The germination paper was placed in a 400 mL beaker with approximately 75 mL of 10 mM CaSO4 solution, covered with a plastic bag and placed in an incubator for four days. Seedlings were transplanted into 20 L tubs filled with an aerated nutrient solution that contained 1 mM CaSO4, 1 mM K2HPO4, 1 mM KH2PO4, 2 mM MgSO4, and 0.2 g L−1 Fe-NaEDTA and micro-nutrients 2HPO4 as the N source, Epstein and Bloom, 2005. The nutrient solution was replaced weekly and an additional 0.2 mM of NO− 3 – or NH4 + − N was added midweek until harvest. The solution volume was maintained by daily addition of deionized water. Solution pH varied between 6.8 and 7.0 for both of the N forms, and the NH4 + and the NO− 3 solutions did not differ by more than 0.1 pH units. The plants were grown in controlled environment chambers set at 23/20˚C day/night at 60–70% relative humidity with a photo period of 15 h. The photosynthetic flux density was 375µmol m−2 s −1 at plant height. Plants were subjected to one of three CO2 concentrations: “sub-ambient” , “ambient” , and “elevated” . sub-ambient CO2 concentrations were maintained by passing air that entered the growth chamber through wet soda lime, a mixture of KOH, NaOH, and Ca2 that was replaced as needed. The elevated CO2 conditions were maintained in an environmental chamber equipped with non-dispersive infrared analyzers for CO2 and valves that added pure CO2 to the incoming air stream to hold the chamber concentration at 720 ppm. The wheat was grown until all above ground parts turned completely yellow. Plant matter was sorted into grain, chaff, shoots,growing strawberries vertically and roots and dried for 48 h at 55˚C. Data on kernel number , kernel mass, number of heads, kernels head−1 , and HI were collected prior to sample preparation for nutrient analysis.

A portion of the grain was analyzed for phytate using a modification of the method as described by Haug and Lantzsch . The remainder of the grain as well as the shoots and chaff was bulked into five repetitions per treatment and sent to the UC Davis Analytical Laboratory for nutrient analysis. The roots of plants for each CO2 × N treatment became entangled within the same tub; therefore, we were unable to separate the roots of the individual plants for analysis. Root data are thus presented as means for each treatment with no standard errors or confidence intervals. Data were analyzed using PROC MIXED . Nitrogen form and CO2 factors were treated as fixed independent variables. We used the Tukey–Kramer Honestly Significant Difference test for mean separation. Probabilities less than 0.05 were considered significant. Because some of the transformed variables did not meet the assumption of homogeneityof variances, but one-way ANOVAs met the ANOVA assumptions, we analyzed the results via one-way ANOVAs to gain some information on the interactions between CO2 and N form.We used a database derived from the United Nation’s Food and Agriculture Organization ’s national food balance sheets to estimate the average daily per capita dietary intake of zinc and phytate from 95 different food commodities in each of 176 countries. This database combines FAO data on per capita intake of food commodities with USDA data on the nutrient or phytate content of each of these commodities. More detailed discussion of the creation of this database for the International Zinc Collaborative Group may be found in Wuehler et al. . Using this database, we produced two datasheets: one containing per capita daily dietary intake of zinc from each food commodity for each country and another containing per capita phytate intake from each food commodity for each country. To calculate total dietary zinc and total dietary phytate per country, we summed across the rows of all food commodities for each respective country. To determine the proportion of a population at risk for zinc deficiency from a hypothetical least developed country , we first calculated TDP and TDZ values for a set of 44 countries defined by the United Nations as being least developed. We took the mean TDP and TDZ values for these countries to represent a hypothetical “less developed country.” To calculate the bio-available zinc portion we used the Miller equation .

Mean TDZ and TDP values were converted to mg mmol−1 and put into the Miller equation to compute the average per capita TAZ in our hypothetical LDC. The variables TDZ, TDP, and TAZ are described above, and Amax, KP, and KR are constants as described in Miller et al. . We made an assumption that our hypothetical LDC receives half of its phytate and half of its zinc from wheat, which is roughly consistent with many of the LDCs in the FAO database. We analyzed the effect of elevated carbon dioxide levels on TDP, TDZ, and TAZ concentrations in a hypothetical LDC population for both NH4 + and NO− 3 -supplied wheat. To calculate a new TAZ for wheat grown under elevated CO2 conditions, we first calculated the percent change in TAZ from ambient to elevated levels for wheat receiving NH4 + or NO− 3 . This computed percent change was then applied to half of the hypothetical TDZ and TDP; meanwhile, the other half of the hypothetical TDZ and TDP remained unmodified. Thus, the total new TDP and TDZ is the sum of the unmodified and modified portions. These new TDP and TDZ values for both NH4 + and NO− 3 -supplied wheat were then put into the Miller equation to compute new hypothetical TAZ values for an LDC. Differences and corresponding percent changes between the new TAZ values and the original TAZ value for a LDC were computed to determine the overall affect of elevated CO2 on TAZ in NH4 + and NO− 3 -supplied wheat for an average developing world population. TAZ, TDP, and TDZ concentrations can only be compared within a single N form across the CO2 concentrations due to methodological constraints of the model. Plants supplied NH4 + vs. NO− 3 nutrition reacted differently to CO2 enrichment . Plants supplied NH4 + differed across CO2 treatments for most of the yield and biomass measurements. The greatest values typically were found at ambient CO2 concentrations. Shoot, chaff, grain yield, number of heads, and KN were greatest at ambient CO2 levels. HI and kernels head−1 showed no change across CO2 treatments. In contrast, biomass and yield measures of NO− 3 -supplied plants did not differ among the three CO2 concentrations.

At sub-ambient CO2, differences between the NH4 + and NO− 3 treatments occurred in shoot biomass and three of the yield components: kernel mass, head number, and kernels head−1 . Ammonium-supplied plants had a larger number of heads while NO− 3 -supplied plants had greater shoot biomass, kernel mass, and kernels head−1 . At ambient CO2, NH4 + -supplied plants had a greater number of heads and greater chaff biomass. Plants supplied NO− 3 had a larger number of kernels head−1 . At elevated CO2, biomass and yield measures did not differ with N treatment. Phytate was relatively insensitive to CO2 concentration. Phytate concentrations were highest at sub-ambient CO2 for NH4 + -supplied plants . Sub-ambient CO2 also produced the lowest phytate concentrations in NO− 3 -supplied plants. NH4 + -supplied plants had greater phytate concentrations than NO− 3 -supplied plants at sub-ambient CO2,best vertical garden system but not at the other CO2 concentrations. Grain from plants grown under NH4 + nutrition had roughly 7, 18, and 8% higher bio-available Zn than NO− 3 -supplied plants at sub-ambient, ambient, and elevated CO2, respectively . Based on this phytate and bio-available Zn data, we modeled how a human population from a LDC would be affected by changes in atmospheric CO2 concentrations . The calculations were based on differences among CO2 concentrations; therefore, modeled TDZ, TDP, and TAZ values cannot be compared between NH4 + and NO− 3 -supplied grain. Grain from plants supplied the different N forms behaved differently as CO2 concentration increased. We found that under NH4 + supply, TAZ would increase 3.6% with the rise in CO2 from sub-ambient to ambient, and decrease 1.6% with the rise from ambient to elevated CO2 . Humans provided NO− 3 -supplied wheat would experience a decrease in TAZ of 3.5% going from sub-ambient to ambient, and an increase 5.6% from ambient to elevated CO2 . Ammonium-supplied plants generally showed a trend toward decreasing nutrient concentrations with increasing CO2 concentration while NO− 3 -supplied plants varied widely across CO2 treatments . The decrease in nutrient concentrations under NH4 + supply corresponded to an increase in root mass. Nitrate supplied plants tended to have their highest nutrient concentrations in the ambient and elevated CO2 treatments. Ammonium supplied plants had higher concentrations of Zn and Mn across all of the CO2 treatments, as well as higher total N and Fe at sub-ambient CO2. Nitrate-supplied plants typically had higher concentrations of the other nutrients at all CO2 concentrations.

The distribution of nutrients and micro-nutrients among plant parts followed similar patterns in both the NH4 + and NO− 3 – supplied plants, although the NH4 + -supplied plant distributions were slightly more variable . Allocations to root and grain usually were greatest at ambient CO2, and those to chaff and shoots at either sub-ambient or elevated CO2. Grain typically contained the largest proportion of total N, P, Zn, and Cu, although the organ with the largest percentage of Cu varied with CO2 treatment among NO− 3 -supplied plants. Plants at sub-ambient and elevated CO2 allocated more Cu to the grain, while those at ambient CO2 allocated more to the roots. In general shoots received the majority of K, S, B,Ca, and Mg for all N and CO2 treatments. Ammonium-supplied plants allocated slightly more Mn to the roots at sub-ambient CO2, but allocated increasing amounts to the shoots at the expense of the roots as CO2 concentration increased. In contrast, NO− 3 -supplied plants allocated most of the Mn to the shoots. Ammonium-supplied plants typically allocated more resources to the chaff while NO− 3 -supplied plants allocated a greater percentage of elements to the roots.No other study to our knowledge has examined the influence of N form on plant nutrient relations at three different atmospheric CO2 concentrations. Overall, N form affected growth, total plant nutrient contents, and nutrient distribution in senescing wheat shoots, grain, and roots. The influence of NH + 4 and NO− 3 on growth and nutrient status were so distinct that they should be treated as separate nutrients and not bundled into a general category of N nutrition. Wheat size and nutrition at senescence responded to CO2 concentration in a non-linear manner. As was previously shown , we found that plants supplied with NH4 + were more responsive to CO2 concentration than those supplied with NO− 3 . Although not explicitly addressed here because of the heterogeneity of variances, interactions between CO2 and N treatments likely existed for a number of the biomass and nutrient measures. Most nutrient concentrations were generally higher in NH4 + – supplied plants, with the exceptions of NO− 3 − N, Mg, B, and Mn, which were generally higher in NO− 3 -supplied plants. Phytate, which hinders human absorption of Zn and Fe , showed little variation at ambient and elevated CO2 between NH4 + and NO− 3 -supplied plants, which, in conjunction with the observed greater bio-available of Zn in NH + 4 -supplied plants, may have consequences for human nutrition. Distribution of nutrients to the shoots, roots, chaff, and grain in response to CO2 concentration and N form was also non-linear and varied by nutrient. The data support our hypothesis that NO− 3 -supplied plants would show a more limited biomass and yield enhancement with CO2 enrichment than NH4 + -supplied plants. Nevertheless, mean biomass and yield decreased from ambient to elevated CO2 in both NO− 3 – and NH4 + -supplied plants in contrast to biomass increases in prior work on wheat seedlings . NO− 3 – supplied plants allocated more biomass to roots and had larger root:shoot ratios than NH4 + -supplied plants regardless of CO2 concentrations as has been reported previously , but increased root mass at elevated CO2 concentration for NO− 3 -supplied plants reported previously were not observed here.