AGRIVITA Journal of Agricultural Science

40 AGRIVITA Journal of Agricultural Science. 2019. 41(1): 40-47

AGRIVITA Journal of Agricultural Science www.agrivita.ub.ac.id

Betacyanin and Growth of Beetroot (Beta vulgaris L.) in Response to Nitrogen Fertilization in a Tropical Condition S. M. Sitompul*) and Ajrina Puspita Zulfati Faculty of Agriculture, Universitas Brawijaya, Malang, East Java, Indonesia

ARTICLE INFO ABSTRACT Keywords: A series of studies was carried out to explore the possibility of beetroot cul- Beetroots tivation at highlands in the tropics. This was initiated by a study on the effect Betacyanin of temperature and duration of beetroot storage on betacyanin content after Nitrogen determination of a wavelength that giving the maximum absorbance of beta- Wavelength cyanin extracts from beetroots. The second study was designed to exam- ine the response of betacyanin and plant growth to nitrogen (N) fertilization. Article History: A randomized block design was used to impose the treatment of N fertilizer Received: 24 October, 2018 (Urea) consisting of 0, 0.15, 0.30, 0.45, and 0.60 g N/plant with five repli- Accepted: 10 January, 2019 cates. The maximum absorbance of betacyanin extracts from fresh beetroots was attained at the wavelength of 536 nm. Betacyanin content of beet- *) Corresponding author: roots increased from 229 ml/l to 639 ml/l during 7 days storage under room E-mail: [email protected] temperature (± 22 0C), and was declined after slight increases when stored at lower temperatures. Nitrogen fertilization with the dosage of 0.6 g N/plant reduced betacyanin content up to 25 % from 351.5 to 257.6 ml/l, while root yield and total dry weight (TDW) increased with the supply of N fertilizer.

INTRODUCTION the beetroots. Beetroots contain compounds exceptionally rich in antioxidants (Clifford, Howatson, Beetroot (Beta vulgaris L.), also known West, & Stevenson, 2015) that capable of inhibiting as red beet, is a cool-weather crop that requires the oxidation of other substances such as blood temperatures between 15-20 oC for optimum vessel walls, protein molecules, carbohydrates, growth (ARC, 2013). Although leaves are edible DNA and lipids (Lobo, Patil, Phatak, & Chandra, and rich in nutrition, this vegetable crop is planted 2010; Yashin, Yashin, Wang, & Nemzer, 2013). for taproots (beetroots) particularly in large The antioxidants, inhibiting the process of oxidation scales as the major yield that are processed into through a set of different reactions, are different various products including pickles. In temperate from antiradicals that react directly with free radicals areas where this crop is widely cultivated, the so that molecules are protected from damage best planting seasons are during spring and (Lobo, Patil, Phatak, & Chandra, 2010; Tirzitis & autumn. The plant growth is highly affected by Bartosz, 2010). However, antioxidant or antiradical environmental conditions. A reduction in yield and activity is a general term used to measure the ability beetroot quality (flavor and texture) was observed to prevent the oxidation of molecules (Halliwell, to occur in addition to growth retardation especially Aeschbach, Löliger, & Aruoma, 1995; Khan, 2016). during prolong high temperature (ARC, 2013). The antioxidant capacity of beetroot juices, In recent years, the beetroots have attracted measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH•) increasing attention due to several reasons inter alia and ferric reducing antioxidant power (FRAP), a global trend in functional foods (Lau, Chan, Tan, was far above that of commonly vegetable juices & Kwek, 2013; Vicentini, Liberatore, & Mastrocola, such as tomato, carrot, and orange juices (Clifford, 2016) and medicinal herbs (Chandrika, Rao, Howatson, West, & Stevenson, 2015; Ryan & Sirisha, & Chandra, 2016) in addition to a better Prescott, 2010; Wootton-Beard, Moran, & Ryan, understanding of the health benefit of antioxidants, 2011). The dominant antioxidants in beetroots, and the presence of powerful antioxidants in

ISSN: 0126-0537 Accredited First Grade by Ministry of Research, Technology and Higher Education of The Republic of Indonesia, Decree No: 30/E/KPT/2018 Cite this as: Sitompul, S. M., & Zulfati, A. P. (2019). Betacyanin and growth of beetroot (Beta vulgaris L.) in response to nitrogen fertilization in a tropical condition. AGRIVITA Journal of Agricultural Science, 41(1), 40-47 . https:// doi.org/10.17503/agrivita.v41i1.2050 41

S.M. Sitompul and Ajrina Puspita Zulfati: Betacyanin and Growth of Beetroot in Response to Nitrogen...... based on analysis using an HPLC (high performance synthesized from amino acid tyrosine. Wang, liquid chromatography) and a spectrophotometer, Lopez-Nieves, Goldman, & Maeda (2017) carried were betalains that consist of 80 % of total pigments out comparative analyses of betalains and their in beetroots (Attia, Moussa, & Sheashea, 2013). precursor, tyrosine, in various beet genotypes to The value was equivalent to 3.80 mg/g fresh identify possible bottlenecks in betalain production. weight of betalains which is within the range of It was found that increased tyrosine levels were 2.50-8.50 mg/g fresh weight reported previously positively correlated with elevated betalain (Zakharova & Petrova, 1997). Betalains, found only accumulation among red but not yellow genotypes. in several families in the order of Caryophyllales, A hypothesis was then proposed that further are a large group of pigments classified as increase in tyrosine production will likely enhance yellow betaxanthins and red-violet betacyanin betacyanin accumulation in red beets (Wang, (Clifford, Howatson, West, & Stevenson, 2015). Lopez-Nieves, Goldman, & Maeda, 2017). In term Many studies have characterized betalains of the important role of protein Tyr phosphorylation as nitrogen-containing water-soluble compounds in many plant processes particularly developmental, derived from amino acid tyrosine and deposited in abiotic and biotic stress responses (Shankar, vacuoles, in term of quantity, antioxidant activity and Agrawal, Sharma, Pandey, & Pandey, 2015), it is other characteristics (Gandía-Herrero, Escribano, likely that an increase in nitrogen supply is followed & García-Carmona, 2016; Khan, 2016). In fact, by an increase in the level of tyrosine in plants. the antiradical activity of betalains characterized The present study was aimed at exploring for the first time in 1998 has led to a renewed a possibility of growing beetroot plants in the interest in beetroots and betalains (Khan, 2016). tropics on areas of high altitudes. The growth Studies conducted thereafter showed a correlation performance of plants, including yield (taproots), between radical−scavenging activity (RSA) and and betacyanin content were used as indicators. betalain content of crude plant extracts (Vulić et The study was designed especially to investigate al., 2014). Other study involving seven different the response of betacyanins and plant growth beetroot varieties cultivated in Upper Austria including yield to nitrogen fertilization. No studies, showed that betalain content, ranging between so far, have been conducted on the performance 0.8 and 1.3 g/l fresh juice, consisted 60 % of beetroots in the tropics particularly in relation betacyanins and 40 % betaxanthins (Wruss et al., to nitrogen fertilization. An initial study was 2015). Up to now, 75 betalains (32 betaxanthins, conducted on the spectrophotometric quantification and 43 betacyanins including betalamic acid of betacyanin involving the storage of beetroots. and betanidin) have been well-characterized MATERIALS AND METHODS (Khan, 2016). Betanin is the major component of betacyanin and may account for 75-95 % of the Study Sites red pigment (Chandran, Nisha, Singhal, & Pandit, A series of studies was carried out in 2016- 2014; Von Ei Be, Sy, Maing, & Gabelman, 1972). 2017 at the Laboratory of Plant Physiology, Faculty An effort to obtain fresh plant materials has of Agriculture, Universitas Brawijaya (UB) in Malang prompted an interest in the cultivation of beetroots (7057’ S, 1120 36’), and at the Agrotechnopark of in the tropics at highlands. These needs decent UB in Cangar (7°44’ S & 112°32’ E). Malang and study due to the lack of information on this crop in Cangar (about 50 km westward of Malang), is the respected region. An intriguing question to study located around 500 and 1700 m asl with daily is related to the response of betacyanin to nitrogen minimum-maximum temperature of about 16- fertilizer that may be mediated by the growth 29 0C and 10-26 0C respectively. During the day, response of plants bringing about a reduction in the air temperature at Cangar was estimated betacyanin content of beetroots. This is based on around 20 0C (Sitompul, Sitawati, & Sugito, 2013). the synthesis of secondary metabolites in general Studies that is suppressed in conditions favorable for plant The first study investigated the wavelength growth (Akula & Ravishankar, 2011). In turn, a giving the maximum absorbance which was positive relationship would be expected between followed by the examination of temperature and betacyanin content and the rate of nitrogen fertilizer duration of tuber storage in relation to betacyanin if the nitrogen supply influences directly betacyanins

S.M. Sitompul and Ajrina Puspita Zulfati: Betacyanin and Growth of Beetroot in Response to Nitrogen...... content in a factorial experiment. Fresh beetroots & Noranizan, 2011; Wong & Siow, 2015). The (tubers) after cleaning as necessary, obtained betacyanin content (BC) was calculated as: from the Agrotechnopark, were stored in different BC (mg/l) = [(A×DF×MW×1000) /(ε×L)] 1) conditions, i.e. open room, refrigerator and freezer with the average temperature of 22, 4 and ≤ 0 0C, Where: A is the absorbance, DF the dilution factor respectively. The second factor was duration of and L the pathlength (1 cm) of the cuvette, MW tuber storage consisting of 0, 3, 4, 5, 6 and 7 days. (molecular weights) = 550 g/mol; and ε (molar

The second study examined the effects of extinction coefficients) = 60,000 L/mol cm inH2O. nitrogen supply on betacyanin content and growth, Plant Dry Weight Measurement including yield (taproots) of beetroot plants. Seeds Plant dry weights were obtained by drying of Beetroot cv. Ayumi 04, purchased from a local samples of plant materials in an oven at 80 0C for agricultural shop, were grown in flexible plastic pots 48-72 h depending upon the types of plant materials. (25 cm in diameter, and 30 cm in height) in an open space at the Agrotechnopark on 3 March 2017. In Statistical Analysis each pot, filled with soil (Andisol, sandy clay loam, Statistical data analyses including analysis pH 6.4), five seeds were sown to obtain a healthy of variance (Anova) were executed in excel plant per pot selected on day 20 after sowing. Each manually or with Data Analysis Tool at the level ≤ pot was supplied with 0.288 g P2O5 (SP36) at sow- of significance of p 0.05. Comparisons of mean ing, and 0.384 K2O (KCl) on day 10 after sowing. values between treatments were analyzed with The treatment of nitrogen fertilizer (urea), applied least significant difference (LSD) at p ≤ 0.05. on day 10, consists of five levels: 0, 0.15, 0.30, RESULTS AND DISCUSSION 0.45, and 0.60 g N/plant (∼38, 76, 114 & 152 kg/ ha, with a population of 250,000 plants/ha). A ran- Air Temperature of Study Site domized block design with five replicates was used Air temperatures (1 m above ground) at the to arrange the imposition of treatments. With six study site measured during the day in February destructive samplings and two plants from each 2017 over five consecutive days at 06:00, 13:00 treatment and replicate, the total number of pots and 17:00, on average, were 18.1, 19.8 and were 5 (N treatments) x 5 (replicates) x 6 (destruc- 18.3 0C respectively. At the final stage of growth tive observations) x 2 (plant samples) = 300 pots. (May), temperatures were 20.3, 21.7 and 19.9 0C respectively. Overall, the air temperatures were Betacyanin Determination roughly within the range of temperatures required The method developed by Stintzing, Schieber, for optimum growth of beetroots (ARC, 2013). & Carle (2003) was used for determination of betacy- Due to cloudy conditions during observations, anin content of Beetroot tubers. All chemicals (Mer- the observed air temperatures deviated from a ck) used in the betacyanin analysis, purchased from normal diurnal fluctuation of air temperatures. a local chemical shop, were analytically graded. A taproot of each treatment and replicate was cleaned, Betacyanin Determination and sliced from the middle part to obtain 1 g sample Wavelength (fresh weight) for betacyanin analysis. Each sam- The maximum absorbance of betacyanin ple was ground with distilled water and then filtered extracts with water, probed by spectrum mode in (Whatman 42) before performing measurements with the present study, was found at 536 nm wavelength a mini UV/VIS spectrophotometer (Shimadzu-1240). independent of taproot samples (Fig. 1). This The measurement of betacyanin was initiated is similar to that used by Wruss et al. (2015) for by running the Spectrum mode of the equipment betacyanin of beetroots who corrected the maximum in the first studies in the range of 200-700 nmto absorbance with absorbance at 650 nm. Other trace a wavelength with a maximum absorbance studies used 538 nm (Stintzing, Schieber, & Carle, to be used in subsequent measurements. The 2003), 538 nm corrected with 600 nm (Jamilah, Shu, absorbance of the diluted samples at 600 nm Kharidah, Dzulkifly, & Noranizan, 2011), 540 nm was also measured for correction of impurities (Reshmi, Aravindhan, & Suganya Devi, 2012), 480 including browning substances (Gonçalves et nm (Ravichandran et al., 2013), and 537 nm (Wong al., 2012; Jamilah, Shu, Kharidah, Dzulkifly, & Siow, 2015). Therefore, it is important to check

S.M. Sitompul and Ajrina Puspita Zulfati: Betacyanin and Growth of Beetroot in Response to Nitrogen...... the wavelength with the maximum absorbance besides betacyanins, extracted from yellow pitaya in every study of betacyanin. The wavelength of peel (Selenicereus megalanthus) (Cejudo-Bastante, 536 nm corrected with 600 nm was then used to Hurtado, Delgado, & Heredia, 2016). This suggests measure absorbance of beetroot extracts in the that beetroots stored at room temperature (± 20 subsequent measurements of the present study. 0C) for a short period (≤ 7 days) are better than that at low temperature (refrigerator or freezer) in term of betacyanin content. It is also important to determine betacyanin content of beetroots around the same time (day) for comparison purposes.

Fig. 1. Absorbance of betacyanin extracts as a function of wevelength. Each line represents an individual sample of beetroots Fig. 2. Changes in betacyanin content of beetroots Storage Duration and Temperature during storage under different temperatures. A question may arise as to the betacyanin Fresh beetroots were stored in a Room (± 22 0C), content of beetroots in relation to duration and a Refrigerator (± 4 0C), or a Freezer ( ± 0 0C) for temperature of storage as it is likely that beetroots 3-7 days. Lines were derived from polynomial are stored for several days before being consumed. regression to outline the general trend of betacyanin It was found that the temperature and duration of content with time during storage. Room: y = -6.673x2 storage influenced significantly (p ≤ 0.01) betacyanin + 100.61*x + 226.53; R² = 0.971**, Refrigerator: content of beetroots that increased with duration y = -11.653*x2 + 104.28*x + 227.21; R² = 0.869*; of storage from 229.2 to 630.7 ml/l when stored at Freezer: y = -6.2781x2 + 44.621 x + 231.71; R² = room temperature (± 22 0C) (Fig. 2). Betacyanin 0.535; (* = significant at p ≤ 0.05; ** = significant at contents of beetroots stored at lower temperatures p ≤ 0.01) (refrigerator, ± 4 0C or freezer, ± 0 0C) were lower than that stored at ± 22 0C, and tended to decline Nitrogen Fertilization after 5 days storage. There was no, however, Nitrogen fertilization influenced significantly (p significant interaction between storage temperature ≤ 0.05) betacyanin content of beetroots that declined and duration. In this study, betacyanin contents with an increase in the rate of nitrogen application of beetroots were also determined at 538 nm (Table 1). The application of 0.6 g N plant-1 (∼150 kg besides those determined at 536 nm. It is clear that N/ha) reduced betacyanin content by > 25 % from betacyanin content of beetroots determined at 536 351.5 mg/l to 257.6 mg/l. A close relationship was nm was significantly higher (434.7 ml/l) than that at obtained between betacyanin content and the rate 538 nm (410.6 ml/l). An increase during storage at of N fertilizer (Fig. 3). In contrast, root fresh weight 20 0C independent of pH was reported previously in (RFW) increased significantly with an increase in the content of betaxanthins, a subclass of betalains the rate of N fertilizer from 49.0 g (no fertilizer) to

S.M. Sitompul and Ajrina Puspita Zulfati: Betacyanin and Growth of Beetroot in Response to Nitrogen......

150.5 g/plant (with 0.6 g N/plant). As a consequence An increase in the yield of beetroots due of large increase in the root weight, an increase in to N fertilizer up to 200 kg N/ha independent of N betacyanin content per plant would be expected with sources was reported previously (Rantao, 2013). N fertilization. The root yield seems to be reasonably The present results suggest that beetroot plants compared with that reported in Bloemfontein (29.0 in response to nitrogen supply follow the general 0S), South Africa (Rantao, 2013). The reported rule of plant life with primary metabolism, support- fresh weight of roots (taproot yield) in Bloemfontein ing growth and development of plants, as the first was only 59.10 g/plant with the application of 200 priority ruling out secondary metabolism import- kg N/ha independent of nitrogen source. Root dry ant for adaptation and survival. Although Arimu- weight (RDW) in the present study varied between ra & Maffei (2017) underlined recently the impor- 3.5 % to 5.8 % of FRW with a tendency of RDW/ tance of plant specialized metabolism (PSMs), a RFW to decline as the rate of N fertilizer increased. substitute term for plant secondary metabolites, beetroots seem to apply a life strategy that accen- tuates the process of growth and development followed by the process of adaptation and survival. Total dry weight (TDW) was also influenced significantly (p < 0.01) by N fertilization, and increased with an increase in the rate of N fertilizer up to the 16.53 g/plant with the highest rate of N fertilizer (0.6 g N/plant). Harvest index (HI = RDW/ TDW), significantly influenced by the N fertilization, showed no clear trend in relation to the rate of N fertilizer. The root dry weight (RDW), however, was closely related to TDW (Fig. 4) with a slope of 0.29 representing an average of HI. These suggest that the production of biomass is the main factor determining the yield of beetroot plants. Tei, Scaife, & Aikman (1996) reported a much higher TDW in Wellesbourne (52.2 0N), UK that reached about 1200 g/m2 on day 90 after emergence (∼43.6 g/plant with 2 Fig. 3. Relationship between betacyanin content of root a population of 27.5 plants/m ). With an average HI yield and nitrogen fertilizer. Vertical bars are standard of 0.29 and RFW/RDW of 23.1 in the present study, deviations of each average betacynanin content the TDW in Wellesbourne would give a RDW of 12.6 g/plant or a FDW of > 290 respectively which is much higher than that observed in the present study.

Table 1. Betacyanin content (BC), root (taproot) fresh weight (RFW,), root dry weight (RDW), total dry weight (TDW), and HI (RDW/TDW) of beetroot plants with different rates of N fertilizer (N)

N BC RFW RDW TDW HI g/plant mg/l g/plant g/plant g/plant 0 351 c 49.0 a 2.8 a 7.83 a 0.36b 0.15 334 bc 69.5 b 3.1 a 10.02 b 0.31a 0.30 318 abc 79.4 b 3.8 b 11.83 c 0.32ab 0.45 253 a 107.4 c 4.0 b 13.91 d 0.29a 0.60 258 ab 150.5 d 5.3 c 16.53 e 0.32ab LSD (5%) 76.9 15.0 0.57 1.52 0.049 Remarks: numbers followed by the same latters in the same column are not significantly different atP ≤ 0.05 by Fisher’s LSD (least significant difference)

S.M. Sitompul and Ajrina Puspita Zulfati: Betacyanin and Growth of Beetroot in Response to Nitrogen......

The development of TDW with time was very by N0, N1, N2, N3 and N4 (0, 0.15, 0.30, 0.45 and slow during the first 30 days and rapid to very rapid 0.6 g/plant). Each point in the figure represents an thereafter depending upon the rate N fertilizer that average of two sample plants of each treatment and can be described by Richard model (Fig. 5). The replicate. Graphs in the figure were derived from significant effect of N fertilization on TDW was Richard model to describe the trend of TDW with observed since 30 days after sowing. A continuous time for beetroot plants. increase up to 150 days after emergence, after a lag phase during about the first 30 days of growth, was CONCLUSION reported in the total dry matter of beetroots grown The results of the present study suggest that in Wellesbourne, UK (Tei, Scaife, & Aikman, 1996). beetroots can be grown in the tropics on highlands with reasonable yields and betacyanin content. The highest yield was obtained from the plants supplied with N fertilizer at the rate of 0.6 g/plant (∼>150 kg N/ha or more). It was also found that the maximum absorbance of betacyanin extracts with water in the present study was 536 nm. ACKNOWLEDGEMENT This work was supported by grants from the Faculty of Agriculture, Universitas Brawijaya, Malang, Indonesia. The authors wish to thank the Agrotechnopark, Universitas Brawijaya for their support in providing research facilities. REFERENCES

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S.M. Sitompul and Ajrina Puspita Zulfati: Betacyanin and Growth of Beetroot in Response to Nitrogen......

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