Impact of elevated CO 2 (free air CO 2 enrichment) was studied on wheat ( Triticum aestivum L. var Kundan) growth, yield and proteome. Elevated CO 2 significantly impacted both underground (+24%) and aboveground (+15%) biomass. Grain weight/plant and harvest index were increased by 35% and 11.4%, respectively under high CO 2. On the other hand, seed protein content was decreased by 19% under CO 2 enrichment while seed starch and soluble sugar contents were increased by 8% and 23%, respectively. Wheat leaf proteomics revealed that 50 proteins were showing differential expression. Twenty proteins were more abundant while 30 were less abundant. Thirty two proteins were identified by MALDI TOF TOF. More abundant proteins were related to defense, photosynthesis, energy metabolism etc. While less abundant proteins were related to glycolysis and gluconeogenesis. Wheat grain proteomics revealed that out of 49 differentially abundant proteins, 24 were more in abundance and 25 were less in abundance in wheat grains under eCO 2 condition. Thirty three proteins were identified and functionally characterized. They were found to be involved mainly in carbon metabolism, storage, defence and proteolysis. Gluten proteins are the major component of wheat storage proteins. Our results showed that both high and low molecular weight glutenins were more in eCO 2 wheat seeds while there was no change in gliadin evels. This might alter wheat dough strength. Concentration of grain Cr and As was increased at eCO 2 while that of Fe, Cu, Zn and Se were found to be decreased. Dynamics of carbon utilization and metabolic abilities of soil microbes under eCO 2 were significantly altered. Our study showed that altered wheat seed composition is cause for concern vis-à-vis nutrition and health and for industries which may have implications for agriculturally dominated country like India.
The rise in atmospheric CO2 concentration is unequivocal as the emissions of CO2 due to anthropogenic activities have increased dramatically within the last 50 years and will continue to increase by almost 3% each year. Future predictions of atmospheric [CO2] estimates concentrations reaching 421 ppm (Representative Concentration Pathways; RCP 2.6) to 1313 ppm (RCP 8.5) by the year 2100 [
Wheat (Triticum aestivum L.) is the world’s foremost food and feed crop and India is second largest producer of this major crop contributing about 12% of the global production. Hogy and Fangmeier (2008) [
In India, CO2 enrichment studies on wheat (Triticum aestivum) in Open Top Chambers (OTCs) showed stimulated growth and yield [
The aim of this study was to investigate the impacts of elevated CO2 on the growth and yield, physiology, proteome and nutrient composition of wheat (Triticum aestivum L. var Kundan) under Free Air CO2 Enrichment (FACE) conditions. Further we studied how elevated CO2 is impacting the structure and activity of the soil microbial community. We report that besides enhanced growth there are indeed some changes in nutrient composition of wheat grains under eCO2.
The experiment was carried out in FACE system of the National Botanical Research Institute, Lucknow (80˚59'E, 26˚55'N, 123 m asl), Uttar Pradesh, India. The NBRI FACE facility consists of three hexagonal CO2 enrichment rings together with their three companion ambient (non-enrichment) rings. The ring has a diameter of 10 m. Each FACE ring is made up of six 3 m long G.I. pipes. Each horizontal tube is grounded in soil. Each horizontal arm is fitted, at three points, with 5 m vertical pipes. These vertical pipes have nozzles to release CO2 inside the ring. CO2 is supplied through 30 kg cylinders fitted with pre-heaters. 300 litre capacity of air compressors are used to pump air mixed with CO2 into FACE ring through GI pipes. Six solenoid valves are used for each arm of FACE ring and one valve is for main CO2 line, therefore total seven solenoid valve were used to control CO2 release inside the ring. The CO2 concentration inside the ring is sampled at 3 places and fed to the infrared gas analyser. Before being fed to the analyser, air is passed through a desiccant and filter to remove moisture and particulate matter. In the middle of the ring, sensors for wind speed and direction, temperature, humidity, light intensity and CO2 are mounted. Signals from these sensors are transmitted toward control room through four core shielded cable. Fully automatic control system (SCADA) for monitoring and regulation of desired CO2 works with inputs form the CO2 analyzer, temperature and anemometer. The control system is operated with microprocessor through in-built timer and data logger input. Online display of temperature, humidity, CO2 concentration in ppm and air velocity is integrated with necessary controls and monitoring station controller. The system has memory backup and real time clock combination and single window operation to monitor temperature, humidity, CO2 level and air velocity with direction. We intended to achieve 500 ppm CO2 concentration but could achieve 472 ppm throughout the experiment (from seed emergence till final harvest).
A local winter wheat cultivar (Triticum aestivum L. cv. Kundan) was grown inside FACE rings. Seeds were manually sown in rows spaced around 15 cm and at depth of 3 - 4 cm. Recommended doses of NPK (Nitrogen: Phosphorus: Potassium at 120:60:60 kg per hectare) were applied as urea, diammonium phosphate (DAP) and potash, respectively. Phosphorus and potassium were applied at the time of seed bed preparation while nitrogen fertilizer was applied in three split doses. One-third nitrogen was applied at the time of seed bed preparation and was thoroughly mixed into soil by ploughing and planking. The second dose (1/3) of nitrogen was applied at the time of 1st irrigation & third dose at the time of 3rd irrigation. The irrigation was maintained regularly throughout the experiment. Weeds were removed manually.
Photosynthetic rate and stomatal conductance were measured using LiCOR model 6400 (Lincoln, Nebraska) equipped with CO2 control modules and LED light sources. Measurements were made on fully expanded leaves two leaves down from the youngest expanding leaf after 8 - 9 weeks of growth for vegetative phase and 12 - 13 weeks of growth for flowering phase.
Plants were harvested for biomass analysis at vegetative phase (8 - 9 weeks of growth) and final harvest (full maturity) in five replicates from each ring. Root and shoot biomass were weighed after drying the plants in oven at 80˚C till constant weight for both the samplings. Spikelet number per inflorescence, inflorescence number per plant and inflorescence weight per plant was also counted. Yield parameters were studied through grain weight per plant, thousand grain weight and harvest index.
Starch was extracted following the perchloric acid method described by Whelan (1955) [
Proteins were extracted initially with extraction buffer 50 mM Tris-HCl, pH 8.0, 25 mM EDTA, 500 mM thiourea and 0.5% 2-mercaptoethanol (BME) after grinding leaf sample in Liquid N2 followed by overnight trichloroacetic acid-acetone precipitation at −20˚C, followed by acetone washing. The pellet was suspended in 0.1 M Tris-HCl, pH 8.0 with 50 mM EDTA and 2% BME followed by phenol-ammonium acetate precipitation overnight at −20˚C. Dried pellet was solubilised in solubilisation buffer 7M urea, 2M thiourea, 2% CHAPS (w/v), 25 mM DTT (Dithiotheritol) for 2 - 3 hours at room temperature subsequently protein was estimated by Bradford method and stored at −20˚C.
Isoelectric focusing (IEF) was performed on 7 cm IPG strips, pH 4 - 7 (Immobilization strip by GE Healthcare) with 120 µg protein in Ettan IPGphor3 unit (GE Healthcare) in triplicates for each treatment. After overnight passive rehydration, focussing was done on Ettan IPGphor under following conditions: 200 V for 20 min, 450 V for 15 min, 750 V for 15 min, and 2000 V for 4 h for a total of 10 kVh. Consequently, equilibration of strips was performed in a buffer containing 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2%(w/v) SDS, 1% (w/v) DTT for 15 min, and another 15 min in the same buffer but with 2.5% (w/v) iodoacetamide replacing DTT. The second dimension was run in Mini-PROTEAN Tetra Cell (BioRad) using 7 × 8 cm homogeneous SDS-PAGE gels of 12% T and 5% C at constant 200 V in standard Tris-Glycine running buffer. Gels were stained with 0.5% Brilliant Blue G-250. Gel images were acquired using HP scanner (SCAN-JET XPA). Image analysis was performed using ImageMaster 2D Platinum 7.0 (GE Healthcare) for protein expression analysis on the basis of relative volume (% volume) as upregulated (1.5 fold increase or more) or downregulated (1.5 fold decrease or more). Different statistical analysis was also performed as facilitated by the software.
Seed protein was extracted following the extraction protocol by Guo et al. (2012) [
Isoelectric focussing (IEF) was performed on 7 cm IPG strips, pH 3 - 10 (Immobilization strip by GE Healthcare) in Ettan IPGphor 3 unit (GE Healthcare) with 60 µg of protein in similar way as described earlier for total protein of leaf with changes in buffer for rehydration which contain 4% CHAPS. Running conditions were: 100 V for 1 hr, 200 V for 30 min, 500 V for 30 min, 1000 V for 2 hr, 5000 V for 2 hr and 8000 V for 5 hr. Equilibration of strip, second dimension, staining of gels and image analysis were performed by following methods described in previous section.
Protein spots were excised from the gels and gel particles were destained overnight by 50% methanol and 0.05 M ABC. Next morning, gels were re-swelled by replacing destain solution with sterilized MQ water for about 5 - 8 min and fresh volume of destain solution were added for upto 3 - 4 h. Gels were washed twice with 0.025 M ABC for 10 min and dehydrated by washing with 2:1 solution of ACN and 0.05 M ABC. The cycle of dehydration was followed by rehydration by 0.025 M ABC three times. Destained gel pieces were dried in a vacuum centrifuge concentrator for 30 min and dried gel pieces were rehydrated in trypsin solution (10 - 20 µl from 20 ng/µl trypsin stock solution) which were added according to 1:20 ratio of protein. Gel particles were immersed in 0.025 M ABC and samples were digested overnight at 37˚C (about 16 - 18 hrs). Peptides were extracted twice with 50% ACN/1% TFA. The recovered peptides were concentrated to a final volume of 10 µl. The identification of protein spots was done through Mass spectrometry (MS) using 4800 Plus MALDI TOF/TOF Analyzer (ABSCIEX, USA). The mono isotopic peptide masses obtained from MALDI-TOF were analyzed by the 4000 Series Explorer software version 3.5 (ABI). On the basis of mass signals, protein identification was performed through Mascot software (http://www.matrixscience.com) against NCBInr protein database. The search criteria in database were as follows: taxonomy, viridiplantae; fixed modification, cysteine carbamidomethylation; variable modification, methionine oxidation; peptide tolerance, ±100 ppm, MS/MS tolerance, ±0.2 Da; peptide charge +1; maximum allowed missed cleavage, 1; instrument type, MALDI-TOF/TOF. The non-probabilistic basis for ranking protein hits and as the sum of the series of peptide scores were protein scores derived from ion scores. The mascot algorithm set the score threshold to achieve p < 0.05 based on the size of the database used in the search. False discovery rate (FDR) for protein identification was set to 1%. The protein spots with MOWSE score above threshold level determined by Mascot were considered and proteins with the confidence interval percentage greater than 95% were considered to represent a positive identification.
1) Isolation of HMW-GS Fractions and Gliadins
HMW Glutenins (alcohol insoluble gluten) fraction was extracted from both AMB and ELE seeds according to Marchylo et al. (1989) [
The extraction of gliadins was performed through modified Osborne fractionation (Wieser et al., 1998) [
2) SDS-PAGE and Image Analysis
SDS-PAGE was carried out according to Lagrain et al. (2013) [
Image analysis was performed through ImageQuant TL 7.0 software (GE Healthcare) with amount of protein calibrated according to Low Molecular Weight Protein Marker from GE Healthcare containing mixture of protein standards (Phosphorylase b, Albumin, Ovalbumin, Carbonic Anhydrase, Trypsin Inhibitor, α Lactalbumin). MS analysis was also done in similar way as described earlier for total leaf protein.
Soil samples (3 samples for each treatment) were collected from the upper layer (0 - 20 cm) in the field and brought to the laboratory in sealed polybags for microbiological and biochemical studies.
Microflora associated with soil samples were determined by the culture enrichment technique [
The patterns of potential carbon source utilization by soil microbial communities under ambient and elevated condition of CO2 were assessed by Biolog Eco and MT plates (Biolog, Inc., Hayward, CA, USA) as described by Campbell et al. (1997) [
All values reported in this work are mean of at least three independent experiments. The means ± SD and the exact number of experiments are given in legends. The significance of differences between control and each treatment was analyzed using Student’s t-test. Principal component analysis was performed on different morphological, physiological and yield parameters using PAST 3 (PAleontological Statistics, Version 3.11).
Our results showed that eCO2 clearly acted as C “fertiliser” positively influencing the biomass accumulation, photosynthesis and yield of wheat plants. Root and shoot weight were increased significantly by 24% and 15%, respectively (
improved yield. The maximum positive loadings on PC1 were of two yield parameters (grain wt/plant and inflorescence wt/plant), stomatal conductance (both stages), photosynthesis (flowering stage), harvest index (HI) followed by root and shoot weight (Supporting Information
The proteomic investigation of leaf and seed revealed increased abundance of proteins involved in carbon metabolism. The non-significant decrease in protein content of leaf was observed under eCO2 (
1) Leaf Proteomics
2-DE resolved a total of 431 proteins in wheat leaf (Supporting Information
2) Seed Proteomics
3) Glutenins and Gliadins
The SDS-PAGE analysis of wheat grains revealed that proteins, five HMW-GS and three LMW-GS, major seed storage proteins, increased under eCO2 condition (
protein synthesis throughout the grain filling period remained unaffected by eCO2, indicating adequate N intermediate supply for protein synthesis. We did not find any change in gliadins (storage protein) levels in response to eCO2 studied through 8 distinct bands on SDS-PAGE (
Nutritional value of food grains should be a matter of concern under changing environment like rise in levels of CO2. In our study, eCO2 caused alterations in the concentrations of microelements in wheat grains (Supporting Information
Soil microbial response under elevated CO2 gives insight about microbial structure and function shift under elevated CO2 environment. In present study, it was observed that the dynamics of carbon utilization and metabolic abilities of microflora under eCO2 were significantly altered. Under ambient condition, the bacterial population and actinomycetes were marginally reduced (0.29 log CFU
and 0.04 log CFU) at the harvesting stage as compared to the initial stage (Zero day) of rhizospheric soil (Supporting Information
Elevated CO2 positively impacted wheat growth and yield. RuBisCO protein was less abundant under eCO2 condition, which might be due to N reallocation and/or photosynthetic acclimation. Seed proteins belonging to carbohydrate and
starch metabolism were more abundant under eCO2 resulting in high seed starch content. Whereas higher glutenin/gliadin ratio under eCO2 will impact visco-elastic dough properties such as higher dough resistance. The decrease in seed Fe and Zn content under eCO2 is a cause for concern in a country like India where child and female anaemia is a major health problem. Microbial study highlighted the necessity and importance of examining the microbial response to eCO2 in agroecosystem and more such long term studies are needed. However, in the present study only one wheat cultivar at one single location and year was examined. Therefore more wheat varieties need to be tested in multi-year studies to determine eCO2-induced changes across different cultivars. Moreover, interactive effects of CO2 enrichment and other abiotic stressors such as temperature and ozone should also be investigated in this part of the world.
Funding for this work was provided by Council of Scientific & Industrial Research (CSIR), New Delhi, India (Grant no. PSC 0112). Senior Research Fellowships provided to MS and SKG by University Grants Commission and Council of Scientific and Industrial Research New Delhi, India, respectively is gratefully acknowledged.
Pandey, V., Sharma, M., Deeba, F., Maurya, V.K., Gupta, S.K., Singh, S.P., Mishra, A. and Nautiyal, C.S. (2017) Impact of Elevated CO2 on Wheat Growth and Yield under Free Air CO2 Enrichment. American Journal of Climate Change, 6, 573-596. https://doi.org/10.4236/ajcc.2017.64029
Tables S1-S8:
https://www.dropbox.com/sh/3j5uoxui04izojc/AADSW0XjiTVFYAEQo687t7h4a?dl=0