Divergent selection for grain protein affects nitrogen use in maize ...
Field Crops Research 100 (2007) 82–90 www.elsevier.com/locate/fcr
Divergent selection for grain protein affects nitrogen use in maize hybrids Martı´n Uribelarrea, Stephen P. Moose, Frederick E. Below * Department of Crop Sciences, 1201 W. Gregory, University of Illinois, Urbana, IL 61801, USA Received 7 July 2005; received in revised form 24 May 2006; accepted 24 May 2006
Abstract The Illinois high (IHP), low (ILP), and corresponding reverse (IRHP, and IRLP) protein–strains of maize represent genetic extremes for differences in grain protein concentration. The objective of this study was to determine how divergent selection for grain protein affects N use in hybrid plants. Inbreds derived from the protein–strains were crossed as males to a common tester and the resultant hybrids evaluated at eight N rates in the field over 3 years. A more than two-fold difference in grain protein concentration was observed among the strain-hybrids with ILP averaging 65 g kg1, IRHP 89 g kg1, IRLP 111 g kg1, and IHP 148 g kg1 of grain protein. Except for IHP at the lowest N levels, the strain-hybrids were similar in their whole shoot biomass production both pre- and post-flowering. Conversely, the strain-hybrids differed markedly in their uptake and accumulation of plant N, and these differences were already evident at flowering before a grain sink was present. Although all hybrids had the same overall N use efficiency at maturity (approximately 24 kg kg1 N), they differed in their N use components with IHP and IRLP exhibiting a higher uptake efficiency, and ILP and IRHP exhibiting high utilization efficiency. The remobilization of leaf N was also more extensive for IHP and IRLP. Changes in grain protein concentration from divergent selection were directly related to changes in uptake and use of N by the plant. # 2006 Published by Elsevier B.V. Keywords: Zea mays; N use efficiency; Biomass accumulation; Illinois protein strains; Maize
1. Introduction Modern agriculture is concerned with yield, the nutritional quality of the crop and the environmental impact of crop production. Efficient use of fertilizer N is therefore critical. Because an adequate N supply is one of the main factors powering yield of cereal crops (Below, 2002), annual applications of fertilizer N are the norm. About half of the 110 kg ha1 annual increase in maize yields over the last half century can be attributed to improved cultural practices, especially N fertilizer use (Duvick, 1992; Sinclair, 1995). Variation in the N supply affects all phases of maize growth, including the development, activity, and senescence of leaves, and the initiation, growth, and composition of ovules (Muchow, 1988; Uhart and Andrade, 1995a, 1995b). Thus, understanding the processes associated with the efficiency of N use (NUE), particularly N uptake and utilization, is of major importance in designing crop management strategies and in developing breeding programs for improved N use.
* Corresponding author. Tel.: +1 217 333 9745; fax: +1 217 333 8377. E-mail address: [email protected] (F.E. Below). 0378-4290/$ – see front matter # 2006 Published by Elsevier B.V. doi:10.1016/j.fcr.2006.05.008
We use NUE here to encompass yield efficiency (the increase in grain yield per unit of applied N fertilizer) and its two components; uptake efficiency (the fraction of fertilizer applied N found in the plant at maturity), and utilization efficiency (the ratio of grain yield to plant N). The N supply can alter the relative importance of these two components, as under high N inputs, NUE is mainly determined by the plant’s ability to acquire N; whereas at low N, the ability to utilize absorbed N is generally more important (Moll et al., 1982; Ma et al., 1998). Many studies show that genotype can also impact NUE (Cerrato and Blackmer, 1990; Smiciklas and Below, 1990; Eghball and Maranville, 1993; Rice et al., 1995; Normand et al., 1997; Muchow, 1998; Ma et al., 1998, 1999; Cassman et al., 2002; Gastal and Lemaire, 2002). Because most modern hybrids are selected according to their yield and N use under high rates of applied N (Castleberry et al., 1984; Bertin and Gallais, 2000), limited genetic variability may exist among commercial hybrids for N utilization, or for the remobilization of N from the stover to the grain (Purcino et al., 1998; Bertin and Gallais, 2000). The Illinois protein strains, which are the result of long term divergent selection for grain protein concentration are unique within the maize germplasm. Illinois high protein (IHP) and
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Illinois low protein (ILP) have been continuously selected for over 100 cycles; whereas the Illinois reverse low protein (IRLP) and Illinois reverse high protein (IRHP) strains are the result of reversing selection in ILP and IHP beginning with cycle 48. Evaluations of the strains during the past 100 cycles have continually demonstrated the effectiveness of this program in altering grain protein level (Woodworth et al., 1974; Dudley et al., 1974; Dudley and Lambert, 1992; Rizzi et al., 1996), as well as a number of other plant traits. The wide variation in protein and dry matter production of these strains must have been accompanied by corresponding changes in N and C metabolism in the plant, and the Illinois protein strains have previously been shown to differ in N metabolism (Wyss et al., 1991; Lohaus et al., 1998; Below et al., 2004). This variation, and the fact that the strains share a common parental background, makes them unique experimental material for studying physiological and biochemical mechanisms associated with differences in maize productivity. Our approach was to make hybrids of each of the strains (including the reverse strains) using inbreds derived from generation 90 crossed to a common tester, then to evaluate these materials in the field for NUE and its main components. Uribelarrea et al. (2004) showed that these hybrids had grain protein concentrations which reflected the strain parents, and that they differed in their use of N (Below et al., 2004). Our objective in this study was to understand how differences in the acquisition, utilization, and remobilization of N are associated with divergent selection for grain protein. 2. Materials and methods 2.1. Field site, cultural practices and treatment arrangements Field experiments were conducted at the Department of Crop Sciences Research and Education Center in Champaign, Illinois during the 2001–2003 growing seasons, on plots that had previously been shown to be responsive to N fertilizer (Gentry et al., 2001). The soil type and cultural practices were as previously reported (Uribelarrea et al., 2004). Briefly, the soil was a Drummer silty clay loam with an average organic matter of 3.7% and a pH of 6.2. The field was under a maize–soybean rotation, with the location of the experimental plots alternated each year. Plots were kept weed-free with chemical control and hand cultivation, and crops were irrigated, when necessary. Hybrids of the Illinois protein strains were produced by crossing inbreds made from generation 90 of IHP and ILP and generation 42 of IRHP and IRLP as males to FR1064 as the tester. Hybrids were over seeded on 26 April 2001, on 25 May 2002, and 22 April 2003 and thinned to a stand density of 65,000 plants ha1. The delay in planting date in 2002 was due to above average precipitation during April and May (270 mm in 2002 compared to a 30-year average of 200 mm). Each of the hybrids was grown under eight rates of fertilizer N (0– 238 kg ha1) in 34 kg increments. The fertilizer was hand applied in a diffuse band down the center of the row as ammonium sulfate and incorporated between the V2 and V3
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growth stages. Treatments consisted of the factorial combination of the four protein-strain hybrids and eight fertilizer rates arranged in a randomized complete block design with four replications. Each experimental unit consisted of four-row plots that were 5.3 m long 3 m wide, with one of the central rows reserved for final yield determination and the other used for destructive plant samplings. 2.2. Crop measurements N acquisition and partitioning were assessed using whole shoots sampled at two growth stages; R1 (i.e. beginning of anthesis and visible silks), and R6 (physiological maturity) when 50% of the plants exhibiting a visible black layer at the base of the kernels. By R6, maize plants are considered to have attained their maximum biomass (Ritchie et al., 1997), and we used shoot dry weight as a relative indicator of net canopy photosynthesis. Because of the large differences in grain composition among these hybrids, we also calculated the energy equivalent (MJ ha1) of the grain biomass using standard caloric values (Hedin et al., 1998) and the respective starch, oil and protein concentrations of the grain (Uribelarrea et al., 2004). At each harvest, four representative plants were separated into leaf, stalk (including leaf sheaths), reproductive support tissues (tassel, husks and cob at R6, or ear-shoot at R1), and grain (only at R6 sampling). Reproductive and grain fractions were placed into a forced-draft oven (75 8C), while the fresh weight of the entire leaf and stalk sample was determined prior to shredding. An aliquot of the shredded material was weighed fresh and then oven-dried (75 8C). The dry weight of each plant fraction was calculated using the fresh weight and the moisture level. Individual plant samples were ground in a Wiley mill to pass a 20 mesh screen, and analyzed for total N concentration (g kg1) using a combustion technique (NA2000 N-Protein, Fisons Instruments). The total N content (g N plant1) was calculated by multiplying the dry weight by the N concentration. Table 1 Significance level of the fixed effects for each of the measured variables, for the protein-strain hybrids grown at Champaign, IL, between 2001 and 2003 Measured variable
Source of variation Hybrid
N rate
Hybrid N rate
R1 biomass R1 N content R1 N uptake
NS 0.0088 0.0001
NS 0.0001 0.0001
0.0001 0.0472 0.0745
R6 biomass R6 N content Post-flowering N uptake
0.0270 0.0725 0.0518
0.0001 0.0001 0.0030
0.0514 0.0001 0.0001
Stover N remobilization Leaf N remobilization Stalk N remobilization
0.0001 0.0002 0.0001
NS 0.0458 NS
NS NS NS
NUE N uptake N utilization
NS 0.0103 0.0783
0.0001 0.0003 0.0198
NS NS NS
A threshold of 0.10 was used to determine a significant effect of the different sources of variation over the measured variables. NS means that term was not significant.
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Table 2 Equation parameters for the effect of N rate on the different measured variables for the Illinois protein-strain hybrids grown at Champaign, IL, between 2001 and 2003 Measured variable
Protein-strain hybrid
Regression equation parameters§ Intercept
Linear term (x)
Quadratic term (x2)
82 93z 96 93
11.6 102 NS NS NS
NS NS NS NS
R1 biomass (g plant1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
R1 N content (g plant1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
0.70 0.90 0.83 0.76
4.1 103 1.6 103 2.2 101 2.4 103
NS NS NS NS
R1 N uptake (kg kg1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
0.65 0.17 0.64 0.45
3.5 103 8.2 104 4.8 103 3.0 103
8.7 106 2.2 106 1.2 106 8.0 106
R6 biomass (g plant1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
6.0 101 5.5 101 3.7 101 6.8 101
1.2 103 1.4 103 6.9 104 2.0 103
R6 N content (g plant1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
1.10 1.20 1.32 1.27
1.2 102 6.8 103 9.5 103 8.5 103
1.0 105 1.0 105 1.0 105 1.0 105
Post-flowering N uptake (g plant1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
0.61 0.39 0.66 0.64
7.2 103 4.1 103 5.4 103 7.2 103
1.0 105 1.0 105 1.0 105 1.0 105
Stover N remobilization (g N plant1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
0.28 0.34 0.21 0.18
3.4 103 NS 2.6 103 NS
8.8 106 NS 1.4 105 NS
Leaf N remobilization (g N plant1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
0.17 0.22 0.15 0.17
2.2 103 NS 1.3 103 NS
6.1 106 NS 4.5 106 NS
Stalk N remobilization (g N plant1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
0.09 0.04 0.02 0.04
NS NS NS NS
NS NS NS NS
NUE (kg kg1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
43 40 44 41
2.0 101 2.0 101 2.0 101 2.0 101
3.3 104 3.3 104 3.3 104 3.3 104
N uptake efficiency (kg kg1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
1.1 103 1.1 103 1.1 103 1.1 103
NS NS NS NS
N utilization efficiency (kg kg1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
9.3 102 9.3 102 9.3 102 9.3 102
NS NS NS NS
§ z
154 184 195 182
0.83 0.55 0.67 0.64 47 65 61 57
x = fertilizer N rate (kg ha1); NS means term was non significant. Value averaged across N rates since parameters from corresponding equation were non significant (P < 0.10).
M. Uribelarrea et al. / Field Crops Research 100 (2007) 82–90 Table 3 Grain protein concentration, grain yield and grain energy equivalent for the protein-strain hybrids grown in Champaign, IL, between 2001 and 2003 Strain-hybrid
Year
Protein concentration (g kg1)
Grain yield (Mg ha1)
Grain energy equivalent (MJ ha1)
FR1064 IHP
2001 2002 2003 Average
170 144 131 148
7.4 7.0 8.2 7.5
223 215 271 236
2001 2002 2003 Average
68 62 64 65
9.0 7.6 9.1 8.6
270 234 302 269
FR1064 IRLP
2001 2002 2003 Average
131 102 100 111
7.8 7.2 9.1 8.0
241 222 312 258
FR1064 IRHP
2001 2002 2003 Average
97 87 84 89
8.8 8.8 9.6 9.1
308 240 279 276
FR1064 ILP
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yield, and plant N content, NUE (kg grain kg1 fertilizer N) and its components, N uptake (kg plant N kg1 fertilizer N) and N utilization (kg grain kg1 plant N), were calculated as shown in Eqs. (1)–(3): NUE ¼
GYX GY0 1000 NRX
(1)
NTX NT0 NRX
(2)
N uptake ¼
N utilization ¼
GYX GY0 1000 NTX NT0
(3)
where GYX and GY0 correspond to the grain yield (Mg ha1) at the X and 0 fertilizer rates (kg ha1); NRX is the fertilizer N rate X (kg ha1), and NTX and NT0 represent the total plant N content at 0 and X N rates (kg ha1). The N remobilization from the leaves, stalk and stover was calculated as the difference in N content in each fraction between R1 and R6. 2.3. Statistical analysis
Values presented are the maximum levels obtained as a function of applied N.
For yield determination, all ears in the unsampled center row of each plot were harvested and mechanically shelled, and weight and moisture level determined. Dry grain yield was expressed as Mg ha1 at 0% moisture. Using the data from grain
Effects of fertilizer N rate for the four hybrids during the 3 years were analyzed with the MIXED procedure in SAS (SAS Institute, 2000), and the parameters of the respective polynomial regressions were also fitted when the effect of N rate was significant. The hybrid and N rate factors were
Fig. 1. Nitrogen rate effect on above-ground biomass (A), N content per plant (B), and N uptake efficiency (C) at flowering (R1) for the Illinois protein-strain hybrids grown at Champaign, IL, between 2001 and 2003. The best polynomial regression model was fitted when the effect of N rate over the corresponding variable was significant. The fitted equation parameters are presented in Table 2.
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Fig. 2. Nitrogen rate effect on above-ground biomass (A), N content per plant (B), and post-flowering N uptake (C) at physiological maturity (R6) for the Illinois protein-strain hybrids grown at Champaign, IL, between 2001 and 2003. The best polynomial regression model was fitted when the effect of N rate over the corresponding variable was significant. The fitted equation parameters are presented in Table 2.
considered fixed, and years (and its interaction with the fixed effects) and replications as random factors. Since there was no interaction between hybrid, N rate and year, we pooled years for a better visualization of the effects of N. Significance levels of fixed effects for each of the measured variables are shown in Table 1, and the regression equation parameters for the response to N rate of the variables are shown in Table 2. 3. Results 3.1. Biomass and N accumulation As previously reported (Uribelarrea et al., 2004), the protein concentrations of the protein-strain hybrids reflected the strain parents, with IHP and ILP having average grain protein concentrations of 148 and 65 g kg1, respectively (Table 3). The two reverse strains were intermediate with IRLP having 111 g kg1 of grain protein and IRHP 87 g kg1. The hybrids with the lowest grain protein concentrations ILP and IRHP out-yielding the higher-protein hybrids (IHP and IRLP) (Table 3). Similar differences were observed among the strain-hybrids for grain energy equivalent, with the two high protein hybrids exhibiting lower average values than the two low protein hybrids (247 MJ ha1 versus 272 MJ ha1).
The protein-strain hybrids all produced similar shoot biomass at flowering, and vegetative biomass was only influenced by the N rate in IHP (Fig. 1A). In contrast, plant N accumulation increased linearly with N rate in each hybrid (Fig. 1B). The IHP-hybrid accumulated the most vegetative plant N (Table 3). The other three protein-strain hybrids all contained similar amounts of plant N at flowering, and all responded to a lesser extent to incremental increases in the N rate than did IHP (Table 3). The N uptake efficiency at flowering (R1) differed among the hybrids, and generally decreased with an increase in N supply (Fig. 1C). The two hybrids with the highest concentrations of grain protein (IHP and IRLP) exhibited higher Table 4 Dry weight harvest index (HI), nitrogen harvest index (NHI), and percentage of total plant N accumulation at flowering for the protein-strain hybrids grown at Champaign, IL, between 2001 and 2003 Strain-hybrid
HI (%)
NHI (%)
N uptake at flowering (%)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
40 46 41 45
68 51 62 62
48 64 48 51
LSD (P < 0.10)
3
4
6
Values are averaged across N rates.
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Fig. 3. Nitrogen rate effect on N remobilization between R1 and R6 for the stover (A), leaf (B), and stalk (C) fractions, for the Illinois protein-strain hybrids grown at Champaign, IL, between 2001 and 2003. Positive values indicate accumulation of N in the plant fraction, while negative values represent N remobilization from the plant fraction. Dashed lines indicate no remobilization or accumulation. The best polynomial regression model was fitted when the effect of N rate over the corresponding variable was significant. The fitted equation parameters are presented in Table 2.
efficiencies of vegetative N uptake, and a sharper negative response to increases in N supply. Conversely, ILP had the lowest N uptake efficiency at R1, which was fairly constant for all N rates. In contrast to R1, at physiological maturity both biomass and N accumulation were affected by the hybrid and by the N rate (Fig. 2). The final biomass of IHP and IRLP responded to N rate with a higher slope, while the lower protein hybrids exhibited a more tempered increase in biomass with N rate (Fig. 2A; Table 3). When grown with the optimum N rate, the final biomass production was relatively similar for all the proteinstrain hybrids. Conversely, the dry weight harvest index (HI) (i.e. the proportion of the total above ground biomass represented by grain) differed among the strain-hybrids with the highest yielding hybrids (ILP and IRHP) having the highest values (Table 4). The response in plant N accumulation was similar to biomass with IHP and IRLP being more responsive to N than the other two hybrids (Fig. 2B; Table 3). The 3.1 g plant1 of N accumulated, on average by IHP and IRLP equates to a seasonal net acquisition of 201 kg N ha1, which was 40 kg more than IRHP and 69 kg more than ILP. The harvest index for grain N (NHI) was not affected by N supply, although the IHP-hybrid had the highest and ILP the lowest NHI (Table 4).
There were minor differences in the timing of N acquisition, with all hybrids except ILP acquiring around 50% of their plant N after flowering (Table 4). The hybrids differed, however, in their magnitude of post-flowering N uptake, with IHP and IRLP exhibiting the highest maximum levels of post-flowering N accumulation and a greater response to increases in N rate (Fig. 2C). The strain-hybrids also differed in remobilization of N from the stover, with IHP having the greatest remobilization, which increased with N supply (Fig. 3A). IHP also exhibited the greatest N remobilization from the leaves (on average 0.33 g N plant1). 1). The remobilization of leaf N was enhanced by N rate in both IHP and IRLP, but not in ILP and IRHP (Fig. 3A). IHP was the only hybrid with measurable remobilization of N from the stalk, which unlike leaves was not affected by the N supply (Fig. 3C). 3.2. Nitrogen use efficiency The overall N use efficiency (NUE) was similar among the hybrids (on average 24 kg kg1 N), and was negatively affected by N rate (Fig. 4A). The hybrids differed, however, in the main components of NUE, N uptake and N utilization (Fig. 4). Differences in N uptake efficiency were related to the levels of grain protein (r = 0.54; P 0.05), as well as to the differences
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Fig. 4. Nitrogen rate effect on N use efficiency (NUE) (A) and its two components: N uptake (B), and N utilization (C), for the Illinois protein-strain hybrids grown at Champaign, IL, between 2001 and 2003. The best polynomial regression model was fitted when the effect of N rate over the corresponding variable was significant. The fitted equation parameters are presented in Table 2.
in grain yield between years (Fig. 4B; Table 3). IHP had the highest overall values of N uptake efficiency and ILP the lowest, with the corresponding reverse-strain hybrids exhibiting intermediate values of N uptake efficiency). In every case, N uptake exhibited a negative linear response to N supply (Table 3). N utilization efficiency was also affected by hybrid and by N rate (Fig. 4C). As opposed to N uptake, IHP exhibited the lowest utilization efficiency, while ILP was the most efficient. The reverse-strains hybrids had similar values of utilization efficiency, falling in between those for IHP and ILP. 4. Discussion A number of distinct differences in plant N use and growth were evident in the strain-hybrids. As reported previously for the IHP and ILP strains (Wyss et al., 1991), the strain-hybrids also produced similar amounts of plant biomass at flowering, indicative of similar levels of light interception and net canopy photosynthesis during the vegetative growth phase. Conversely, plant N accumulation and N uptake efficiency were markedly affected by both the hybrid and by the N rate. The much greater N accumulation by the IHP-hybrid than the other hybrids is in agreement with the idea that the IHP strain is enhanced in the
absorption and translocation of N (Lohaus et al., 1998; Rizzi et al., 1996). An adequate N rate was needed for the IHP-hybrid to manifest its superior N accumulation, as at N levels of 100 kg ha1 or lower it accumulated the same or lower amounts of vegetative N as did the other hybrids. The IHP- and IRLP-hybrids also exhibited more efficient pre-flowering N uptake than the two low-protein hybrids, and a greater magnitude of post-flowering N accumulation. The difference in pre-flowering N uptake shows that the genetic differences in N metabolism are manifested before the grain is developed. A positive relationship between pre-flowering leaf NO3 and grain yield and N concentration has been reported by Hirel et al. (2001), who proposed that a high capacity to store nitrate during vegetative growth was associated with yield improvement. Similarly, our results show that N accumulation by the vegetative biomass can be enhanced by selection for grain protein. The strain-hybrids also produced the same biomass at physiological maturity, even though IHP appeared to have poorer growth when N was deficient. Post-flowering N uptake was greatest in IHP and IRLP, intermediate in IRHP, and lowest in the ILP-hybrid, which is in agreement with differences in their sink demand for N (Uribelarrea et al., 2004). Similar to the strains themselves (Wyss et al., 1991; Rizzi et al., 1996; Below
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et al., 2004), the IHP-hybrid exhibited substantial remobilization of N from the leaves and stalk, and had the lowest grain yield and HI. The strain-hybrids had the same overall NUE, but differed markedly in the strategy they employed to achieve it with IHP and IRLP having higher efficiencies of N uptake, and ILP and IRHP being more efficient in N utilization. We expect that high N uptake efficiency would be associated with roots and nitrate uptake, and high utilization efficiency with the degree of kernel set and starch synthesis. Previous studies of the strains themselves showed that roots of IHP are more efficient at absorbing, reducing, and translocating N than ILP, while the grain of ILP has higher levels of sugars and enhanced activities of enzymes associated with starch synthesis than does IHP (see review by Below et al., 2004). N uptake efficiency declined with N rate to a greater extent than did N utilization efficiency, which may be because high levels of available N saturate the root uptake system before they saturate the pathways for N assimilation and utilization (Bertin and Gallais, 2000; Presterl et al., 2003). Selection for grain protein has clearly altered N use by the maize plant, and these differences appear to be highly heritable in hybrids. The large difference in N use strategy employed by these hybrids makes them unique genetic materials for additional research. Studies with ILP should focus on its uniquely high ability to utilize N for dry matter production, while those with IHP should highlight its superior ability to uptake N. Acknowledgements This study was part of project no. 15-0390 of Agric. Exp. Stn., College of Agricultural, Consumer and Environmental Sciences, University of Illinois at Urbana Champaign. It was supported in part by the Illinois C-FAR program project no. 021-081-5D. The authors express their gratitude to Juliann Seebauer, Martha Schneerman, John Meharry, and Mark Harrison for laboratory and field assistance, and Dr. Matı´as Ruffo for a critical review of this paper. We also thank Illinois Foundation Seed, Champaign, IL for providing seed of the FR1064 inbred. References Below, F.E., 2002. Nitrogen metabolism and crop productivity. In: Pessarakli, M. (Ed.), Handbook of Plant and Crop Physiology. 2nd ed. Marcel Dekker Inc., New York, pp. 385–406. Below, F.E., Seebauer, J.R., Uribelarrea, M., Schneerman, M.C., Moose, S.P., 2004. Physiological changes accompanying long-term selection for grain protein in maize. In: Janick, J. (Ed.), Plant Breeding Reviews, vol. 1. John Wiley & Sons Inc., New Jersey, pp. 133–151. Bertin, P., Gallais, A., 2000. Genetic variation for nitrogen use efficiency in a set of recombinant maize inbred lines. I. Agrophysiological results. Maydica 45, 53–66. Cassman, K.G., Dobermann, A., Walters, D.T., 2002. Agroecosystems, nitrogen-use efficiency, and nitrogen management. Ambio 31 (2), 132–140. Castleberry, R.M., Crum, C.W., Krull, C.F., 1984. Genetic yield improvement of U.S. maize cultivars under varying fertility and climatic environments. Crop Sci. 24, 33–36.
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Cerrato, M.E., Blackmer, A.M., 1990. Comparison of models for describing corn yield response to nitrogen fertilizer. Agron. J. 82, 138– 143. Dudley, J.W., Lambert, R.J., Alexander, D.E., 1974. Seventy generations of selection for oil and protein in the maize kernel. In: Dudley, J.W. (Ed.), Seventy Generations of Selection for Oil and Protein in Maize.Crop Science Society of America, Madison, WI, pp. 181–211. Dudley, J.W., Lambert, R.J., 1992. Ninety generations of selection for oil and protein in maize. Maydica 37, 81–87. Duvick, D.N., 1992. Genetic contributions to advances in yield in U.S. maize. Maydica 37, 69–79. Eghball, B., Maranville, J.W., 1993. Root development and nitrogen influx of corn genotypes grown under combined drought and nitrogen stresses. Agron. J. 85, 147–152. Gastal, F., Lemaire, G., 2002. N uptake and distribution in crops: an agronomical and ecophysiological perspective. J. Exp. Bot. 53, 789–799. Gentry, L.E., Below, F.E., David, M.B., Bergerou, J.A., 2001. Source of the soybean N credit in maize production. Plant Soil. 236, 175–184. Hedin, P.A., Williams, W.P., Buckley, P.M., 1998. Caloric analyses of the distribution of energy in corn plants Zea mays L. J. Agric. Food Chem. 46, 4754–4758. Hirel, B., Bertin, P., Quillere, I., Bourdoncle, W., Attagnant, C., Dellay, C., Gouy, S., Retailliau, C., Falque, M., Gallais, A., 2001. Towards a better understanding of the genetic and physiological basis for nitrogen use efficiency in maize. Plant Physiol. 125, 1258–1270. Lohaus, G., Bu¨ker, M., Hubmann, M., Soave, C., Heldt, H.-W., 1998. Transport of amino acids with special emphasis on the synthesis and transport of asparagine in the Illinois low protein and Illinois high protein strains of maize. Planta 205, 181–188. Ma, B.L., Dwyer, L.M., Gregorich, E.G., 1999. Soil nitrogen amendment effects on nitrogen uptake and grain yield of maize. Agron. J. 91, 650–656. Ma, B.L., Dwyer, L.M., Tollenaar, M., Smith, D.L., 1998. Stem infusion of nitrogen-15 to quantify nitrogen removilization in maize. Commun. Soil Sci. Plant Anal. 29 (3/4), 305–317. Moll, R.H., Kamprath, E.J., Jackson, W.A., 1982. Analysis and interpretation of factors which contribute to efficiency of nitrogen utilization. Agron. J. 74, 562–564. Muchow, R.C., 1988. Effect of nitrogen supply on the comparative productivity of maize and sorghum in a semi-arid tropical environment. I. Leaf growth and leaf nitrogen. Field Crop. Res. 18, 1–16. Muchow, R.C., 1998. Nitrogen utilization efficiency in maize and grain sorghum. Field Crop. Res. 56, 209–216. Normand, B., Recous, S., Vachaud, G., Kengni, L., Garino, B., 1997. Nitrogen15 tracers combined with tensio-neutronic method to estimate the nitrogen balance of irrigated maize. Soil Sci. Soc. Am. J. 61, 1508–1518. Presterl, T., Seitz, G., Landbeck, M., Thiemt, E.M., Schmidt, W., Geiger, H.H., 2003. Improving nitrogen-use efficiency in european maize: estimation of quantitative genetic parameters. Crop Sci. 43, 1259–1265. Purcino, A.A.C., Arellano, C., Athwal, G.S., Huber, S.C., 1998. Nitrate effect on carbon and nitrogen assimilating enzymes of maize hybrids representing seven eras of maize breeding. Maydica 43, 83–94. Rice, C.W., Havlin, J.L., Schepers, J.S., 1995. Rational nitrogen fertilization in intensive cropping systems. Fertil. Res. 42, 89–97. Ritchie, S.W., Hanway, J.J., Benson, G.O., 1997. How a corn plant develops, Special report no. 48, Iowa State University of Science and Technology Cooperative Extension Service, Ames, p. 20. Rizzi, E., Balconi, C., Bosio, D., Nembrini, L., Morselli, A., Motto, M., 1996. Accumulation and partitioning of nitrogen among plant parts in the high and low protein strains of maize. Maydica 41, 325–332. SAS Institute, 2000. SAS user’s guide. SAS Institute, Cary, NC. Sinclair, T.R., 1995. Effect of nitrogen supply on maize yield. I. Modeling physiological responses. Agron. J. 87, 632–641. Smiciklas, K.D., Below, F.E., 1990. Influence of heterotic pattern on nitrogen use and yield of maize. Maydica 35, 209–213. Uhart, S.A., Andrade, F.H., 1995a. Nitrogen deficiency in maize. I. Effects on crop growth, development, dry matter partitioning, and kernel set. Crop Sci. 35, 1376–1383.
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Uhart, S.A., Andrade, F.H., 1995b. Nitrogen deficiency in maize. II. Carbon– nitrogen interaction effects on kernel number and grain yield. Crop Sci. 35, 1384–1389. Uribelarrea, M., Below, F.E., Moose, S.P., 2004. Grain composition and productivity of maize hybrids derived from the Illinois protein strains in response to variable nitrogen supply. Crop Sci. 44, 1593– 1600.
Woodworth, C.M., Leng, E.R., Jugenheimer, R.W., 1974. Fifty generations of selection for oil and protein in corn. In: Dudley, J.W. (Ed.), Seventy Generations of Selection for Oil and Protein in Maize. Crop Science Society of America, Madison, WI, pp. 121–132. Wyss, C.S., Czyzewicz, J.R., Below, F.E., 1991. Source-sink control of grain composition in maize strains divergently selected for protein-concentration. Crop Sci. 31, 761–766.
Divergent selection for grain protein affects nitrogen use in maize hybrids Martı´n Uribelarrea, Stephen P. Moose, Frederick E. Below * Department of Crop Sciences, 1201 W. Gregory, University of Illinois, Urbana, IL 61801, USA Received 7 July 2005; received in revised form 24 May 2006; accepted 24 May 2006
Abstract The Illinois high (IHP), low (ILP), and corresponding reverse (IRHP, and IRLP) protein–strains of maize represent genetic extremes for differences in grain protein concentration. The objective of this study was to determine how divergent selection for grain protein affects N use in hybrid plants. Inbreds derived from the protein–strains were crossed as males to a common tester and the resultant hybrids evaluated at eight N rates in the field over 3 years. A more than two-fold difference in grain protein concentration was observed among the strain-hybrids with ILP averaging 65 g kg1, IRHP 89 g kg1, IRLP 111 g kg1, and IHP 148 g kg1 of grain protein. Except for IHP at the lowest N levels, the strain-hybrids were similar in their whole shoot biomass production both pre- and post-flowering. Conversely, the strain-hybrids differed markedly in their uptake and accumulation of plant N, and these differences were already evident at flowering before a grain sink was present. Although all hybrids had the same overall N use efficiency at maturity (approximately 24 kg kg1 N), they differed in their N use components with IHP and IRLP exhibiting a higher uptake efficiency, and ILP and IRHP exhibiting high utilization efficiency. The remobilization of leaf N was also more extensive for IHP and IRLP. Changes in grain protein concentration from divergent selection were directly related to changes in uptake and use of N by the plant. # 2006 Published by Elsevier B.V. Keywords: Zea mays; N use efficiency; Biomass accumulation; Illinois protein strains; Maize
1. Introduction Modern agriculture is concerned with yield, the nutritional quality of the crop and the environmental impact of crop production. Efficient use of fertilizer N is therefore critical. Because an adequate N supply is one of the main factors powering yield of cereal crops (Below, 2002), annual applications of fertilizer N are the norm. About half of the 110 kg ha1 annual increase in maize yields over the last half century can be attributed to improved cultural practices, especially N fertilizer use (Duvick, 1992; Sinclair, 1995). Variation in the N supply affects all phases of maize growth, including the development, activity, and senescence of leaves, and the initiation, growth, and composition of ovules (Muchow, 1988; Uhart and Andrade, 1995a, 1995b). Thus, understanding the processes associated with the efficiency of N use (NUE), particularly N uptake and utilization, is of major importance in designing crop management strategies and in developing breeding programs for improved N use.
* Corresponding author. Tel.: +1 217 333 9745; fax: +1 217 333 8377. E-mail address: [email protected] (F.E. Below). 0378-4290/$ – see front matter # 2006 Published by Elsevier B.V. doi:10.1016/j.fcr.2006.05.008
We use NUE here to encompass yield efficiency (the increase in grain yield per unit of applied N fertilizer) and its two components; uptake efficiency (the fraction of fertilizer applied N found in the plant at maturity), and utilization efficiency (the ratio of grain yield to plant N). The N supply can alter the relative importance of these two components, as under high N inputs, NUE is mainly determined by the plant’s ability to acquire N; whereas at low N, the ability to utilize absorbed N is generally more important (Moll et al., 1982; Ma et al., 1998). Many studies show that genotype can also impact NUE (Cerrato and Blackmer, 1990; Smiciklas and Below, 1990; Eghball and Maranville, 1993; Rice et al., 1995; Normand et al., 1997; Muchow, 1998; Ma et al., 1998, 1999; Cassman et al., 2002; Gastal and Lemaire, 2002). Because most modern hybrids are selected according to their yield and N use under high rates of applied N (Castleberry et al., 1984; Bertin and Gallais, 2000), limited genetic variability may exist among commercial hybrids for N utilization, or for the remobilization of N from the stover to the grain (Purcino et al., 1998; Bertin and Gallais, 2000). The Illinois protein strains, which are the result of long term divergent selection for grain protein concentration are unique within the maize germplasm. Illinois high protein (IHP) and
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Illinois low protein (ILP) have been continuously selected for over 100 cycles; whereas the Illinois reverse low protein (IRLP) and Illinois reverse high protein (IRHP) strains are the result of reversing selection in ILP and IHP beginning with cycle 48. Evaluations of the strains during the past 100 cycles have continually demonstrated the effectiveness of this program in altering grain protein level (Woodworth et al., 1974; Dudley et al., 1974; Dudley and Lambert, 1992; Rizzi et al., 1996), as well as a number of other plant traits. The wide variation in protein and dry matter production of these strains must have been accompanied by corresponding changes in N and C metabolism in the plant, and the Illinois protein strains have previously been shown to differ in N metabolism (Wyss et al., 1991; Lohaus et al., 1998; Below et al., 2004). This variation, and the fact that the strains share a common parental background, makes them unique experimental material for studying physiological and biochemical mechanisms associated with differences in maize productivity. Our approach was to make hybrids of each of the strains (including the reverse strains) using inbreds derived from generation 90 crossed to a common tester, then to evaluate these materials in the field for NUE and its main components. Uribelarrea et al. (2004) showed that these hybrids had grain protein concentrations which reflected the strain parents, and that they differed in their use of N (Below et al., 2004). Our objective in this study was to understand how differences in the acquisition, utilization, and remobilization of N are associated with divergent selection for grain protein. 2. Materials and methods 2.1. Field site, cultural practices and treatment arrangements Field experiments were conducted at the Department of Crop Sciences Research and Education Center in Champaign, Illinois during the 2001–2003 growing seasons, on plots that had previously been shown to be responsive to N fertilizer (Gentry et al., 2001). The soil type and cultural practices were as previously reported (Uribelarrea et al., 2004). Briefly, the soil was a Drummer silty clay loam with an average organic matter of 3.7% and a pH of 6.2. The field was under a maize–soybean rotation, with the location of the experimental plots alternated each year. Plots were kept weed-free with chemical control and hand cultivation, and crops were irrigated, when necessary. Hybrids of the Illinois protein strains were produced by crossing inbreds made from generation 90 of IHP and ILP and generation 42 of IRHP and IRLP as males to FR1064 as the tester. Hybrids were over seeded on 26 April 2001, on 25 May 2002, and 22 April 2003 and thinned to a stand density of 65,000 plants ha1. The delay in planting date in 2002 was due to above average precipitation during April and May (270 mm in 2002 compared to a 30-year average of 200 mm). Each of the hybrids was grown under eight rates of fertilizer N (0– 238 kg ha1) in 34 kg increments. The fertilizer was hand applied in a diffuse band down the center of the row as ammonium sulfate and incorporated between the V2 and V3
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growth stages. Treatments consisted of the factorial combination of the four protein-strain hybrids and eight fertilizer rates arranged in a randomized complete block design with four replications. Each experimental unit consisted of four-row plots that were 5.3 m long 3 m wide, with one of the central rows reserved for final yield determination and the other used for destructive plant samplings. 2.2. Crop measurements N acquisition and partitioning were assessed using whole shoots sampled at two growth stages; R1 (i.e. beginning of anthesis and visible silks), and R6 (physiological maturity) when 50% of the plants exhibiting a visible black layer at the base of the kernels. By R6, maize plants are considered to have attained their maximum biomass (Ritchie et al., 1997), and we used shoot dry weight as a relative indicator of net canopy photosynthesis. Because of the large differences in grain composition among these hybrids, we also calculated the energy equivalent (MJ ha1) of the grain biomass using standard caloric values (Hedin et al., 1998) and the respective starch, oil and protein concentrations of the grain (Uribelarrea et al., 2004). At each harvest, four representative plants were separated into leaf, stalk (including leaf sheaths), reproductive support tissues (tassel, husks and cob at R6, or ear-shoot at R1), and grain (only at R6 sampling). Reproductive and grain fractions were placed into a forced-draft oven (75 8C), while the fresh weight of the entire leaf and stalk sample was determined prior to shredding. An aliquot of the shredded material was weighed fresh and then oven-dried (75 8C). The dry weight of each plant fraction was calculated using the fresh weight and the moisture level. Individual plant samples were ground in a Wiley mill to pass a 20 mesh screen, and analyzed for total N concentration (g kg1) using a combustion technique (NA2000 N-Protein, Fisons Instruments). The total N content (g N plant1) was calculated by multiplying the dry weight by the N concentration. Table 1 Significance level of the fixed effects for each of the measured variables, for the protein-strain hybrids grown at Champaign, IL, between 2001 and 2003 Measured variable
Source of variation Hybrid
N rate
Hybrid N rate
R1 biomass R1 N content R1 N uptake
NS 0.0088 0.0001
NS 0.0001 0.0001
0.0001 0.0472 0.0745
R6 biomass R6 N content Post-flowering N uptake
0.0270 0.0725 0.0518
0.0001 0.0001 0.0030
0.0514 0.0001 0.0001
Stover N remobilization Leaf N remobilization Stalk N remobilization
0.0001 0.0002 0.0001
NS 0.0458 NS
NS NS NS
NUE N uptake N utilization
NS 0.0103 0.0783
0.0001 0.0003 0.0198
NS NS NS
A threshold of 0.10 was used to determine a significant effect of the different sources of variation over the measured variables. NS means that term was not significant.
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Table 2 Equation parameters for the effect of N rate on the different measured variables for the Illinois protein-strain hybrids grown at Champaign, IL, between 2001 and 2003 Measured variable
Protein-strain hybrid
Regression equation parameters§ Intercept
Linear term (x)
Quadratic term (x2)
82 93z 96 93
11.6 102 NS NS NS
NS NS NS NS
R1 biomass (g plant1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
R1 N content (g plant1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
0.70 0.90 0.83 0.76
4.1 103 1.6 103 2.2 101 2.4 103
NS NS NS NS
R1 N uptake (kg kg1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
0.65 0.17 0.64 0.45
3.5 103 8.2 104 4.8 103 3.0 103
8.7 106 2.2 106 1.2 106 8.0 106
R6 biomass (g plant1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
6.0 101 5.5 101 3.7 101 6.8 101
1.2 103 1.4 103 6.9 104 2.0 103
R6 N content (g plant1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
1.10 1.20 1.32 1.27
1.2 102 6.8 103 9.5 103 8.5 103
1.0 105 1.0 105 1.0 105 1.0 105
Post-flowering N uptake (g plant1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
0.61 0.39 0.66 0.64
7.2 103 4.1 103 5.4 103 7.2 103
1.0 105 1.0 105 1.0 105 1.0 105
Stover N remobilization (g N plant1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
0.28 0.34 0.21 0.18
3.4 103 NS 2.6 103 NS
8.8 106 NS 1.4 105 NS
Leaf N remobilization (g N plant1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
0.17 0.22 0.15 0.17
2.2 103 NS 1.3 103 NS
6.1 106 NS 4.5 106 NS
Stalk N remobilization (g N plant1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
0.09 0.04 0.02 0.04
NS NS NS NS
NS NS NS NS
NUE (kg kg1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
43 40 44 41
2.0 101 2.0 101 2.0 101 2.0 101
3.3 104 3.3 104 3.3 104 3.3 104
N uptake efficiency (kg kg1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
1.1 103 1.1 103 1.1 103 1.1 103
NS NS NS NS
N utilization efficiency (kg kg1)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
9.3 102 9.3 102 9.3 102 9.3 102
NS NS NS NS
§ z
154 184 195 182
0.83 0.55 0.67 0.64 47 65 61 57
x = fertilizer N rate (kg ha1); NS means term was non significant. Value averaged across N rates since parameters from corresponding equation were non significant (P < 0.10).
M. Uribelarrea et al. / Field Crops Research 100 (2007) 82–90 Table 3 Grain protein concentration, grain yield and grain energy equivalent for the protein-strain hybrids grown in Champaign, IL, between 2001 and 2003 Strain-hybrid
Year
Protein concentration (g kg1)
Grain yield (Mg ha1)
Grain energy equivalent (MJ ha1)
FR1064 IHP
2001 2002 2003 Average
170 144 131 148
7.4 7.0 8.2 7.5
223 215 271 236
2001 2002 2003 Average
68 62 64 65
9.0 7.6 9.1 8.6
270 234 302 269
FR1064 IRLP
2001 2002 2003 Average
131 102 100 111
7.8 7.2 9.1 8.0
241 222 312 258
FR1064 IRHP
2001 2002 2003 Average
97 87 84 89
8.8 8.8 9.6 9.1
308 240 279 276
FR1064 ILP
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yield, and plant N content, NUE (kg grain kg1 fertilizer N) and its components, N uptake (kg plant N kg1 fertilizer N) and N utilization (kg grain kg1 plant N), were calculated as shown in Eqs. (1)–(3): NUE ¼
GYX GY0 1000 NRX
(1)
NTX NT0 NRX
(2)
N uptake ¼
N utilization ¼
GYX GY0 1000 NTX NT0
(3)
where GYX and GY0 correspond to the grain yield (Mg ha1) at the X and 0 fertilizer rates (kg ha1); NRX is the fertilizer N rate X (kg ha1), and NTX and NT0 represent the total plant N content at 0 and X N rates (kg ha1). The N remobilization from the leaves, stalk and stover was calculated as the difference in N content in each fraction between R1 and R6. 2.3. Statistical analysis
Values presented are the maximum levels obtained as a function of applied N.
For yield determination, all ears in the unsampled center row of each plot were harvested and mechanically shelled, and weight and moisture level determined. Dry grain yield was expressed as Mg ha1 at 0% moisture. Using the data from grain
Effects of fertilizer N rate for the four hybrids during the 3 years were analyzed with the MIXED procedure in SAS (SAS Institute, 2000), and the parameters of the respective polynomial regressions were also fitted when the effect of N rate was significant. The hybrid and N rate factors were
Fig. 1. Nitrogen rate effect on above-ground biomass (A), N content per plant (B), and N uptake efficiency (C) at flowering (R1) for the Illinois protein-strain hybrids grown at Champaign, IL, between 2001 and 2003. The best polynomial regression model was fitted when the effect of N rate over the corresponding variable was significant. The fitted equation parameters are presented in Table 2.
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Fig. 2. Nitrogen rate effect on above-ground biomass (A), N content per plant (B), and post-flowering N uptake (C) at physiological maturity (R6) for the Illinois protein-strain hybrids grown at Champaign, IL, between 2001 and 2003. The best polynomial regression model was fitted when the effect of N rate over the corresponding variable was significant. The fitted equation parameters are presented in Table 2.
considered fixed, and years (and its interaction with the fixed effects) and replications as random factors. Since there was no interaction between hybrid, N rate and year, we pooled years for a better visualization of the effects of N. Significance levels of fixed effects for each of the measured variables are shown in Table 1, and the regression equation parameters for the response to N rate of the variables are shown in Table 2. 3. Results 3.1. Biomass and N accumulation As previously reported (Uribelarrea et al., 2004), the protein concentrations of the protein-strain hybrids reflected the strain parents, with IHP and ILP having average grain protein concentrations of 148 and 65 g kg1, respectively (Table 3). The two reverse strains were intermediate with IRLP having 111 g kg1 of grain protein and IRHP 87 g kg1. The hybrids with the lowest grain protein concentrations ILP and IRHP out-yielding the higher-protein hybrids (IHP and IRLP) (Table 3). Similar differences were observed among the strain-hybrids for grain energy equivalent, with the two high protein hybrids exhibiting lower average values than the two low protein hybrids (247 MJ ha1 versus 272 MJ ha1).
The protein-strain hybrids all produced similar shoot biomass at flowering, and vegetative biomass was only influenced by the N rate in IHP (Fig. 1A). In contrast, plant N accumulation increased linearly with N rate in each hybrid (Fig. 1B). The IHP-hybrid accumulated the most vegetative plant N (Table 3). The other three protein-strain hybrids all contained similar amounts of plant N at flowering, and all responded to a lesser extent to incremental increases in the N rate than did IHP (Table 3). The N uptake efficiency at flowering (R1) differed among the hybrids, and generally decreased with an increase in N supply (Fig. 1C). The two hybrids with the highest concentrations of grain protein (IHP and IRLP) exhibited higher Table 4 Dry weight harvest index (HI), nitrogen harvest index (NHI), and percentage of total plant N accumulation at flowering for the protein-strain hybrids grown at Champaign, IL, between 2001 and 2003 Strain-hybrid
HI (%)
NHI (%)
N uptake at flowering (%)
FR1064 IHP FR1064 ILP FR1064 IRLP FR1064 IRHP
40 46 41 45
68 51 62 62
48 64 48 51
LSD (P < 0.10)
3
4
6
Values are averaged across N rates.
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Fig. 3. Nitrogen rate effect on N remobilization between R1 and R6 for the stover (A), leaf (B), and stalk (C) fractions, for the Illinois protein-strain hybrids grown at Champaign, IL, between 2001 and 2003. Positive values indicate accumulation of N in the plant fraction, while negative values represent N remobilization from the plant fraction. Dashed lines indicate no remobilization or accumulation. The best polynomial regression model was fitted when the effect of N rate over the corresponding variable was significant. The fitted equation parameters are presented in Table 2.
efficiencies of vegetative N uptake, and a sharper negative response to increases in N supply. Conversely, ILP had the lowest N uptake efficiency at R1, which was fairly constant for all N rates. In contrast to R1, at physiological maturity both biomass and N accumulation were affected by the hybrid and by the N rate (Fig. 2). The final biomass of IHP and IRLP responded to N rate with a higher slope, while the lower protein hybrids exhibited a more tempered increase in biomass with N rate (Fig. 2A; Table 3). When grown with the optimum N rate, the final biomass production was relatively similar for all the proteinstrain hybrids. Conversely, the dry weight harvest index (HI) (i.e. the proportion of the total above ground biomass represented by grain) differed among the strain-hybrids with the highest yielding hybrids (ILP and IRHP) having the highest values (Table 4). The response in plant N accumulation was similar to biomass with IHP and IRLP being more responsive to N than the other two hybrids (Fig. 2B; Table 3). The 3.1 g plant1 of N accumulated, on average by IHP and IRLP equates to a seasonal net acquisition of 201 kg N ha1, which was 40 kg more than IRHP and 69 kg more than ILP. The harvest index for grain N (NHI) was not affected by N supply, although the IHP-hybrid had the highest and ILP the lowest NHI (Table 4).
There were minor differences in the timing of N acquisition, with all hybrids except ILP acquiring around 50% of their plant N after flowering (Table 4). The hybrids differed, however, in their magnitude of post-flowering N uptake, with IHP and IRLP exhibiting the highest maximum levels of post-flowering N accumulation and a greater response to increases in N rate (Fig. 2C). The strain-hybrids also differed in remobilization of N from the stover, with IHP having the greatest remobilization, which increased with N supply (Fig. 3A). IHP also exhibited the greatest N remobilization from the leaves (on average 0.33 g N plant1). 1). The remobilization of leaf N was enhanced by N rate in both IHP and IRLP, but not in ILP and IRHP (Fig. 3A). IHP was the only hybrid with measurable remobilization of N from the stalk, which unlike leaves was not affected by the N supply (Fig. 3C). 3.2. Nitrogen use efficiency The overall N use efficiency (NUE) was similar among the hybrids (on average 24 kg kg1 N), and was negatively affected by N rate (Fig. 4A). The hybrids differed, however, in the main components of NUE, N uptake and N utilization (Fig. 4). Differences in N uptake efficiency were related to the levels of grain protein (r = 0.54; P 0.05), as well as to the differences
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Fig. 4. Nitrogen rate effect on N use efficiency (NUE) (A) and its two components: N uptake (B), and N utilization (C), for the Illinois protein-strain hybrids grown at Champaign, IL, between 2001 and 2003. The best polynomial regression model was fitted when the effect of N rate over the corresponding variable was significant. The fitted equation parameters are presented in Table 2.
in grain yield between years (Fig. 4B; Table 3). IHP had the highest overall values of N uptake efficiency and ILP the lowest, with the corresponding reverse-strain hybrids exhibiting intermediate values of N uptake efficiency). In every case, N uptake exhibited a negative linear response to N supply (Table 3). N utilization efficiency was also affected by hybrid and by N rate (Fig. 4C). As opposed to N uptake, IHP exhibited the lowest utilization efficiency, while ILP was the most efficient. The reverse-strains hybrids had similar values of utilization efficiency, falling in between those for IHP and ILP. 4. Discussion A number of distinct differences in plant N use and growth were evident in the strain-hybrids. As reported previously for the IHP and ILP strains (Wyss et al., 1991), the strain-hybrids also produced similar amounts of plant biomass at flowering, indicative of similar levels of light interception and net canopy photosynthesis during the vegetative growth phase. Conversely, plant N accumulation and N uptake efficiency were markedly affected by both the hybrid and by the N rate. The much greater N accumulation by the IHP-hybrid than the other hybrids is in agreement with the idea that the IHP strain is enhanced in the
absorption and translocation of N (Lohaus et al., 1998; Rizzi et al., 1996). An adequate N rate was needed for the IHP-hybrid to manifest its superior N accumulation, as at N levels of 100 kg ha1 or lower it accumulated the same or lower amounts of vegetative N as did the other hybrids. The IHP- and IRLP-hybrids also exhibited more efficient pre-flowering N uptake than the two low-protein hybrids, and a greater magnitude of post-flowering N accumulation. The difference in pre-flowering N uptake shows that the genetic differences in N metabolism are manifested before the grain is developed. A positive relationship between pre-flowering leaf NO3 and grain yield and N concentration has been reported by Hirel et al. (2001), who proposed that a high capacity to store nitrate during vegetative growth was associated with yield improvement. Similarly, our results show that N accumulation by the vegetative biomass can be enhanced by selection for grain protein. The strain-hybrids also produced the same biomass at physiological maturity, even though IHP appeared to have poorer growth when N was deficient. Post-flowering N uptake was greatest in IHP and IRLP, intermediate in IRHP, and lowest in the ILP-hybrid, which is in agreement with differences in their sink demand for N (Uribelarrea et al., 2004). Similar to the strains themselves (Wyss et al., 1991; Rizzi et al., 1996; Below
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et al., 2004), the IHP-hybrid exhibited substantial remobilization of N from the leaves and stalk, and had the lowest grain yield and HI. The strain-hybrids had the same overall NUE, but differed markedly in the strategy they employed to achieve it with IHP and IRLP having higher efficiencies of N uptake, and ILP and IRHP being more efficient in N utilization. We expect that high N uptake efficiency would be associated with roots and nitrate uptake, and high utilization efficiency with the degree of kernel set and starch synthesis. Previous studies of the strains themselves showed that roots of IHP are more efficient at absorbing, reducing, and translocating N than ILP, while the grain of ILP has higher levels of sugars and enhanced activities of enzymes associated with starch synthesis than does IHP (see review by Below et al., 2004). N uptake efficiency declined with N rate to a greater extent than did N utilization efficiency, which may be because high levels of available N saturate the root uptake system before they saturate the pathways for N assimilation and utilization (Bertin and Gallais, 2000; Presterl et al., 2003). Selection for grain protein has clearly altered N use by the maize plant, and these differences appear to be highly heritable in hybrids. The large difference in N use strategy employed by these hybrids makes them unique genetic materials for additional research. Studies with ILP should focus on its uniquely high ability to utilize N for dry matter production, while those with IHP should highlight its superior ability to uptake N. Acknowledgements This study was part of project no. 15-0390 of Agric. Exp. Stn., College of Agricultural, Consumer and Environmental Sciences, University of Illinois at Urbana Champaign. It was supported in part by the Illinois C-FAR program project no. 021-081-5D. The authors express their gratitude to Juliann Seebauer, Martha Schneerman, John Meharry, and Mark Harrison for laboratory and field assistance, and Dr. Matı´as Ruffo for a critical review of this paper. We also thank Illinois Foundation Seed, Champaign, IL for providing seed of the FR1064 inbred. References Below, F.E., 2002. Nitrogen metabolism and crop productivity. In: Pessarakli, M. (Ed.), Handbook of Plant and Crop Physiology. 2nd ed. Marcel Dekker Inc., New York, pp. 385–406. Below, F.E., Seebauer, J.R., Uribelarrea, M., Schneerman, M.C., Moose, S.P., 2004. Physiological changes accompanying long-term selection for grain protein in maize. In: Janick, J. (Ed.), Plant Breeding Reviews, vol. 1. John Wiley & Sons Inc., New Jersey, pp. 133–151. Bertin, P., Gallais, A., 2000. Genetic variation for nitrogen use efficiency in a set of recombinant maize inbred lines. I. Agrophysiological results. Maydica 45, 53–66. Cassman, K.G., Dobermann, A., Walters, D.T., 2002. Agroecosystems, nitrogen-use efficiency, and nitrogen management. Ambio 31 (2), 132–140. Castleberry, R.M., Crum, C.W., Krull, C.F., 1984. Genetic yield improvement of U.S. maize cultivars under varying fertility and climatic environments. Crop Sci. 24, 33–36.
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