The current study uses multiple time point sampling of an expanded profile of mineral concentrations and contents of all shoot organs in NAM knockdown and control lines. This sampling allows the quantification of N and mineral remobilization as contributors to final grain protein and mineral content, and provides a better understanding of the physiological effects of the NAM genes. In addition, experimental treatments have been included to test the remobilization capacity of Fe and Zn in these lines, and radiolabelled Zn was used to quantify the short-term translocation to grain. The information presented here will inform future genomic and systems level studies designed to understand genes and processes that can be targeted to increase grain mineral concentrations for the bio-fortification of foods.The lines used in this study were the NAM RNAi lines designed to reduce expression of all NAM family members and its non-transgenic control . The transformed line is Bobwhite, a semi-dwarf, hard, white, spring, common wheat variety. Wheat seeds were imbibed at 4 C for 3 d and allowed to germinate in darkness at room temperature for 4 d. Seedlings were planted in commercial potting mix and vermiculite at a 2:1 ratio in 17 cm diameter, 17 cm tall pots, three plants per pot, and placed in a growth chamber . Pots were placed in trays with two pots of each line per tray. Plants were watered as needed by sub-irrigation with a nutrient solution of the following composition: 1.2 mM KNO3, 0.8 mM Ca2, 0.8 mM NH4NO3, 0.3 mM KH2PO4, and 0.2 mM MgSO4. Plants were sampled at anthesis of the first emerged head ,25 liter round pot and at 14, 28, 35, 42, and 56 d after anthesis , with an additional harvest at 70 DAA for the RNAi line.
At each sampling, the number of tillers was noted, and plants were cut with a scalpel into the following parts: heads, peduncles, stems, lower leaves, and flag leaves. For each plant, organs from all tillers were pooled and a total of 4–6 plants from two separate pots were analysed per time point. Tissues were dried for 48 h in a drying oven at 60 C, and dry weights were obtained. After drying, heads were separated into grain, rachis, and florets, grains were counted, and these parts were weighed.Wheat seeds were imbibed as described above. Seedlings were planted in plastic cups with plastic beads for support, then placed in lids over 4.5 l containers, 10 plants per pot, in a growth chamber with the settings as described above. Plants were sampled at anthesis of the first emerged head , and at 42 DAA for the Fe deficiency and control treatments . Since 0 Zn plants matured more rapidly than control or 0 Fe plants, plants were sampled at 35 DAA for Zn deficiency and control treatments. At each sampling, the number of tillers was noted, and plants were cut with a scalpel into the following parts: heads, peduncles, stems, lower leaves, and flag leaves. Usually, all tillers from a plant were collected, although the occasional late-emerging tiller was discarded. Thus, 2–5 tillers from a minimum of two plants per time point were collected. Organs from each tiller were analysed separately, then average values were calculated. Tissues were dried for 48 h in a drying oven at 60 C, and dry weights were obtained. After drying, grain was removed from heads, and weighed separately.For mineral analysis of potting mix-grown plants, above ground organs were dissected and organs from all tillers of each plant were pooled as described above. All tissues except florets were ground in a stainless-steel coffee mill.
Duplicate sub-samples of approximately 250 mg were weighed into glass tubes, and digested in nitric:perchloric acid for 1 h at 100 C, then gradually to 200 C until the sample was taken to dryness. Samples were then resuspended in 15 ml 2% nitric acid. All acids were trace metal grade and water was filtered through a MilliQ system to 18 MX resistivity. Mineral concentrations were determined by ICP-OES . The mineral content of the tissues was calculated by multiplying tissue DW by each mineral concentration. For mineral analysis of hydroponically grown plants, a different digestion procedure was used . Whole organs were weighed into glass tubes, and digested in 2.0 ml nitric acid overnight, then at 125 C for 1.5 h. 1.5 ml 30% H2O2 was added and samples were digested for 1 h. A second 1.5 ml volume of H2O2 was added and samples were digested for 1 h. The temperature was then increased to 200 C and samples were evaporated to dryness. Residues were dissolved in 15 ml 2% nitric acid. Mineral concentrations were determined by ICP-OES. The mineral content of the tissues was calculated by multiplying tissue DW by each mineral concentration. For N analysis, tissue samples were dried to constant weight and ground to a fine powder using a ball mill. The samples were analysed for N concentration by a continuous-flow mass spectrometer at the University of California, Davis Stable Isotope Facility.Wheat plants were grown in hydroponics as described above. The dates of anthesis were noted, and at mid-grain fill , plants were moved from a complete nutrient solution to a complete solution spiked with 65Zn at 1 lCi l 1 for continuous labelling experiments, and at 4 lCi l 1 for pulse labelling experiments. All labellings were initiated between 3–4 h into the photoperiod. Plants were not removed from the growth chamber during the labelling period. For continuous labelling, the plants remained in the labelling solution for up to 24 h. For pulse-labelling, the plants were removed from the labelling solution after 3 h and rinsed for 10 min in complete, unlabelled nutrient solution, then placed in fresh complete, unlabelled nutrient solution. At 12 h or 24 h after the commencement of labelling, shoots were excised and cut into lower leaves, stems, flag leaf, peduncle, and heads.
Heads were ovendried for 4–12 h, then the grains were removed. All tissues were quantified for 65Zn by gamma counting.Control and RNAi plants grew similarly in terms of appearance and total plant size . This similarity was also true for individual plant organs, although some tissues differed at some time points. Total grain weight, on a per head basis, was similar between the two lines. Weight of individual kernels was nearly identical and reached maximum values by 35–42 DAA, although total grain dry weight continued to increase as a result of increased seed numbers at later time points. Across sampling points, RNAi lines had a higher number of grains per head, although these differences were significant only at 14 DAA and 35 DAA . It was observed that, as described previously by Uauy et al. ,greenhouse ABS snap clamp the most notable difference between the two lines was delayed leaf yellowing of the RNAi line. The complete data set of mineral contents for the potting mix-grown plants is presented in Supplementary Table S1 at JXB online. At anthesis, Fe and the contents of most other minerals were similar in the vegetative organs of both lines , suggesting that NAM genes had no effect on the content of these minerals to this point in development. Total Zn content was slightly lower in the RNAi line at anthesis . At grain maturity , the total shoot contents of Fe, Zn, and N were similar in both lines , indicating that total uptake and accumulation of each mineral was not significantly affected by the NAM genes. Total vegetative Fe content decreased between anthesis and maturity in the control line . Although the decrease in content between anthesis and maturity was not significant, the RNAi line total vegetative Fe content did not decrease, but rather increased significantly . Total vegetative Zn decreased only in the control line . Comparing the quantity of mineral remobilized from all vegetative tissues to the quantity of mineral in the grain pool at maturity , the net remobilized Fe and Zn could account for between 13.0% of grain Fe and 42.6% of total grain Zn content in the control line, assuming that all of each mineral demonstrating net remobilization was translocated to the grain . In the control line, Fe content decreased over time in lower and flag leaves, stems, peduncle, and rachis, indicating net remobilization. Contrary to this, Fe remained constant or accumulated over time in all tissues of the RNAi line . This was especially marked in the peduncle of RNAi plants, which accumulated 286% of the initial Fe content. Zinc content decreased significantly in all vegetative tissues of the control line In the RNAi line, Zn was remobilized from both the flag leaf and lower leaves, but the percentage change was lower than the control . Zinc decreased in the RNAi line until 35 DAA in stem, peduncle, and head tissues, after which the content increased .
At anthesis and grain maturity, total N contents were similar between both lines , suggesting that reduced NAM expression did not alter total N uptake and accumulation. Throughout grain development, total and individual grain N content were significantly lower in the RNAi line than in the control line . As with Zn, RNAi plants remobilized N from flag leaves and lower leaves over the time-course, but to a lesser extent than in the control plants . The greatest differences in flag leaf N remobilization occurred between 35 DAA and 56 DAA . Comparing N content of all shoot vegetative organs between these time points , total shoot N increased by similar quantities between the RNAi and control lines . However, grain of the control line gained 14 mg of N, while grain of the RNAi line increased only by 2.7 mg. Vegetative tissues of control plants showed a net remobilization of 6.6 mg of N, while those of the RNAi line did not show a net remobilization of N, but rather N increased by 5.3 mg. From anthesis to maturity, N was remobilized from the control stem and peduncle , while N content increased in these tissues of the RNAi line . This resulted in significant differences between control and RNAi lines for both grain N and vegetative N at maturity .To characterize further the mineral remobilization in the RNAi lines under different levels of Fe and Zn, plants were grown using hydroponic conditions, either in a complete nutrient solution for the duration of the experiment, or in an Fe- or Zn-deficient solution from anthesis onwards. Withholding Fe and Zn forced plants to rely solely on stored Fe and Zn to supply the grain. This treatment was used to assess potential net remobilization from the vegetative tissue to the grain while preventing uptake and xylem translocation of these minerals during grain fill, although some root-associated Fe and Zn may have supplied a finite quantity of residual mineral content. If the RNAi line was capable of remobilizing minerals, a net loss of Fe or Zn content would be detected in vegetative tissues between anthesis and maturity. The full data set is presented as Supplementary Table S2 available at JXB online. Leaf yellowing was delayed in the RNAi line in hydroponics, similar to potting mix-grown plants. The control and RNAi lines had similar Fe and Zn vegetative contents at anthesis . For total shoots , neither line exhibited significant net remobilization of Fe or Zn when grown on complete nutrient solution, but both remobilized significant quantities of these minerals when grown on deficient nutrient solutions . Iron-deprived RNAi plants had significantly less net remobilization, with a decrease of 40.2% of vegetative Fe content as compared to 65.6% for the control line . Zinc-deprived RNAi plants showed a net remobilization of 69.6% of Zn content compared to 74.8% for the control line, but the difference between lines was not significant . The quantities of Fe and Zn remobilized were more than enough to account for grain mineral content , although total Fe or Zn content in the grain for each line was significantly lower than when these minerals were supplied continuously . Some shoot Fe and Zn may have been translocated to roots to maintain root growth. Roots of both lines grown in complete nutrient solution did not decrease in Fe or Zn content during grain fill .