Anthocyanin Accumulation and Color Development in Seedless Barberry (Berberis vulgaris L.) Fruits: The Role of Altitude and Sun Light - the Preliminary Results

ABSTRACT

The present research was conducted to investigate the effect of direct sun-light on color development of seedless barberry fruit. The selected orchards were in different locations (with different altitude). On some trees, some inflorescences were chosen to cover as bagged (excluded from direct sun-light). Some cases also used as non-bagged. Bagging was done in different times during growing season. Results showed that in all regions, day temperatures were higher than 20°C, and night was lower than 30°C. The lowest difference of day-night temperatures observed in Marvak. Bagging showed a significant effect on anthocyanin (ant), hue angle (h*), lightness and redness (a*). Bagging time significantly influenced chroma (C*) and b/a values. Ant, C*, a*, and a/b values significantly affected by location characteristics specifically altitude, and the highest ant and a* value observed in Marvak. In all locations, the highest value of ant and h* observed with not-bagged fruits. The highest value of a* obtained in not-bagged fruits. It is concluded that direct sun is needed for color development of barberry fruits, however, night-cool temperatures also are important for anthocyanin accumulation of this fruit.

KEYWORDS:

• Altitude
• anthocyanin
• bagging
• barberry
• redness
• temperature

Introduction

Fruit color of many plants is resulted from the presence of anthocyanins. These are water-soluble pigments, leading to a wide range of colors including orange, red to purple/blue (Tanaka et al., 2008). There are some biological functions proposed for anthocyanin pigments including recruitment of pollinators and seed dispersers, having roles in the signaling between plants and microorganisms, plant defense mechanisms, auxin transport, UV protection (Koes et al., 2005; Winkel-Shirley, 2001), and because of their high antioxidant activity, are valuable to human health (De Pascual-Teresa and Sanchez-Ballesta, 2008; Hou et al., 2004). There is a great interest to supply anthocyanin in the food industries, for replacing artificial coloring agents (Fan et al., 2008; Laleh et al., 2006).

Nowadays, seedless barberry (Berberis vulgaris L.) has received much attention because of its high level of medicinal properties. This is a medicinal shrub, dicotyledonous and perennial (Ebadi et al., 2010; Shamsa et al., 1999), which is cultivated as a domestic plant in south Khorasan province of Iran, since more than 200 years ago. Seedless barberry is an important source accounted for anthocyanin pigments (Fallahi et al., 2010). Accumulation of anthocyanin in the barberry fruit might also be essential for protection against abiotic stresses, although is economically important as fruit red color is attractive to consumers.

There are some data showing that many environmental factors such as sun light and temperature significantly influence some qualitative indices of berries and fruits including color development (Bergqvist et al., 2001; Cortell and Kennedy, 2006; Dokoozlian and Kliewer, 1992; Gao and Cahoon, 1992; Kliewer, 1970; Marais et al., 2001; Saure, 1990). Solovchenko et al. (2006) stated that sun light elevation induces breakdown of chlorophyll and increases carotenoid synthesis. Low autumnal temperatures promote anthocyanin biosynthesis (Marais et al., 2001; Saure, 1990). Compared to the average day temperature, average night temperature is more closely correlated to the amount of pigments in fruits. Recent research works demonstrated that light increases anthocyanin content in berry skin regardless of temperature, however, act synergistically with low temperatures (15°C) on the expression of related genes (Azuma et al., 2012). Anthocyanin biosynthesis has different patterns among different plants. Those which accumulate anthocyanins both in their skin and flesh, those which accumulate anthocyanins only in their skin and those that accumulate anthocyanins in their skin only as a response to light stimulus (Jaakola, 2013). About biosynthesis of anthocyanin in apple fruit, one occurs in young fruitlets and the other in ripening fruit (Honda et al., 2002; Saure, 1990). In grape, biosynthesis of anthocyanin started only when the ripening of the berry begins and continues then after (Boss et al., 1996). About pear, there are some cultivars attain their highest anthocyanin concentrations between anthesis and harvest (Dussi et al., 1997; Marais et al., 2001). About barberry fruit, and based on findings by Fallahi et al. (2010) and Maraki et al. (2014), it seems that anthocyanin biosynthesis begins when fruit growth ceases (September) and increases then after.

Although sunlight is important for color development of some fruits, however, it is not clear that if this factor is essential for color development and anthocyanin biosynthesis of barberry fruit. In addition, it is not understood if day-night temperature influence color development of barberry fruit. Thus, the main aims of the present research were to (1) evaluate the effect of fruit exposure to direct sun-light on anthocyanin biosynthesis and color parameters, and (2) assessment the effect of altitude and day-night temperatures on color development of barberry fruits.

Material and Methods

Plant Selection and Treatments

The experiment was conducted at different commercial plantations (Table 1) in Birjand suburban, Iran, on 20 years old seedless barberry (Berberis vulgaris L.) trees in 2015 and 2016 growing seasons. Three locations including Marvak (33° 7.5ʹ N), Marak (32° 58.09ʹ N), and Amirabad (32° 56ʹ N) were selected. Study was done on 189 uniform trees (150 ± 20 cm height), planted at 3–4 m * 2–3 m spacing based on location and five shoots chose on each tree.

Table 1. Characteristics of different locations used in this experiment

Location	Altitude^a (m)	Mean Temperature (°C)			Soil
		Yearly	Day	Night	pH	EC (dS/m)
Marvak	2079	20.49	23.87	16.94	8.17	17.62
Marak	1648	23.34	31.97	14.69	7.50	16.70
Amirabad	1480	25.01	30.54	22.82	7.80	16.72

^aAltitude represents rising of each location above the sea level.

Mean temperatures obtained from data logger and the nearest weather station.

The selected trees were treated according to the conventional farm management, for example, pruning, irrigation, fertilization, and manuring. The soil type was deep, loamy and plants were fertilized using manure, urea, and ammonium phosphate at essential amount regarding soil analysis results.

Temperature data obtained from nearest weather stations, however, data logger (Extech Instruments, Model RHT20, Humidity and Temperature Data logger, USA) were used for more confidence. Monthly, day and night mean temperatures and also day/night differences of each month calculated using these data (Table 2).

Table 2. Temperature-related parameters (Monthly, day, night, and difference between day and night mean temperatures) of different locations used in this experiment

Location	Mean T^a	Month
		April	May	June	July	August	September	October
Marvak	Monthly	21.10	20.70	22.80	25.00	20.20	20.90	12.70
Marak		15.40	19.50	26.65	28.20	27.70	24.00	21.90
Amirabad		16.85	21.25	28.15	29.80	29.80	26.20	23.00
Marvak	Day	25.88	24.24	26.46	28.68	22.76	23.83	15.21
Marak		22.80	27.50	35.60	36.50	36.30	34.10	31.00
Amirabad		25.66	34.12	36.09	34.62	32.97	27.79	22.50
Marvak	Night	14.58	17.63	19.33	21.44	17.34	17.81	10.46
Marak		8.00	11.50	17.70	19.80	19.20	13.90	12.70
Amirabad		15.77	22.38	28.33	27.56	22.52	24.26	18.90
Marvak	Difference	11.30	6.61	7.13	7.24	5.42	6.02	4.75
Marak		14.80	16.00	17.90	16.70	17.10	20.20	18.30
Amirabad		9.89	11.74	11.09	10.79	10.45	17.50	18.30

^aMean T represents mean temperature; Difference represents any difference between day and night temperatures calculating as followed: day-night.

Bagging was done using reflective aluminum foils, 0.1 mm thickness, with some prepared pores and opened-lower side for better aeration around cluster. Pores designed on north-side of bags to prevent direct sun light perceived by clusters. Fruit set was the basal time used for bagging. One month after fruit set, the first bagging was done and continues monthly, up to the color change of fruit (from yellow-green to pink). Cumulative heat units also calculated for each location and also for each bagging time (Table 3).

Table 3. Calculation of cumulative heat units for each location and each bagging time

Location	Bagging time	Cumulative heat units^a (degree days)
Marvak	1	31.00
	2	424.60
	3	900.78
Marak	1	30.70
	2	415.00
	3	1507.80
Amirabad	1	21.65
	2	408.45
	3	1753.15

^aCumulative heat units calculated up to each bagging time.

Anthocyanin Content Evaluation

Determination of spectroscopy-based anthocyanins was performed based on the pH difference. The method consists of two buffer systems: potassium chloride and sodium acetate. A solution of 0.025 M of potassium chloride was produced and then pH 1 was adjusted using HCl. Sodium acetate buffer was produced using 0.4 M sodium acetate and pH 4.5 was adjusted using acetic acid. Anthocyanin content was estimated as pelargonidin 3-glucoside at 510 nm, and data showed as mg/L (Swain, 1965).

Color Parameter

Hunter color values including a*, b*, L*, chroma, and hue were evaluated (McGuire, 1992). There is another derived color calculation including ratio of yellowness over redness ($\mathrm{Y}=\frac{\mathrm{b}}{a}$) (Batu, 2004). Moreover, the ratio of redness over yellowness ($\frac{a}{b}$) estimated.

Experimental Design and Data Analysis

The experiment was conducted as split plot design, with different locations as the main plot and bagging time as subplot. Three replications with 21 trees in each were used (63 trees for each location). On each tree, five shoots selected and all clusters covered for bagging treatments or not covered as control. Statistical analysis of data was performed using ANOVA to determine statistically different values at a significance level of 0.05. All statistical analyses were performed using SAS version 9.2.

Results and Discussion

Temperature Data Assessment

Regarding data (Table 2), it is clear that in all regions, day temperatures were higher than 20°C, although Marvak date temperature was cooler than others. A fluctuation was observed in day temperature of Marvak, and the fruit growth and development were done in cooler date of Marvak, compared with other regions. In all regions, the night temperature was lower than 30°C, and the highest range was observed with Amirabad. In other words, fruit growth and development in Amirabad were done under temperatures more than 20°C, with the exception of October that was lower than it. Monthly mean temperature of these regions also showed lower amounts for Marvak, compared with others, especially with duration of fruit growth and color development (Table 2). The lowest differences between day and night temperatures also obtained with Marvak, which may result from cooler days (Table 2).

Calculation of heat units (Table 3) during fruit growth and development also indicated that all regions were similar about this trait up to second bagging, however, about the third bagging a different manner observed (Table 3). From these data, it is clear that perceived heat units not important for barberry color change.

Anthocyanin Accumulation

Simple effect of bagging, bagging time, and location indicated in Table 4. Data showed that bagging significantly inhibited the anthocyanin accumulation in barberry fruit and light exposure positively influenced anthocyanin accumulation in it that was in agreement with Niu et al. (2010) in chinese bayberry and Løvdal et al. (2010) in tomato fruits. We suggested that light perception is needed for color development in barberry, however, color change starts with or without direct sun light, which may result from other environmentally or physiologically factors. There are some studies showing that exclusion of fruits from sun light lead to suppressed expression of flavonoid pathway genes, so anthocyanin accumulation is inhibited (Wei et al., 2011). Spayd et al. (2002) stated that exposure to sun-light increases anthocyanin concentrations in grape skin regardless of ambient temperature. Time of bagging unaffected this variable, which may define that duration of direct sun light perception not significant for barberry color change or development, although it seems that light is needed for that. Moreover, it is clear that light-related factors needed for barberry color change are activated or supplemented within the fruit during the first month after fruit set, if just sun light is contributed for color change.

Table 4. Simple effects of bagging, time of bagging, and location on accumulation of anthocyanin

Variable	Anthocyanin content (mg/L)
Bagging
Not bagged	402.0a
Bagged	16.9b
SE	13.61
Bagging time
1	210.1a
2	208.2a
3	210.0a
SE	2.8
Location
Marvak	274.5a
Marak	139.2c
Amirabad	214.6b
SE	38.99

Bagging time calculated based on cumulative heat unit perception

Mean values in each column followed by the same letter are not significantly different by the LSD (P < 0.05).

As bagging was started one-month after fruit set, some other factors may be contributed to color change. Significant influence of this variable showed in different location and the highest anthocyanin accumulation obtained in Marvak, followed by Amirabad and Marak. The accumulation of anthocyanins is sensitive to light quality received by plants, and shorter wavelength in the range of blue and UV-light shows the most prominent effect in the accumulation of this variable, possibly by increasing the expression of related genes (Kadomura-Ishikawa et al., 2013). Barnes et al. (1987) stated that altitude has an effect on the contents of secondary metabolites in higher plants. Additionally, to incurring many climatic differences, quality of radiation also affected, especially UV-B. On the other hand, stress caused by UV light is known to enhance the production of ROS, and some flavonoids are reported to be highly effective to scavenge ROS. Coblentz and Stair (1935) found that an increase of 40% to 50% in intensity in the band of wavelength between 2900 and 3130 A is observed in rising 2 Km above sea level. It is suggested that more UV light perceived by plants in Marvak, possibly because of more altitude, compared with others. Temperature may be also important and possibly effective. Cooler temperatures specifically night temperatures in Marvak may be effective in this case. In addition, lower monthly mean temperatures also obtained with this region, compared with others. As all bagged fruits received at least 1-month low temperatures, it seems that temperature is effective in this manner.

However, Azuma et al. (2012) stated that light exposure increases anthocyanin accumulation in grape berry skin regardless of ambient temperature. Choi et al. (2009) stated that low temperatures induce anthocyanin synthesis in various plants, although in cold temperatures is light dependent and in the absence of light, low temperatures prevent anthocyanin biosynthesis, as we have seen in our study.

Interaction of location and bagging showed that more anthocyanin accumulated in not bagged treatments, those perceived direct sun, and the lowest rate of this variable observed in bagged clusters (Table 5). Moreover, in bagged clusters, the highest amount observed with Marvak, followed by Amirabad and Marak. About bagged clusters, the highest rate indicated with Marvak, followed by Amirabad and Marak. We suggest that temperature is also contributing to anthocyanin accumulation in barberry fruit, as can be seen here. From data presented here, lowest amount of this trait indicated with Marak, in both bagged and not-bagged clusters. Regarding calculated data, differences between day and night temperatures showed the lowest values with Marvak and Amirabad and the highest with Marak region (Table 2). Lower night temperatures in Marak is the cause of this difference, as Choi et al. (2009) stated that low temperature in the absence of light prevents anthocyanin accumulation. We think that the accumulation of anthocyanin in barberry fruits needs direct sun light and cool temperatures more than 15 and lower than 25°C.

Table 5. Interaction of bagging × location on accumulation of anthocyanin in the fruit seedless barberry

	Bagging
Location	Not bagged	Bagged
Marvak	516.0a	33.0d
Marak	273.3c	5.1e
Amirabad	416.6b	12.5e
SE	42.40

Mean values with the same letters are not significantly different by the LSD (P < 0.05).

Color Development

Results indicated that bagging led to the lowest hue angle (1.49; representing pink color) and the highest lightness (L*) value (30.89), although chroma (C*) unaffected (Table 6). The highest L* value under bagging condition was in agreement with the findings of Hudina and Stampar (2012) on pear fruit. Hue angles from 0 to 30 represent red color, and regarding data, not bagged clusters were more reddish than bagged ones, which shows importance of light perception by barberry fruits for color development. Lightness range between 0 and 100, and not-bagged clusters showed the lowest L*, indicating that bagging leads to more brightness and reduce appearance. Moreover, simple effect of bagging time and location showed no significant influence on hue and L* value, but, C* significantly affected. Chroma represents color saturation, varying from dull colors (low chroma values) to vivid colors (high chroma values) (Ergunes and Tarhan, 2006). Data showed the most C* in third bagging time, indicating that more saturated color obtained with time consequence. Evaluation of different locations indicated that more rate of this variable obtained with Marvak, compared with others, which related with more saturations (Table 6). We suggest that as more altitude correlated to denser UV-light and more stresses of this spectrum, so more saturated color obtained in Marvak. Interaction between location and bagging also showed that C* and L* values unaffected by these factors that shows overlapping influence of location and bagging, however, hue significantly influenced.

Table 6. Simple effects of bagging, bagging time, and location on hue, chroma, and L*

Variable	Hue	Chroma	L*
Bagging
Not bagged	5.30a	58.2a	21.05b
Bagged	1.49b	55.9a	30.89a
SE	0.461	4.37	2.58
Bagging time
1	3.15a	54.6b	26.95a
2	3.51a	55.6b	26.46a
3	3.52a	60.8a	24.49a
SE	0.435	2.23	4.11
Location
Marvak	2.61a	62.5a	29.10a
Marak	3.61a	55.2b	24.73a
Amirabad	3.97a	53.4b	24.07a
SE	1.637	3.38	7.34

Bagging time calculated based on cumulative heat unit perception

Mean values in each column followed by the same letter are not significantly different by the LSD (P < 0.05).

Results (Table 7) showed more hue with not bagged clusters in all locations, compared with bagged fruits. As data related to hue showed here, all data are between 1.34 and 6.53 that is related to red color. However, more data observed with not-bagged fruits, which indicated rich red color. We suggest that only light perceived by clusters is effective on hue value of barberry fruits, and not temperature. There is an overlapped influence between location and bagging observed about C*, which led to non-significant results, although higher values observed with Marvak. We think that in addition to sun light perception, other parameters related to location are effective on this variable. Regarding data presented in Table 6, it is clear that the best C* obtained with Marvak, which may be related to lower night temperatures. Table 8 indicates the simple effect of bagging, time of bagging, and location on a*, b*, a/b, and b/a ratios. Data showed that bagging significantly affected all variables above, and the lowest a* and a/b and the highest b* and b/a observed with bagging. As a is related to redness, it is suggested bagging inhibited the red color development of fruits, however, yellowness developed. Bagging time unaffected a*, b*, and a/b variables, however, b/a significantly influenced. The lowest rate of b/a observed with third bagging time, which resulted from redness development during fruit growth and development. Location also significantly affected a* and a/b variables, however, b* and b/a unaffected.

Table 7. Interaction of bagging × location on hue, chroma, and L*

Variable	Hue		Chroma		L*
	Bagging
Location	Not bagged	Bagged	Not bagged	Bagged	Not bagged	Bagged
Marvak	3.49a	1.72b	63.5a	61.5a	24.76a	33.44a
Marak	5.87a	1.34b	53.5a	56.9a	19.41a	30.05a
Amirabad	6.53a	1.42b	57.6a	49.1a	18.96a	29.18a
SE	2.08		15.84		17.11

Mean values in each column followed by the same letter are not significantly different by the LSD (P < 0.05).

Table 8. Simple effects of bagging, bagging time, and location on a*, b*, a/b, and b/a

Variable	a*	b*	a/b	b/a
Bagging
Not bagged	28.74a	5.62b	5.84a	0.195b
Bagged	17.66b	11.57a	1.56b	0.704a
SE	4.46	3.27	2.16	0.21
Bagging time
1	21.81a	8.27a	3.93a	0.469a
2	22.92a	8.51a	3.83a	0.489a
3	24.87a	9.00a	3.33a	0.390b
SE	4.32	2.54	1.24	0.073
Location
Marvak	26.15a	9.30a	3.20b	0.409a
Marak	21.83b	8.48a	3.71a	0.472a
Amirabad	21.63b	8.01a	4.18a	0.468a
SE	2.69	1.94	0.48	0.11

Bagging time calculated based on cumulative heat unit perception

Mean values in each column followed by the same letter are not significantly different by the LSD (P < 0.05).

Data showed that the highest a* and the lowest a/b obtained with Marvak (Table 8). We think that other factors relating to environment are effective in this manner, and temperature is important. Low night and low differences between day and night temperatures may be effective on redness development of barberry fruit under different locations, which observed in Marvak, with higher a* value (Table 8).

The a/b value significantly influenced with the interaction of bagging and location, although other variables including a*, b*, and b/a unaffected (data not shown). Results showed that bagging in different location reduced a/b ratio and the lowest observed in Marak and Amirabad, compared with Marvak. Although red (a value) color development observed in bagged and not bagged fruits, however, more redness observed with not-bagged cluster, in different locations (data not shown), which may be related to temperature.

Interaction between bagging time and bagging (Table 9) showed the highest a value with not bagged clusters at both second and third times. The lowest rate of this value also observed with bagged fruit clusters in both first and second times, which may result from low color development under bag.

Table 9. Interaction of Bagging time × bagging on a*, b*, a/b, and b/a

Variable	a*		b*		a/b		b/a
	Bagging
Bagging time	Not bagged	Bagged	Not bagged	Bagged	Not bagged	Bagged	Not bagged	Bagged
1	0.563b	0.182d	1.44d	6.43a	11.55a	4.99a	21.15b	27.36a
2	0.794a	0.184d	1.40d	6.26a	11.42a	5.61a	15.56 c	30.27a
3	0.755a	0.218 c	1.83 c	4.82b	11.74a	6.27a	16.27 c	28.60a
SE	0.35		0.41		8.45		4.93

Mean values in each column followed by the same letter are not significantly different by the LSD (P < 0.05).

From data showed here, it is concluded that the critical time important for color development specifically anthocyanin accumulation in barberry fruit is the first half of growth season. On the other hand, b* value showed the lowest rate with not bagged clusters at first-two times and the highest amount observed with bagging at these times (Table 9). The a/b value unaffected by interaction of bagging time and bagging, however, b/a significantly influenced (Table 9). Data indicated the highest rate of this ratio with bagged clusters under all times, although the reducing manner observed with not-bagged clusters after second time.

Table 10 indicates the interactive effects among location, bagging time, and bagging on a, b*, a/b, and b/a values. The a*, b*, and b/a unaffected with interaction of these factors, however, a/b significantly affected at 5% of confidence. Results showed the highest a/b value with not bagged clusters in Amirabad during first and second times. On the other hand, the lowest rate of this value observed in all regions and all times with bagged fruits (Table 10).

Table 10. Interaction of location × bagging time × bagging on a*, b*, a/b, and b/a

Location	Time	Bagging	a*	b*	a/b	b/a
Marvak	1	No	30.85a	6.58a	4.78 c	0.213a
Marvak	1	Yes	19.11a	11.54a	1.67d	0.622a
Marvak	2	No	34.38a	7.44a	4.62 c	0.219a
Marvak	2	Yes	20.26a	11.64a	1.77d	0.586a
Marvak	3	No	32.61a	7.01a	4.69 c	0.216a
Marvak	3	Yes	19.68a	11.59a	1.70d	0.596a
Marak	1	No	24.51a	4.52a	6.71b	0.190a
Marak	1	Yes	13.91a	12.00a	1.17d	0.937a
Marak	2	No	28.11a	5.82a	5.29b	0.207a
Marak	2	Yes	15.26a	11.72a	1.32d	0.786a
Marak	3	No	25.80a	4.51a	5.80b	0.175a
Marak	3	Yes	23.37a	12.30a	1.97d	0.537a
Amirabad	1	No	26.72a	3.88a	7.81a	0.145a
Amirabad	1	Yes	15.78a	11.11a	1.47d	0.708a
Amirabad	2	No	28.31a	3.57a	8.88a	0.124a
Amirabad	2	Yes	11.17a	10.89a	1.11d	1.011a
Amirabad	3	No	27.37a	7.28a	3.98 c	0.264a
Amirabad	3	Yes	20.40a	11.33a	1.82d	0.554a
SE			24.18	10.81	0.93	0.11

Mean values in each column followed by the same letter are not significantly different by the LSD (P < 0.05).

Conclusion

As data show, day temperatures were higher than 20°C, and night was lower than 30°C, in all three locations. Altitude difference among locations (from 1480 to 2079 m above sea level) may be effective on inducing different light quality. Bagging showed a significant effect on anthocyanin, hue angle, L* and a*, and the highest anthocyanin accumulation, hue angle, and redness observed with not bagged fruits. Bagging time significantly influenced C* and b/a values, although unaffected anthocyanin biosynthesis. Location showed significant effect on anthocyanin, C*, a*, and a/b values and the highest anthocyanin and a* value observed in Marvak. Interaction of bagging × location showed the highest value of anthocyanin accumulation with not bagged fruits, especially in Marvak, followed by other locations. The highest hue angle observed with not-bagged fruits in all locations. Interaction of bagging × bagging time showed the highest value of a* with not bagged fruits, compared with bagged ones. We conclude that direct sun is needed for color development of barberry fruit, however, cool temperatures especially cool nights are important for color development and anthocyanin accumulation of barberry fruit, although very cool night was not useful, as could be seen with Marak region.

Acknowledgments

We gratefully acknowledge all who co-operate ours to find new insights into physiological behaviors of barberry shrubs.