Elucidating the effects on polyphenol oxidase activity and allelic variation of polyphenol oxidase genes on dough and whole wheat-derived product color parameters

ABSTRACT

Polyphenol oxidase (PPO) activity is a primary cause of the development of unattractive dark brown discoloration of wheat-based end products. The present study aims to evaluate a set of 41 diverse wheat genotypes grown at three different locations in India for grain phenol color reaction, PPO activity and molecular marker-based characterization of alleles of PPO genes. Relationships among these parameters were analyzed along with the effects of grain PPO activity on dough and chapati color at different time intervals. The mean PPO activity ranged from 7.42 to 27.57 min⁻¹ g⁻¹ 10⁻³ among the genotypes and it showed a significant negative correlation with color brightness (L*) of dough rested for 0 min (r = -0.406), 15 min (r = -0.406), 2 h (r = -0.502) and 4 h (r = -0.551) and whole wheat flour-derived chapati rested for 2 h (r = -0.267) and 4 h (r = -0.424). The overall quality color score was negatively correlated with PPO activity (r = -0.863) and showed a positive correlation with both dough and chapati visual color measured at different time intervals. PPO activity in the genotypes carrying different alleles was found to be Ppo-A1a>Ppo-A1b; Ppo-B2d>Ppo-B2a; and Ppo-D1b>Ppo-D1a. The allelic constitution Ppo-A1bPpo-B2aPpo-D1a and Ppo-A1bPpo-B2dPpo-D1 was found to produce the lowest PPO activity, and thus these alleles are recommended to be used in marker assisted breeding for low PPO activity genotypes to minimize the discoloration of wheat-based end-products.

KEYWORDS:

• PPO activity
• dough and chapati color
• color analyzer
• phenol color test
• PPO genes

Introduction

Bread wheat (Triticum aestivum L.) is a major staple food crop growing around the world. It serves as one of the most important sources of protein and calories for most people in emerging and developing countries.^[1] Wheat is used as a basic raw material in the industrialization of food production. Wheat is generally consumed after being processed into a variety of products, the most common being breads, biscuits, noodles, pasta, and flat breads like chapati.^[2–4] India, Pakistan, and some other Asian nations consume more than 85% of the wheat produced in the country in the form of chapati, which is their traditional staple meal and is the least processed.^[5] In fact, Chapati is a whole wheat product and its variants such as tandoori roti, kulcha, naan, etc. are produced from very fine whole wheat flour or flour (locally called Maida). Asian countries like China, Japan, and Korea consume wheat in the form of noodles or steamed bread, while baked bread is the staple product in Western countries.^[6]

Wheat grain contains a number of compounds necessary for germination as well as defense from pathogens. The phenolic compounds are present majorly in the bran but also in the endosperm. Peroxidase, polyphenol oxidases, and catechol oxidase are the enzymes responsible for oxidizing plant phenols into brown pigments.^[7,8] Polyphenol oxidase (PPO) is by far the most investigated enzymes among these. It is a class of ubiquitous enzymes and has been found to be involved as a major cause of browning and discoloration of the products such as pasta,^[9] pan bread,^[10] steamed bread,^[11] chapati,^[12] noodles,^[3,13,14] and other wheat products. PPO activity is predominantly found in the aleurone layer.^[15] Bran and endosperm contain a variety of phenolic acids (ferulic acid, sinapic acid, and vanillic acid) that act as PPO substrates.^[16] The phenolic compounds present in the bran/endosperm react with endogenous PPO during wet milling or kneading, causing browning of wheat end products.^[17] Flour PPO activity and phenolic content rise with flour extraction rate.^[18,19] PPO activity variations were found to be highly associated with milling fractions.^[20] As a result, wheat end products made from whole wheat flour (WWF) such as chapati, suffer more from PPO effects of discoloration.^[21] Genotypes and growing regions^[22] as well as quality characteristics^[19] have also been found to influence discoloration. End products such as noodles, chapati dough as well as chapati develop a dark brownish color on storage which affects the consumer's acceptance of these products.^[13,21,23] Chapati flour is traditionally produced by dry grinding to get WWF, as a result of which there is no discoloration of the flour. However, when WWF is mixed with water to make dough for chapati, the PPO gets activated by water and reacts with the phenolic compounds present in the bran thus producing a browning of the dough and chapati. Chapatis with a light creamy color, a soft texture that is more pliable, puff nicely, and possess a distinctive wheaty scent are preferred by the consumers.^[24] Therefore, little or no PPO activity is a highly desirable trait in wheat used for making chapati dough. Browning discoloration caused by PPO activity must be reduced or eliminated in order to improve wheat end product quality. Although the food processing industry can use reaction inhibitors, physical treatments, by-product extracts, and a controlled environment to prevent enzymatic browning,^[25] reduction of PPO activity by breeding for low PPO types is considered the most eco-friendly and sustainable approach.

The phenol color test, which is a measure of polyphenol oxidase activity, is a quick and easy way to sort wheat and rice genotypes based on grain color discoloration.^[26–28] However, it is a visual test and can be interpreted differently by observers. Application of molecular markers is important for accurate discrimination of divergent alleles in marker assisted selection.^[29]

The genes coding for PPO activity are spread over different chromosomes and are multiple allelic system.^[30] Many studies show that PPO activity is controlled by paralogous genes Ppo-1 and Ppo-2 members.^[31,32] Quantitative trait loci on the 2A, 2B, and 2D chromosomes have been implicated in the control of the PPO enzyme activity.^[12,33] The family of paralogous genes Ppo-1 members Ppo-A1, Ppo-B1, and Ppo-D1 are situated on the long arms of the respective homeologous chromosomes 2A, 2B, and 2D.^[31,32] The newly identified paralogous genes Ppo-2 members Ppo-A2, Ppo-B2, and Ppo-D2 are also found on the long arm of chromosomes 2A, 2B, and 2D, respectively.^[34] Both these paralogous gene groups were found to be separated by approximately 10 cM distance.^[31,32,34] Sequence tagged site (STS) markers for genes PPo-A1, PPo-B2, and PPo-D1 have been developed for genetic characterization of polyphenol oxidase activity.^[30–32] Singh et al.^[27] characterized the 57 Indian wheat genotypes for PPO activity using Ppo-A1 and Ppo-D1 genes. Many durum and bread wheat germplasms were characterized using Ppo-B1 and Ppo-B2 for PPO activity.^[32] Forty-one diverse genotypes of wheat were chosen for this study after evaluating the core group of 200 genotypes for phenol color reaction. The present work was designed with the following goals in mind:

1. Investigate the relationship between PPO activity and dough and Chapati color of wheat genotypes recorded visually and with color analyzer

2. Investigate the efficacy of Phenol reaction test by investigating its relation with PPO activity and dough and Chapati color recorded visually and with the color analyzer

3. Identify the polyphenol oxidase coding genes and alleles in the genotypes

The study of influence of genotype, environment, and GXE on the PPO activity in these genotypes was also investigated and is being communicated separately.

Materials and methods

Experimental materials and field trials

The experimental materials used in this study comprised 41 diverse wheat genotypes, including both new and old elite Indian varieties, exotic lines, and synthetic derivatives. These materials were evaluated at three different locations during the rabi crop season in 2019–20. Among the locations, two were situated in the North Western Plain Zone (NWPZ), namely Delhi (ICAR-Indian Agricultural Research Institute, Experimental Farm, New Delhi, 28°38’N, 77°09’ E, and 228.61 m AMSL) and Pantnagar (Govind Ballabh Pant University of Agriculture and Technology, Research Farm, Uttarakhand, 29°N, 79°31′ E, and 243.8 m AMSL). The third location was the ICAR-IARI Regional Station in Indore, located at 22° 44’N, 75° 50’ E, and 553 m AMSL, known for producing wheat with superior grain quality in the country. Each genotype was grown in five row plots of 2.5 m each; with a row-to-row distance of 0.25 m following a randomized complete block design. Standard agronomic practices were followed to raise the crop. Plant materials were harvested when the grains reached physiological maturity and were completely dry in the field. The genotypes used in the present study and their parentage were provided in the Supplementary table S1.

Phenol color reaction test

The sound and cleaned 100–150 wheat grains were soaked with distilled water in petri plates for 15–16 h. Subsequently, the grains were placed in a specimen tube containing 5 ml of a 1% aqueous phenol solution and left at room temperature (between 15°C and 45°C) for 4 h to facilitate the reaction. After treatment with phenol, the grains were air-dried by placing them on a petri dish with filter paper.^[27,35] A visual assessment was conducted by a five-member scientific panel, who assigned a visual score (ranging from 1 to 10) based on the degree of color discoloration observed after the grains had dried during two separate evaluation sessions. Grains with darker color intensity received a higher score.^[28]

Grain PPO activity assay

The assessment of PPO activity in wheat grains followed the procedure outlined in Anderson and Morris.^[6] A buffer solution containing 4.5 ml of 10 mM L-DOPA (3, 4-dihydroxyphenyl alanine) in 50 mM MOPS (3-(N-morpholino) propane sulfonic acid) at a pH of 6.5 was prepared. In a 25 ml centrifuge tube, 4.5 ml of the MOPS buffer solution was added along with 15–20 sound and clean wheat grains. The tubes were then continuously rotated for 30 min to allow the reactions to occur. L-DOPA buffer solutions and other chemicals were freshly prepared each day. To measure the absorbance (A475), 1.0 ml of the incubated solution was analyzed using a UV 2450 UV–VIS spectrophotometer (Shimadzu, Kyoto, Japan) at a wavelength of 475 nm. The PPO activity was quantified as the change in absorbance (A475) unit per minute per gram (min⁻¹g⁻¹ 10⁻³) over a 1-cm path at 475 nm. Each grain sample was analyzed twice, and the mean value from three different locations was used for statistical analysis.

Production of whole wheat flour

Wheat grain samples, weighing between 500 and 800 gm, were thoroughly cleaned and adjusted to a moisture content of 9.5–10% for each wheat cultivar. These samples were then ground using an electric Atta maker or Atta chakki equipped with grinding stones (Natraj, India) to obtain whole wheat flour. The machine was fitted with a 1 mm sieve to ensure a 98% extraction rate. Subsequently, the whole wheat flour (WWF) samples were stored in airtight containers in a refrigerator until they were used for further experiments.

Preparation of dough and chapati

Dough was prepared from mixing 100 gm of whole wheat flour with water (Based on the water absorption capacity of flour measured with Brabender farinograph) for 2.30 min in dough knead mixer (National manufacturing company, Lincoln, USA) at beater speed of 55–60 rpm. The dough was divided into 20 gm pieces, and each piece of dough was shaped into a sphere and placed on a steel plate covered with a moist cloth to rest for 15 min before start of making chapati. After the resting time, the dough was sheeted to a thickness of 1.5–2.0 mm using specially designed steel Prestige Roti Maker PRM 3.0. The sheeted dough was baked on the electric hot plate (Prestige Roti Maker PRM 3.0) 20 sec in one side and 15 sec of other side. Maintaining the electric hot plate’s temperature between 210° and 225°C for all test samples to ensure uniform baking and sheeting. The sheeted chapatis were placed on a heated gas flame for 10 sec on one side and 5 to 10 sec for other to allow maximum puffing.^[36] The gas flame-puffed chapati was cooled and examined.

Color measurements of PTG, RWF, WWF, dough and chapati

Color parameters whiteness/brightness (L*), redness/greenness (a*), and yellowness/blueness (b*)^[37,38] of phenol treated grains (PTG), refined wheat flour (RWF), and whole wheat flour (WWF) were measured using 3nh NH310 Portable Colorimeter (China). Samples at intervals of 0 min, 15 min, 2 h, and 4 h of preparing dough were used. Stored chapatis samples were removed after two and four hours and were cut into spherical shapes of about 2.5 cm for measuring their L*, a*, and b* values.

Visual evaluation of whole wheat flour dough and chapati

A trained panel of five scientific members with knowledge of chapati color consumer preferences gave each test sample a visual scoring (out of 15) based on the changes in color of dough and Chapati after each interval in two separate sittings. An average of five member’s panel values was then obtained as the final visual score. The quality color score was determined by adding visual scores of dough and chapati color after 4 h.

STS molecular marker analysis

Total genomic DNA was isolated from leaf tissues of young seedlings of wheat genotypes using the CTAB method of Murray and Thompson.^[39] Molecular characterization of PPO genes at Ppo-A1, Ppo-B2, and Ppo-D1 loci was carried out using Sequence tagged site (STS) markers.^[31,32] A 25 µl volume containing 2 µl of 50 ng of genomic DNA, 2.5 µl of 10× PCR buffer, 1.5 µl of 1.5 mM of MgCl2, 1 µl of 200 mM of dNTP, 1 µl of 0.2 mM of each primer: forward and reverse, 0.25 µl of 1.0 unit of Taq DNA polymerase and 16.25 µl of nuclear free water was used to perform the polymerase chain reaction (PCR). PCR conditions were as follows: 94°C for 4 min for initial denaturation, followed by 30 cycles of 94°C for 60s, 64°C for 60s for primer annealing, and 72°C for 60s with a final extension of 72°C for 10 min. Electrophoresis in a 1.5% agarose gel separated the PCR products. Ethidium bromide was used to stain the bands, which were then visualized under UV light. The primers list and sequence information was furnished in Supplementary table S2.

Statistical analysis

All the tests were done in replicates. Mean values from three different locations were used for statistical analysis. The significance of mean values was assessed using one-way ANOVA and Duncan’s multiple-range tests with a statistical significance level of p < .05. Pearson’s correlation among various color parameters of RWF, WWF, PTG, dough, chapati, and PPO activity was performed using SPSS software (version 29).

Results and discussion

Phenol color responses of grains of diverse wheat genotypes

One of the distinctive tests for determining the genetic purity of cereal seeds is the phenol color reaction of grain kernels.^[26] The wheat kernel responds to the phenol substrate by producing color, which is measured to categorize the grains into various color classes. When the grain comes in contact with the phenol/substrate solution, the PPO enzyme (Tyrosinase family), found in the bran/aleurone layer, gets activated and causes oxidation of externally supplied phenol to produce the dark color melanin pigment.^[26,40,41] This trait is genetically controlled and can serve as a biochemical marker. Abrol and Uprety^[26] identified six classes: black, dark black, dark brown, brown, light brown, and non-reactive to phenol. In our study, genotypes showed significant variation in grain color discoloration after phenol treatment (Figure 1 and Supplementary table S6). The visual scores were grouped into four categories based on the degree of darkening of grain after phenol treatment (Table 1 , column 7; Supplementary table S6). The visual scores >8 were categorized into very dark (++++), those scoring 6 to 8 were into dark (+++), 4.5 to 6 were into slightly dark (++), and <4.5 as light (+) (Supplementary table S6). A higher visual score implies a stronger reactivity to the phenol treatment and darker grain color. Among the 41 genotypes examined 17 genotypes showed very dark and 11 genotypes showed dark color reactions, respectively. On the other hand, 13 genotypes exhibited weak color reaction, resulting in colors ranging from “slightly dark” (9) to “light” (4). Older genotypes such as HD2851, C306, C273, and C518 had the lightest colored grains after the phenol reaction, while the new and popular varieties like DBW187, DBW222, HD3059, HD3226, and HD3249 had the darkest grains (Table 1). Different studies have evaluated the phenol reaction of a large range of durum and bread wheat genotypes. Singh et al.,^[27] Nair and Tomar,^[41] Singhal and Prakash,^[42] Singhal et al.,^[43] and Watanabe et al.^[44] tested Indian-origin wheat varieties and accessions with varying ploidy levels for phenol reaction on grains. In these and other studies,^[45] durum wheat genotypes exhibited significantly lower or nil discoloration of grain on phenol treatment, while a complete range was observed for bread wheat accessions. The test has also been widely used to discriminate between the lines of Oryza indica and Oryza japonica.^[46]

Figure 1. Depicting the variation in color of dough, baked chapati, and phenol-treated grains of different wheat genotypes.

Table 1. Molecular characterization of polyphenol oxidase genes and their mean activity and phenol color reactions in diverse wheat genotypes.

Sl. No	Genotypes	PPO 18	MG33	PPO 16	PPO 29	Phenol color Reaction	PPO activity
		Ppo-A1	Ppo-B2	Ppo-D1a	Ppo-D1b
1	2	3	4	5	6	7	8
1	PBW752	a	d	―	+	++++	27.08
2	PBW725	a	d	―	+	++++	26.22
3	PBW771	a	a	―	+	++++	22.83
4	DBW16	a	d	+	―	+++	21.57
5	DBW173	a	a	―	+	++++	20.88
6	DBW187	a	d	―	+	++++	23.63
7	DBW222	a	d	―	+	++++	21.23
8	CS5	a	d	―	+	+++	16.89
9	CS28	b	a	+	―	+++	12.25
10	CS46	a	d	―	+	++++	18.24
11	CS86	a	a	―	+	++++	27.57
12	CS110	b	a	+	―	++	7.42
13	DBW51	b	a	+	―	++	11.53
14	QBP-13-9	a	a	―	+	++++	24.35
15	QBP-19-1	a	d	―	+	++++	20.90
16	QBP-19-4	a	d	―	+	++++	21.32
17	QBP-19-5	a	a	―	+	++++	21.99
18	BOB WHITE	b	a	+	―	++	8.51
19	SEMONG 2	b	a	+	―	++	8.47
20	K1006	a	d	―	+	++++	17.71
21	YANG-MAI-6	b	a	+	―	++	13.08
22	VL907	a	a	―	+	++++	25.56
23	4HPAN61	a	d	+	―	+++	17.91
24	4 HPAN84	a	a	―	+	+++	26.22
25	ASOCMAP173	a	d	―	+	+++	22.70
26	HUW666	a	d	―	+	+++	21.66
27	HI1531	b	d	+	―	++	9.63
28	HD2851	b	d	+	―	+	8.12
29	HD2982	b	d	+	―	++	8.92
30	HD2985	a	d	―	+	+++	17.41
31	HD3059	a	a	―	+	++++	21.20
32	HD3226	a	d	―	+	++++	17.73
33	HD3249	a	a	―	+	++++	21.26
34	C306	b	a	+	―	+	9.87
35	G322	b	a	―	+	+++	17.59
36	HD3086	a	a	―	+	+++	19.83
37	HI1544	b	a	+	―	++	17.25
38	C273	b	d	+	―	+	12.56
39	C518	b	a	+	―	+	10.20
40	C591	b	a	+	―	++	10.41
41	HD2967	a	a	―	+	+++	16.81

Color measurements of PTG, RWF, WWF, dough, and chapati

A key advancement in color science was the nearly global approval of the International Commission on Illumination (CIE) 1976 approach for determining and expressing color using L*, a*, and b* values.^[47] L* represents “lightness” (black = 0, white = 100), a* is the green-red axis (green negative, red positive), and b* is the blue-yellow axis (blue negative, yellow positive). When both a* and b* are equal to zero, true neutral gray is depicted. The measurement of flour and wheat end products color is also possible with this triaxial technique and has been utilized by many wheat research centers to large scaled wheat millers.^[48] Significant variation was observed for the color parameters L*, a* and b* of PTG, RWF, WWF, dough and chapati measured from colorimeter (color analyzer) (Supplementary table S4, S5, S6). The variation for PTG L*, a* and b* values ranged from 37.9 to 42.78, 0.8 to 3.9 and 0.8 to 6.5, respectively (Supplementary table S4). The genotypes HI1531, C518, C591, Semmong2, Yangmai6, and HD2851 had the highest L*, a*, and b* values and very light discoloration upon phenol treatment, while genotypes CS46, K10006, QBP19-1and CS86 had the lowest L*, a* and b* values and very dark discoloration upon phenol treatment. Figure 1 shows the visual variation in color of dough and baked chapatis of different wheat genotypes.

The dough prepared from WWF rested for different time intervals of 0 min, 15 min, 2 h, and 4 h was shown to have significant variation for the color parameters L*, a*, and b* (Supplementary table S5). Dough color L* value ranges from 61.01 to 73.22 for 0 min, 59.29 to 71.22 for 15 min, 57.07 to 68.43 for 2 h, and 52.84 to 66.67 for 4 h, respectively. The results show that there is a consistent decrease in the L* value and dough becoming darkened over a period of time (Figure 2). The rate changes in color brightness were reported to be controlled genetically and it varies with the genotypes.^[49] In our study, we observed a change in the value of L*(0–4 h) ≥ 10 for some genotypes (CS46, DBW187, CS86, 4HPAN84, PBW 752, and PBW 725) whereas others (C306, HD2851, Semmong2, Yang mai6, and Bobwhite) that already had higher L* values showed very little decrease in brightness, L*(0–4 h) was ≤7 (Figure 2; Supplementary table S5). Baik et al.,^[50] Seib et al.^[51] and Lagassé et al.^[52] observed a similar pattern of changes in L*, a*, and b* color parameters of noodles.

Figure 2. Depicting the effects of resting time on the chapati dough and chapati color brightness (L*) of different wheat genotypesGrain PPO activity assay.

Chapati color parameters followed the same pattern as dough. This was the first report measuring chapati color using a color analyzer. Earlier, several researchers have measured chapati color visually by assigning visual scores.^[4,53,54] Chapati color parameter L* ranged from 64.48 to 72.73 and from 62.79 to 71.93 for 2 h and 4 h, respectively. Parameters a* and b* values varied from 5.2 to 7.9 and 13.6 to 21.25 for 2 h and 4.9 to 7.45 and 12.99 to 19.88 for 4 h, respectively. The differences in the L* and b* (2–4 h) among the genotypes were substantial (L * ~62 ‒ 73; b*~13 ‒ 22), whereas differences in a* were slight or insignificant (a * ~5 ‒ 8). It was observed that there were common genotypes possessing higher L* values, lesser rate of change in L* values, having the lightest grain color upon phenol treatment, brighter RWF and WWF color, and less darkening of dough and Chapati on resting for different periods of time. This indicates that the different tests used in the present study are equally effective in predicting the color of the final product.

A wide range of grain PPO activity was observed in the studied genotypes. The mean range of PPO activity was found to be between 7.42 min⁻¹ g⁻¹ 10⁻³ (CS110) and 27.57 min⁻¹ g⁻¹ 10⁻³ (CS86) with an average value of 17.72 min⁻¹ g⁻¹ 10⁻³(Table 1). Out of 41 genotypes studied, 13 genotypes were found to have a low PPO activity (≤15 min⁻¹ g⁻¹ 10⁻³), 10 genotypes were found to have intermediate PPO activity (15–20 min⁻¹ g⁻¹ 10⁻³) while remaining 18 genotypes showed very high PPO activity (≥20 min⁻¹ g⁻¹ 10⁻³). The genotypes CS110, C518, C306, HD2851, HI1531, HD2982, Bob White, and Semmong2 showed very low PPO activity (≤10 min⁻¹ g⁻¹ 10⁻³). Interestingly, some of the soft wheat varieties like Yangmai6, Semmong2, and CS110 showed low PPO activity (≤15 min⁻¹ g⁻¹ 10⁻³) while the hard wheat varieties PBW 725, PBW752, DBW187, and DBW 173 showed ≥20 min⁻¹ g⁻¹ 10⁻³. The highest activity was observed for QBP-13-9 (24.35 min⁻¹ g⁻¹ 10⁻³). Salaria et al.^[15] revealed a wide range of PPO activity in the 115 Indian wheat cultivars studied, with values ranging from 2.14 to 37.19 min⁻¹ g⁻¹ 10⁻³. A higher range of PPO activity was determined to be between 14.2 and 41.4 min⁻¹ g⁻¹ 10⁻³by Chang et al.^[55] and between 15.7 and 40.5 min⁻¹ g⁻¹ 10⁻³by He et al.^[31] Zhai et al.^[56] recorded a very low PPO activity in the flour of the RIL population, with a mean of 4.47 min⁻¹ g⁻¹ 10⁻³. This is because most phenolic compounds are concentrated in the bran component of the grain and PPO is present predominantly in the aleurone layer, which were removed to a large extent during milling to obtain the flour. Hence, PPO activity is expected to increase with increasing flour extraction rate.

Relationship among phenol color reaction visual score, phenol treated grain color parameters and PPO activity

The genotypes that showed higher phenol reaction grain visual score had low L*, a* and b* values, indicating that grains showing darkening on phenol treatment will have low brightness level. This was evident by the negative significant correlation of phenol treated grain visual score with phenol treated grain color parameters L* (r = −0.888), a* (r = −0.902) and b* (r = −0.901), respectively (Supplementary table S7). The PPO activity showed a strong positive significant correlation (r = 0.86) with phenol treated grain visual score, indicating that higher PPO activity leads to greater darkening of the grains. As expected, PPO activities showed significant negative correlation with phenol treated grain color parameters (r = −0.857), a* (r = −0.853) and b* (r = −0.880). This association shows that the higher the PPO activity, the higher will be the grain darkening and the lower will be the grain brightness and vice versa. Hence, phenol reaction test can be used effectively for preliminary screening and as a predictive test for grain PPO activity.

Identification of allelic variation in polyphenol oxidases genes and their relationship with phenol reaction and PPO activity

It is vital to examine and understand the genetic basis of polyphenol oxidase activity in wheat in order to target this trait in quality breeding. PPO activity is a complex quantitative trait controlled by several genes and also highly influenced by environment.^[22,57] Hence, the PPO genes and the genetics of the phenol color reaction in wheat have long been the subject of research. Several researchers reported that PPO activity-encoding genes in wheat are part of the Ppo-1 and Ppo-2 paralogous gene groups.^[32] The homologous genes Ppo-A1, Ppo-B1, and Ppo-D1 make up the first gene family (Ppo-1), and numerous studies suggest that Ppo-A1 and Ppo-D1 are crucial for PPO enzymatic activity, explaining 83% of variance in bread wheat.^{[6,9,12,31,58]} He et al.^[31,59] found two allelic variants for Ppo-D1 (a and b) and five allelic variants for Ppo-A1 (a, b, e, f, and g). Ppo-A1b, Ppo-A1e, and Ppo-A1g were shown to be related to low PPO activity, whilst Ppo-A1a and Ppo-A1f were found to be associated with high PPO activity.^[60,61] It was found that Ppo-D1a and Ppo-D1b alleles were related to low and high PPO activity, respectively. Despite the discovery of QTLs for PPO activity on chromosome 2B, the mapping and role of Ppo-B1 in modulating PPO activity is unknown.^{[12,30,44,62]}

Ppo-A2, Ppo-B2, and Ppo-D2 are recent members of a new paralogous Ppo gene family that was identified by Beecher and colleagues.^[30,34] Ppo-A2 (a, b, and c) and Ppo-D2 (a and b) have been found to have three and two alleles, respectively. Despite the expression of Ppo-A2b in growing wheat kernels, Ppo-A2 was not known to influence PPO activity in mature grain. There was evidence attributing Ppo-D2a and PpoD2b to low and high PPO activity, respectively. Four allelic variants (a, b, c, and d) of the Ppo-B2 gene (located on chromosome 2B) have been identified by Beecher et al..^[30] Ppo-B2b was not associated with PPO activity; however, Ppo-B2a and Ppo-B2c were connected to high and low PPO activity, respectively.^[30,34]

The complementary dominant functional STS markers PPO16 and PPO29 were developed by He et al.,^[31] which can effectively discriminate two haplotypes of Ppo-D1 gene. In addition, in order to detect Ppo-A1 loci a new STS codominant marker PPO18 was developed, which can amplify PCR fragments larger than PPO33 which act as complementary marker for PPO18.^[31,58] The marker combination of PPO18/PPO16 or PPO33/PPO16 can be efficiently used to identify genotypes with low PPO activity in wheat breeding. According to Watanabe et al.,^[44] a locus on chromosome 2BL appears to contribute to kernel PPO activity, but this may only be a small contributing element, as demonstrated in T. dicoccum. However, Taranto et al.^[32] used MG33 for Ppo-B2 and MG08 for Ppo-B1 codominant STS marker to identify the low and high PPO activity among large wheat accessions.

In our study, wheat genotypes were characterized using PPO18, PPO16, PPO29, and MG33, markers in order to identify allelic variations for the PPOA1, PPOD1, and PPOB2 genes (Table 1 ; column 3, 4, 5, 6). Both PPO18 and MG33 act as codominant markers. PPO18 detected two different electrophoresis patterns: a 685-bp fragment in 26 genotypes, 876-bp fragment in 15 genotypes while MG33 detected a 555-bp fragment in 22 genotypes and 549-bp fragment in 19 genotypes, respectively (supplementary figure A1a–c). The 685-bp and 876-bp fragments were deduced as Ppo-A1a and Ppo-A1b respectively.^[31] The 555-bp and 549-bp fragments were deduced as Ppo-B2a and Ppo-B2d, respectively.^[32] PPO16 and PPO29 serve as dominant markers to determine whether Ppo-D1 alleles are present or absent.^[31] A 713-bp fragment was present in 16 genotypes signifying the presence of Ppo-D1a allele, and a 490-bp fragment was present in 25 genotypes signifying Ppo-D1b allele when PPO16 and PPO29, respectively, were used as primers for amplification. The genotypes and the alleles of different PPO genes present in them are depicted in Table 1 and supplementary figure A1a–c.

“ a” and “b” indicates the shorter and longer fragments, respectively, amplified by PPO18; “a” and “d” indicates the longer and shorter fragments, respectively, amplified by MG33; PPO 16 produces a 713-bp fragment in genotypes carrying the allele PPO-D1a, while PPO 29 produces a 490-bp fragment in the genotypes carrying the PPO-D1b allele

Since there is more than one gene involved in determining the overall PPO activity, the genotypes were grouped into different classes and these were compared for the PPO activity. A very high level of significance was found in the statistical analysis that examined the relationship between allele combinations and mean PPO activity when compared between classes (Table 2; columns 2, 3, 4).

Table 2 . Classification of the different allelic combinations and the no. of genotypes in each class with their mean PPO activity in diverse wheat genotypes.

Sl. No	Allele combinations^a	No of genotypes with the allelic combination	Mean PPO activity (with comparison between classes)^b	Standard Deviation
1	2	3	4	5
1	A1a	26	21.56a	3.23
	A1b	15	11.05b	3.08
2	B2a	22	17.04a	6.9
	B2d	19	18.49a	5.5
3	D1a	16	11.73a	3.8
	D1b	25	21.41b	3.25
4	aab	11	22.59a	3.12
5	adb	13	20.97b	3.33
6	ada	2	19.74b	2.05
7	bab	1	17.59c	0
8	baa	10	10.89d	3.52
9	bda	4	9.80d	1.93
10	bdb		No such class was found
11	aaa		No such class was found

^aFirst letter “a” and “b” indicates the allelic variations of Ppo-A1; Second letter “a” and “d” indicates the allelic variations of Ppo-B2; Third letter “a” and “b” indicates the allelic variations of Ppo-D1.

^bDifferent letters followed the mean PPO activity indicates highly significant differences among different genotypes (P < .01).

Having a Ppo- A1b allele significantly reduced the PPO activity over the Ppo-A1a allele. Ppo-D1a allele also significantly reduced the PPO activity over the Ppo-D1b allele; however, Ppo-B2a and Ppo-B2d allele’s classes did not differ significantly for PPO activity. Therefore, the class, Ppo-A1aPpo-B2a based on the above observations is expected to have the highest PPO activity which was again found to be the case. Also, the genotypic combination Ppo-A1bPpo-B2dPpo-D1a written as “bda” and Ppo-A1b-Ppo-B2aPpo-D1a written as “baa” should have the least PPO activity. This was indeed found to be the case for “bda” and “baa,” where a low PPO activity of 9.80 and 10.89 min⁻¹ g⁻¹ 10⁻³was observed that was significantly lower than the other classes (Table 2, columns 2 and 4). The genotypes with these genotypic combinations also showed weak phenol reaction and fell into the lighter grain color category (Table 1). Genotypes with “bdb” and “aaa” genotypic constitution were not observed in the present study. He et al.^[31] also reported Ppo-A1b and Ppo-D1a having low mean PPO activity 15.7 min⁻¹ g⁻¹ 10⁻³ compared to other gene combinations among the Chinese cultivars. Ppo-A1a and Ppo-D1b were identified as the two primary alleles of genes with high PPO activity, whereas Ppo-A1b and Ppo-D1a alleles indicated low PPO activity in other studies also.^{[12,31,63,64]}Based on these observations, it is summarized that genotypes with Ppo-A1b and Ppo-B2a or Ppo-B2d and Ppo-D1a alleles are more desirable for a lower PPO activity in wheat genotypes.

Relation of PPO activity with RWF and WWF color parameters

The color parameters L* a* and b* value of RWF and WWF showed the significant association with PPO activity (Table 3). The L* value was showed strong significant negative association with a*(r = −0.943, r = −0.932) and b* (r = −0.703, r = −0.572) values of both RWF and WWF, respectively. This result was in close agreement with Peterson et al.^[65] Conversely, color parameters a* (r = 0.740) and b*(r = 0.748) value shows very strong significant positive associations among them in both RWF and WWF. These results suggest that greater whiteness/brightness of RWF or WWF with a reduction in redness and yellowness; it is highly desirable for producing end products with excellent color stability and high consumer acceptance.

Table 3. Correlation between PPO activity, wet milled RWF, and WWF color parameters recorded with a color analyzer.

		Refined wheat flour			Whole wheat flour			PPO Activity
		L*	a*	b*	L*	a*	b*
Refined wheat flour	L*	1
	a*	-0.943**	1
	b*	-0.703**	0.678**	1
Whole wheat flour	L*	0.525**	0.495**	-0.18	1
	a*	-0.582**	0.593**	0.258	-0.932**	1
	b*	-0.675**	0.740**	0.608**	-0.572**	0.748**	1
PPOActivity		-0.138	0.186	-0.178	-0.389*	0.420**	0.299	1

**Correlation is significant at the 0.01 level (2-tailed).

*Correlation is significant at the 0.05 level (2-tailed).

The PPO activity showed a negative relation with RWF color parameters L*, a*, and b* but non-significantly. While it has shown a significant negative association with WWF color L* (r = −0.38) and positive correlation with a* (r = 0.42), but with low magnitude level (Table 3). This may be due to the fact that PPO activity is primarily found in the bran and, more particularly, in the aleurone layer of wheat kernels, which are largely eliminated during the wet milling process but not the dry milling process.^[66] The extraction rate of the flour also affects the quality of the color of the flour; a high extraction rate raises the bran content of the flour, which in turn results in relatively higher PPO activity in the flour.^[18,67]

Relation of grain PPO activity with dough and chapati color parameter

The end product’s color is a crucial quality factor for consumer appeal and acceptability. Many studies have reported the negative impact of PPO activity on colors of dough,^[4,50,60,68] chapati,^[69] noodles,^[68] Chinese steamed bread,^[11] and other wheat end products. In the present study, grain PPO activity was shown to have significant association with dough and chapati color parameters L*, a*, and b* measured at different time intervals of storage (Table 4). PPO activity showed a highly significant negative correlation with dough color L* at 0 min (r = −0.4) and 15 min (r = −0.4), 2 h (r = −0.5), and 4 h (r = −0.55) with increasing correlation as time elapsed. This result suggests that PPOs impact on dough color prediction is high at later stages of storage. Kruger et al.^[13] and Baik et al.^[50] also showed a correlation between the rate of change in lightness and the amount of PPO activity as well as different phenolic compounds in dough. This trend also continued with chapati color parameters.

Table 4 . Correlation between the PPO activity, dough, and chapati color parameters.

			Dough color 0 min			Dough color 15 min			Dough color 2 h			Dough color 4 h			Chapati color 2 h			Chapati color 4hr
		PPO Activity	L*	a*	b*	L*	a*	b*	L*	a*	b*	L*	a*	b*	L*	a*	b*	L*	a*	b*
PPOActivity		1
Dough color 0 min	L*	−0.406**	1
	a*	0.057	−0.724**	1
	b*	0.265	0.083	−0.138	1
Dough color 15 min	L*	−0.406**	0.966**	−0.722**	0.084	1
	a*	0.048	−0.685**	0.980**	−0.13	−0.704**	1
	b*	0.061	0.415**	−0.394*	0.500**	0.372*	−0.375*	1
Dough color 2 h	L*	−0.502**	0.872**	−0.565**	0.049	0.892**	−0.561**	0.248	1
	a*	0.027	−0.605**	0.904**	−0.172	−0.630**	0.939**	−0.363*	−0.601**	1
	b*	−0.378*	0.613**	−0.436**	0.254	0.600**	−0.442**	0.577**	0.652**	−0.409**	1
Dough color 4 h	L*	−0.551**	0.726**	−0.521**	0.017	0.728**	−0.499**	0.092	0.784**	−0.479**	0.443**	1
	a*	−0.171	−0.410**	0.822**	−0.109	−0.414**	0.863**	−0.18	−0.376*	0.910**	−0.21	−0.389*	1
	b*	−0.413**	0.657**	−0.508**	0.241	0.662**	−.490**	0.458**	0.705**	−0.447**	0.835**	0.686**	−0.28	1
Chapati color 2 h	L*	−0.267	0.497**	−0.427**	−0.016	0.541**	−0.422**	0.247	0.613**	−0.410**	0.569**	0.757**	−0.226	0.681**	1
	a*	−0.106	−0.495**	0.800**	−0.134	−0.554**	0.803**	−0.371*	−0.422**	0.784**	−0.245	−0.352*	0.745**	−0.259	−0.277	1
	b*	0.226	−0.036	−0.095	0.123	−0.106	−0.097	0.131	−0.061	−0.127	0.031	−0.096	−0.136	−0.063	−0.016	0.085	1
Chapati color 4 h	L*	−0.424**	0.495**	−0.400**	−0.082	0.551**	−0.393*	0.183	0.609**	−0.359*	0.543**	0.780**	−0.157	0.670**	0.964**	−0.242	−0.084	1
	a*	−0.143	−0.404**	0.748**	−0.068	−0.459**	0.761**	−0.328*	−0.326*	0.757**	−0.188	−0.23	0.741**	−0.131	−0.133	0.910**	0.004	−0.08	1
	b*	0.156	−0.017	−0.095	0.166	−0.081	−0.107	0.135	−0.005	−0.154	0.057	−0.019	−0.164	−0.001	0.069	0.051	0.938**	0.018	0.057	1

**Correlation is significant at the 0.01 level (2-tailed).

*Correlation is significant at the 0.05 level (2-tailed).

The chapati color L* at 2 h showed a non-significant negative correlation with PPO activity, but at 4 h, it showed a highly significant negative correlation (r = −0.42) with PPO activity. Bhattacharya et al.^[68] also observed a significant decrease in noodles brightness with increasing interval over a 24 h time period and reported involvement of PPO in darkening of noodles. This result reveals that PPO activity prediction in both dough and chapati will be more effective in later stages of storage (i.e., after 2 and 4 h for dough and 4 h for chapati). The genotypes viz. Bobwhite, Semmong2, and Yangmai-6 showed greatest dough color brightness (>70 L* value) at 0 min and retained the maximum lighter color (>65 L* value) even after 4 h resting period. Simultaneously, these genotypes also showed low PPO activity and less darkening during rest periods, whereas PBW752, PBW725, CS86, and VL907 genotypes had lower brightness ≥65 (L*) value at 0 min that reduced to ≤55 (L* value) after 4 h. These genotypes showed very high PPO activity ≥25 min⁻¹ g⁻¹ 10⁻³. Data for visual evaluation of dough and chapati color scores, overall quality color score, and its relation with PPO activity (Supplementary table S6; Table 5) also showed similar results.

Table 5 . Correlation between PPO activity, dough, and chapati visual color scores.

	PPO Activity	Dough color 0 min	Dough color 15 min	Dough color 2 h	Dough color 4 h	Chapati color 2 h	Chapati color 4 h	Quality color score
PPOActivity	1
Dough color 0 min	−0.500**	1
Dough color 2 h	−0.683**	0.901**	1
Dough color 15 min	−0.775**	0.784**	0.936**	1
Dough color 4 h	−0.845**	0.732**	0.877**	0.944**	1
Chapati color 2 h	−0.697**	0.692**	0.801**	0.855**	0.811**	1
Chapati color 4 h	−0.837**	0.624**	0.793**	0.864**	0.898**	0.854**	1
Quality color score	−0.863**	0.692**	0.855**	0.925**	0.970**	0.856**	0.978**	1

**Correlation is significant at the 0.01 level (2-tailed).

*Correlation is significant at the 0.05 level (2-tailed).

Impact of PPO activity level of wheat flour on color changes in dough during storage was observed by Yadav et al..^[21] Asian noodles, pasta, pan bread, steamed bread, chapati, and many other wheat-based end products are influenced by the PPO activity.^[3,9–14] Hence, our results confirm that genotypes with higher PPO activity will negatively impact the color parameters of the wheat end products, ultimately reducing consumer appeal and acceptability. Therefore, breeding genotypes with low PPO levels in mature grain are an important objective in wheat quality improvement breeding.

Conclusion

Polyphenol oxidase (PPO) activity plays a crucial role in causing undesirable dark discoloration in wheat-based end products, particularly those made from whole wheat grain flour or high extraction rate flours. The present study confirms the detrimental effect of PPO activity on the brightness of dough and chapati color. Moreover, the study demonstrates that the correlation between PPOs and color brightness increases over time, indicating that PPOs have a stronger impact on dough and chapati color prediction in later stages of storage. Additionally, the study reveals a significant correlation between the phenol color reaction test, PPO activity, and specific alleles of PPO genes in the grain. The study concludes that the traditional phenol color reaction test can be employed as a rapid method for selecting low PPO activity genotypes in segregating populations, as it provides similar results to the time-consuming PPO activity assay. However, both tests are destructive and not suitable for use in a breeding program, especially at F₂ and F_2:3 generations. Fortunately, molecular markers for the PPO genes and most of their alleles are now available. The allelic combinations “baa” (Ppo-A1bPpo-B2aPpo-D1a) and “bda” (Ppo-A1bPpo-B2dPpo-D1a) are identified as having the lowest PPO activity in this study. Consequently, in wheat quality breeding for these combinations using markers is recommended as an effective strategy to produce genotypes with reduced PPO activity. The PPO activity assay and phenol color reaction can be utilized in fixed generations to validate the selection based on molecular markers.

Author’s contributions

A.M.S. conceptualized the investigation, planned and supervised the conduct of experiments and edited the manuscript. R.H. conducted the experiment and prepared the draft of the manuscript. A.K.A., J.P.J., and J.B.S. facilitated the field trials. R.H., S.N., and A.K.A. performed the chemical analysis and laboratory experiments. S.K.S., S.S., A.P.B., and B.B. contributed in recording the experimental data. R.H., S.N., and R.R.K. did the statistical analysis and interpretation of data. S.N. and S.K.S. assisted in proof reading of the manuscript. All the authors contributed to this article and approved the submitted version.

Acknowledgment

R.H. acknowledges the Council of Scientific and Industrial Research (CSIR), New Delhi, and ICAR-Indian Agricultural Research Institute (IARI), New Delhi, for providing the fellowship to complete this work as part of Ph.D. thesis. R.H. also acknowledges Grain Quality Laboratory technical and non-technical staff for assistance and support in completing the work.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/10942912.2023.2252196