An optimistic future of C₄ crop broomcorn millet (Panicum miliaceum L.) for food security under increasing atmospheric CO₂ concentrations

Site description

The pot experiments were carried out in 2013, 2015, 2016 at two OTC facilities, where located in Shanxi Agricultural University (37.42°N, 112.55°E), Taigu, Shanxi, China. The height and diameter of each OTC were 2.5 and 4.0 m respectively. The upper part of the OTC had a frustum of 0.5 m at 2.5 m height, to reduce the dilution of CO₂ by air current inside the chamber and to keep it open and close to natural conditions. Detailed facility operational procedures can be found in Hao et al. (2017). The average temperature of each OTC during the broomcorn millet growing season were 23.4 °C, 23.1 °C and 22.9 °C in 2013, 2015 and 2016. The average relative humidity was 69.0%, 67.4% and 71.2% in 2013, 2015 and 2016 respectively.

Experimental design

Different CO₂ concentrations were set in the two OTC facilities. An a CO₂ (approximately 400 µmol mol⁻¹CO₂) as the control (CK) treatment and another eCO₂ (aCO₂ + 200 µmol mol⁻¹, approximately 600 µmol mol⁻¹CO₂ as EC treatment) were used from crop emergence to harvest.

HuachiRuan Red millet (Panicum miliaceum L.) from Huachi county, Gansu province, were sown on 13 June 2013, 17 June 2015 and 22 June 2016 in 40 cm × 60 cm pots (28 cm depth). The soil was obtained from a nearby cropland (0–20 cm surface soil). The soil type was clay loam, and the soil total N content, organic C content and pH value was 0.12%, 1.37% and 8.3 respectively. The soil was sieved, homogeneously mixed and packed in the pots. Five holes were made to drain away water in each pot bottom.

Fertilizers were applied at the elongation stage of millets at 1.7 g N and 1.15 g P₂O₅ per pot. Ten plants were grown in each pot and 10 pots were included in every chamber. Irrigation equivalent to 10–20 mm of rainfall was applied every 3–5 d to keep the soil water content in 60–80% of relative water content after seedling emergence. The water content was measured by wet sensor (KZSF, China).

Harvesting and chemical analyses

At maturity, broomcorn millet plants were hand-harvested on 18 September 2013 (99 days after sowing), 25 September 2015 (100 days after sowing) and 1 October 2016 (103 days after sowing). The seeds were separated from the spikes after air drying, and air-dried at room temperature to constant weight, and then the yield was weighed. All of the seeds were ground into fine powder in preparation for chemical analyses.

The Kjeldahl method (China agricultural trade standard NY/T 3, 1982) was used to measure the protein concentration of seeds. 0.1 g of powdered seed was put into a Kjeldahl bottle for acidolysis and three mL concentrated sulfuric (18.4 mol L⁻¹) acid was added. Then the liquid was heated by electric stove to keep it boiling for 1 h. 0.05 mol L⁻¹ HCl was titrated until the color of liquid changed from blue–green to grayish purple. The crude protein concentration was calculated based on the dosage of HCl and a conversion factor of 6.25 was used.

The total oil concentration of seeds was evaluated by extraction with petroleum ether (60–80° C), with a soxhlet apparatus, and fat content was obtained by China agricultural trade standard NY/T 4 (1982). Total starch concentration was analyzed by enzyme hydrolysis methods (Mcleary, Solah & Gibson, 1994).

The seed was milled in a Willey-type mill. N was measured from the digestion of samples in H₂SO₄, distillation with the Kjeldahl distiller and titration in H₂SO₄ solution (Bataglia et al., 1983).

From the digestion of the samples in HNO₃ and perchloric acid solution, P was determined from the phosphovanadomolybdic complex formed in the reaction of P with the solution of molybdovanadate at 450 nm in a 722S spectrophotometer (INESA, Shanghai, China) (Bataglia et al., 1983). 0.5 g of dried seed was digested in 10 mL of HNO₃ and 2.5 mL HClO₄ (v/v 4:1) acid for 24 h at room temperature until clear liquid was obtained; subsequently, the liquid was diluted to 25 mL. The concentration of K, Fe, Mn, Ca, Cu, Zn, Mg and B was analyzed following the procedure described in Zarcinas, Catwright & Spouncer (1987) by using inductively-coupled plasma atomic emission spectrometry (ICP-AES) (Optima 5300DV, PerkinElmer, Waltham, Massachusetts, USA). Grain mineral accumulation was calculated from the yield per m² of plot surface area (g m⁻²) and grain mineral concentration.

Metabolite extraction and metabolite profiling analysis

The seed was crushed using a mixer mill (MM 400, Retsch, Haan, Germany) with a zirconia bead for 1.5 min at 30 Hz. 100 mg powder was weighted and extracted overnight at 4 °C with 1.0 ml 70% aqueous methanol. The extracts were centrifugated at 10,000g for 10 min, then absorbed (CNWBOND Carbon-GCB SPE Cartridge, 250 mg, 3 ml; ANPEL, Shanghai, China) and filtrated (SCAA-104, 0.22 mm pore size; ANPEL, Shanghai, China) before LC-MS analysis. The sample extracts were analyzed for metabolites using an LC-ESI-MS/MS system (HPLC, Shim-pack UFLC SHIMADZU CBM30Asystem, Kyoto, Japan; MS, Applied Biosystems 6500 quadrupole-linear ion trap mass spectrometer Q TRAP, San Diego, California, USA) according to the method of Chen et al. (2013). Metabolite quantification was based on the MWDB (metware database) and public database of metabolite information. Metabolite intensity was conducted using multiple reaction monitoring mode (MRM) analysis (Fraga et al., 2010). The supervised multivariate method, orthogonal partial least squares discriminant analysis (OPLS-DA), was used to maximize the metabolome differences between the pair of samples. The relative importance of each metabolite to the OPLS-DA model was conformed using the parameter called variable importance in projection (VIP). Metabolites with VIP ≥1 and —log₂ (fold change)—>1 were set as differential metabolites for group discrimination. For pathway annotation, all the metabolites were manually checked for their Kyoto Encyclopedia of Genes and Genomes (KEGG) name and number using KEGG database (Kanehisa et al., 2016), thereafter, classified in component classes and putative pathways.

Statistical analysis

An analysis of variance (ANOVA) with two-way ANOVA by SAS System 8.1 (SAS Institute Inc., USA) was used to test the effects of (CO₂) and year on yield, grain nutrients concentration, and grain accumulations of protein, starch, oil, and nutrients in broomcorn millet. Duncan’s multiple range tests at P = 0.05 were used to compare treatments.

Response of yield, grain protein, starch and oil concentration to eCO₂

Compared with aCO₂, eCO₂ significantly increased the yield of broomcorn millet by 19.4% in 2015 and 29.9% in 2016 (P < .001) (Fig. 1). Grain protein concentration ranged from 10.98% to 15.58%. It was significantly decreased under eCO ₂ by 7.8%, averaged across three years (P < .001). Elevated CO₂ had no significant effect on the concentration of starch and oil in broomcorn millet for three years (Fig. 1). A significant CO₂ and year interaction was detected for the concentration of protein, starch, and oil.

Response of grain mineral concentration to eCO₂

Elevated CO₂ significantly increased grain Mn, B, and Mg concentration across three years on average by 14.6%, 21.2%, and 27.3%, respectively (Table 1), while it had no significant effect on grain P, K, Fe, Ca, Cu or Zn concentration for three years. N concentration was significantly decreased by 7.8%, averaged across three years. Fe concentration was increased on average by 3.8% across three years. A significant CO₂, year, and CO₂x year interaction was detected for Mg concentration. Only significant CO₂x year interaction was reported in K concentration which was attributable to the increased values in 2013 compared with 2015 and 2016 under eCO₂.

Response of the accumulations of grain nutrients, protein, starch, and oil in broomcorn millet to eCO₂

Elevated CO₂ significantly increased the grain mineral accumulations of P (+23.87%), K (+29.5%), Mn (+40.08%), Ca (+22.58%), Mg (+51.31%), Zn (+40.95%) and B (+48.54%) on average for three years (Table 2). Elevated CO₂ significantly increased the grain accumulation of starch and oil for three years on average by 16.96% and 28.37%, respectively (Table 3). Grain protein accumulation ranged from 33.22 g m⁻² to 83.02 g m⁻². Elevated CO₂ had no significant effect on the grain protein accumulations in 2013, and significantly increased it by 13.08% in 2015, 26.59% in 2016, respectively.

Metabolic changes in response to eCO₂

There were 36 metabolites with significant change under eCO₂, among which 18 metabolites were increased and the others were reduced (Table 4). According to KEGG pathways analysis, eCO₂ affected the synthesis of some important amino acids such as aminoacyl-tRNA biosynthesis, D-glutamine and D-glutamate metabolism, lysine biosynthesis and also affected protein digestion and absorption (Fig. 2). Elevated CO₂ affected not only carbon metabolism (pentose phosphate pathway, glyoxylate and dicarboxylate metabolism) but also N metabolism (Fig. 2). Gluconic acid, pantothenol, and D-Mannitol all increased under eCO₂ (Table 4). Flavone and flavanol biosynthesis were also affected by eCO₂. Most flavonoids metabolites including eriodictyol, apigenin, luteolin, chrysoeriol, kaempferide were substantially accumulated under eCO₂ (Fig. 3).

Among the reduction of 18 metabolites, L-Glutamine and L-Lysine both involved in many pathways such as aminoacyl-tRNA biosynthesis, biosynthesis of amino acids, protein digestion and absorption, microbial metabolism in diverse environments and biosynthesis of plant secondary metabolites (Table 5). L-Glutamine participated in D-Glutamine and D-glutamate metabolism, mineral absorption, nitrogen metabolism etc.

Elevated CO₂ induced the increase in yields in broomcorn millet

There is broad consensus that eCO₂-induced increase in the yields of C₃ crops is higher than that of C₄ crops. However, both our previous work (yield increased by 31.4% in 2013, (Hao et al., 2017) and the present study illustrated that the yield of broomcorn millet were significantly enhanced by eCO₂ (13.6–34.2%), and the yields of foxtail millet increased on average by 21.5% in two years (Li et al., 2019). This was in contrast to the common belief that C₄ crops (maize and sorghum) did not show yield response to eCO₂, because the photosynthesis of C₄ crops was saturated at current atmospheric (CO₂) (Leakey et al., 2006; Markelz, Strellner & Leakey, 2011). While, our previous work proved that photosynthetic rate and intrinsic water use efficiency of broomcorn millet were significantly increased under eCO₂ (Hao et al., 2017; Zhang et al., 2020). Similar responses of millets were observed by Sage & Zhu (2011). This may be conducive to an increase in yield of broomcorn millet. This study further suggested that eCO₂ affected pentose phosphate pathway, glyoxylate and dicarboxylate metabolism, fructose and mannose metabolism, which in turn increased accumulation of carbohydrates such as glucose acid, leading to an increase in grain yield of broomcorn millet.

Effect of eCO₂ on grain concentration and accumulations of protein, starch and oil

Broomcorn millet had a higher protein content more than 11% (dry basis), which was higher than the 7.2% protein found in rice and 10.4% found in sorghum (Jha et al., 2013). The protein quality of broomcorn millet (Essential Amino Acid Index) was higher (51%) compared to wheat (Kalinova & Moudry, 2006). Elevated CO₂ reduced N concentrations by 4%–21% in non-legumes such as wheat, cotton and sorghum at elevated CO₂ (540–958 µmol mol⁻¹) (Prior et al., 1994), while it was reduced by only 1.5%-−2.1% in legumes (Lam et al., 2012). The effect of eCO₂ on the protein concentration of C₄ crops was less studied. Our study suggested that eCO₂ significantly reduced the protein concentration of broomcorn millet by 7.8% over an average of three years. Elevated CO₂ decreased N concentrations in plants through: (1) increasing plant biomass, the so-called ‘dilution effect’ (Reich et al., 2006); (2) reducing the prevalent protein Rubisco concentrations (Long et al., 2004); (3) changing the rhizosphere environment and limiting the N available for plant uptake (Reich et al., 2018); and (4) inhibiting plant N metabolism, especially the assimilation of nitrate of C₃ plants (Butterly et al., 2015). Elevated CO₂-induced inhibition of N uptake and assimilation would decrease organic N production and retard growth of C₃ plants (Bloom et al., 2012). In the present study, eCO₂ also affected the grain N metabolism of broomcorn millet (Fig. 2). At the same time, eCO₂ affected synthesis of some important amino acids such as aminoacyl-tRNA biosynthesis, D-glutamine and D-glutamate metabolism, lysine biosynthesis and also affected protein digestion and absorption, thus influencing the protein content (Fig. 2). In addition, eCO₂ had reduced the specific nitrogenous metabolites (L-lysine metabolites and L-glutamine), which were the most represented amino acid in grain proteins. The fact that lysine recycling was perturbed by eCO₂ suggested that sustain amino acid metabolism and protein synthesis was inhibited. It was also reported that a typical decrease in glutamine under eCO₂ in wheat grain (Soba et al., 2019; Högy et al., 2009) and soybean seed (Li & Siddique, 2018). Some studies revealed that glutamine had been reported to accumulate under eCO₂ (Yu et al., 2012; Watanabe et al., 2014; AlJaouni et al., 2018). Hence, impact of eCO₂ on glutamine was expected to show some kind of complexity. The effects of eCO₂ on metabolites were different among species. The decrease in protein was also caused by the carbon dilution effect due to the significant increase in grain yield for three years. Elevated CO₂ significantly increased protein accumulation due to the significant increase in grain yield except in 2013. Jobe et al. (2020) revealed that C₄ plants required less total N, had higher N use efficiency, and maintain N levels under eCO₂.

In our study, eCO₂ had no significant effect on the grain oil concentration of broomcorn millet, while significantly increased the grain oil accumulation as a result of the significant increase in grain yield. Elevated CO₂ had been shown to increase oil concentrations in soybean seed (Hao et al., 2014; Li & Siddique, 2018) and oilseed rape (Högy et al., 2010). Enhanced oil synthesis and storage in plants under eCO₂ had been linked to carbon and energy supply (Rawsthorne, 2002; Bates, Stymne & Ohlrogge, 2013). Singh et al. (2016) also stated that the increase in seed oil concentration under eCO₂ was attributed to the enhancement of photosynthesis. Starch was the major component in proso millet grain, accounting for 58.1%–77.9% of the total grain weight (Yang et al., 2018). In our study, the effect of eCO₂ on the starch content and starch accumulation of broomcorn millet was similar to that of oil, which also proved that starch quality was improved under eCO₂. Mao et al. (2020) also reported that eCO₂ alone also had no significant effects on starch content in rice grains, which is consistent with our findings.

Effect of eCO₂ on grain mineral quality

Dietary deficiency of Zn and Fe is a major global public health problem. Myers et al. (2015) predicted the risk of global Zn deficiency would increase under eCO₂. Their meta-analysis results demonstrated that eCO₂ significantly decreased the concentrations of Zn and Fe in all C₃ grasses and legumes investigated, but not for C₄ crops (maize and sorghum) (Myers et al., 2014). Our research also confirmed that eCO₂ had less effect on Zn and Fe concentrations of C₄ millet, suggesting that C₄ crops had more desirable nutritional traits for combating hidden hunger. Millets are considered as high-energy yielding nourishing foods which help in addressing malnutrition (Nithiyanantham et al., 2019). Consumption of broomcorn millet is associated with reduced risk of type-2 diabetes mellitus because whole grains are a rich source of Mg (Pathak, 2013). Mg acts as a co-factor in a number of enzymatic reactions that regulate the secretion of glucose and insulin. Mg can also reduce the frequency of migraine headaches and heart attacks, thus it is beneficial for people suffering from atherosclerosis and diabetic heart disease (Shobana & Malleshi, 2007; Gélinas et al., 2008). Elevated CO₂ significantly increased grain Mg concentration across the three years on average by 27.3%. This exceptional nutritional properties of broomcorn millet make it an excellent crop, essential for food, nutrition, and health security under climate change scenarios. We also observed a significant increase in the concentration of Mn in broomcorn millet. In contrast, eCO₂ decreased the Mn content in nine diverse rice cultivars by 53% (Ujiie et al., 2019). A previous study revealed that consumption of a Mn-deficient diet (0.11 mg/day; about 3% of RDA) for 39 days caused a mild form of dermatitis among five of seven men (Friedman et al., 1987). Boron was involved in enzyme reactions (Seth & Aery, 2017), as well as in photosynthesis and carbohydrate metabolism (Han et al., 2008; Han et al., 2009; Teija et al., 2011), flavonoid synthesis, and nitrogen (Camacho-Cristóbal & González-Fontes, 2007) and phenol metabolism (Teija et al., 2011). A significant increase in B was observed in broomcorn millet under eCO₂. Carbohydrate dilution (Reich et al., 2006) and decreased mass flow due to reduced transpiration (McGrath & Lobell, 2013; Houshmandfar et al., 2018) may all be relevant to explain this phenomenon of decreased grain mineral concentration value under eCO₂. The carbon dilution effect due to the significant increase in grain yield across the three years did not occur in Mn, Mg, and B. In our previous study, transpiration rate in broomcorn millet was increased by an average of 12.0% and 21.2% in 2013 and 2014, respectively (Hao et al., 2017). Increases in transpiration may stimulate translocation of nutrients from the root to shoot, and then to the grain of broomcorn millet. Pérez-López et al. (2014) suggested that the increase in growth under eCO₂ could be attributed to the stimulation of metabolic activity in plants, and, accordingly, to the requirement for nutrients that serve as enzyme cofactors in metabolic reactions (Mg and Mn). In the present study, the increase in concentration of Mn, Mg, and B of broomcorn millet observed in response to eCO₂ suggests improved nutritional value of soybean under the scenario of elevated CO₂ concentration. The influence of eCO₂ on elemental content varied among years, possibly because the elemental content of grains was affected by environmental factors during the ripening period, such as air temperature (Seo & Chamura, 1980). While no significant changes were found in the concentrations of K, Ca, Cu and Zn under eCO₂ on average for three years in broomcorn millet, similar to the findings of Myers et al. (2014). This confirmed that increase in atmospheric (CO₂) would have less effect on nutrient content of C₄ crops. Grain Zn accumulation were increased by eCO₂ in the present, in agreement with this study (Fernando et al., 2012). The increase in the grain mineral accumulations of K, Mn, Ca, Mg, Zn, B for three years might be explained by the stronger increase (34.2%) in grain yield in 2013 (Hao et al., 2017), compared with the increase of 19.4% and 29.9% in 2015 and 2016, respectively. Thus, besides possibly decreased nutrient concentrations, increased grain nutrient accumulations should be considered in assessing the impacts of future e CO₂ on human nutrition. As said in Jobe et al. (2020), broomcorn millet, as an orphan crop, has great potential to deliver sufficient nutrients for human food and health. More effort is needed to understand the control of nutrient fluxes and homeostasis in broomcorn millet to ensure that this will also be true in the coming decades.

Effect of eCO₂ on flavonoids

Millet grain was abundant in health-beneficial phenolic compounds compromise phenolic acids, flavonoids, and tannins, which were beneficial to human health (Hassan, Sebola & Mabelebele, 2021). Millets flavonoids had shown a remarkable spectrum of therapeutic properties for medical and clinical applications, such as anti-inflammatory, anti-cancer, antihy- pertensive, diuretic, analgesic, and hypolipidemic effects (Banerjee et al., 2012; Chethan, 2008; Edge, Jones & Marquart, 2005; Ekta & Sarita, 2016; Li et al., 2022). A recent study revealed that the contents and types of flavonoids compounds in minor grains (sorghum, foxtail millet, and broomcorn millet) were more than those in staple crops (wheat, maize, and rice) (Tang et al., 2022). Kaempferol and apigenin were major flavonoids in raw millets (Pradeep & Sreerama, 2015). In this study, they were substantially accumulated under eCO₂. In addition, eriodictyol, luteolin, and chrysoeriol were also accumulated under eCO₂. Weisshaar & Jenkins (1998) stated that approximately 20% of the carbon from photosynthesis was used to synthesize the phenolic compounds found in nature, including flavonoids. Many studies have shown that eCO₂ significantly stimulates plant growth and improves photosynthetic products of plants (Li et al., 2017; Gong et al., 2021), thus increasing precursors of flavonoids, and consequently, increasing the flavonoid content in plants (Karimi, Jaafar & Ghasemzadeh, 2016; Sgherri et al., 2017; Hozzein et al., 2020; Zhang et al., 2021). The previous study revealed that CO₂ enrichment lead to increased photosynthesis rates in broomcorn millet (Hao et al., 2017). Therefore, larger amounts of carbon could be obtained to generate flavonoids from broomcorn millet under eCO₂. In the present study, the increase in flavonoids of broomcorn millet in response to eCO₂ indicated that the nutritional value of broomcorn millet was improved under the scenario of eCO₂. Therefore, broomcorn millet could be used as nutritional supplements in the area of therapy. Similarly, eCO₂ enhanced the production of flavonoids in soybean (Li & Siddique, 2018; O’Neill et al., 2010; Kretzschmar et al., 2009), while it decreased the total flavonoid content in rice grain (Goufo et al., 2014). These studies suggested that eCO₂-induced flavonoid changes exhibited some degree of variability, depending on the plant species.

Cultivating broomcorn millet promotes access to better nutrition for communities, especially in the developing regions of the world. Food security can be further improved by diversifying our staple foods to provide more options for combating climate change. The United Nations Food and Agriculture Organization has announced the year 2023 as ‘International Year of Millets’ (https://news.un.org/en/story/2021/05/1092492), recognizing the potential of this crop. By that time, it may be expected to incentivize increased millet production and achieve success in combating hunger and malnutrition among the vulnerable population in any future aberrant conditions.