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SCIENCE ADVANCES   RESEARCH ARTICLE
ECOLOGY
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Carbon dioxide (CO2) levels this century will alter the protein,
micronutrients, and vitamin content of rice grains with potential
health consequences for the poorest rice-dependent countries
Chunwu Zhu,1 Kazuhiko Kobayashi,2 Irakli Loladze,3 Jianguo Zhu,1 Qian Jiang,1 Xi Xu,1 Gang Liu,1
Saman Seneweera,4 Kristie L. Ebi,5 Adam Drewnowski,6 Naomi Fukagawa,7 Lewis H. Ziska8*

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Declines of protein and minerals essential for humans, including iron and zinc, have been reported for key crop species
in response to rising atmospheric carbon dioxide concentration [CO2]. For the current century, initial estimates of the
potential human health impact of these declines range from 138 million to 1.4 billion, depending on the nutrient in
question. However, potential changes in plant-based vitamin content in response to [CO2] have not been elucidated.
Inclusion of vitamin information would substantially improve estimates of health risks over this century. Among key
crop species, rice is the primary food source for more than 2 billion people. We used multiyear, multilocation in situ
AQ6 FACE (free-air CO2 enrichment) experiments for 18 genetically diverse rice lines, including Japonica, Indica, and hybrids
currently grown throughout Asia. We report for the first time the integrated nutritional impact of those changes (protein, micronutrients, and vitamins) for the 10 countries that consume the most rice as part of their daily caloric supply.
Whereas our results confirm the declines in protein, iron, and zinc, we also find consistent declines in vitamins B1, B2,
B5, and B9 and, conversely, an increase in vitamin E. A strong correlation between the impacts of elevated [CO2] on
vitamin content based on the molecular fraction of nitrogen within the vitamin was observed. Finally, potential health
risks associated with anticipated CO2-induced nutritional deficits of protein, minerals, and vitamins in rice were directly
correlated to the lowest overall gross domestic product per capita for the highest rice-consuming countries, suggesting
potential consequences for a global population of approximately 600 million.
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One of the consequential impacts of rising [CO2] and climate change is
expected to be on food security (1). This is due, in part, to the vulnerability of the global population to food supply (that is, depending on
definition, up to 1 billion people are deemed food insecure) (2). For example, harvests of staple cereal crops, such as rice and maize, could decline by 20 to 40% as a function of increased surface temperatures in
tropical and subtropical regions by 2100 without considering the impacts of extreme weather and climate events (3). Overall, there has been
a directed effort to understand the consequences of [CO2] and climate
on agricultural production (4, 5).
However, the connection between food security and well-being
extends beyond production per se; for example, dietary quality has a substantial influence on human health (6). Globally, insufficient micronutrients, protein, vitamins, etc. can contribute to nutritional deficiencies
among 2 billion people in developing and developed countries (7). These
deficiencies can directly (cognitive development, metabolism, and immune system) and indirectly (obesity, type 2 diabetes, and mellitus) affect
human health on a panoptic scale (8).
The elemental chemical composition of a plant (that is, ionome) reflects a balance between carbon, obtained through atmospheric [CO2],
and the remaining nutrients, obtained through the soil. As evidenced by
over a hundred individual studies and several meta-analyses, projected
increases in atmospheric [CO2] can result in an ionomic imbalance for

1
State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science,
Chinese Academy of Sciences, Nanjing 210008, P. R. China. 2University of Tokyo,
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. 3Bryan College of Health Sciences, Bryan
Medical
Center, Lincoln, NE, USA. 4Centre for Crop Health, University of Southern
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Queensland, Toowoomba, Queensland 4350, Australia. 5Center for Health and the
Global Environment (CHanGE), University of Washington, Seattle, WA, USA. 6Center
for Public Health Nutrition, University of Washington, Seattle, WA 98195, USA. 7U.S. Department of Agriculture–Agricultural Research Service (USDA-ARS), Beltsville Human
Nutrition Center, Beltsville, MD, USA. 8USDA-ARS, Adaptive Cropping Systems Laboratory, Beltsville, MD, USA.
*Corresponding author. Email: l.ziska@ars.usda.gov

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Zhu et al., Sci. Adv. 2018; 4 : eaaq1012

Copyright © 2018
The Authors, some
rights reserved;
exclusive licensee
American Association
for the Advancement
of Science. No claim to
original U.S. Government
Works. Distributed
under a Creative
Commons Attribution
NonCommercial
License 4.0 (CC BY-NC).

most plant species whereby carbon increases disproportionally to soilbased nutrients (9–11). This imbalance, in turn, may have significant
consequences for human nutrition (12, 13) including protein and micronutrients. However, at present, no information is available regarding a key constituent of nutrition and vitamin content; as a result, no
integrated assessment (protein, micronutrients, and vitamins) is available.
The consequences of CO2-induced qualitative changes may be exacerbated where food diversity is limited, that is, where populations rely
heavily on a single plant-based food source. In this regard, rice supplies
approximately 25% of all global calories, with the percentage of rice consumed varying by socioeconomic status, particularly in Asia (14). Rice is
considered among the most important caloric and nutritional sources
particularly for low- and lower-middle–income Asian countries (15).
Therefore, for those populations that are highly rice-dependent, any
CO2-induced change in the integrated nutritional value of rice grains
could disproportionally affect health. We use a multiyear, multilocation,
multivarietal evaluation of widely grown, genetically diverse rice lines at
ambient and anticipated end-of-century CO2 concentrations to (i) quantify varietal response to changes in dietary components, including protein,
iron, calcium, zinc, vitamin E, and the vitamin B complex, and (ii) socioeconomically calculate any CO2-induced deficits in these nutritional
parameters for the 10 most rice-centric countries globally, as a function
of gross domestic product (GDP) per capita.
Although end-of-century [CO2] projections vary, it is very likely that
actual atmospheric CO2 concentrations will reach 570 mmol mol−1
before the end of this century (16). Global [CO2] is expected to reach
these levels even as additional steps are taken to decrease emissions, due,
in part, to the projected energy usage, the longevity of the CO2 molecule
in the atmosphere, and the temporal delay in reducing [CO2] emissions
before mid-century (17). Overall, the experimental concentrations used
here for the elevated [CO2] treatment (568 to 590 mmol mol−1) reflect
the reality that those born today will be eating rice grown at CO2 concentrations of 550 mmol mol−1 (or higher) within their lifetimes.
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Teaser: Rising CO2 levels may induce nutritional deficits (protein, minerals, and vitamins) in the highest rice-consuming countries.

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SCIENCE ADVANCES   RESEARCH ARTICLE
When grown under field conditions at these anticipated CO2 concentrations, a significant reduction (an average of −10·3%) in protein
relative to current CO2 concentrations was observed for all rice cultivars
F1 (Fig. 1). Similarly, significant reductions in iron (Fe) and zinc (Zn) were
also observed (−8·0 and −5·1%, respectively) among all rice cultivars
F2 tested (Fig. 2). On the basis of [CO2] assessment per se, there were no
significant site difference effects on rice grain quality between Japan and
China (P = 0.26, 0.17, and 0.10 for protein, iron, and zinc, respectively).
The rice lines chosen reflect a wide genotypic and phenological
range, suggesting that the declines in nutrient parameters observed here
are representative of rice in toto. However, a larger sample size would be
of benefit both to confirm these findings and, if possible, to determine
whether any lines may be preferred for improving protein or microAQ16 nutrient availability as [CO2] increases.
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Regarding the B vitamin complex, significant reductions in vitamins
B1 (thiamine), B2 (riboflavin), B5 (pantothenic acid), and B9 (folate)
were observed in response to projected CO2 levels with average declines
AQ18 F3 among cultivars of −17·1, −16·6, −12·7, and −30·3%, respectively (Fig. 3).
As observed for protein and minerals, no increase in these parameters
was detected for any of the 18 rice lines evaluated; in addition, no significant [CO2] by cultivar interactions were noted (Fig. 3). In contrast, increases were observed on average for vitamin E (a-tocopherol) (fig. S1).
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Although these data indicate that [CO2] affects nutrient composition, the impact of these qualitative changes on health will vary as a
function of rice consumed relative to the total caloric intake. Previous
calculations of the impact of rising CO2 on human nutrition relied on

Food and Agriculture Organization (FAO) food balance sheets com- AQ20
bined with Monte Carlo simulations run on the range of projected declines of zinc, protein, and iron (12, 13). Here, we also rely on FAO food
balance sheets but use an economic approach whereby average qualitative
changes observed with [CO2] as a function of rice consumption for the
top 10 rice-consuming countries as of 2013 are compared with per capita
GDP of that country. In this context, any protein and mineral deficits AQ21
(Fe + Zn), associated with higher CO2 values, are observed to be greater
for those countries with the lowest overall GDP per capita (for example,
Bangladesh and Cambodia) (Fig. 4). The reductions in vitamin B (B1, B2, F4
B5, and B9) availability were greatest for these same countries (Fig. 4).
Similarly, the increase in vitamin E with higher CO2 levels and the subsequent consumption is proportionally greater for those poorer countries
that ingest greater quantities of rice (Fig. 4).
There is growing evidence demonstrating a clear link between crop
growth at projected increases in [CO2] and changes in nutritional quality
including, but not limited to, protein, secondary compounds, and
minerals (for example, Zn) (9–11, 18, 19). The basis for the CO2-induced
changes in crop quality is still being elucidated, in part, because increasing [CO2] influences several biophysical processes (20). However, AQ22
for near-term projections of [CO2], the qualitative decline can be reasonably (given the accuracy of the current data) approximated as linear
(for example, protein) (21).
The nutritional data reported here for elevated [CO2] confirm that
deficits in protein, zinc, and iron may occur even among genetically diverse rice lines grown in different countries (11, 22). In addition, the

Fig. 1. Average reduction in grain protein at elevated relative to ambient
[CO2] for 18 cultivated rice lines of contrasting genetic backgrounds grown
in China and Japan using FACE technology. A country by [CO2] effect on proAQ76AQ77tein reduction was not significant (P = 0.26). Bars are +SE. *P < 0.05 and **P < 0.01
AQ78 (see Methods for additional details).

Fig. 2. Average reduction in grain micronutrients, iron (Fe), and zinc (Zn) concentration at elevated relative to ambient [CO2] for 18 cultivated rice lines of contrasting genetic backgrounds grown in China and Japan using FACE technology.
A country by [CO2] effect was not significant for either micronutrient [P = 0.17 and 0.10
for iron (Fe) and zinc (Zn), respectively] so data from both locations are shown. Bars are
+SE. *P < 0.05 and **P < 0.01 for a given cultivar. CO2; **P < 0.01 is based on all cultivars AQ79
(see Methods for additional details).

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Zhu et al., Sci. Adv. 2018; 4 : eaaq1012

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Fig. 2

Teaser: Rising CO2 levels may induce nutritional deficits (protein, minerals, and vitamins) in the highest rice-consuming countries.

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SCIENCE ADVANCES   RESEARCH ARTICLE

Fig. 3. CO2-induced reductions in vitamins B1 (thiamine), B2 (riboflavin), B5 (pantothenic acid), and B9 (folate) by cultivar. No significant effect was observed for vitamin
AQ80 B6 (pyridoxine), and results are not shown. Analysis was conducted only for the China FACE location. Bars are +SE. *P < 0.05 and **P < 0.01 for a given cultivar. CO2; **P < 0.01 is
based on all cultivars (see Methods for additional details).

current data indicate, for the first time, a pattern in the changes in vitamin content, that is, the extent of observed variation between vitamin
B (B1, B2, B5, B6, and B9) and vitamin E (a-tocopherol).
Variation among [CO2]-induced changes in secondary compounds,
such as vitamins, may relate to the well-established decline of nitrogen
in plants exposed to elevated [CO2] [for example, see the study of
AQ23 Taub et al. (9)]. The effect of increasing levels of [CO2] on vitamin levels
could therefore be inversely correlated with the molecular fraction of
nitrogen within the vitamin. This was observed for rice in the current
F5 study (r2 = 0.82) (Fig. 5), consistent with the carbon-nutrient balance
hypothesis (23); at least in the context of rapid increases in atmospheric
AQ24 [CO2] and carbon availability [but see the study of Hamilton et al. (24)],
the levels of nitrogen containing vitamins decreased (B vitamin group),
whereas the level of carbon-based compounds (vitamin E) increased.
Additional information regarding the effects of [CO2] on nutritional
quality is obviously desired; however, this relationship could provide
initial guidance as to the aspects of rice grain chemistry affected by
increasing atmospheric [CO2].
As of 2013, approximately 600 million individuals, primarily in
Southeast Asia [the countries of Bangladesh, Cambodia, Indonesia,
AQ25 Lao People’s Democratic Republic (PDR), Madagascar, Myanmar, and
Vietnam], consume >50% of their per capita dietary energy and/or protein directly from rice (25, 26). The data shown here provide the first
integrated assessment of [CO2]-induced changes in nutritional quality
(protein, minerals, and vitamins) for many of the most widely grown
Zhu et al., Sci. Adv. 2018; 4 : eaaq1012

rice lines; as such, they indicate that, for key dietary parameters, the CO2
concentration likely to occur this century will add to nutritional deficits
for a large segment of the global population.
In assessing the outcome of the [CO2]-induced dietary changes for
rice in the current study, it is evident (Fig. 4) that the bulk of these
changes, and the greatest degree of risk, will occur among the highest
rice-consuming countries with the lowest GDP. However, as income
increases, consumers prefer more diverse caloric sources, with a greater
emphasis on protein from fish, dairy, and meat as per western foods
(27). Therefore, future economic development could potentially limit
future CO2-induced changes in rice nutrition. For example, in Japan,
rice accounted for 62% of total food energy consumption in 1959,
but that share fell to 40% by 1976 and, in recent years, is <20% (28);
in South Korea, per capita rice consumption almost halved since
1975 (29). However, strong, sustained economic growth cannot be
assumed for all rice-consuming countries. For example, in Bangladesh,
75% of the total caloric supply per capita came from rice in 1990;
23 years later, in 2013, it was 70% (http://faostat.fao.org/beta/en/
#data/FBS); in Madagascar, the percentage of rice consumption has
increased since 1990 (25). In addition, other countries, such as Guinea,
Senegal, and Côte d’Ivoire, become more reliant on rice as a percentage
of their caloric supply (20 to 40% as of 2011) (30). Overall, although
the top rice-consuming countries are likely to change in the coming
decades, the continuing reliance on rice globally as a dietary staple
will continue.
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Fig. 3

Teaser: Rising CO2 levels may induce nutritional deficits (protein, minerals, and vitamins) in the highest rice-consuming countries.

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SCIENCE ADVANCES   RESEARCH ARTICLE

Fig. 4. Projected [CO2]-induced deficits in protein and minerals (Fe and Zn) and cumulative changes in vitamin B and cumulative changes in vitamin E derived from
rice as a function of GDP per capita. Data are based on 2011/2013 FAO food balance sheets for rice consumption and 2011/2013 World Bank estimates of GDP per capita
per country.

Fig. 5. Average change in vitamin concentration (as percentage) in response
AQ81 to anticipated, relative to current, [CO2] + SE as a function of the ratio of the
molecular weight of nitrogen (N) to the molecular weight of the vitamin.
There was a highly significant correlation between the amount of N present in
the vitamin and the overall decrease or increase in response to higher [CO2].
Zhu et al., Sci. Adv. 2018; 4 : eaaq1012

Specific health outcomes of consuming rice with reduced nutritional
quality are also difficult to forecast. Staple foods, such as rice, are widely
available and affordable for most of the world’s population, particularly
the poor. It is understood that undernutrition can put people at risk in
low-income countries for a wide range of other adverse health outcomes, particularly stunting, diarrheal disease, and malaria (31). For example, Kennedy et al. (15) found that the percentages of children under
5 years of age who suffer from stunting, wasting, or are underweight are
generally high in countries with very high per capita rice consumption.
Overall, the current data suggest that, for these countries, any [CO2]induced change in nutritional quality would likely exacerbate the overall
burden of disease and could affect early childhood development.
Overall, it is difficult, without a great deal of additional socioeconomic data at the country level (which is often unavailable), to provide exact estimates of nutritional deficits (protein, minerals, and
vitamins) and associated health consequences likely to incur for ricedependent populations. However, CO2-induced reductions in these
qualities and associated risks of undernutrition or malnutrition are likely
to transcend the entire food chain, from harvest to consumption, especially for the poorest people within a country or region.
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Fig. 5

Teaser: Rising CO2 levels may induce nutritional deficits (protein, minerals, and vitamins) in the highest rice-consuming countries.

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Table 1. Characteristics of rice lines used.

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Cultivar

Origin

Subgroup

Comments

86Y8

China

Hybrid

Bred for disease-resistance; high ripening rate

Bekoaoba

Japan

Japonica

Hokuriku 193

Japan

Indica

Hoshiaoba

Japan

Japonica

Philippines

Indica

Japan

Japonica

Widely grown in Japan

United States

Japonica

Semi-dwarf grown in Mississippi Delta

Milyang 23

Korea

Indica

High-yielding, cadmium accumulator

Momiroman

Japan

Japonica

Medium grain, high-yielding variety

Nipponbare

Japan

Japonica

Genome-sequenced

Liang You 084

China

Hybrid

Grown extensively in southeast China

Takanari

Japan

Indica

Widely grown in Japan

Wuyunjing 21

China

Japonica

Grown extensively in East China

Wuyunjing 23

China

Japonica

Grown extensively in East China

Yangdao 6 hao

China

Indica

Grown extensively in East and Central China

Yliangyou

China

Hybrid

Recently introduced (2008) hybrid line

Yong you 2640

China

Hybrid

Widely planted in lower Yangtze River

Zhonghua 11

China

Japonica

Disease-resistant line used in breeding

IR 72
Koshihikari
Lemont

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Is there a way then to reduce—or negate—this risk? Cultivar selection, either through traditional breeding or genetic modification, to provide nutritionally superior rice with additional CO2 is an obvious strategy.
The current data for a genetically diverse set of rice lines suggest that, at
least for some characteristics (for example, protein and vitamin B2), many
additional lines would need to be screened; furthermore, at present, it can
take many years, even decades, to identify, cultivate, and distribute new
cereal lines that are adapted to a changing climate (32). In addition, other
aspects of climate change, especially temperature, would need to be
considered. For example, previous work indicated that rising temperature
per se can also reduce protein concentration in rice (33). Although the
extent of future surface temperatures would vary depending on location,
temperature and [CO2] should also be evaluated concurrently regarding
rice nutritional impacts in future assessments.
In addition, management could include application of mineral fertilizers or postharvest biofortification. On the consumer side, education
about the role of rising [CO2] on nutrition, including opportunities to
implement favorable nutrition practices and food fortification, may also
provide opportunities to maintain nutritional integrity. Finally, there is
an obvious need for the research community, including agronomists,
physiologists, nutritionists, and health care providers, to accurately
quantify the exact nature of the [CO2]-induced changes in human nutritional status and their associated health outcomes.
Whereas much remains to be done, the current study provides the
first evidence that anticipated CO2 concentrations will result in significant reductions in integrated rice quality, including protein, minerals,
and vitamin B, for a genetically diverse and widely grown set of rice
Zhu et al., Sci. Adv. 2018; 4 : eaaq1012

Bred for lodging resistance, used in silage
High-yielding, blast-resistant
Cultivar used for silage and bioenergy
Semi-dwarf, often used as check cultivar

lines. Occurrence of these nutritional deficits will most likely affect
the poorest countries that are the most rice-dependent. Overall, these
results indicate that the role of rising [CO2] on reducing rice quality
may represent a fundamental, but underappreciated, human health
effect associated with anthropogenic climate change.
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METHODS

Free-air CO2 enrichment sites
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The multiyear study was conducted at free-air CO2 enrichment (FACE)
facilities in two countries: (i) China [at Zhongcun Village (119°42′0″E,
32°35′5″N), Yangzhou City, Jiangsu Province; as part of the Yangtze AQ35
River Delta region, a typical rice growing region (34)] and (ii) Japan
[at Tsukuba (35°58°N, 139°60′E), in Ibaraki Prefecture within farmer’s
fields (35)]. Eighteen rice lines representing varietal groups of cultivated AQ36
rice (Indica and Japonica) and new hybrid lines were chosen. These AQ37
lines were, for the most part, representative and widely grown in the
geographical regions where the FACE facilities were located (Table 1). T1
CO2 and environmental parameters
A complete description of CO2 control for the China and Japan locations can be found in the studies of Zhu et al. (34) and Hasegawa et al.
(35), respectively. The operation and control systems for the China
FACE facilities were the same as those at the Japan FACE site. Briefly,
each site consisted of identical octagonal rings imposed on farmer’s
fields with three rings (China) or four rings (Japan) receiving pure
CO2 supplied from polyethylene tubing installed horizontally on
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Teaser: Rising CO2 levels may induce nutritional deficits (protein, minerals, and vitamins) in the highest rice-consuming countries.

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the periphery of the FACE ring at 30 cm above the rice canopy (elevated
CO2 treatment), with additional rings (three and four, respectively) that
did not receive supplemental CO2 (ambient CO2 treatment). The concentration of CO2 was monitored at the center of each ring, and using
the ambient CO2 concentration as the control, a proportional-integralderivative algorithm was used (relative to the ambient control) to regulate the injection and direction of CO2 in the elevated ring. Rings were
spaced at 90-m intervals to prevent CO2 contamination between plots.
Ring diameters varied between locations (14 and 17 m for the Tsukuba
and Zongcun sites, respectively); [CO2] was controlled to within 80% of
the set point for >90% of the time during the growing season for each
location and year. For the China location, the average daytime [CO2]
levels at canopy height for the elevated treatment were 571, 588, and
590 mmol mol−1 for 2012, 2013, and 2014, respectively; for the Japan
location, the season-long daytime average CO2 was 584 mmol mol−1
(2010, Tsukuba); ambient [CO2] varied from 374 to 386 regardless of
location.
Rice fields in all locations were flood-irrigated and grown as “paddy”
rice, as consistent with local practices. For the China location, the average growing season temperature was 24.4°, 24.8°, and 22.1°C for 2012,
2013, and 2014, respectively; for Japan, the growing season temperature
was 24.6°C for the Tsukuba location in 2010. The soil type in the China
location was classified as Shajiang-Aquic Cambiosol with a sandy loam
texture. The soil type at Tsukuba, Japan is Fluvisols, typical of alluvial
areas. Fertilizer was applied at rates to maximize commercial yield,
consistent with location; any additional pesticides were consistent with
cultural agronomic practices for the given region. Sowing and transplanting methods are described elsewhere (34, 35). At seed maturity,
1 to 2 m2 per CO2 ring, per cultivar, per year, and per location were
harvested for yield assessment.
Nutrient analysis
For the China FACE, a subsample (500 g) of grain was frozen before
analysis. Dehusked (unpolished) brown (raw and uncooked) rice
(100 g) was homogenized to a fine powder using a Mix/Mill Grinder,
sifted through a 100-mesh sieve, and then dried to a constant weight at
70°C. A 0·5-g sample was added to a graphite tube for digestion, 0.2 ml
of pure deionized (DI) water was added, followed by 8 ml of HNO3, and
digested for 24 hours. An additional 2 ml of HClO4 was then added.
Digestion temperature was regulated until clear color was obtained.
Finally, DI water was added to increase any remaining solution to
50 ml. Inductively coupled plasma (ICP) atomic emission spectrometry
(Optima 8000, PerkinElmer) was used to determine Ca content,
whereas ICP–mass spectrometry (MS) (7700, Agilent) was used to
determine Fe and Zn content. Elemental analyses for the samples
from the Tsukuba FACE location are described by Dietterich et al.
(36). Briefly, the air-dried husked (but unpolished) brown rice grains
were air-dried and ground as described previously. Nitrogen was
analyzed with a Leco TruSpec CN analyzer. Fe, Zn, and Ca were
determined with an ICP optical emission spectrophotometer. Note
that brown rice was analyzed because previous publications [for example, the study of Myers et al. (11)] had used brown rice as the standard for CO2 effects on nutrition.
Elemental concentrations of carbon and nitrogen were determined for an additional 30 mg of harvest sample using an elemental
analyzer (Vario, MAX CN, Element). Nitrogen content and carbon
content were determined as a percentage of the dry weight of the sample. A factor of 5·61 was used for converting nitrogen to protein concentration in rice, consistent with previous studies (37).
Zhu et al., Sci. Adv. 2018; 4 : eaaq1012

Vitamin extraction and analysis
Although rice does not supply the complete vitamin B complex, it is
known to provide B1, B2, B5, B6, and B9, as well as vitamin E. These
were extracted from dehusked, unpolished brown rice seed for the nine
rice cultivars at the China FACE location. Brown rice (100 g) was
homogenized to a fine powder using the previously described method;
then, frozen sample was lyophilized using a VFD-1000 freeze dryer
(Bilon). Lyophilization occurred in two cycles; drying at −20°C for
48 hours, followed by secondary drying at 0°C for 3 hours.
For thiamine, riboflavin, pantothenic acid, and pyridoxine determination, 0·05 g of ascorbic acid was added to homogenized samples
(0·5 g) as an antioxidant and then followed by 10 ml of extracting
solution (methanol/water/phosphoric acid = 100:400:0·5, v/v/v). After AQ48
the suspension was vortexed, it was autoclaved at 100°C for 20 min and
then incubated under ultrasonic conditions for 30 min. The solution
was allowed to cool to room temperature and then centrifuged at
11,945g for 15 min. Blank controls were generated following the same
process without rice samples. The final supernatant was filtered through
a 0·22-mm filter before high-performance liquid chromatography
(HPLC)–MS analysis.
Folate determination was per Blancquaert et al. (38): 4 ml of extraction buffer was added to 0.5 g of homogenized samples, and the capped
tube was placed at 100°C for 10 min. A tri-enzyme treatment with 80 ml
of a-amylase (20 min), 350 ml of protease (1 hour at 37°C), and 250 ml of
conjugase (2 hours at 37°C) was used to degrade the starch matrix, to
release protein-bound folates, and to deconjugate polyglutamylated
folates. To stop protease and conjugase activity, additional heat treatments were carried out, followed by cooling on ice. The resulting solution was ultrafiltrated at 11,958g for 15 min. The final solution was AQ49
filtered through a 0·22-mm filter before analysis.
Vitamin E (a-tocopherol) was extracted using an improved method,
as described by Zhang et al. (39). One gram of the homogenized fine
powder was saponified under nitrogen in a screw-capped tube with
1 ml of potassium hydroxide (600 g/liter), 5 ml of ethanol, 1 ml of sodium
chloride (10 g/liter), and 2.5 ml of ethanolic pyrogallol (60 g/liter)
added as antioxidants. Tubes were placed in a 70°C water bath and
mixed at 5-min intervals during saponification. Following alkaline
digestion at 70°C for 30 min, the tubes were cooled in an ice bath, and
5 ml of sodium chloride (10 g/liter) was added. The suspension was
extracted twice with 8 ml of n-hexane/ethyl acetate (4:1, v/v). The organic
layer was collected and was dried using pure nitrogen (EVA 30A,
Polytech Co.) and then dissolved in n-hexane/methanol (20:80, v/v; 1·0 ml).
A similar procedure was used to generate a blank control. The final
solution was filtered through a 0·22-mm filter before analysis.
HPLC–tandem MS (Thermo Finnigan TSQ) was used to quantify AQ50
vitamin content. Column oven temperature was maintained at 25°C,
and the autosampler was maintained at 4°C. Two separate Phenomenex
Kinetex C18 columns (4.6 mm × 100 mm × 2.6 mm and 4.6 mm × AQ51
30 mm × 5 mm) were used for vitamins B and E, respectively. Injection
volume was 20 ml. For gradient elution, the mobile phase consisted of
eluent A (methyl alcohol) and eluent B (0·1% formic acid in water), with
each eluent pumped at a flow rate of 0.6 ml min−1. The mobile phase
was linearly adjusted to separate the different vitamins (table S1).
For the MS setting, source conditions were optimized for vitamin B AQ52
as follows: ion source, electrospray ionization; spay voltage, 3500 V; AQ53
vaporizer temperature, 400°C; capillary temperature, 350°C; sheath gas
pressure, 50; auxillary gas pressure, 10; scan type, selected reaction
monitoring (SRM); collision pressure, 1.0-mtorr Ar. For vitamin E, AQ54AQ55
the source conditions were optimized as follows: ion source, atmospheric
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Teaser: Rising CO2 levels may induce nutritional deficits (protein, minerals, and vitamins) in the highest rice-consuming countries.

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pressure chemical ionization; discharge current, 10 mA; vaporizer temperature, 300°C; capillary temperature, 350°C; sheath and auxiliary gas
pressure, 50 and 10, respectively; scan type, SRM; collision pressure,
1.0-mtorr Ar (table S2). Known standards for vitamin B1 (thiamine HCl),
vitamin B2 (riboflavin), vitamin B5 (calcium-D-pantothenate), vitamin B6
(pyridoxine HCl), vitamin B9 (folic acid), and vitamin E (a-tocopherol)
were purchased from Sigma-Aldrich Co. as standards. All vitamin analyses
were performed in duplicate. Before sample analysis, the instrument
was calibrated using seven standards (six standards and the blank control).
Estimate of nutritional deficits
The 10 most rice-dependent countries were determined on the basis of
the largest consumption of rice as a fraction of total available calories
[Bangladesh, Cambodia, China, Indonesia, Lao PDR, Madagascar,
Myanmar, Philippines, Thailand, and Vietnam (23)]. FAO food balance
sheets (http://faostat.fao.org/beta/en/#data/FBS; food supply quantity,
kilogram per capita per year and food supply, and kilocalorie per capita
per day) from either 2011 (Cambodia and Lao PDR) or 2013 (all other
countries) were used to determine rice consumption along with the
U.S. Department of Agriculture (USDA) Nutrient Database for Standard
Reference data for raw brown long-grain rice (http://ndb.nal.usda.
gov/ndb/foods/show/6505?fgcd=&manu=&lfacet=&format=&
count=&max=35&offset=&sort=&qlookup=rice%2C+brown) to quantify any CO2-induced differences in qualitative nutritional characteristics by individual country.
With respect to nutritional characteristics, we used a holistic approach to assess changes in a number of qualitative parameters including protein, minerals (Fe, Ca, and Zn), and vitamins B1 (thiamine), B2
(riboflavin), B5 (pantothenic acid), B6 (pyridoxine), B9 (folic acid), and
E (a-tocopherol). Inadequate intake of the vitamins and minerals assessed were associated with specific physiological conditions and clinical
manifestations (40). Data for protein and minerals were available for all
three experimental locations; however, vitamin analysis was only conducted for the rice lines from the China location. Because income level is
the most important determinant of per capita rice consumption (25),
and because of the wide range of per capita incomes of the countries
assessed, any significant CO 2-induced change in a nutritional
characteristic was characterized with respect to GDP per capita (from
2013) for the 10 countries examined (https://data.worldbank.org/
indicator/NY.GDP.PCAP.CD)

and 0.1 for protein, iron, and zinc, respectively). Because our purpose
was to elucidate the effect of [CO2] on rice, but not on geographic area,
cultivar effects are inclusive for the figures. Seasonal (yearly) variation
was not significant for a given location and, consequently, was averaged
across years for each FACE site. To aid reproducibility, our original data
AQ62AQ63
and code are available in the Supplementary Materials.

SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/
content/full/4/5/eaaq1012/DC1
table S1.
table S2. Compound parameters for vitamins B1, B2, B5, B6, B9 and E.
fig. S1. As for Fig. 3, but for vitamin E (a-tocopherol) (see Methods for additional details).

Zhu et al., Sci. Adv. 2018; 4 : eaaq1012

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REFERENCES AND NOTES

Statistics
All field experiments at each location represented a completely randomized design with either three (China) or four (Japan) replicates. All
measured and calculated parameters were analyzed using a two-way
analysis of variance (ANOVA) with [CO2] and cultivar as fixed effects
(Statview Software). Coefficient of determination (r2) was calculated for
protein, mineral (Fe and Zn), and vitamin (B1, B2, B5, B6, B9, and E)
deficits as a function of CO2 concentration and GDP per capita. Each
value is the mean ± SE. **P < 0.01; *0.01 ≤ P < 0.05; †*0.05 ≤ P < 0.1; ns,
not significant (P ≤ 0.1). The figures were generated using Systat
Software (SigmaPlot 10.0, Systat Software Inc.). No significant differences for CO2 concentration or [CO2] by cultivar interaction were
found for calcium (Ca) or vitamin B6; consequently, these data are
not shown separately. Every cultivar was grown only at a single site,
which does not allow separation of cultivar effects from site effects.
However, when averaged for all cultivars within a single location (Japan
or China), no significant country interaction was observed for [CO2]
impacts on noted reductions in protein, iron, or zinc (P = 0.26, 0.17,

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Teaser: Rising CO2 levels may induce nutritional deficits (protein, minerals, and vitamins) in the highest rice-consuming countries.

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AQ68
Acknowledgments: We thank G. Kordzakhia of the U.S. Food and Drug Administration for his
contributions. Funding: This work was supported by the National Basic Research Program of China
(973 Program, 2014CB954500), Natural Science Foundation of Jiangsu Province in China
(BK20140063), Youth Innovation Promotion Association of Chinese Academy of Sciences
(CAS; member no. 2015248), and the frontier projects for 13th 5-year plan of CAS (Y613890000 to
C.Z.). Author contributions: C.Z., L.H.Z., and K.K. designed the study. I.L., S.S., and K.L.E. did the
literature review. I.L., C.Z., and L.H.Z. did the analysis with contributions from K.L.E., N.F., and A.D. J.Z.,
Q.J., X.X., and G.L. contributed data. L.H.Z. wrote the report. All authors interpreted the results,
commented on the draft version of the report, and approved the submission draft. Competing
interests: A.D. has received grants, honoraria, and consulting fees from numerous food, beverage,
and ingredient companies and other commercial and nonprofit entities with an interest in diet
quality and nutrient content of foods. The University of Washington receives research funding from
public and private sectors. N.F. is the Editor-in-Chief of Nutrition Reviews, an International Life
Sciences Institute publication, and has received honoraria from Monsanto and the National Dairy
Council before employment by the USDA. The other authors declare that they have competing
interests. Data and materials availability: All data needed to evaluate the conclusions in the
paper are present in the paper and/or the Supplementary Materials. Additional data related to this
paper may be requested from the authors.
Submitted 1 October 2017
Accepted 6 April 2018
Published 25 May 2018
10.1126/sciadv.aaq1012
Citation: C. Zhu, K. Kobayashi, I. Loladze, J. Zhu, Q. Jiang, X. Xu, G. Liu, S. Seneweera,
K. L. Ebi, A. Drewnowski, N. Fukagawa, L. H. Ziska, Carbon dioxide (CO2) levels this century
will alter the protein, micronutrients, and vitamin content of rice grains with potential health
consequences for the poorest ricedependent countries. Sci. Adv. 4, eaaq1012 (2018).

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RAaaq1012/EA/ECOLOGY

minacay

4-23-2018 / 20:42

PE's:

AA's:

Comments:

Art no:
8

Teaser: Rising CO2 levels may induce nutritional deficits (protein, minerals, and vitamins) in the highest rice-consuming countries.

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