Metabolic Effects of Elevated CO2 on Wheat Grain Development and

Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Metabolic Effects of Elevated CO2 on Wheat Grain Development and Composition David Soba,† Sinda Ben Mariem,† Teresa Fuertes-Mendizab́ al,‡ Ana María Meń dez-Espinoza,§ Françoise Gilard,∥,⊥ Carmen Gonzaĺ ez-Murua,‡ Juan J. Irigoyen,# Guillaume Tcherkez,∇ and Iker Aranjuelo*,†,‡

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†

Instituto de Agrobiotecnología (IdAB), Consejo Superior de Investigaciones Científicas−Gobierno de Navarra, Avenida Pamplona 123, 31006 Mutilva, Spain ‡ Department of Plant Biology and Ecology, University of the Basque Country (UPV/EHU), 48940 Bilbao, Spain § Plant Breeding and Phenomic Center, Faculty of Agricultural Sciences, Universidad de Talca, Talca 3460000, Chile ∥ Plateforme Métabolisme-Métabolome, Institut de Biologie des Plantes, CNRS UMR 8618, Université Paris-Sud, Bâtiment 630, 91405 Orsay Cedex, France ⊥ INRA, UMR INRA/UCBN 950 Ecophysiologie Végétale, Agronomie et Nutritions NCS, IFR 146 ICORE, Institut de Biologie Fondamentale et Appliquée, Université de Caen Basse-Normandie, 14032 Caen, France # Grupo de Fisiología del Estrés en Plantas (Departamento de Biología Ambiental), Unidad Asociada al CSIC, EEAD, Zaragoza e ICVV, Logroño, Facultades de Ciencias y Farmacia, Universidad de Navarra, Irunlarrea 1, 31008 Pamplona, Spain ∇ Research School of Biology, Joint College of Sciences, Australian National University, 2601 Canberra, Australian Capital Territory, Australia S Supporting Information *

ABSTRACT: The increase in the atmospheric CO2 concentration is predicted to influence wheat production and grain quality and nutritional properties. In the present study, durum wheat (Triticum durum Desf. cv. Sula) was grown under two different CO2 (400 versus 700 μmol mol−1) concentrations to examine effects on the crop yield and grain quality at different phenological stages (from grain filling to maturity). Exposure to elevated CO2 significantly increased aboveground biomass and grain yield components. Growth at elevated CO2 diminished the elemental N content as well as protein and free amino acids, with a typical decrease in glutamine, which is the most represented amino acid in grain proteins. Such a general decrease in nitrogenous compounds was associated with altered kinetics of protein accumulation, N remobilization, and N partitioning. Our results highlight important modifications of grain metabolism that have implications for its nutritional quality. KEYWORDS: durum wheat, atmospheric CO2, N metabolism, phenology, quality, nutritional parameters, Triticum durum

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INTRODUCTION

are currently more frequently observed in intensive wheat cultivation areas, such as Western Europe and Australia. The specific effects of elevated CO2 on wheat have been well-studied because of potential issues for future food security.3 In plants with C3 photosynthetic metabolism, such as wheat, the ambient CO2 mole fraction (≈415 μmol mol−1) is a limiting factor, and thus, in principle, photosynthesis and growth in wheat are expected to be enhanced by elevated CO2, resulting in a higher yield.3−12 However, the improvement in the grain yield may occur at the expense of grain quality traits, such as nitrogen (N) and protein contents, mineral composition, or starch properties.5,8,13 Durum wheat is the preferred raw material for the production of pasta worldwide. For pasta making, dough properties are crucial aspects of quality, and they are mostly determined by storage proteins of the wheat endosperm [here,

The atmospheric carbon dioxide (CO2) mole fraction has increased dramatically since pre-industrial times, and a further substantial increase is expected by the end of the next century. CO2 is the major greenhouse gas of anthropogenic activity and is believed to participate in climate change, whereby global temperature and precipitation patterns will be altered. At the plant level, an increasing CO2 mole fraction tends to increase photosynthesis but feedback effects are so that plant primary production may not increase accordingly. Also, in crops, such as cereals, both the grain yield and quality may change, and this must have consequences for food production and industrial processing of harvested grains. Wheat is one of the most important crops at the global scale, and in 2016, the worldwide production of wheat was 749 million metric tons, representing a surface of 220 million ha globally.1 Despite a continuous increase in wheat grain production and yield (which increased from ≈1 metric tons ha−1 in the 1960s to 3.4 metric tons ha−1 presently), grain production in wheat is considerably affected by climatic events, such as high temperature and drought,2 that © XXXX American Chemical Society

Received: March 11, 2019 Revised: June 12, 2019 Accepted: June 18, 2019

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DOI: 10.1021/acs.jafc.9b01594 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

close to field conditions. Plants were watered with a complete Hoagland solution22 twice a week and with water once a week to avoid excessive salt accumulation. Half of the plants were grown in greenhouses with atmospheric CO2 (400 μmol mol−1), while the other half was transferred to greenhouses where the CO 2 concentration was increased to 700 μmol mol−1 by injecting pure CO2 with a valve placed at the two inlet fans. CO2 was provided by Air Liquide (Bilbao, Spain). The CO2 concentration was continuously monitored using a Guardian Plus gas monitor (Edinburgh Instruments, Ltd., Livingston, U.K.). The gas monitor signal was transferred to a proportional−integral−derivative controller that regulated valve opening (within a 10 s cycle). The pot position was changed weekly to avoid edge effects, and the pots were changed of greenhouse every month to avoid differences derived from chamber effects. The harvest for metabolomics and protein determinations was carried out 2 weeks after anthesis, defined when at least 50% of the spikes in a pot showed protruding anthers. This period was selected because growth conditions between 10 day pre- and 20 day post-anthesis are considered to be a critical period for the grain yield.23 Plant sampling was always carried out 4 h after the onset of the photoperiod. Nitrogen isotope labeling was conducted for 1 week, starting 1 week after anthesis (time at which >50% of spikes showed protruding anthers), coinciding with the critical time frame for the grain yield12 and the onset of protein remobilization in leaves.24 Labeling was conducted on a randomized subset of plants at each CO2 condition, by replacing KNO3 (1.22 g L−1) of the Hoagland solution by K15NO3 (same concentration). A total of 500 mL of 15N-enriched (2% 15N) Hoagland solution was poured at the first, third, and fifth days of the labeling week in each pot. The nitrogen isotope composition (δ15N) of the natural (non-labeled) Hoagland solution was −1.53‰ (i.e., 0.37% 15N). Isotope measurements were conducted in samples collected immediately after the end of the labeling period. Plant Growth Determinations. Ears, flag leaves, and stems were collected 2 times, 2 weeks after anthesis (grain filling) and at maturity (eight plants per treatment combination). The samples were dried at 60 °C for 48 h in an oven, and the dry weight (DW) was determined. The harvest index (HI) was calculated as the ratio between the grain DW and total DW. Total DW included ear, flag leaf, and shoot dry matter. Metabolic Analyses. For analyses of metabolites, flag leaf and grain samples (20 mg) were ground in liquid nitrogen and then 2 mL of 80% methanol (v/v) was added and mixed. Ribitol (100 μmol L−1) was added as an internal standard. After centrifugation at 15 000 rpm for 15 min at 4 °C, the supernatant was collected and centrifuged again. Then, the supernatants were spin-dried under vacuum and stored at −80 °C until analysis. Metabolomics analyses were carried out using gas chromatography coupled to time-of-flight mass spectrometry (GC−MS). Sample derivatization and GC−MS analyses were carried out as described.25 To quantify the grain starch content, samples were homogenized in 80% ethanol (v/v) and sonicated for 25 min at 30 °C using an Ultrasons-H ultrasonic bath (Selecta, Spain). The hydroalcoholic phase was evaporated through a Turbovap evaporator (Zymark, Carmel, IN, U.S.A.) and resuspended with 4 mL of distilled water. Starch was determined using an amyloglucosidase-based test kit (RBiopharm AG, Darmstadt, Germany). Protein Analysis by Capillary Electrophoresis. The method used was similar to the Osborne method, with modifications.26,27 Albumins and globulins were first removed from 50 mg of flour with 1 mL of 50 mM Tris−HCl buffer (pH 7.8) containing 50 mM KCl and 5 mM ethylenediaminetetraacetic acid (EDTA). After 10 min of mixing and centrifugation at 15 000 rpm for 5 min, the supernatant was discarded and the precipitate was resuspended in 1 mL of water. After 10 min of mixing and centrifugation again at 15 000 rpm for 5 min, the supernatant was discarded and the surface of the pellet was rinsed twice with water. The gliadin extract was obtained by mixing the precipitate with 1 mL of 50% propan-1-ol, vortexing for 30 min, and centrifuging at 15 000 rpm for 5 min. The remaining pellet was washed twice with 50% propan-1-ol, and supernatants were discarded. Glutenins were extracted by mixing the precipitate with 1 mL of 50%

“quality” refers to nutritional and end-use properties that can be influenced by the genetic background (wheat line) as well as culture management and environmental conditions]. In effect, the grain protein concentration and composition are major determinants of the grain nutritional value as well as flour functional properties.13 Additional factors, such as starch and non-starch polysaccharides and non-gluten proteins, can also play a role.14 Flour and dough quality might change because of high CO2-induced reduction in grain N and protein contents. In fact, numerous studies described in the literature have shown a general decrease in gluten protein fractions.8,9,15,16 Along with a lowered protein concentration in grain, the amino acid composition has also been shown to be modified under elevated CO2.17 The grain N content has been found to decrease as a result of a lower N pool available for remobilization from leaves.18 In addition, during the grain filling phase, both the atmospheric CO2 concentration and high temperature have been shown to have important consequences on grain quality traits.19 Taken as a whole, elevated CO2 leads to lower flour quality, mostly via a decline in nitrogenous components in mature grains. However, mechanisms affecting grain quality induced by CO2 enrichment have not yet been completely elucidated, and data on potential changes during grain filling (i.e., not only on properties of mature grains at harvesting) are lacking. In wheat, the metabolic demand during grain filling is met by both CO2 and N assimilation in leaves (in addition to refixation of respired CO2 by glumes) and remobilization of stem reserves formed before anthesis.20 At later phenological stages, the contribution of remobilization increases, including remobilized material from leaves. During the pre-anthesis growth phase, absorbed nutrients (including N) are primarily used for shoot development and synthesis of the photosynthetic machinery.21 After anthesis and during grain filling, assimilated N is mostly allocated to grains, and as a result, much of the mature (dry) grain N content eventually comes from remobilization. In other words, the grain filling phase is crucial for grain elemental composition, and grain quality traits greatly depend upon remobilization. However, little is known about the impact of elevated CO2 on metabolism and N acquisition in developing grains when remobilization occurs. The aim of the present study was to examine the effects of elevated CO2 on grain metabolism, including both during the grain filling phase and at maturity. We quantified yield parameters (grain yield, biomass, harvest index, and thousand grain weight) and quality parameters (elemental composition and grain protein content) and did isotopic labeling and metabolomics analyses at two development stages (grain filling and grain maturity) in durum wheat (cv. Sula) exposed to either a normal (ambient) or an elevated (700 μmol mol−1) CO2 mole fraction.

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MATERIALS AND METHODS

Plant Material, Experimental Design, and 15N Labeling. Experiments were carried out with the durum wheat (Triticum turgidum Desf.) cultivar Sula, which is extensively cultivated in the Mediterranean region. Seedlings were vernalized for 4 weeks at 4 °C and then transplanted to 13 L pots containing a substrate filled with 2:2:1 (v/v/v) vermiculite/perlite/peat. Eight pots of each combination with four plants per pot were used in the experiment. After sowing, the plants were transferred to four CO2-controlled greenhouses located at the campus of the Universidad de Navarra (42.80 N, 1.66 W; Pamplona, Spain). Inside greenhouses, pots were placed in holes made in the soil to generate natural temperature fluctuations B

DOI: 10.1021/acs.jafc.9b01594 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry Table 1. Growth and Grain Parameters of Durum Wheat (cv. Sula) Cultivated under Ambient and Elevated CO2a stage

trait

ambient

elevated

CO2 effect (%)

p value

grain filling

grain DW total DW harvest index grain DW total DW harvest index TGW tiller number ear number

1.14 ± 0.17 13.36 ± 1.76 0.086 ± 0.017 7.15 ± 1.74 15.94 ± 2.47 44.57 ± 7.10 48.14 ± 7.84 6.67 ± 1.97 5.67 ± 1.03

1.19 ± 0.60 15.04 ± 3.56 0.079 ± 0.034 14.59 ± 1.87 27.30 ± 3.82 53.56 ± 2.24 50.58 ± 13.50 9.00 ± 1.97 6.33 ± 1.86

+4.10 +12.60 −8.77 +104.11 +71.29 +20.17 +5.08 +34.93 +11.64

0.858 0.324 0.638 0.001