Mutation increasing β-carotene concentrations does not adversely affect concentrations of essential mineral elements in pepper fruit

Vitamin and mineral deficiencies are prevalent in human populations throughout the world. Vitamin A deficiency affects hundreds of millions of pre-school age children in low income countries. Fruits of pepper (Capsicum annuum L.) can be a major dietary source of precursors to Vitamin A biosynthesis, such as β-carotene. Recently, pepper breeding programs have introduced the orange-fruited (of) trait of the mutant variety Oranzheva kapiya, which is associated with high fruit β-carotene concentrations, to the mutant variety Albena. In this manuscript, concentrations of β-carotene and mineral elements (magnesium, phosphorus, sulphur, potassium, zinc, calcium, manganese, iron and copper) were compared in fruit from P31, a red-fruited genotype derived from the variety Albena, and M38, a genotype developed by transferring the orange-fruited mutation (of) into Albena. It was observed that fruit from M38 plants had greater β-carotene concentration at both commercial and botanical maturity (4.9 and 52.7 mg / kg fresh weight, respectively) than fruit from P31 plants (2.3 and 30.1 mg / kg fresh weight, respectively). The mutation producing high β-carotene concentrations in pepper fruits had no detrimental effect on the concentrations of mineral elements required for human nutrition.


Introduction
Fruits and vegetables are an important dietary source of many of the vitamins and minerals required by humans [1][2][3][4]. Plant carotenoids, such as β-carotene, are precursors of Vitamin A biosynthesis and provide a major source of Vitamin A to human diets [5][6]. It is estimated that Vitamin A deficiency affects up to 190 million pre-school age children and 19 million pregnant women in countries with a Gross Domestic Product (GDP) < 15 000 $ per capita in PLOS ONE | DOI: 10 [7]. Vitamin A deficiency is associated with increased risk of ocular disorders, degenerative diseases, respiratory, urinary and intestinal disorders, cardiovascular disease, and certain types of cancer [7][8][9]. Dietary intake of carotenoids can mitigate against these risks [6]. In addition, β-carotene improves the bioavailability of zinc and iron, which are also often lacking in human diets [10][11]. Thus, increasing β-carotene in food is of great importance. Pepper fruits can often be a major dietary source of carotenoids, especially β-carotene [12][13][14]. There is considerable natural variation in β-carotene concentrations in pepper fruit [15][16][17][18][19][20][21]. In addition, induced mutations have increased β-carotene concentrations in pepper fruit [22][23][24][25][26][27]. For example, the orange-fruited variety Oranzheva kapiya, whose fruit has a high βcarotene concentration, was developed after subjecting dry seeds of a red-fruited variety, Pazardzhishka kapiya 794, to X-ray mutagenesis [22,28]. The mutation conferring the orangefruited (of) trait of Oranzheva kapiya appears to be in the 3'-terminal region of the CrtZ gene, which encodes an enzyme that converts β-carotene to β-cryptoxanthin [24]. Recently, this mutation was introduced into the variety Albena, which possesses the anthocyanin-free (al) mutation, has early and high yield, attractive fruit and better flavour [29], and a promising genotype (M38) with excellent agronomic characteristics and fruit quality has been developed [30]. The M38 genotype was registered in the working collection at the Maritsa Vegetable Crops Research Institute. The fruit from lines with orange fruit arising from the same breeding program as M38 have high β-carotene concentrations [24,31]. However, it is important that the mutation that has resulted in high β-carotene concentrations has not affected other nutritional attributes of the fruit. Thus, the aim of this study was to check that the induced mutations producing high β-carotene concentrations in the fruits of M38 plants had no unforeseen effects on the concentrations of essential mineral elements in these fruit.

Plant genotypes
The sweet pepper (Capsicum annuum L.) genotypes analysed in this study were P31, a redfruited genotype derived from the variety Albena [29], and M38 (Okal38) (Fig 1), an orangefruited genotype obtained by transferring the orange-fruited mutation (of) from the variety Oranzheva kapiya into Albena [28] and advancing to M 8 isogenic lines. Both P31 and M38 are early ripening, anthocyanin-free (al), have high yield potential, and attractive fruit with an excellent flavour [25].

Growth conditions and harvested portions
Ten plants of each genotype were grown in the field at the Maritsa Vegetable Crops Research Institute, Plovdiv, Bulgaria. The plants were grown in furrows following conventional practice for mid-early pepper production in this region. For mineral analyses, six ripe fruit from each plant were collected as green fruit at commercial maturity. The shape (length and diameter), thickness of pericarp, and fresh weight (FW) of each fruit was determined. Fruit were dried at 105˚C for one hour then at 60˚C to a constant weight to determine their dry weight (DW). For analyses of β-carotene, nineteen green fruit from P31 and nine green fruit from M38 were analysed at commercial maturity, and fifteen red fruit from P31 and ten orange fruit from M38 were analysed at botanical maturity. Fruit of each maturity x genotype were of uniform size and colour.

Beta-carotene analysis
Biochemical analyses were performed on the day on which fruit were collected. Carotenoids were extracted in duplicate from 5 g FW of green fruit or 2 g FW of red or orange fruit into acetone: ethanol: hexane (1: 1: 2) with MgCO 3 as neutralizing agent, sonicated for 1 min, and centrifuged at 3000 rpm for 3 min, as described by Periago et al. [33]. The pellet was reextracted following the same procedure until the supernatant was colorless. The extract was dried using a rotary vacuum evaporator (Büchi Labortechnik, Flawil, Switzerland) at 30˚C at 210 mbar increasing gradually to 1400 mbar. The extract was redissolved in 1 mL acetone containing 0.1% butylhydroxytoluene (BHT) as antioxidant, and then filtered through 0.45 μm Nylon membrane (EMD Millipore, Billerica, MA, USA) into dark vials. Samples were extracted and stored in the dark under nitrogen. Most of the mature fruit samples were diluted 2-3 fold before analysis of β-carotene concentration. Beta-carotene content was determined on redissolved extracts using high performance liquid chromatography (HPLC). A 50 μl sample was injected into an Agilent Model 1200 HPLC (Agilent Technologies, Santa Clara, CA, USA), equipped with a quaternary pump and two detectors (a diode array detector set at 450 nm and a refractometer). Separation was performed for 22 min per analysis on a ZORBAX Eclipse XDB-C18 column (150 x 4.6 mm ID; Agilent Technologies) at flow rate of 0.8 ml /min. The column temperature was maintained at 20˚C during the separation process. The mobile phase consisted of: A (acetonitrile and 0.1% BHT), B (acetone and 0.1% BHT), and C (water). The gradient used to separate β-carotene, targeted in the present study, and lycopene was: in 1 min 60% А and 40% С, in 10 min 35% В and 12% С, in 12 min 100% В.
Compounds were identified by their retention time and absorption spectra compared to those of known standards (CaroteNature, Ostermundigen, Switzerland). Data analysis was performed using ChemStation software (Agilent Technologies). All chemicals were HPLC grade and obtained from Merck (Darmstadt, Germany).
Six point calibration curves (n = 3 replicates) for both lycopene and β-carotene were linear in the working range of 0.04-5 μg / mL (correlation coefficients > 0.99). The limits of detection (LOD), defined as the concentration of the carotenoid resulting in a peak height three fold greater than the baseline noise, were 0.0171 μg / mL for lycopene and 0.0117 μg / mL for β-carotene. The limits of quantification (LOQ), set at 2.5 fold the LOD [34], were 0.0428 μg / mL for lycopene and 0.0293 μg / mL for β-carotene. The average short-term reproducibility (the same sample determined within a day), calculated as the coefficient of variation, was 6.0%. The average long-term reproducibility (the same sample determined 10 times over the following two days) was 7.4%. To determine the recovery of added analytes, samples were spiked with concentrations of each carotenoid from 0.07 to 0.5 μg / mL. The recoveries of the carotenoids were 97% for lycopene and 95% for β-carotene.

Statistical analysis
Data were expressed as mean ± standard error of the mean (SE) from n determinations, unless otherwise specified. Student's t-test was used to determine the significance of the difference between two sets of data. Statistical calculations were performed using Microsoft Office Excel (Microsoft Corporation, Redmond, WA, USA) or SPSS Version 17.0 (SPSS, Chicago, IL, USA).

Results
Fruits from P31 and M38 plants differed in their morphological characteristics (Table 1).
However, fruits from P31 and M38 plants had different dry weights (p = 0.651; Table 1). The β-carotene concentration of green fruit from M38 plants was greater than that of green fruit from P31 plants at commercial maturity, and the β-carotene concentration of orange fruit from M38 plants was greater than that of the red fruit from P31 plants at botanical maturity (Table 1).
At commercial maturity the average β-carotene concentration in green fruit of M38 was 4.9 mg / kg FW and that of P31 was 2.3 mg / kg FW. The average β-carotene concentration in orange fruit from M38 plants was 52.7 mg / kg FW and that in the red fruit from P31 plants was 30.1 mg / kg FW.
Fruits from P31 and M38 plants also differed in their mineral composition (Table 2).
To avoid the vagaries of hydration, the concentrations of mineral elements in fruits from P31 and M38 plants were compared on a DW basis (cf. [11]). Although the concentrations of magnesium (Mg), potassium (K), zinc (Zn), calcium (Ca), manganese (Mn) and copper (Cu) were similar in fruit from P31 and M38 plants, the sulphur (S), phosphorus (P) and iron (Fe) concentrations in fruit from M38 plants were significantly (P < 0.01) greater than those in fruit from P31 plants.

Discussion
Morphological characteristics are consistent with previous observations that the variety Albena had longer fruits with more pericarp than lines with orange fruit arising from the same breeding program as M38 used in this study [35]. The compared accessions have similar dry weight. The greatest difference between P31 with red fruits and M38 with orange fruits is that the βcarotene concentration of orange fruit exceeds significantly the β-carotene of red fruits. Both P31 and M38 belong to the "kapiya" type of sweet pepper that is consumed as green fruit and, because they ripen early, they are suitable for early market production. The values of β-carotene concentration in green fruit (M38 outperforms that of P31) compare favourably with green and red peppers available in the US, which contain 2.08 mg / kg FW and 16.24 mg / kg FW β-carotene, respectively [36](USDA 2012) and yellow, green and red peppers available in the UK, which contain 1.85 mg / kg FW, 2.65 mg / kg FW and 38.4 mg / kg FW β-carotene, respectively [37].
The US Recommended Dietary Allowance (RDA) for men and women is 900 and 700 μg retinol activity equivalents (RAE) / day, respectively [38]. The UK Reference Nutrient Intake (RNI) values for men and women are slightly lower, at 700 and 600 μg RAE / day, respectively [39]. Assuming that 12 μg β-carotene is equivalent to 1 μg RAE, then the US RDA for men and women could be provided by 10.8 mg β-carotene and 8.4 mg β-carotene, respectively. Thus, five to six of the orange fruit from M38 plants, or six to eight of the red fruit from P31 plants, would be sufficient to supply the US RDA. Thus, the induced mutations producing high β-carotene concentrations in the fruits of M38 plants had no detrimental effects on the concentrations of mineral elements required for human nutrition in these fruit.
The concentrations of mineral elements found in the fruit of P31 and M38 plants grown in this study were similar to those of green and red peppers available in the US [36], and slightly lower (S, Ca, Mn), similar to (Mg, Fe) or slightly greater than (P, K, Zn, Cu) those reported for green and red peppers available in the UK [38]. Previous studies have indicated that fruit from different pepper varieties can vary significantly [40][41], but that the concentrations of most mineral elements often do not change during the transition from green to botanically mature fruit [42]. The concentrations of most mineral elements found in the fruit of P31 and M38 plants grown in this study were within the range found in previous studies (Mg, P, S, K, Zn, Fe), with the exception of Cu, which was higher than generally observed [36,[40][41][42][43][44].

Conclusions
The present study demonstrates that β-carotene concentrations can be increased in pepper fruit without adverse effects on their mineral composition. This should not only improve the Vitamin A status of humans, but is also likely to increase the bioavailability of zinc and iron in the diet.
Supporting information S1 File. Original data characterizing two mutant lines. (XLS)