Bleaching color‐loss of green field pea: An investigation on inference of genotypic‐resistance based on chlorophyll and phenolic acid content

Color‐loss of green field pea, commonly referred to as bleaching, can occur prior to harvest once the grain has ripened or during grain storage and is thought to be the result of chlorophyll depletion in the cotyledon. However, the mechanisms of bleaching‐resistance, exhibited within some genotypes, are not well understood. The antioxidant activity of phenolic compounds can inhibit chlorophyll degradation, and therefore, the presence of phenolic compounds may improve bleaching‐resistance. In this study, five green pea genotypes, differing in resistance to bleaching, were assessed for grain‐color as well as content of chlorophyll and major phenolic acids, in both the hull and cotyledon. For each genotype, resistance to bleaching was inferred by the range of color scores observed. Chlorophyll and phenolic acid contents both decreased overall as the whole‐grain color became lighter (bleached); however, there was no direct relation found between these compounds and the known resistance of each genotype. Chlorophyll content in the cotyledon was correlated (r = −0.735, p < 0.001) with cotyledon color but not with whole‐grain color (r = −0.212, p = 0.01). Furthermore, the ratio of cotyledon chlorophyll A/B and the ratio of phenolic acid content (hulls) to total chlorophyll content (cotyledon) were not directly correlated with resistance. Although the total chlorophyll and phenolic acid contents were both depleted as the extent of bleaching increased, neither could fully explain differences in bleaching‐resistance between genotypes. Furthermore, the green color and color‐loss of whole‐grain samples could not be fully attributed to chlorophyll pigments. Therefore, it is likely that other phenolic compounds contribute to both the grain color and the resistance to bleaching.

2 Canada," and "No. 3 Canada," respectively (Canadian Grain Commission, 2018; Khan & Croser, 2004). Similarly, Australian Pulse Standards allow a maximum of 1% (by weight) off-color hull or kernel to qualify for the market class of "No. 1 grade" (Grain Trade Australia, 2018). Higher graded field peas are marketed for human consumption and attract premium prices compared with the lower grades, which are used as livestock feed.
Chlorophyll is the main pigment contributing to the color of green field pea, and it is reported to be depleted during cooking and processing of the grains (Cheng et al., 2004;Steet & Tong, 1996).
However, within many green-pea genotypes, there is also a susceptibility toward degradation of chlorophyll content in unprocessed grains resulting in the loss of green color. This discoloration is known as bleaching and can occur preharvest due to adverse environmental conditions or postharvest due to storage conditions (Gubbels & Ali-Khan, 1990). It is generally understood to be a cosmetic defect affecting marketability rather than functionality (Phelps, 2015); however, bleaching has been linked to loss of seed vigor and increased seed coat (hull) permeability for sugars, amino acids, and organic acids (Browning & George, 1981;Maguire, Kropf, & Steen, 1973). Chlorophyll degradation during bleaching has been attributed mainly to chlorophyllase activity; however, the activity of this enzyme was not found to be significantly different between bleaching-resistant and bleaching-susceptible green-pea genotypes (Cheng et al., 2004).
Phenolic compounds within the hulls of pulse grains are also closely linked to the expressed color (Williams & Singh, 1988). These compounds are understood to form a protective layer over the cotyledon due to their antioxidant and antimicrobial properties which can act to inhibit oxidative enzyme activity, effectively slowing the degradation of chlorophylls (Amarowicz et al., 2010;Dueñas, Estrella, & Hernández, 2004;Yamauchi, Funamoto, & Shigyo, 2004;Zhang et al., 2015). Dry field pea classes with darker colored hulls, such as dun-type peas, generally have much higher concentrations of phenolic compounds compared with the opaque hulls which are characteristic of most green pea genotypes (Magalhães et al., 2017;Maharjan, Penny, Partington, & Panozzo, 2019;Williams & Singh, 1988).
Despite the comparatively low concentration of phenolic compounds in the hulls of green pea compared with other field pea types, some green pea genotypes exhibit a level of resistance to bleaching. It has been suggested that the ratio of phenolic compounds to chlorophyll content may be related to genotypic resistance because significant differences in this ratio were observed between a resistant and susceptible genotype (Ubayasena et al., 2013).
Understanding the relation between genotypic resistance to bleaching and grain composition would be a valuable tool within field pea breeding programs in developing new cultivars with improved color stability and market acceptance. Therefore, the aim of the present study was to observe changes in chlorophyll and phenolic acid content of green peas during postharvest bleaching and determine whether a relationship exists between the presence of these compounds and the known resistance to bleaching of these genotypes.
Five genotypes were included in the study, and the resistance of each to bleaching was assessed by the range of whole-grain colors observed. Color scores, chlorophyll content, and phenolic acid content were analyzed separately for hull and cotyledon to account for the role of each component.

| Samples
Green-pea samples were sourced from a field trial grown at Rupanyup in western Victoria, Australia, and stored in dark conditions immediately after harvest. There were 15 green pea samples, consisting of five genotypes (Aragorn, Excell, OZB1308, OZB1309, and OZB1316) replicated three times within the field trial. From previous studies, Excell was known to be susceptible to bleaching, and OZB1308 was known to exhibit some resistance (Brand, 2016;McDonald et al., 2019). In the following sections, the methods for producing bleached grains, assessing color, and quantifying composition constituents (chlorophyll and phenolic acids) are outlined.

| Bleaching green peas
The pea samples were first sieved to obtain uniform grain size (6-7 mm) and to remove any dust or contaminants. There was no visible sign of mold or microbial activity. Samples were then sorted into visibly uniform grain color within each genotype-replicate to obtain a homogeneous sample of 150 to 200 grains. Each color-sorted sample was divided evenly between two Petri dishes and stored according to the method outlined in McDonald et al. (2019); one Petri dish was wrapped in aluminum foil to block out light (and avoid bleaching), and the other Petri dish was left unwrapped (to stimulate bleaching). All samples were stored at room temperature (22 C and 40% RH) and exposed to light intensity of 9,000 lux.
The loss of color occurred over a storage period of 24 weeks and subsampling (15 grains) was undertaken at 6-week intervals from the beginning of the storage period to capture grain exhibiting various degrees of bleaching. The sampled grain was placed in an airtight bag then wrapped in foil and stored at −18 C until the completion of the 24 weeks.

| Image capture
Following the 24-week storage period all subsampled grain was imaged on a matte-white background using a Nikon D7200 camera fixed to a copy stand (macro lens, f8 aperture, 1/200 s shutter

| Moisture analysis
The moisture content of each hull and cotyledon sample was determined by the Karl Fischer method using the automated Metrohm Karl

| Statistical analysis
A one-way ANOVA with the post hoc Tukey test was used to identify significant differences between genotypes for green color scores and chlorophyll concentrations and phenolic acid concentrations. The T A B L E 1 Descriptive statistics of color scores for whole grain, cotyledon, and hull Note. The whole-grain color score range for each genotype was used as the relative measure of bleaching-resistance. Smaller range values indicated greater bleaching-resistance. Significant differences of means calculated by using the Tukey method and 95% confidence. Groups are labelled in descending order of mean value. were computed for cotyledon color, as the cotyledons were significantly darker than the whole grains. In the context of this study, only whole grain color scores were used for quantifying the extent of, and resistance to bleaching, but the cotyledon color values were still considered appropriate for observing trends and differences.

T A B L E 2 Descriptive statistics for the concentration (mg/100 g DB) of chlorophyll in green pea cotyledon and hull samples
Color scores varied between genotypes independent of any bleaching, as some genotypes are naturally darker than others. However, greater variability in color scores within a genotype indicated a greater extent of bleaching and therefore, the range of color scores (measured on whole grain samples) within each genotype was taken as a relative score for susceptibility to bleaching. In order of increasing susceptibility (i.e. decreasing resistance), the genotypes in this study were ranked as follows: OZB1308, OZB1316, Aragorn, Excell and OZB1309 (Table 1). These results support previous reports wherein Excell and OZB1308 were observed to be relatively susceptible and resistant respectively (Brand, 2016;McDonald et al., 2019).
Resistance to bleaching was not strongly related to the mean color of the whole-grain, hull, or cotyledon samples (Table 1). The least resistant genotype (i.e., the genotype with the greatest range of whole grain color scores) had the darkest mean whole-grain color and was also significantly darker than all other genotypes in the mean hull-color. However, there was no relation overall between the grain color and resistance to bleaching. For cotyledon-color scores, the least resistant (OZB1309) was statistically indifferent from the most resistant genotype (OZB1308) but significantly lighter than Aragorn, which ranked third in resistance (Table 1).
Although grain was sampled at regular intervals, the rate of bleaching over the storage period was not calculated. This is because bleaching does not occur uniformly within green pea genotypes (McDonald et al., 2019), and therefore, the subsamples would be too small to make an assumption of uniformity for that purpose. Measured concentrations of phenolic acids and chlorophyll were related to grain color (i.e., extent of bleaching) rather than to the time spent in storage.

| Chlorophyll content
Typically, the whole-grain color of green field pea is attributed to the chlorophyll content in the cotyledon because concentrations of chlorophyll in the hull are very low and often below the levels of detection (Table 2). Only one of the five genotypes, OZB1309, had colored hulls (Figure 1) and contained detectable concentrations of chlorophyll in the hull ( Table 2). The chlorophyll content within the hull of OZB1309 was predominantly composed of chlorophyll B and was much lower in concentration than in the cotyledon.
Total chlorophyll concentration measured in the cotyledon samples ranged from 3.1 to 23.8 mg/100 g and was predominantly composed of chlorophyll A ( Table 2). The concentration of total chlorophyll (cotyledon) was significantly higher (p < 0.05) for Aragorn F I G U R E 2 Relationship between cotyledon chlorophyll content and (a) cotyledon color score, (b) whole grain color score, and (c) whole grain color score grouped by genotypes with higher mean phenolic acid content, that is, >100 mg/kg, (black dots) and with lower mean hull phenolic content, that is, <100 mg/kg, (grey triangles) than any other genotype, and the lowest chlorophyll concentrations were observed in Excell and OZB1308.
In agreement with the results of Cheng et al. (2004), the total chlorophyll concentration (cotyledon) was correlated (r = −0.735, p < 0.001) with the cotyledon color ( Figure 2a). However, the chlorophyll concentration was not highly correlated (r = −0.212, p = 0.01) with the whole-grain color (Figure 2b) and therefore could not account fully for the differences in green color between genotypes or for bleaching within a genotype. Cheng et al. (2004) reported significant differences between a susceptible and resistant green pea cultivar in both the chlorophyll content and chlorophyll A/B ratio. Although the results of the present study do show that there are significant differences in both chlorophyll content and the chlorophyll A/B ratio between genotypes, there was no correlation of these values to resistance of the genotype (Table 3, Figure 3c,d). The chlorophyll A/B ratio for Excell, which is susceptible to bleaching, was not significantly different from that of the most resistant genotype (Figure 3c).

| Phenolic acid content
The phenolic acid compounds detected in the highest concentrations were o-coumaroyl malic acid, p-coumaroyl malic acid, and feruloyl malic acid, where feruloyl malic acid is reported as the sum of its cis and trans isomers ( Table 3). The sum of the three main phenolic acids was taken to be representative of the total phenolic acid content because the total peak area of these phenolic acids, within the 280 nm PDA chromatograms, constituted the majority of all phenolic acids present. For example, o-coumaroyl malic acid, p-coumaroyl malic acid, and feruloyl malic acid accounted for approximaately 90% of all phenolic acids for OZB 1309 (Figure 4). These compounds were also observed within a previous study as the phenolic acids occurring in the greatest concentrations in white pea, which have similar opaque hulls to green pea (Maharjan et al., 2019).
Phenolic acids in the field pea hulls were typically in higher concentrations than those observed in the cotyledons. The p-coumaroyl malic acid content varied widely between genotypes but was the most prevalent phenolic compound overall, measured at concentrations between 0.1 and 17.4 mg/kg for cotyledon and 0.0 to 144.4 mg/kg for hull. The concentrations of phenolic acids observed in the hulls of all green pea genotypes was comparable to other studies, although there is a large variation in the concentration of phenolic compounds in field peas reported within the literature, due largely to differences between market classes (e.g., dun pea compared with white pea) (Dueñas et al., 2004;Magalhães et al., 2017;Maharjan et al., 2019;Marles, Warkentin, & Bett, 2013;Padhi, Liu, Hernandez, Tsao, & Ramdath, 2017;Parikh & Patel, 2018;B. Singh et al., 2017).
The concentration of phenolic acids in the hulls was significantly higher (p < 0.05) for OZB1308 and OZB1309 (Table 3); however, in the same two genotypes, there was also the greatest depletion of phenolic acids within the hull as the whole grain color score increased, that is, the phenolic content of the hull decreased as the grain bleached ( Figure 5b). Phenolic acid content in the cotyledon showed no significant correlation, within each genotype, to the whole grain color ( Figure 5a) and was significantly higher (p < 0.05) for OZB1308 than all other genotypes in both mean value and range (Table 3). In the present study, there were significant differences between genotypes in the phenolic acid/chlorophyll ratio ( Figure 3a) Figure 3b). However, the genotypes which had a higher total concentration of phenolic compounds in the hull (>100 mg/kg) tended to also have a slower degradation of chlorophyll content in the cotyledon as bleaching color scores increased (Figure 2c). Nevertheless, the color of the whole grains still bleached even with the reduced degradation of chlorophyll, which supports the earlier finding that color changes in the wholegrains may also be attributed to more than chlorophyll degradation in the cotyledon. Although OZB1309 had the greatest phenolic acid content (hulls), it was also the least resistant to bleaching, and although bleaching in this genotype is due partially to the depletion of chlorophyll in the cotyledon and hull, it is likely that other compounds in the hull also contribute to the grain color and degradation thereof.

| CONCLUSIONS
Green field pea genotypes can vary widely in color, composition, and resistant to bleaching. This study has shown that, within genotypes, there is an association between the extent of bleaching (whole-grain color) and the concentration of phenolic acids and chlorophylls in the hull and cotyledon. The concentrations of total chlorophyll and phenolic acids decreased as bleaching increased. However, degradation of total chlorophyll in the cotyledon did not account fully for the observed changes in whole-grain color.
The concentrations of chlorophylls and total phenolic acids as well as ratios between these constituents could not account fully for anecdotal observations of resistance to bleaching of green pea genotypes. Therefore, it is possible that there are genotypic differences in the concentration of minor phenolic acids or other compounds which regulate color stability in green peas. Furthermore, it is not understood whether structural properties of the grain, such as hull adherence to cotyledon, could impact the bleaching response or whether preharvest conditions can impact the resistance of grain to postharvest bleaching. Therefore, further examination of several green pea genotypes harvested across differing environments would be a valuable contribution to the study of bleaching resistance.