Seed specific expression of PvFERRITIN and constitutive expression of OsNAS2 in wheat
Single insertion Fer-BW, Nas-BW, and FerNas-BW transgenic lines (Figs. 1, S1) were analyzed for transgene expression in grains and leaves. PvFERRITIN was specifically expressed in the grains of all Fer-BW and FerNas-BW lines, except for two lines, Fer-BW 51 and FerNas-BW 30, which also showed expression in leaves (Figs. 2a, c, S3). These results confirm the seed-specific expression of PvFERRITIN under the control of the rice OsGLOBULIN promoter in most wheat lines. OsNAS2-specific primers (Table S1) were used to analyze the expression of the transgene in Nas-BW leaves and both leaves and grains of FerNas-BW lines (Fig. 2b, d). All Nas-BW lines showed OsNAS2 expression in the leaves. In FerNas-BW lines OsNAS2 expression was also detected in grains, except for FerNas-BW 17. However, OsNAS2 expression was many folds higher in leaves than grains, indicating that the ZmUBIQUITIN promoter is not very active in wheat endosperm.
Expression of PvFERRITIN increases iron and zinc content in whole grains and flour
The single insertion Fer-BW lines 18, 47, 51, 62, and 69 were analyzed for their metal content. Iron content was significantly increased in all lines except Fer-BW 69. Iron content in the T4 grains of these lines ranged between 42.0 and 61.9 µg/g DW, as compared to 38.5 µg/g DW in Bobwhite (Fig. 3). Fer-BW 51 had the highest iron content (61.9 µg/g) with an increase of 1.6-fold as compared to the control grains. The iron content in the flour (the endosperm of the grains) of Fer-BW transgenic lines ranged from 20.2 to 33.9 µg/g DW compared to 20.4 µg/g DW in Bobwhite. Flour from grains of line 47 had the highest iron content, with a 1.7-fold increase (33.6 µg/g) as compared to the control (Fig. 3). In addition, whole grains of lines 18, 47, 51 and 62 had significantly increased zinc content ranging from 59.1 to 72.6 µg/g DW as compared to 43.1 µg/g DW in Bobwhite (Figs. 3, S4). Zinc content in the flour ranged from 30.7 to 35.3 µg/g DW in the transformed lines as compared to 23.0 µg/g DW in the control. Line Fer-BW 47, which contained the highest iron content in the flour, also contained the highest zinc content in whole grains (72.6 µg/g) as well as in flour (35.3 µg/g), representing 1.7-fold and 1.5-fold increases, respectively, as compared to the control. In addition, copper, manganese, and magnesium levels were also measured in whole grains and flour. Copper was increased in grains of four of the Fer-BW lines, except for line 69 (Fig. S5), and ranged from 6.9 to 8.3 µg/g DW compared to 6.5 µg/g DW in the control line. A similar trend was observed in the copper content of flour which ranged from 4.8 to 5.7 µg/g DW in the transformed lines, compared to 3.91 µg/g DW in Bobwhite. With few exceptions, manganese and magnesium content was not altered in the transformed lines (Fig. S5). Phenotypic greenhouse performance of the Fer-BW lines were similar to Bobwhite for plant height and 1000 GW, while they showed some variation for days to flowering (DTF) and tiller number (Table. S2).
OsNAS2 overexpression increases iron and zinc content to dietary significant levels
Ten single insertion Nas-BW lines expressing rice OsNAS2 had significant increases in iron and zinc content in comparison to Bobwhite (Fig. 4). The iron content in T4 whole grains ranged from 59.6 to 93.1 µg/g DW, as compared to 42.7 µg/g DW in the control. Moreover, iron content in the flour of these lines ranged from 30.1 to 53.3 µg/g DW, as compared to 21.4 µg/g DW in the control. Line 65 had the greatest increase in iron (2.1-fold) in whole grains and a 2.5-fold increase in the flour as compared to Bobwhite. The Nas-BW lines had relatively higher iron content in comparison to Fer-BW lines and all of these lines also surpass the suggested target levels of 59 µg/g DW iron in whole grains to meet the 30 % estimated average requirement of human diets. The iron increases in the NAS-BW lines correlated well with the transgene expression (Fig. 2) in these plants, with line 65 having the highest OsNAS2 expression in leaves.
The zinc content in whole grains of the transformed lines ranged from 54.2 to 140.6 µg/g DW and in the flour from 31.9 to 94.1 µg/g DW, as compared to 37.9 and 22.4 µg/g DW in Bobwhite, respectively (Fig. 4). Line 40 had the greatest zinc levels with a 3.7-fold increase (140.6 µg/g) in whole grains and a 4.2-fold increase (94.1 µg/g) in flour, as compared to control. Increases in copper, magnesium, and manganese content were also measured in most of the transformed lines (Fig. S6). All lines had significantly higher copper content in whole grains as well as flour. The manganese content also increased significantly in whole grains and flour in most of the lines except in whole grains of lines 38, 53, 66 and 81 and flour of line 66. As an exception, line 91 showed significantly lower manganese content in both whole grains and flour. The magnesium content in whole grains as well as flour was significant increased except in whole grains of lines 42 and 66 and flour of line 91.
Importantly, most of the Nas-BW lines were phenotypically indistinguishable from Bobwhite for DTF, plant height, 1000 GW, and tiller number (Table 1). Lines 40 and 91 flowered earlier, and 1000 GW of lines 40 and 42 was significantly increased but decreased in line 81 as compared with Bobwhite.
Iron and zinc levels in lines expressing both PvFERRITIN and OsNAS2
Wheat lines expressing PvFERRITIN and OsNAS2 from a single construct (FerNas-BW lines) were analyzed to determine if the transgenes act synergistically leading to higher iron and zinc increases in comparison to plants expressing single genes (Fer-BW and Nas-BW lines). The iron content in whole grains of five single insertion FerNas-BW lines ranged from 35.6 to 60.3 µg/g DW, as compared to 42.7 µg/g DW in Bobwhite (Fig. 5). The flour of these transgenic lines had iron content in the range of 24.9–45.3 µg/g DW, compared to 21.4 µg/g DW in Bobwhite. FerNas-BW lines had a maximum 1.7-fold increased of iron (60.3 µg/g in line 15) in whole grains and of 1.8-fold increased iron in the flour (45.3 µg/g in line 3), as compared to Bobwhite. Among the FerNas-BW lines, line 3 had the highest zinc content in whole grains (82.4 µg/g DW) as well as in flour (55.7 µg/g DW) (Fig. 5). However, in contrary to the expectations, these increases for iron and zinc content are smaller than those observed in plants expressing either PvFERRITIN or OsNAS2 alone. These lower increases in iron and zinc content possibly correlate to a relatively lower level of FERRITIN expression in the FerNas-BW lines as compared to that in the Fer-BW lines.
Copper, manganese and magnesium contents were variable among the different FerNas-BW lines (Fig. S7). Lines 3, 15, and 42 showed significantly increased copper and magnesium content in both whole grains as well as the flour. In contrast, lines 17 and 30 had reduced copper content in comparison to the control. Manganese content was also significantly higher in lines 3 and 15 for both the whole grains and the flour, as compared with Bobwhite. FerNas-BW lines were phenotypically similar to Bobwhite for DTF, 1000 GW and tiller number as well as plant height, except for line 42 which was significantly taller (Table S3).