Dec 18, 2015 - measurements can, in some cases, give indirect information on ..... MS information using commercial reference compounds. However, in this ...
Article pubs.acs.org/JAFC
Nondestructive Optical Sensing of Flavonols and Chlorophyll in White Head Cabbage (Brassica oleracea L. var. capitata subvar. alba) Grown under Different Nitrogen Regimens Giovanni Agati,*,† Lorenza Tuccio,† Barbara Kusznierewicz,§ Tomasz Chmiel,§ Agnieszka Bartoszek,§ Artur Kowalski,# Maria Grzegorzewska,# Ryszard Kosson,# and Stanislaw Kaniszewski# †
Istituto di Fisica Applicata ‘N. Carrara’ − CNR, via Madonna del Piano 10, 50019 Sesto Fiorentino (Florence), Italy Department of Food Chemistry, Technology and Biotechnology, Chemical Faculty, Gdansk University of Technology, Narutowicza 11/12, 80-233 Gdansk, Poland # Research Institute of Horticulture, Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland §
S Supporting Information *
ABSTRACT: A multiparametric optical sensor was used to nondestructively estimate phytochemical compounds in white cabbage leaves directly in the field. An experimental site of 1980 white cabbages (Brassica oleracea L. var. capitata subvar. alba), under different nitrogen (N) treatments, was mapped by measuring leaf transmittance and chlorophyll fluorescence screening in one leaf/cabbage head. The provided indices of flavonols (FLAV) and chlorophyll (CHL) displayed the opposite response to applied N rates, decreasing and increasing, respectively. The combined nitrogen balance index (NBI = CHL/FLAV) calculated was able to discriminate all of the plots under four N regimens (0, 100, 200, and 400 kg/ha) and was correlated with the leaf N content determined destructively. CHL and FLAV were properly calibrated against chlorophyll (R2 = 0.945) and flavonol (R2 = 0.932) leaf contents, respectively, by using a homographic fit function. The proposed optical sensing of cabbage crops can be used to estimate the N status of plants and perform precision fertilization to maintain acceptable crop yield levels and, additionally, to rapidly detect health-promoting flavonol antioxidants in Brassica plants. KEYWORDS: Brassica oleracea, chlorophyll fluorescence, flavonols, mapping, nitrogen, optical sensor, white head cabbage
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INTRODUCTION Flavonoids and chlorophylls are two important classes of compounds to be monitored in Brassicaceae. The former are antioxidants, able to scavenge reactive oxygen species (ROS), and possess antimicrobial properties; chlorophyll can be used as a parameter for maturity and storability evaluation. The major antioxidants in Brassica vegetables are phenolic compounds and vitamin C, accounting for >80% of their total antioxidant capacity,1 with flavonols being among the strongest antioxidants.2 Nosek et al.3 reported on the distribution of primary antioxidant compounds from the outside to the inside of the cabbage (Brassica oleracea L. var. capitata f. alba) head. They also showed that ROS distribution was not uniform, with H2O2 having the highest concentration in the outer leaves. The presence of antioxidant flavonoids in the external leaves can help to scavenge H2O2 when the primary antioxidant system is not sufficient for this task. Controlling and stimulating flavonoid biosynthesis can reduce crop damage caused by plant pests and pathogens4 to alleviate the growing concern about the increasing amount of agricultural byproducts. On the other hand, an index of flavonoids on waste leaves can be useful to evaluate and drive the recovery of phenolic compounds from this discharged material. In fact, considering several varieties of B. oleracea, a good correlation between total flavonoids and total phenolics was found.5 In view of the increased interest of consumers6 and food industries 7 in functional foods, the level of bioactive © 2015 American Chemical Society
compounds in raw Brassica materials should be maximized by using suitable agricultural and postharvest practices to enhance their potential benefits for human health.8,9 Chlorophyll content is a fundamental quality parameter to be controlled for the monitoring of Brassicaceae storability.10 The retention of chlorophyll is important for the green appearance of leafy vegetables, but it can also have beneficial effects on human health, for example, by limiting the injurious activity of carcinogenic aflatoxins.11 Chlorophyll detection can be used to control the level of senescence and the related reduction of nutritional quality occurring during storage.12 In general, both chlorophyll and flavonoid contents in leaves are good indicators of the plant nitrogen (N) status independent of the species. Chlorophyll level was found to be positively correlated to the leaf N content in many plant species.13 On the contrary, a clear inverse relationship was observed between leaf flavonoid concentration and nitrogen availability.14,15 Fallovo et al. confirmed the presence of this relationship also in Brassica rapa and Brassica juncea in a greenhouse experiment.16 Because of the opposite N dependency of chlorophyll and flavonoids, the ratio of these two components was expected to Received: Revised: Accepted: Published: 85
October 13, 2015 December 17, 2015 December 17, 2015 December 18, 2015 DOI: 10.1021/acs.jafc.5b04962 J. Agric. Food Chem. 2016, 64, 85−94
Article
Journal of Agricultural and Food Chemistry
July 3, 2014. Harvest of cabbages was performed at the end of October 2014 (23−24). The monthly mean irradiance during the growing season was maximal in July (195 W/m2) and minimal in October (52 W/m2). Accordingly, the monthly mean air temperature ranged between 9.2 °C in October and 20.8 °C in July. The sum of precipitation on a month basis reached 92.6 mm in May and got down to 7.6 mm in October. Because irradiation stimulates the biosynthesis of flavonoids,32 to increase the variability in flavonoids content, a shade treatment of 35% of sunlight, by using a green polyethylene net, was added to the extreme 0 and 400 kg/ha N fertilization rates. Only two rows of plants were included, 14 plants/treatment, and no replicates were used. Description of the Dualex Sensor. The Dualex Scientific+ (Force-A, Orsay, France) was an updated version of the Dualex 4 Scientific leaf-clip optical sensor described earlier.33 It provided indices of flavonols, anthocyanins, and chlorophyll. The leaf chlorophyll content is assessed by measuring light transmission at 710 nm, absorbed by chlorophyll, and in the near-infrared at 850 nm, to take into account the effects of leaf structure. The chlorophyll Dualex index is given by the formula
be a more robust and sensitive index of the plant N status than considering only a single class of compounds. Indeed, this was proved to be true in several studies using the combined chlorophyll-to-flavonoids optical proxy of leaf N in crops17−19 and turfgrasses.20,21 The simultaneous monitoring of chlorophyll and flavonoids was therefore suggested as an innovative tool for the sustainable management of N fertilization. Optical tools for the in situ monitoring of compounds in plants are advantageous because they are nondestructive and rapid. However, they are limited to the detection of superficial compounds and of those with particular optical properties of absorbance and fluorescence. On the other hand, optical measurements can, in some cases, give indirect information on other compounds (internal compounds) for which abundance is correlated to those detected on the sample surface. Optical methods exist to detect nondestructively flavonoids and chlorophyll in leaves17,22,23 and fruits.24−27 Chlorophyll-meters, based on leaf transmittance measurements, have been largely used to assess the chlorophyll content in many species,28 including cabbage.29 Fluorescence-based methods were previously presented to estimate the flavonol content in curly kale (B. oleracea L. var. acephala)30 and broccoli (B. oleracea L. var. italica) flower buds.31 This study was aimed at testing the potential of a relatively new optical sensor for the nondestructive contemporary estimate of flavonols and chlorophyll in cabbage plants grown under different fertilization treatments. Accurate calibration of the sensor against wet chemistry was possible by selecting samples with the largest range in concentrations of the target compounds, induced by the diverse N rates and two light levels. Besides the presentation of the specific important application of the sensor to estimate crop N status, this work indicates a more general use of optical indices in detecting quality and healthpromoting compounds in Brassica plants.
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CHL = [(I850/I0,850)/(I710/I0,710)] − 1
(1)
where I and I0 are the signals measured with and without the leaf sample in the leaf clip, respectively. The Dualex (Dx) measures leaf epidermal flavonols or anthocyanins at 375 and 520 nm, respectively, using the chlorophyll fluorescence (ChlF) screening method32 and equalizing the ChlF signals under these excitation wavelengths and that under red excitation at 650 nm, as reference.34 Compounds present in the epidermis of leaves attenuate the incident radiation, before this can reach the first chlorophyll layer present in the mesophyll, depending on their absorption spectrum. Flavonols are the main flavonoids in dicotyledons absorbing UV radiation at 375 nm; therefore, the intensity of the ChlF induced by this radiation (ChlF_UV) will be inversely proportional to the epidermal flavonols concentration. Using a red light excitation, not attenuated by flavonols, a ChlF signal (ChlF_R) independent of the flavonols concentration is obtained. This signal is used as a reference. By comparing the ChlF signals from the two different excitations, the index of flavonols can be calculated (in accordance with the Beer− Lambert law) as the logarithm of the ratio between the ChlF under red light and that under UV radiation:
MATERIALS AND METHODS
Plants and Experimental Design. The study was carried out at the Research Institute of Horticulture (InHort, Skierniewice, Poland) using cabbage (B. oleracea L. var. capitata subvar. alba) plants of the Transam and Typhoon cultivars. These cultivars are largely cultivated by producers for both processing and storage purposes, presenting high yields and long storability. Cabbage plants were sown on April 25, 2014, into PV 96 multipots (VEFI, Drammen, Norway) filled with prepared peat substrate (“Select”, Klasmann-Deilman, Geeste, Germany) and placed in a greenhouse under 20−23 °C and 17−18 °C day and night temperature, respectively. On May 28, 2014, transplants at the stage of 5−6 fully expanded leaves were made into the field, with a planting density of 33000 plants/ha. Plant distances were 0.6 and 0.5 m within and between rows, respectively. The experiment was established on podzolic soil containing 15−17% of floatable parts in the Ap horizon (0−25 cm) and 1.15% organic matter, with a pH of 7.0. The experimental design was a randomized block with four replicates for each of four different nitrogen treatments and the two cultivars, each covering about 18 m2 area. Before fertilization, the soil contents of mineral nutrients were 9, 70, and 176 mg/L for nitrate-nitrogen, phosphorus, and potassium, respectively. The amounts of phosphorus and potassium were supplemented to the level required by cabbage, that is, 80 and 200 mg/L, respectively. The low content of nitrate-nitrogen was similar for all blocks. The N fertilization treatments were 0, 100, 200, and 400 kg/ha N. Preplant N fertilization occurred on May 26, 2014. The highest N rate was supplied as the sum of two doses of 200 kg/ha N, the first applied with all the others at the preplant N fertilization, whereas a second application of the same rate was supplied as sidedress fertilization on
FLAV = log(ChlF_R/ChlF_UV)
(2)
The same concept applies to the determination of anthocyanins using a green light, absorbed by anthocyanins, instead of UV radiation.35 In addition to the above indices, the Dx sensor calculates the nitrogen balance index (NBI)
NBI = CHL/FLAV
(3)
as the ratio between the chlorophyll and flavonols indices that can be used as a proxy of the crop leaf nitrogen level.17 Protocols of Measurements. The first nondestructive measurements were performed on August 21, 2014, by using the Dx sensor on detached leaves. Twelve leaves/cultivar, three from each of the four N treatments, were collected, choosing the leaves closest to the heads (Figure 1A). Detached leaves were immediately transported to the laboratory, kept at room temperature (22 °C), and measured within 2 h of harvest. For each leaf, Dx measurements were taken at three different positions: apical, middle, and basal (Figure 1B) and for each point from both adaxial (AD) and abaxial (AB) sides, avoiding main veins. From October 16 to 18, 2014, Dx mapping of the whole experimental site, measuring one leaf/cabbage head (on the apical position, recording both AD and AB side data), was performed. The average time to measure a plot of 500 cabbage plants was 1 h and 50 min. At cabbage harvest (October 23−24, 2014), several leaf samplings derived from different N treatments and two light levels were collected to perform the calibration of the sensor. From each leaf, a square of 8 × 8 cm2 was cut from a region between the apical and middle 86
DOI: 10.1021/acs.jafc.5b04962 J. Agric. Food Chem. 2016, 64, 85−94
Article
Journal of Agricultural and Food Chemistry
HPLC Analysis of Phenolic Compounds. Chemicals. Kaempferol and quercetin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Hydrochloric acid (≥37%) was purchased from Fluka Chemie GmbH (Buchs, Switzerland). Methanol, formic acid, and Folin−Ciocalteu’s phenol reagent were obtained from Merck KGAa (Darmstadt, Germany). All solvents were of HPLC grade, and water was of Milli-Q quality. Extract Preparation. The freeze-dried cabbage powders (60 mg) were placed in centrifuge tubes. To each tube was added 1 mL of methanol/water (7:3, v/v), and the samples were sonicated for 10 min. The samples were centrifuged (3000 rpm, 15 min) and clear supernatants collected. The extraction procedure was repeated with a new portion of solvent (1 mL). The supernatants from two repeated extractions were combined to give about 2 mL of the final extract. Half of the final extracts (1 mL) were used for acidic hydrolysis, as well as for the analysis by high-performance liquid chromatography (HPLC) with a diode array detector (DAD) followed by a mass spectrometer (MS) using electrospray ionization (ESI). Acidic Hydrolysis. To determine the content of flavonol aglycones, the methanolic cabbage extracts were subjected to acidic hydrolysis.37 The extracts (1 mL) were mixed with 3.233 M hydrogen chloride (0.59 mL) and kept in a thermoblock at 90 °C for 120 min. The hydrolysates were filtered through a 0.45 μm Millex HV filter (Merck Millipore, Warsaw, Poland) and immediately analyzed by HPLCDAD-ESI-MS. LC-DAD-ESI-MS Analysis. Phenolic compounds in the methanolic cabbage extracts and in the hydrolysates were characterized using a 1200 series HPLC system with DAD and an ESI interface coupled to a 6130 quadrupole LC-MS (Agilent Technologies, Santa Clara, CA, USA). Chromatographic separation was carried out using a 150 mm × 4.6 mm, 5 μm, Phenomenex Kinetex XB-C18 100A column. The separation of phytochemicals was carried out using a mobile phase composed of 0.1% formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B) at a flow rate of 1 mL/min; the injection volume of all samples was 20 μL. The elution gradient profile used was 10−100% B in 30 min, followed by 5 min 100% B. The column was allowed to equilibrate between the injections for 5 min with the initial composition of mobile phase. Absorbance spectra were recorded between 190 and 700 nm every 2 s with a bandwidth of 4 nm, whereas the chromatograms were monitored at 270 and 350 nm. MS parameters were as follows: capillary voltage, 3000 V; fragmentor, 120 V; drying gas temperature, 350 °C; gas flow (N2), 12 L/min; nebulizer pressure, 35 psig. The instrument was operated in both positive and negative ion modes, scanning from m/z 100 to 1200. Individual phenolic compounds were identified by comparing their retention times with those for standards or on the basis of available literature data and UV and mass spectra. Aglycones and flavonol glycosides remaining after hydrolysis were quantitated on the basis of external standards of quercetin and kaempferol (standard line in the concentration range of 6−60 μg/mL) with UV detection at 350 nm.
Figure 1. (A) Arrows indicate the kind of leaves collected for the sensor measurements and calibration. (B) Circles indicate the spots where the Dualex measurements (adaxial + abaxial) were taken.
positions, depending on the size of the leaf, avoiding as much as possible the main veins of the leaf. The basal parts of the leaves containing smaller amounts of both chlorophyll and flavonols were also collected. Each disk was measured by the Dx on three different points. Both AD and AB sides were measured. After that, samples were frozen until freeze-drying. Additional whole leaves/N treatments from each cultivar were used to determine the leaf N content by using the Kjeldahl method. Elaboration of Data and Statistics. For each positional sensor measurement on the cabbage leaves, the CHL index was calculated as the average of the AD and AB measurements, the FLAV index was calculated as the sum of the AD and AB measurements, and the NBI was the ratio between CHL and FLAV indices. On August 21, 2014, for each N treatment of sun-exposed cabbages, the average (±SD) of the Dx indices was calculated over 12 leaves, 3 for each of the 4 replicates. For shaded cabbages, the Dx indices were averaged over 12 leaves from the single replicate for the 0 and 400 kg/ha N treatments. On October 16−18, 2014, for all Dx measurements in the field, the mean (±SD) of indices was calculated on the total of the leaves measured that was between 228 and 252/treatment/cultivar under sunlight and 14/treatment/cultivar under shade. For each leaf disk used for the sensor calibration, the three Dx measurements/leaf side were averaged, and then the FLAV was calculated as the sum of the AD and AB values, and the CHL was calculated as the average of AD and AB values. Statistical analysis and curve fitting were carried out with SigmaPlot for Windows version 12.5 software (Systat Software, Inc., San Jose, CA, USA). The coefficient of determination (R2), the BIAS, and the standard error of estimate (SEE) were the parameters used to evaluate the fitting quality. Mean values of data underwent ANOVA and were compared by the all pairwise multiple comparison Tukey test. P values of
