Fig 1.
Changes of grain dry weight (mg DW per spike) against thermal time after anthesis (cumulative average daily air temperature exceeding 0°C, °C d) and days post-anthesis (DPA) (above axis) of durum wheat in 2011 (a) and 2012 (b). Wheat plants were exposed to control conditions (unfertilized CONTROL, black triangle) and to two N fertilization treatments with urea (UREA, white rhombus) and calcium nitrate (NITRATE, white circle) at the rate of 150 kg N ha-1 (mean value ± standard errors, n = 3 independent replicates; split-plot ANOVA over thermal time after anthesis; effects: (a) treatment, P < 0.01; time, P < 0.01; treatment x time, P< 0.01; (b) treatment, P < 0.01; time, P < 0.01; treatment x time, P< 0.01).
Fig 2.
Changes of leaves dry weight (mg DW per plant) and stems dry weight (mg DW per plant) against thermal time after anthesis (cumulative average daily air temperature exceeding 0°C, °C d) and days post-anthesis (DPA) (above axis) of durum wheat in 2011 (a and c, respectively) and 2012 (b and d, respectively). Wheat plants were exposed to control conditions (unfertilized CONTROL, black triangle) and to two N fertilization treatments with urea (UREA, white rhombus) and calcium nitrate (NITRATE, white circle) at the rate of 150 kg N ha-1 (mean value ± standard errors, n = 3 independent replicates; split-plot ANOVA over thermal time after anthesis; effects: (a) treatment, P < 0.01; time, P < 0.01; treatment x time, P = 0.08; (b) treatment, P < 0.01; time, P < 0.01; treatment x time, P = 0.33; (c) treatment, P < 0.05; time, P < 0.01; treatment x time, P < 0.01; (d) treatment, P < 0.05; time, P < 0.01; treatment x time, P < 0.05).
Table 1.
Plant height (cm), ear lenght (cm) and grain nitrogen utilization efficiency (grain-NutE; kg- DM kg-N-1) as recorded at harvest, in 2011 and 2012.
Wheat plants were exposed to control conditions (unfertilized CONTROL) and to two N fertilization treatments with urea (UREA) and calcium nitrate (NITRATE) at the rate of 150 kg N ha-1. Data are averages ± standard errors, for n = 3 independent replicates. Different letters indicate significant differences at p<0.05 (Fisher’s LSD test).
Fig 3.
Increment of gliadins and glutenins in durum wheat mature grains under UREA and NITRATE fertilisation treatments respect to CONTROL.
Percentage increments of gliadins (grey chart) and glutenins (diagonal lines; LMW-GS plus HMW-GS fractions) with respect to CONTROL (white chart) in durum wheat fertilized with urea (UREA) and calcium nitrate (NITRATE) at the rate of 150 kg N ha-1 in 2011 (a) and 2012 (b). Data are averages ± standard errors, for n = 3 independent replicates.
Fig 4.
Gliadin and glutenin contents in in durum wheat mature grains under CONTROL and UREA and NITRATE fertilisation treatments.
Gliadins (mg g-1 flour; grey chart) and glutenin (GS) fractions (mg g-1 flour; horizontallines: LMW-GS fraction; diagonal cross: HMW-GS fraction) in durum wheat grains fertilized with urea (UREA) and calcium nitrate (NITRATE) at the rate of 150 kg N ha-1 plus unfertilized CONTROL in 2011 (a) and 2012 (b). Data are averages ± standard errors, for n = 3 independent replicates. Different letters indicate significant differences at p<0.05 (Fisher’s LSD test).
Fig 5.
Changes of soil-plant analysis development (SPAD) against thermal time after anthesis (cumulative average daily air temperature exceeding 0°C, °C d) and days post-anthesis (DPA) (above axis) of durum wheat in 2011 (a) and 2012 (b). Wheat plants were exposed to control conditions (unfertilized CONTROL, black triangle) and to N fertilization treatments with urea (UREA, white rhombus) and calcium nitrate (NITRATE, white circle) at the rate of 150 kg N ha-1 (mean value ± standard errors, n = 3 independent replicates; split-plot ANOVA over thermal time after anthesis; effects: (a) treatment, P < 0.01; time, P < 0.01; treatment x time, P < 0.05; (b) treatment, P < 0.01; time, P < 0.01; treatment x time, P< 0.01).
Fig 6.
Canopy reflectance of durum wheat plants (averaged over DC65, DC71, DC75 and DC77 phenological stages).
Durum wheat plants were exposed to control conditions (unfertilized CONTROL, black triangle) and to N fertilization treatments with urea (UREA, white rhombus) and calcium nitrate (NITRATE, white circle) at the rate of 150 kg N ha-1, in 2012.
Table 2.
Pearson’s correlation coefficients between grain protein content (GPC, %) or total gluten protein content (GLUTEN, mg g-1 flour) of durum wheat grains at harvest (DC92) and nine VIs calculated from reflectance data recorded at different growth stages, during grain-development (DC65, DC71, DC75 and DC77, corresponding to 0, 6, 13 and 21 days post-anthesis (DPA)) in 2012.
Fig 7.
Two dimensional principal component analysis (PCA) in 2012.
(a) loading plot for the 9 variables (VIs indices: NDVI, GNDVI, WI, OSAVI, SIPI, NRI, SR, MCARI and TVI); (b) score plot for the 12 treatments (CONTROL_0DPA, CONTROL_6DPA, CONTROL_13DPA, CONTROL_21DPA, UREA_0DPA, UREA_6DPA, UREA_13DPA, UREA_21DPA, NITRATE_0DPA, NITRATE_6DPA, NITRATE_13DPA, NITRATE_21DPA) obtained as a combination of the 3 N treatments (CONTROL, UREA and NITRATE) at 4 sampling dates during grain development (0, 6, 13 and 21 days post-anthesis (DPA)). Further explanations on VIs, sampling dates/phenological stages and N fertilization treatments are provided in S2 Table and in the text.
Fig 8.
2D-GE pattern of the LMW-GS from durum wheat (cv Achille) in 2011 and 2012.
Comparison of 2D-GE maps of the LMW-GS fractions extracted from mature grains of durum wheat fertilized with urea (UREA) and calcium nitrate (NITRATE) at the rate of 150 kg N ha-1 plus unfertilized CONTROL in 2011 and 2012. The marked spots were analysed by LC/MS for protein identification.
Table 3.
Quantitative densitometry analysis of LMW-GS from Group 1 and Group 2 extracted from wheat grains deriving from plants exposed to control conditions (unfertilized CONTROL) and to two fertilization treatments with Urea (UREA) and Calcium Nitrate (NITRATE) at the rate of 150 kg N ha-1.
Table 4.
2D-GE LMW-GS protein identification by LC-ESI-MS/MS analysis.
Fig 9.
2D-GE pattern of total protein extracted from durum wheat (cv Achille) immature grains.
Immature grains—derived from plants fertilized with urea (UREA) and calcium nitrate (NITRATE) at the rate of 150 kg N ha-1 plus unfertilized CONTROL—were sampled at DC75 phenological stage corresponding to 15 DPA and 13 DPA in 2011 and 2012 and total protein extracts were analysed by 2D-GE. The marked spots were analysed by ESI-LC-MS/MS for protein identification. The enlargement illustrates some of the spots showing differential abundance between CONTROL and UREA and NITRATE fertilized treatments.
Fig 10.
ESI-LC-MS/MS identification and differential abundance of proteins from durum wheat immature grains.
(a) Venn diagram showing the number of unique proteins in the 15 and 13 DPA (in 2011 and 2012, respectively) phase of grain filling, which were up-regulated in the different treatments, (b) Venn diagram showing the number of unique proteins in the 15 DPA and 13 DPA (in 2011 and 2012, respectively) phase of grain filling, which were down-regulated in the different treatments, (c) Heat map showing protein abundance by UREA (U) and NITRATE (N) fertilization treatments compared with unfertilized CONTROL (C). Data on single protein sequence and function obtained by LC-ESI-MS/MS analyses are listed in S3 Table. Fold variation between data was normalised as follows: up-regulation 2, 3- fold bright red, > 3 fold dull red, down-regulation 2, 3-fold bright green, >3 fold dull green.