The Form in Which Nitrogen Is Supplied Affects the Polyamines

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The form in which nitrogen is supplied affects the polyamines, amino acids, and mineral composition of sweet pepper fruit under an elevated CO2 concentration. Maria Carmen Piñero, Ginés Otálora, Manuel E. Porras, María-Cruz SánchezGuerrero, Pilar Lorenzo, Evangelina Medrano, and Francisco M del Amor J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04118 • Publication Date (Web): 11 Jan 2017 Downloaded from http://pubs.acs.org on January 12, 2017

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Journal of Agricultural and Food Chemistry

The form in which nitrogen is supplied affects the polyamines, amino acids, and mineral composition of sweet pepper fruit under an elevated CO2 concentration. Maria C. Piñeroa, Ginés Otáloraa, Manuel E., Porrasb, Mari C. Sánchez-Guerrerob, Pilar Lorenzob, Evangelina Medranob, Francisco M. del Amorb* a

Departamento de Hortofruticultura. Instituto Murciano de Investigación y Desarrollo

Agrario y Alimentario (IMIDA), C/Mayor s/n, 30150 Murcia, Spain b

Agricultural Research and Development Centre of Almería (IFAPA-Almería), Autovía

del Mediterráneo, Sal. 420, Paraje San Nicolás, 04745 La Mojonera, Almería, Spain. *Corresponding author: Complete full name: Francisco M. del Amor Saavedra Telephone: +34 968 366748 E-mail: [email protected]

1 ACS Paragon Plus Environment


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Abstract

2

We investigated the effect of supplying nitrogen, as NO3- or as NO3-/NH4+, on the

3

composition of fruits of sweet pepper (Capsicum annuum L. cv. Melchor) plants grown

4

with different CO2 concentrations ([CO2]): ambient or elevated (800 µmol mol-1). The

5

results show that the application of NH4+ and high [CO2] affected the chroma related to

6

the concentrations of chlorophylls. The concentrations of Ca, Cu, Mg, P, and Zn were

7

significantly reduced in the fruits of plants nourished with NH4+, the loss of Fe being

8

more dramatic at increased [CO2], which was also the case with the protein

9

concentration. The concentration of total phenolics was increased by NH4+, being

10

unaffected by [CO2]. Globally, the NH4+ was the main factor that affected fruit free

11

amino acid concentrations. Polyamines were affected differently: putrescine was

12

increased by elevated [CO2], whilst the response of cadaverine depended on the form of

13

N supplied.

14 15

Keywords: pepper fruit; CO2 enrichment; climate change; nitrate; ammonium; mineral

16

composition.

17 18 19 20 21


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INTRODUCTION

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Nitrogen (N) is one of the most important nutrients that limits net primary production,

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but is also an important water contaminant co-responsible for the eutrophication of

25

many agricultural areas worldwide.1 The great increase in human food demand under

26

conditions of climate instability requires more efficient use of resources such as water

27

and nutrients.2 Thus, a climate change scenario involving a significantly increased CO2

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concentration implies the need for substantial adjustments to the way horticultural food

29

is produced, as well as changes in the composition of the products. An elevated CO2

30

concentration (e[CO2]) has been found to have negative effects, such as reduced tissue

31

concentrations of N and proteins and reduced uptake of nutrients, resulting in lower

32

nutritional values of crops.3 The exact mechanisms by which the reduction in the

33

concentration of N in plants occurs are still unknown, but three potential scenarios are

34

considered: (i) the buildup of carbohydrates and other organic compounds as a result of

35

effect of CO2 in the photosynthesis,4 (ii) decreased N uptake under high CO2 due to

36

reduction of stomatal conductance, which causes lower transpiration rates,5 and (iii)

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alteration nitrate (NO3-) assimilation related with declines in the photorespiration

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pathway at e[CO2], as found in C3 species such as wheat and tomato.6 Ellsworth et al. 7

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noted that the N availability to plants is the main factor that determines their responses

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to e[CO2],, and photorespiration supplies a significant part of the energy for NO3-

41

assimilation in C3 plants.8 This implies that e[CO2] can inhibit the photorespiration-

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dependent NO3- assimilation in the shoots of many species.9 Therefore, in order to

43

overcome the envisaged reduction of N uptake under e[CO2], which implies a

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deleterious effect on protein concentration in major crops,10 we hypothesized that the

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additional supply of NH4+ to the nutrient solution (instead of using NO3- as the sole N

46

source) may partially or totally overcome the predicted effect on fruit quality. 3 ACS Paragon Plus Environment


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Consequently, proper management of N fertilization could help to reduce the negative

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effects of e[CO2]. In general, plants show preference for NO3- over NH4+ ions, and

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others can growth better if they have access to both ions.11 However, each plant species

50

has an optimum NO3-/NH4+ ratio, which also depends on the stage of development and

51

the environmental conditions.12

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Sweet peppers are popular fruits, with growing importance in the human food due to

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their versatility: they can be consumed fresh in salads, in cooked meals, or dehydrated

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for spices.13 Furthermore, they have important nutritional properties, providing

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carbohydrates, proteins, vitamins, minerals, carotenoids, and phenolic compounds,

56

important antioxidants that are able to protect our cells from free radicals.14 Several

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investigations of the antioxidant compounds of peppers have indicated beneficial effects

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regarding the prevention of several disease states, including cardiovascular and

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neurodegenerative disorders and cancer.15 However, these nutritional values can be

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disturbed by the growing conditions.16 Consequently, our aim was to study the effects of

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different forms of N fertilization, under ambient and e[CO2], on the quality and

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nutritional value of sweet pepper fruits, in order to elucidate a fertilization strategy that

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will be effective under the current climate change scenario.

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MATERIALS AND METHODS

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This study was conducted at IFAPA center “La Mojonera” (Almería, Spain, latitude

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36°48’ N, longitude 2°41’ W), in two adjacent greenhouses with an area of 720 m2

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each. The greenhouse heights were 4.7 m and the cover was thermal polyethylene

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(0.2 mm thick). The greenhouses were provided with controlled ventilation by two roof


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vents (opening area: 1 × 30 m) and two side vents (opening area: 1.5 × 26 m) for each

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structure. The climate control system (CDC, INTA S.A.) was also used to measure the

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temperature and humidity (sensors HMP45C, Campbell Sci.), and CO2 concentration

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(IRGAs GMD-20, Vaisala). In both greenhouses, pepper seedlings (Capsicum annuum

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L. cv. Melchor) were transplanted on 19 August 2013, two plants into each 27-L

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container filled with perlite, with a density of 2.5 plants per m2. The cultivation was

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carried out according to a type of Dutch pruning; two stalks were left on each plant.

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Enrichment with CO2 was applied in one of the two greenhouses (elevated), through

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emitters located on the surface of the containers. The other greenhouse, without CO2

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enrichment, was the reference greenhouse (ambient). The CO2 began to be applied 14

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days after transplanting. It was applied only during the daytime period. A variable

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strategy was established according to the greenhouse ventilation.17 The aim was to

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maintain a concentration of 800 µmol mol-1 when the windows were closed and 380

83

µmol mol-1 when the opening was greater than 30%. The period of CO2 supply during

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the daytime began 15 min before sunrise and ended 75 min before sunset.

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A drip irrigation controlled system (CDN, INTA S.A.) was used to supply water and

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nutrients. The nutrient solution was applied with one dripper (3 L h-1) per container.

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Nitrogen was supplied as NO3- (N) or NO3-/NH4+ (A). To adjust the N input to the crop

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demand, two phases were established. In the first phase (until October 31) the N inputs

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were: N) 12 mM NO3- and A) 10 mM NO3- + 2 mM NH4+; and in the second phase

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(October 31 to the end of the cycle) the contributions were: N) 10 mM NO3- and A) 8

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mM NO3- + 2 mM NH4+. The volume of nutrient solution supplied by each irrigation

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event was 500 mL per container. The irrigation frequency fluctuated between one and

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five times per day depending on the needs of the plants, maintaining approximately

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40% drainage. A surplus of water uptake is necessary to avoid nutrient toxicities and 5 ACS Paragon Plus Environment


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imbalances in the rhizosphere18. The harvest period was between 28/10/2013 and

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24/02/2014, the fruits being harvested once they had reached commercial maturity (red

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color). Twelve fruits per treatment were processed by measuring color, chlorophylls,

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lycopene, β-carotene, mineral content, total proteins, total phenolic compounds, amino

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acids, and polyamines. Two fruits were considered a sample; therefore, analyses were

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carried out using six replicates per treatment.

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Skin color

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Fruit color was measured with a Konica-Minolta CR-300 colorimeter (Konica-Minolta,

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Kyoto, Japan) with a D65 illuminant, and making three readings along the equatorial

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perimeter of the fruit. The color data are showed as CIEL*a*b* coordinates, as

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previously described by McGuire19.

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Fruit chlorophylls, lycopene, and ß-carotene

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The β-carotene, lycopene, and chlorophylls were extracted from 1 g of frozen pepper

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fruits (-80ºC) with 25 mL of acetone–hexane (2:3) solvent. Samples of pepper fruit

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were homogenized using a polytron and centrifuged at 3,500 rpm for 6 min, at 4ºC.

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Subsequently,

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spectrophotometrically at wavelengths of 663, 645, 505, and 453 nm. The contents of

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chlorophylls a and b, lycopene, and β-carotene were determined according to the

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Nagata and Yamashita 20 equations:

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Chlorophyll a (mg 100 mL-1) = 0.999 * A663 – 0.0989 * A645

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Chlorophyll b (mg 100 mL-1) = - 0.328 * A663 + 1.77 * A645

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Lycopene (mg 100 mL-1) = - 0.0458 * A663 + 0.204 * A645 + 0.372 * A505 - 0.0806 *

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A453.

the

optical

density

of

the

supernatant

was

measured


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β-Carotene (mg 100 mL-1) = 0.216 * A663 - 1.22 * A645 - 0.304 * A505 + 0.452 * A453.

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Mineral content

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Sweet pepper fruits were dried for 72 h at 65 ºC in a heater. The cations were extracted

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by acid digestion from ground material (0.1 g) using an ETHOS ONE microwave

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digestion system (Milestone Inc., Shelton, CT, USA). The Ca, K, Mg, B, Cu, Fe, Mn, P,

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and Zn concentrations in the dry matter of the fruits were analyzed with an inductively-

124

coupled plasma (ICP) spectrometer (Varian Vista MPX, Palo Alto, CA, USA).

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Total protein

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The total protein was analyzed in the dry matter (after at least 72 h at 65ºC) using a

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combustion nitrogen/protein determinator (LECO FP-528, Leco Corporation, St.

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Joseph, MI, USA)21.

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Total phenolic compounds

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The total phenolic compounds were measured from 0.5 g of frozen sweet pepper fruits

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(−80ºC) with 5 mL of 80% acetone. The homogenate was centrifuged at 10,000 rpm at

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4ºC, for 10 min. Folin–Ciocalteu reagent was used, diluted with Milli-Q water (1:10). 1

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mL of the diluted reagent was mixed with 100 µL of supernatant and 2 mL of Milli-Q

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water, and 5 mL of sodium carbonate (20%) were then added. The mixture was kept for

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30 min in the dark. The absorbance was measured at 765 nm according to the

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methodology of Kähkönen et al. 22. The total phenolic content was expressed as gallic

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acid equivalents, in mg g−1 fresh weight.

138 139

Free amino acids



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The free amino acids were extracted from fruits (frozen at −80ºC): the sap was

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extracted, after vortexing at 5,000 rpm (10 min, 4ºC), and analyzed by the AccQ·Tag-

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ultra ultra-performance liquid chromatography (UPLC) method (Waters, UPLC Amino

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Acid Analysis Solution, 2006). For derivatization, 70 µL of borate buffer were added to

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10 µL of the fruit sap, and 20 µL of reagent solution. The reaction mixture was mixed

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instantly and heated at 55ºC for 10 min. After lowering temperature an aliquot of the

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reaction mixture was used for injection. The column was an Acquity BEH C18 1.7 µm,

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2.1 mm ×100 mm (Waters), and the wavelengths were set at 266 nm (excitation) and

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473 nm (emission). The solvent system consisted of two eluents: (A) AccQ·Tag-ultra

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eluent A concentrate (5%, v/v) and water (95%, v/v); (B) AccQ·Tag-ultra eluent B. The

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following elution gradient procedure was used for the analysis: 0–0.54 min, 99.9% A–

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0.1% B; 5.74 min, 90.9% A–9.1% B; 7.74 min, 78.8% A–21.2% B; 8.04 min, 40.4%

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A–59.6% B; 8.05–8.64min, 10% A–90% B; 8.73–10 min, 99.9% A–0.1% B. The

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injection volume was 1 µL, and a flow rate of 0.7 mL min−1. The temperature of the

154

column was maintained at 55ºC. External standards (Thermo Scientific) were used for

155

the quantification of the amino acids, and Empower 2 (Waters) software for data

156

acquisition and processing.

157 158

Polyamine analysis

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Free polyamines were extracted by homogenizing 1.0 g of fruit in 10 mL of 5%

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perchloric acid, and quantified according to the benzoylation method as previously

161

described Serrano et al.

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As an internal standard, 1,6-hexanediamine (100 nmol (g fresh weight)-1 of tissue) was

163

used, and standard curves of cadaverine, histamine and putrescine were prepared. The

164

results are expressed as nmol (g fresh weight)-1 (mean ± SE).

23

, using a liquid chromatography (HPLC) (Hewlett-Packard).


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Statistical analysis

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Six completely randomized blocks with 12 plants per block, were selected for each

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treatment. The data were tested for homogeneity of variance and normality of

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distribution. Analysis of variance (ANOVA) was performed and means were separated,

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using Duncan’s multiple range test at P ≤ 0.05, using Statgraphics Centurion® XVI

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statistical package (Statpoint Technologies, Inc.). Four combinations of treatments were

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used, involving two N forms (NO3- (N) or NO3-/NH4+ (A)) and two ambient CO2

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concentrations (380 µmol mol-1 (a[CO2]) and 800 µmol mol-1 (e[CO2])), with six

174

replications per combination.

175 176

RESULTS AND DISCUSSION

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Color, chlorophylls, lycopene, and ß-carotene

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The color of the fruit is the main visual feature that the consumer uses to accept or reject

179

it,24 being indicative of the quality of the fruit. The color change in the pepper fruits can

180

be attributed to lower chlorophylls concentrations and an increase in carotenoids

181

concentrations, which are influenced by the ambient conditions to which the fruits are

182

exposed.16 Our results for pepper fruits show that L* and hab were not affected by N

183

form or [CO2]. However, the parameters a*, b*, and C* had higher values in the fruits

184

that grew in the reference greenhouse (a[CO2]) with NO3- as the sole N source. The

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values of these parameters were reduced by the application of NH4+ and CO2. Thus,

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fruits grown without NH4+ supply in the reference greenhouse had greater red color (a*

187

= 26.59 and b* = 12.62) and a more intense and vivid color (C* = 29.51), although it

188

was impossible to perceive this with the naked eye (Table 1). In turn, these fruits

189

showed lower chlorophylls concentrations and an increase in the formation of lycopene 9 ACS Paragon Plus Environment


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and β-carotene (Figure 1). The chlorophyll pigments (Chl a and Chl b) exhibited similar

191

behaviors; both were increased at e[CO2]. Additionally, the combination of NH4+ and

192

e[CO2] had a synergic effect, giving higher concentrations of chlorophylls in the

193

pericarp of the fruit (Figure 1). The total content of chlorophyll (a + b) is also related to

194

the ripening process in pepper

195

significant effect on this at e[CO2]. The β-carotene concentration was only affected by

196

the application of NH4+ in the nutrient solution: it was reduced from 146.53 to 84.12 mg

197

kg-1 FW at a[CO2], and from 142.02 to 83.90 mg kg-1 FW at e[CO2] in the plants treated

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with NH4+. Moreover, the lycopene concentration was not affected by the [CO2] or N

199

form (Figure 1). These results are in agreement with those of Pérez-López et al. 16, who

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found a similar pattern when studying the effects of different agricultural practices on

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the quality of sweet peppers. These authors reported that the β-carotene values were

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greatest in the fruits with the highest values of the color parameters.

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Mineral content

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The concentrations of minerals such as Ca, Cu, Mg, P, and Zn were significantly

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reduced in the fruits of plants treated with NH4+ in the nutrient solution (Table 2), the

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declines of Ca and Cu being particularly sharp at a[CO2]. Although the Ca content in

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the fruit is considered an important factor in the appearance of blossom-end rot (BER),

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in the present experiment BER incidence was not correlated with Ca content in the

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pepper fruits. NH4+ caused a similar increase in the percentage of BER by 6.7% and

210

6.5%, at ambient and e[CO2], respectively (data not shown). These results are agreed

211

with those finding reported by Borgognone et al.

212

correlation between Ca content in tomato fruits and BER incidence. However, NH4+ had

213

no significant effects on marketable yield, but it was increased by 21.5 % when CO2

25

and the change in the N supply (NH4+) had a

26

, who even found a negative


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was supply (data not shown). Other researchers reported that the uptake of Ca and Mg

215

was reduced when the proportion of NH4+ in the nutrient solution increased,26 which

216

suggests that NH4+ was competing with these minerals in pepper.27 Furthermore, the

217

concentrations of Cu, Fe, Mg, and K were also reduced in plants grown at e[CO2]. Thus,

218

the rise in [CO2] reduced the concentrations of these nutrients in plants grown with

219

NO3- as the only source of N (Table 2). Seneweera and Conroy

220

observed reductions in the foliar nutrients of rice and wheat at e[CO2]. McGraLobell

221

suggested that the reductions in the leaf concentrations of nutrients at e[CO2] may be

222

due to several mechanisms, including dilution of non-carbon compounds by the

223

increased concentrations of carbohydrates arising from enhanced photosynthesis and

224

limitations to the transpiration-driven mass flow of nutrients due to decreased stomatal

225

conductance.31 This effect of e[CO2] on the sweet pepper fruit Fe concentration could

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not be effectively overcome by adjusting the N fertilization strategy; however, NH4+

227

proved effective with regard to increasing the Mn concentration. In contrast, provision

228

of NH4+ should be avoided at a[CO2], to avoid dramatic Ca reductions in pepper fruits,

229

but NH4+ did not have a detrimental effect on this nutrient when applied at e[CO2].

230

Total protein

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As for the rest of the nutrients, the fruit total protein concentration was reduced by

232

e[CO2] (from 116 to 105 g kg-1 DW) (Figure 2A). This result was similar to the

233

reductions described by Taub et al.

234

15%). Rubio et al.

235

mitochondrial respiration in the light and protein synthesis. Thus CO2 reduces

236

conversion of NO3- into protein during daytime. This was also observed in pasture and

237

cereal plants by authors like Wieser et al.

238

alterations may produce significant ecological, economic, and nutritional consequences.

33

32

28

29

and Loladze

also 30

in wheat, barley, and rice (ranging from 10% to

pointed out that e[CO2] during the daytime decreases plant

34

and Weigel and Manderscheid

35

. Such



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However, the improvement in N nutrition when supplying NH4+ under e[CO2] was able

240

to recover the fruit protein levels to those of plants grown at a[CO2] and nourished with

241

NO3- as the sole source of N.

242

Total phenolic compounds

243

The fruits of C. annuum cv. Melchor plants supplied with NO3- alone had a total

244

phenolics concentration of nearly 0.88 mg g-1 FW, while those of the plants also

245

receiving NH4+ showed a higher concentration (1.06 mg g-1 FW) (Figure 2B). These

246

results are in agreement with those obtained by Leja et al.

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accumulation of phenolic compounds in pepper fruits when NO3-: NH4+: NO2- was

248

applied instead of NO3- alone. On the other hand, Abu-Zahra 37 noted that differences in

249

the concentrations of total phenols were due to nutrient availability, which coincides

250

with our results. The high content of phenolic compounds in the peppers could be

251

attributed to a decreased availability of plant nutrients; although all plants had the same

252

availability of nutrients in the solution, when they were supplied with NH4+ the uptake

253

of cations was reduced. Horchani et al. 38 observed that NH4+ toxicity led to antagonism

254

in cation uptake and/or alterations in the osmotic balance, which lowered the uptake of

255

cations.

256

Free amino acids

257

Amino acid metabolism is one of the main biochemical processes in plants. The role of

258

free amino acids in synthesis of proteins and other compounds, such as glucosinolates

259

and phenolics, confers them great importance in the plant-environment interactions and

260

human health.39 Bialczyk et al.

261

organs may reflect the intensity of NH4+ uptake and its assimilation in roots. Our data

262

show (Figure 3) that, of the free amino acids of the pericarp of pepper fruits, proline and

40

36

, who indicated a higher

reported that the free amino acids content in plant


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aspartic acid were the second and third most abundant, respectively, in red fruits.

264

Glycine was the most abundant free amino acid in all fruits, with the highest relative

265

content (approximately 43% and 37%) in fruits of plants irrigated with NO3-/NH4+ or

266

NO3-, respectively, at both elevated and ambient [CO2]. The NH4+ supply was the main

267

factor that affected free amino acid concentrations (Figure 3). It reduced the

268

concentrations of the majority of amino acids, while the treatment which yielded the

269

highest concentration of total amino acids was the irrigation with NO3- as the sole

270

source of N under e[CO2], since there was an interaction between these two factors.

271

This effect of NH4+ could be partially attributed to the observed decline in fruit K+ -

272

which agrees with the data of Armengaud et al.

273

was a rise of 6.4% in the concentration of total amino acids, from 3477 ± 68 mg L-1 at

274

a[CO2] to 3699 ± 65 mg L-1 at e[CO2]. The highest increases were found for glycine

275

(13.1%) and aspartic acid (38.3%), which are non-essential amino acids. In addition,

276

essential amino acids like leucine, isoleucine, valine, methionine, and cysteine were also

277

increased at e[CO2].

278

Polyamines

279

Putrescine, cadaverine, and histamine were present in pepper fruit. However, histamine

280

was scarcely detectable; therefore, only the data for putrescine and cadaverine are

281

presented (Figure 4A). Increases in the levels of polyamines, especially putrescine, have

282

been proposed as a general response of plant tissues to different stresses, such as

283

salinity,42 water stress,43 nutritional stress,44 UV radiation,45 CO2 stress,46 and chilling

284

injury.47 In this study, putrescine levels in pepper fruits were significantly increased at

285

e[CO2] (Figure 4). In fruits of plants grown with NO3-, at a[CO2] the putrescine

286

concentration was 66 nmol g-1 FW, while at e[CO2] it was 108.9 nmol g-1 FW High

287

concentrations of polyamines may also have positive effects on fruits post-harvest.

41

. Regarding CO2 enrichment, there



48

49

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288

Botella et al.

289

polyamines in the fruits could confer greater firmness, as they hinder the access of

290

degradative enzymes to the cell wall and inhibit the enzyme activity that degrades pectic

291

acids, thus reducing the rate of softening during storage. On the other hand, the

292

cadaverine levels showed an inverse pattern in relation to [CO2] (Figure 4B), being

293

reduced by e[CO2] when only NO3- was supplied and increased when NH4+ was added,

294

for the same [CO2].

295

The present study shows the combined effects of different N fertilization regimes and

296

CO2 enrichment on the external appearance and nutritional quality of sweet peppers.

297

The results show that the application of NH4+, e[CO2], or a combination of both reduced

298

the lycopene accumulation and a* value, although this reduction in the color was not

299

noticeable visibly. In addition, the e[CO2] and NH4+ application reduced the uptake of

300

minerals. This was caused mainly by the e[CO2], although we found a different effect

301

for the calcium concentration: its decrease caused by e[CO2] was overcome by the

302

supply of NH4+ to the roots. Furthermore, the combination of these two factors

303

increased the levels of total phenols and polyamines, and maintained the total protein

304

concentration at the level found under control conditions. It appears that the NH4+

305

application maintained the diurnal conversion of the N forms into proteins. On the other

306

hand, the total amino acids concentration was increased by the combination of e[CO2]

307

and the supply of N as NO3- alone. The results of this work highlight the importance of

308

adequate fertilization to mitigate the deleterious effect of atmospheric composition

309

changes. The modified nutrient solution assayed for sweet pepper resulted moderately

310

effective, and further work on this topic should be done due to its importance for human

311

nutrition.

and Martínez-Romero et al.

observed that elevated levels of

312 14 ACS Paragon Plus Environment

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ACKNOWLEDGEMENTS

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M.C. Piñero and M.E. Porras are the recipients of a pre-doctoral fellowship from the

315

INIA-CCAA. The authors thank M. Marín for technical assistance, and Dr. David J.

316

Walker for assistance with the correction of the English. This work has been supported

317

by the Instituto Nacional de Investigaciones Agrarias (INIA), through project

318

RTA2011-00026-C02-01. Part of this work was also funded by the European Social

319

Fund.

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321

REFERENCES

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Tables and figures Table 1. Effect of two different N forms (N, NO3- and A, NO3-/NH4+ combined) under an elevated CO2 concentration on sweet pepper fruits: CIEL*a*b* Color Coordinates. Data are means ± SE (n=6). [CO2] 380 800

Nitrogen formc N A N A

ANOVAb NFc CO2 NF x CO2

L*

a*

b*

C*

hab

31.75 ± 0.13 a

26.59 ± 0.50 b

12.62 ± 0.26 b

29.51 ± 0.53 b

26.06 ± 0.41 a

31.25 ± 0.32 a

24.01 ± 0.59 a

11.56 ± 0.32 a

26.73 ± 0.64 a

26.06 ± 0.20 a

31.83 ± 0.27 a

24.31 ± 0.23 a

11.58 ± 0.20 a

26.69 ± 0.40 a

25.74 ± 0.35 a

31.37 ± 0.25 a

25.08 ± 1.03 ab

10.81 ± 0.40 a

26.44 ± 1.57 a

26.33 ± 0.31 a

ns

ns

*

ns

ns

ns

ns

*

ns

ns

ns

*

ns

ns

ns

a

Different letters within a column indicate significant (P ≤ 0.05) differences between treatments. b Analysis of variance: ns. not significant; *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.001. c Nitrogen form: (N) NO3- and (A) NO3-/NH4+.



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Table 2. Effect of two different N forms (N, NO3- and A, NO3-/NH4+ combined) under an elevated CO2 concentration on sweet pepper fruits: mineral contents (in dry matter). Data are means ± SE (n=6). [CO2] 380 800

Nitrogen P c form (mg kg-1) N 2263 ± 33.4 c

K Ca Mg B Mn Fe Zn Cu -1 -1 -1 -1 -1 -1 -1 (mg kg ) (mg kg ) (mg kg ) (mg kg ) (mg kg ) (mg Kg ) (mg kg ) (mg kg-1) 22984 ± 817 b 411.4 ± 18.2 b 1275 ± 55.3 c 9.96 ± 0.27 ab 10.13 ± 0.6 ab 26.17 ± 0.70 b 12.89 ± 2.47 b 2.77 ± 0.17 d

A

1807 ± 49.4 a

19784 ± 283 a

10.22 ± 10.2 a

N

2238 ± 14.5 c

20589 ± 284 a

387.8 ± 19.9 b 1146 ± 21 ab 9.77 ± 0.46 ab 8.92 ± 0.38 a 21.59 ± 0.37 a 8.58 ± 1.54 ab 2.10 ± 0.11 c

A

2014 ± 84.0 b

19145 ± 661 a

390.8 ± 11.6 b 1180 ± 39 bc

1055 ± 5.1 a

10.5 ± 0.22 b

9.21 ± 0.25 a 26.19 ± 0.23 b 7.96 ± 1.19 a

9.25 ± 0.10 a 11.36 ± 0.55 b 22.87 ± 1.05 a 7.87 ± 0.49 a

ANOVAb *** ** *** * ns ns NFc ns * *** ns * ns CO2 * ns *** * ns * NF x CO2 a Different letters within a column indicate significant (P ≤ 0.05) differences between treatments. b Analysis of variance: ns, not significant; *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.001. c Nitrogen form: (N) NO3- and (A) NO3-/NH4+.

0.08 ± 0.07 a 1.54 ± 0.23 b

ns

ns

***

**

ns

*

ns

ns

***

24

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Figure 1. Effect of two different N forms (N, NO3- and A, NO3-/NH4+ combined) under an elevated CO2 concentration on sweet pepper fruits: (A) chlorophyll a, (B) chlorophyll b, (C) lycopene and (D) β-carotene. Data are means ± SE (n=6). Data with the same letter were not significantly different at P ≤ 0.05 (Duncan’s multiple range test).

Figure 2. Effect of two different N forms (N, NO3- and A, NO3-/NH4+ combined) under an elevated CO2 concentration on sweet pepper fruits: (A) total protein and



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(B) total phenolics concentration. Data are means ± SE (n=6). Data with the same letter were not significantly different at P ≤ 0.05 (Duncan’s multiple range test).

Figure 3. Effect of two different N forms (N, NO3- and A, NO3-/NH4+ combined) under an elevated CO2 concentration on sweet pepper fruits: amino acid profiles. Data are means ± SE (n=6). (*) denotes significant differences between 400 and 800 µmol CO2 (P ≤ 0.05), and (+) denotes significant differences between NO3- and NO3-/NH4+ combined (P ≤ 0.05) (Duncan’s multiple range test).


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Figure 4. Effect of two different N forms (N, NO3- and A, NO3-/NH4+ combined) under an elevated CO2 concentration on sweet pepper fruits: (A) Putrescine and (B) Cadaverine levels. Data are means ± SE (n=6). Data with the same letter were not significantly different at p ≤ 0.05 (Duncan’s multiple range test).



Abstract Graphic 254x190mm (96 x 96 DPI)

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The Form in Which Nitrogen Is Supplied Affects the Polyamines

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