonoids and phenylpropanoids, PAL gene is considered a key gene that regulates the synthesis of most phenolic substances, including anthocyanins, flavanols, and flavanols³⁵. Given all these facts, if we consider the effect produced by the different doses of phenylalanine, it seems that, since the phenolic compounds are produced by the flavonoid synthesis pathway and that the most crucial precursor of this pathway is phenylalanine, phenylalanine could have an influential role on the enhancement of phenolic compounds through the effect on the expression level of associated genes.

CHS is considered one of the important genes of the phenylpropanoid pathway, mainly regulating the synthesis of phenolic compounds such as stilbenes and phenolic acids³⁵. According to the present study, CHS gene expression was significantly affected by foliar application of phenylalanine at both sampling time points, especially at 500 µM (Fig. 5). Similar to the transcription level of the PAL gene, higher doses of phenylalanine did not show more influence on this level of gene expression.

These results are consistent with the conclusions of Cheng et al.⁹ on Cabernet Sauvignon grape variety. They found that foliar application of phenylalanine during veraison significantly increased associated gene expressions in the phenolic synthesis pathway and resulted in the regulation of phenolic biosynthesis and promoting antioxidant activities.

The present work supports the view that an increased quantity of phenolic compounds by phenylalanine application can be attributed, at least in part, to phenylalanine-induced expression and activation of phenylpropanoid pathway genes, such as CHS gene.

Plant material. This study was performed in a commercial vineyard (West Azarbaijan province, Iran, 37.63425333 N, 45.00784833 E; 1370 m above sea level) in the summer of 2019 on 15 grapevines (Vitis vinifera L. cv. Qızıl Uzum) which were 12-year-old grown on their own roots, irrigated 3 times during the growing season (Fig. 1). Grapevines were sprayed at the berries veraison stage and repeated one week later with phenylalanine at five concentrations (0, 100, 500, 1000, and 2000 µM). Fruits were harvested on 10 October at their optimum technological maturity based on maturity index (ºBrix, %18) at the full coloring stage of berries. Four clusters from the middle parts of the vine were selected and harvested. Then a mixture of berries from these clusters was taken to the laboratory for testing. The experimental design was a completely randomized design with 3 replications. Each of the experimental samples was homogeneous.

Determination of Antioxidant activity. The antioxidant activity of methanolic extracts of fruit samples was estimated using the free radical scavenging method of 2,2-diphenyl-1-picrylhydrazyl (DPPH)³⁶. 0.1 ml of the extract of the whole part of 12 berries (3 berries from each 4 clusters) was added to 1.9 ml of a 0.1 mM methanolic solution of DPPH and incubated for 30 minutes at room temperature in the dark. The absorbance was recorded at 517 nm wavelength using a spectrophotometer (UNICO UV-2100, Shanghai, China). The following formula calculated the percentage of DPPH radical inhibition:

1) DPPH inhibition=[(Acontrol − Asample)/Acontrol]·100 where A_control is the absorbance of the DPPH solution without the extract

Determination of total phenolics and flavonoids. To determine total phenols and total flavonoids, fruit samples (1 g) were extracted with 0.1% acid methanol hydrochloride 80% (10 ml) and then centrifuged (12000 × g, 20 minutes, 4 ° C). The phenolic value of all extracts was measured by Folin-Ciocalteu reagent method³⁷, evaluated based on the standard gallic acid curve, and finally expressed in mg gallic acid (GAE) per gram fresh weight. The flavonoid content of all extracts was evaluated according to Zareei et al.³⁸ method and stated in mg quercetin per fresh weight.

Determination of total anthocyanin content. Total anthocyanin level (mg g^− 1 FW) was determined using spectrophotometric pH differential method³⁹ and molar absorption coefficient of 29600 (cyanidin-3-glucoside) and expressed in mg of cyanidin-3-glucoside in gram fresh mass according to the following equation:

2) A= (A520 nm at pH = 1 − A700 nm at pH = 1)−(A520 nm at pH = 4.5 − A700 nm at pH = 4.5)

Catalase (CAT) enzyme activity. The CAT activity of the treated fruits was measured according to the method of Aebi⁴⁰ with some changes using monitoring of H₂O₂ disappearance by recording the absorption decrease at 240 nm. The reaction mixture contained 2.5 mL of sodium phosphate buffer (50 mM, pH 7.0) and 0.3 mL of enzymatic extract. The reaction was started by adding 0.2 ml of 1% H₂O₂. One unit (U) of enzyme activity was expressed as the amount of enzyme that catalyzes the breakdown of 1µmol H₂O₂ per minute at 30 ° C per mg of protein under assay conditions.

PAL enzyme activity. PAL enzyme was extracted based on Karthikeyan et al.⁴¹ method. One gram fruit samples were homogenized in 3 ml of 0.1 M ice-cold sodium borate buffer, containing 2-mercaptoethanol 1.4 mM and 0.1 g insoluble polyvinylpyrrolidone, pH 7.0. The extract was filtered through a cheesecloth and centrifuged at 16,000 g at 40 ° C for 15 minutes. The supernatant was used as the source of the enzyme. PAL activity was determined as the conversion rate of L-phenylalanine to trans-cinnamic acid. Samples containing 0.4 ml of enzyme extract were incubated with 0.5 ml of 0.1 M borate buffer, pH 8.8, and 0.5 ml of 12 mM L-phenylalanine in the same buffer for 30 min at 30 ° C. The optical density was recorded at 290 nm, and the amount of trans-cinnamic acid formed was calculated using its extinction coefficient of 9630 M^− 1. Enzyme activity was stated as nM trans-cinnamic acid min^− 1mg^− 1 protein.

Extraction and HPLC analysis of phenolic compounds. A 100 mg sample of berries in 10 ml of methanol was extracted for 20 minutes in an ultrasonic bath in three replications and then centrifuged at 2000 g for 15 minutes. Isolation and determination of phenolic acids were performed using a high-performance liquid-photodiode chromatography photodiode array (HPLC-PDA, Water Alliance 2695 Separation Module), equipped with a C18 column (250mm × 4.6mm, 5µm, Waters) and a UV detector (Water 2487). Water and methanol were used as mobile phases A and B, both of which contained trifluoroacetic acid (0.02%) and a flow rate of 0.5 ml min^− 1. The following gradient was used: initial conditions were 20% B, B concentration increased from 20–30% B in 10 min. 10–30 min B increased to 60% B, reached 100% B in 35 minutes, and remained in this condition for another 5 minutes. The injection volume was 10 µl. A stock solution for each phenol and resveratrol was prepared separately in pure acetonitrile and then diluted to draw a standard curve in the concentration range (1-100 µg ml^− 1). The peaks were monitored at 254, 275, 305, and 320 nm wavelengths. The injection volume was 20 µl, and the temperature was maintained at 25° C. All injections were repeated three times (n = 3).

RNA extraction and gene expression analysis. Total RNA was extracted and purified from 1 g of mixed berry powder according to the method described by Reid et al.⁴². The concentration of extracted RNA was measured by a Nanodrop 2000c spectrophotometer (Thermo Scientific), and its integrity was checked on a 1.2% agarose gel. RNAs were treated with DNase I to remove any traces of genomic DNA. For cDNA synthesis, according to the manufacturer’s instructions, 2 µg of purified, DNase I-treated RNA was reverse transcribed with 0.5 µM of oligo T primer (18 mers) and M-MLV reverse transcriptase (Promega). The relative expression of genes was evaluated using the Rotor-Gene Q-Pure Detection-Qiagen Model 6000 (Qiagen Corporation, USA). For real-time PCR, the Maxima SYBR Green/Fluorescein qPCR Master Mix (2x) # K0241 kit (Thermo Scientific, Lithuania) was used. Each reaction (10 µl) contained 5 µl of SYBR Green, 250 nM of each primer, and 2 µl of 1:10 diluted cDNA. The reactions were subjected to the following thermal conditions: 95°C for 3 min, followed by 40 cycles of 95°C for 10 seconds, annealing temperature (60°C) for 1 min and 72°C for 40 seconds in 96-well optical reaction plates.

Statistical analysis. The experiment was designed as a completely randomized design with five phenylalanine concentrations treatment. Each treatment was conducted in three replications. The statistical analysis was performed by SAS software (9.4). The differences among means of data were compared by Duncanʼs Multiple Range Test. Differences at P ≤ 0.05 or P ≤ 0.01 were considered significant.

In the present study, foliar application of phenylalanine during veraison significantly increased antioxidant activity and anthocyanin content. There was a positive correlation between antioxidant activity and increased phenolic composition of phenylalanine-treated fruits, such that increased anthocyanins and related phenolic compounds led to the development of the antioxidant activity. Moreover, after the application of phenylalanine, the activity of the PAL enzyme was significantly increased and led to a greater accumulation of phenolic compounds. Moreover, according to the results of the real-time quantitative RT-PCR assay, the highest PAL and CHS gene expression was observed at 500 µM phenylalanine. This study suggests that the use of phenylalanine as a main precursor for the synthesis of phenolic compounds, can improve the phenolic composition of grapes and could be a practical approach to advance fruit quality in the production of grape cv. Qizil Uzum.

Acknowledgments

Urmia University, Urmia, Iran, supports this research, so we thank the research directors of this university.

Contributions

A.A.O.: performing all field operations, biochemical and molecular experiments and preparing the manuscript. L.N. and A.A.: supervised the experiment, prepared the figures, and revised the manuscript. R.D.: designed molecular experiments and data analysis. S.N.E.: performing HPLC analysis and interpreting them.

Additional Information

Competing interests

The authors declare no competing interests.

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Phenylalanine promotes phenolic compounds and quality of Qızıl Uzum grape (Vitis vinifera L.) fruit by regulating expression patterns of PAL and CHS genes and modulating the antioxidant system

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