106
DOI: 10.1002/jpln.200625147
J. Plant Nutr. Soil Sci. 2007, 170, 106–114
Leaf senescence induced by nitrogen deficiency as indicator of genotypic
differences in nitrogen efficiency in tropical maize
Gunda Schulte auf’m Erley1, Nazma Begum1, Mosisa Worku1, Marianne Bänziger2, and Walter J. Horst1*
1
2
Institute of Plant Nutrition, University of Hannover, Herrenhäuser Str. 2, 30419 Hannover, Germany
CIMMYT-Kenya, P.O. Box 25171, Nairobi, Kenya
Dedicated to Professor Dr. Burkhard Sattelmacher, who passed away on November 21, 2005
Accepted November 17, 2006
Summary
Nitrogen efficiency is a complex trait. Identification of secondary plant traits correlating with N efficiency would facilitate the
breeding for N-efficient cultivars. Sixteen tropical maize cultivars differing in grain yield at low N supply (N efficiency)
under field conditions in Zimbabwe exhibited a significant
negative correlation between N efficiency and leaf senescence during grain filling. The same cultivars were studied for
leaf senescence under N deficiency in a short-term nutrientsolution experiment. Leaf chlorophyll contents as estimated
by SPAD values and photosynthesis rates were used as
measures for leaf senescence. Cultivars differed both in
SPAD values and photosynthesis rates of the older leaves
during N deprivation. Significant negative correlations were
found between SPAD values, photosynthesis rates in the
1 Introduction
Nitrogen (N) is the element required in largest quantities for
growth and development of crops. However, when high
amounts are supplied, environmental pollution like nitrate
leaching into the groundwater and nitrous oxide emission into
the atmosphere may occur. On the other hand, access to N
fertilizers and their application is very unequally distributed
among regions of the world. Low-N stress is one of the major
abiotic stresses causing yield reductions in the tropics. To
solve the problems under both conditions, low-input strategies for N fertilization need to be developed (Raun and Johnson, 1999; Horst et al., 2003). Among these strategies,
breeding and cultivation of N-efficient cultivars (cultivars with
an above-average yield under low-N conditions) represent an
important approach (Sattelmacher et al., 1994; Lafitte and
Edmeades, 1994).
For the breeding of N-efficient cultivars, selection under
low-N conditions is necessary (Bänziger et al., 1997; Presterl
et al., 2003). Because of the high environmental variability
under such conditions, the use of secondary plant traits for
the selection process has been suggested (Blum, 1988).
Suitable secondary plant traits are particularly those, which
play a physiological role in achieving N efficiency.
* Correspondence: Prof. Dr. W. Horst;
e-mail: [email protected]
nutrient-solution experiment, and leaf-senescence scores in
the field experiments, and positive correlations were found
between photosynthesis rates and grain yield under low-N
conditions in the field. Relationships between physiological
root parameters, which were also investigated in the nutrientsolution experiment, and N uptake or grain yield of the cultivars in the field could not be established. It is concluded, that
the assessment of the capacity of a genotype to maintain a
higher photosynthetic capacity of older leaves during N deficiency–induced senescence at the seedling stage is a suitable selection parameter for the N efficiency of tropical maize
cultivars.
Key words: chlorophyll / cultivars / N-uptake kinetics / photosynthesis /
SPAD / Zea mays L.
A negative relationship between leaf senescence and N efficiency of both tropical and temperate maize cultivars was
found, indicating that cultivars with delayed leaf senescence
have higher grain yields under low-N conditions (Lafitte and
Edmeades, 1994; Bänziger et al., 1997; Rajcan and Tollenaar, 1999a; Paponov et al., 2005). In studies including a
further physiological characterization of N-efficient cultivars
with delayed leaf senescence, these cultivars also displayed
a high N uptake and dry-matter accumulation after flowering
(Ma and Dwyer, 1998; Rajcan and Tollenaar, 1999b). The
causal relationship between these traits and their importance
for N efficiency is unclear (Ma and Dwyer, 1998). A delayed
leaf senescence may lead to a better availability of carbon
assimilates, which enhances root growth and N uptake. On
the other hand, cultivars may differ in their root growth and
ability for N uptake during later growth stages, leading to a
delay of leaf senescence. In this case, leaf senescence is not
the cause for N efficiency, but an indicator of a higher root
activity (growth and N uptake).
In recent years, CIMMYT (International Maize and Wheat Improvement Center) maize breeders and physiologists have
made a major effort to increase grain yield under low-N by
exploiting the genetic variability in adaptation to low-N stress
in tropical maize germplasm (Bänziger et al., 1997; Friesen
et al., 2002). Progress has been made in developing N-efficient maize cultivars, which perform better than the controls
under all fertility conditions in east and southern Africa
(Friesen et al., 2002; Bänziger et al., 2005). The physiological
mechanisms of the N efficiency of these cultivars were
 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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J. Plant Nutr. Soil Sci. 2007, 170, 106–114
Leaf senescence induced by N deficiency as indicator of N efficiency 107
explored in field studies conducted in Zimbabwe and Kenya
(Worku, 2005). The N-efficient cultivars were characterized
by delayed leaf senescence and a high dry-matter accumulation and N uptake after anthesis.
The objective of this study was to investigate N deficiency–induced leaf senescence and parameters of N uptake of the
same tropical maize cultivars in short-term nutrient-solution
experiments in order to identify secondary traits contributing
and/or related to N efficiency under field conditions.
DAG). The nutrient solution was replaced when the N concentration of the nutrient solution fell below 0.5 mM measured
daily with nitrate test-strips (Merck, Germany). Twenty-three
days after germination, the N concentration in the nutrient
solution was decreased from 0.5 mM (2 d) to 0.4 mM (2 d) to
0.2 mM (1 d) to 0.1 mM (5 d) and finally to 0 mM (3 d). The
nutrient solution was replaced at least every other day. The
experiment was arranged as a completely randomized block
design with five replications.
2.2 Measurements and plant analysis
2 Material and methods
2.1 Plant material and growing conditions
Plants were grown in a greenhouse at the Institute of Plant
Nutrition, University of Hannover, Germany from May 12 to
June 18, 2003. Sixteen tropical maize cultivars provided by
CIMMYT were used in the study (Tab. 1). The classification of
the cultivars as N-efficient and N-inefficient was given by the
breeders and confirmed in field studies in Kenya and Zimbabwe as described by Worku (2005). The experimental conditions for the nutrient-solution experiment were established
in a preliminary experiment with two contrasting cultivars.
Seeds were germinated between moistened filter paper. After
germination, two seedlings of each cultivar were transplanted
into continuously aerated complete nutrient solution in 5 L
plastic pots. The composition of the nutrient solution was:
500 lM K2SO4, 100 lM KH2PO4, 325 lM MgSO4, 50 lM
NaCl, 8 lM H3BO3, 1 lM MnSO4, 0.4 lM ZnSO4, 0.4 lM
CuSO4, 0.1 lM MoNa2O4, and 85 lM Fe-EDDHA. As nitrogen source, Ca(NO3)2 was used. The plants were precultured
at high N supply (2 mM) until the fifth leaf counted from the
base of the plants was fully expanded (23 d after germination,
One plant per pot was harvested 23 DAG (harvest 1) and 37
DAG (harvest 2). Plants were separated into shoot and roots.
At harvest 2, leaf 8 counted from the base of the plant was
harvested separately. Total leaf area of this leaf was measured by a leaf-area meter (LI-3100, LI-COR, Lincoln, NE,
USA), and ten leaf discs with a diameter of 1.1 cm were
obtained from each leaf and frozen in liquid nitrogen for
chlorophyll analysis.
For leaf-chlorophyll analysis, the leaf discs were ground in
liquid nitrogen and extracted in 80% acetone. After centrifugation at room temperature, absorbance of the supernatant
at 664 nm and 647 nm wavelength was measured spectrophotometrically. Chlorophyll a and b contents were calculated
on a leaf-area basis according to Lichtenthaler (1987).
Dry weight of the plant fractions was measured after drying
the samples at 70°C until they attained a constant weight.
N concentrations of the dried and ground plant fractions were
determined using a CNS analyzer (Vario EL, Elementar Analysensysteme, Hanau, Germany).
Table 1: Maize cultivars/hybrids used in this study.
No.
Cultivar (hybrid)
Source
N efficiency
C1
CML444/CML445//CML440
CIMMYT-Zimbabwe
efficient
C2
CML395/CML444//CML440
CIMMYT-Zimbabwe
efficient
C3
CML202/CML395//CML205
CIMMYT-Zimbabwe
inefficient
C4
SC515
Seed-CO-Zimbabwe
inefficient
C5
CML395/CML444//CML442
CIMMYT-Zimbabwe
efficient
C6
CML444/CML197//CML443
CIMMYT-Zimbabwe
efficient
C7
SC633
Seed-CO-Zimbabwe
inefficient
C8
CML181/CZL01005//CZL01006
CIMMYT-Zimbabwe
inefficient
C9
CML181/CML182//CML176
CIMMYT-Zimbabwe
efficient
C10
CML144/(16304/6303Q)-B-6-1-3-3-B*6
CIMMYT-Mexico
inefficient
C11
CML247//CML254
CIMMYT-Mexico
efficient
C12
CML78/CML373
CIMMYT-Mexico
efficient
C13
CML264/CML311//CML334
CIMMYT-Mexico
inefficient
C14
CML442/CML444//[MSRXPL9]C1F2-205-1(OSU23i)-1-1-X-1-X-B-B
CIMMYT-Kenya
efficient
C15
LPSC4F273-2-2-1-B-B-B/CML202//CML384
CIMMYT-Kenya
efficient
C16
CML312/CML247//CML78
CIMMYT-Kenya
efficient
 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Schulte auf’m Erley, Begum, Worku, Bänziger, Horst
J. Plant Nutr. Soil Sci. 2007, 170, 106–114
N-uptake kinetics were determined prior to harvest 2 according to Claassen and Barber (1974). The plant roots were
washed in distilled water, and the plants were transferred to
5 L pots filled with nutrient solution containing 150 lM
Ca(NO3)2. Samples 10 mL of nutrient solution were removed
every 15 min and after 2 h every 10 min until no further
decrease in the nitrate concentration could be detected.
Nitrate concentration of the samples was measured using an
autoanalyzer (Technicon Autoanalyzer II, Bran+Lübbe, Norderstedt, Germany). The depletion of nitrate (Q) over time (t)
is proportional to the nitrate-uptake rate I (influx rate)
[pmol cm–1 s–1] and inversely proportional to the root length
L [cm]. The influx rate is dependent on the ion concentration
in the solution and can be described by MichaelisMenten kinetics using the following three parameters: Imax
[pmol cm–1 s–1], Km [lM], and E [pmol cm–1 s–1]; Imax
describes the maximum uptake rate under saturated nutrient
concentration, Km is the nutrient concentration where the
actual uptake equals half of Imax, and E is the efflux, the
movement of ions out of the roots into the external solution.
Thus, the following equation can be used to describe the ion
depletion from the solution with a constant volume (v):
"
#
dQ
Imax Q=v
ˆ L
E
(1)
dt
Km ‡ Q=v
Table 2: Simple linear correlations of leaf-senescence score at
anthesis, 14 d after anthesis and 28 d after anthesis with grain yield
under low-N stress conditions in Kenya in 2003 and Zimbabwe in
2003 and 2004 for 16 maize cultivars. + , *, ** = significant at p < 0.1,
0.5, and 0.01, respectively (n = 16).
Equation 1 was integrated numerically, and Imax and Km were
obtained using a Newton iteration scheme by the SAS program (Seidel and Hothorn, pers. commun. 2003).
Root length was measured at harvest 2. The fresh roots were
cut into 1–2 cm pieces, mixed in water, and collected on a
sieve. Three subsamples (each of 2–3 g on weight basis)
were taken. The samples were carefully spread in a thin layer
of water (2 mm) on a transparent tray, avoiding an overlapping of individual pieces, and scanned. Root length was estimated by the software WinRHIZO-2003 (Regent Instruments,
Canada). From these subsamples, total root length was calculated taking the total fresh weight of the root system into
account.
Nondestructive measurements of the chlorophyll contents of
leaf 5 (counted from the base of the plants) were taken by a
portable chlorophyll meter (SPAD-502, Minolta, Japan).
Three readings per leaf were taken. The measurements were
repeated on the same leaves every 2–3 d between 23 and
37 DAG.
Photosynthesis rates were measured using a portable gasexchange system (LI-6400, LI-COR, Lincoln, NE, USA) on three
dates between 23 and 37 DAG on leaf 6 counted from the base
of the plants. Photosynthetic photon-flux density (1500 lmol
m–2 s–1) and the incoming CO2 concentration (400 lmol
mol–1) were held constant during the measurements.
2.3 Field data
Data on grain yields, total biomass, and leaf-senescence
scores under low-N stress conditions in the field were taken
from Worku (2005). A detailed description of the experiments
can be found there. Briefly, the experiments were conducted
at the CIMMYT research station at Harare, Zimbabwe (2003
 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Date of senescence-score
evaluation
Kenya
Zimbabwe
2003
2003
2004
Anthesis
–0.26
–0.47+
–0.13
14 d after anthesis
–0.38
–0.60*
–0.48+
28 d after anthesis
–0.50*
–0.74**
–0.44+
and 2004) and at the Kenya Agricultural Research Institute
substation at Kiboko (2003). The cultivars were tested under
three N levels (low, medium, and high N) at both sites. Low-N
stress conditions in the field experiments were attained by
using nonfertilized plots previously depleted of nitrogen.
Phosphorus and potassium were applied uniformly based on
the recommendation of each center prior to planting. The
trials at Harare were irrigated to field capacity at planting
using sprinkler irrigation. A second irrigation of 20–30 mm
was applied 6–7 d after planting to facilitate germination.
Thereafter, trials were irrigated to field capacity whenever soil
moisture was less than 40% of field capacity. Similar procedures were followed for trials at Kiboko. A plot size of 4 m
length by 4.5 m width with six rows per plot was used.
Spacing was 0.75 m and 0.25 m between rows and plants,
respectively. A plant density of 53,333 plants per hectare was
kept after thinning. Leaf-senescence score was estimated on
a scale of 0 (0% of the plot leaf-area senescent) to 10 (100%
of the plot leaf-area senescent) at anthesis and 14 and 28 d
after anthesis.
For the determination of grain yield and total aboveground
biomass, an area of 5.65 m2 corresponding to 32 plants in
the central four rows was harvested immediately after physiological maturity.
2.4 Statistical analysis
Statistical analysis of the data was performed using the
PROC GLM procedure of SAS (SAS Institute, 2003) followed
by a Tukey test for estimation of the least-significant differences between means. Pearson correlation coefficients were
calculated using the PROC CORR procedure of SAS.
3 Results
3.1 Leaf-senescence scores in the field
experiments
In the field experiments, significant correlations between leafsenescence scores and grain yield under low-N stress (N efficiency) were found (Tab. 2). With the progress in leaf senescence after anthesis, both the height and the levels of significance of the correlation coefficients increased. The closest
relationships were found for the leaf-senescence scores 28 d
after anthesis in all three environments. Since Additive Main
effect and Multiplicative Interaction (AMMI) analysis (Ebdon
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J. Plant Nutr. Soil Sci. 2007, 170, 106–114
Leaf senescence induced by N deficiency as indicator of N efficiency 109
and Gauch, 2002) revealed that the Kenya environment was
very different from Zimbabwe (Worku, 2005), the relationship
for the individual cultivars is only shown for the means of the
Zimbabwe experiments (Fig. 1). Most of the N-efficient cultivars had lower leaf-senescence scores than the inefficient
ones. However, for some of the efficient cultivars (C1, C2,
C5, and C14), leaf-senescence scores were in the same
range as for the inefficient ones (Fig. 1).
at both harvests (r = 0.90*** and r = 0.94*** for harvest 1 and
harvest 2, respectively). Nitrogen uptake between the two
harvests was very low and even negative in some cases suggesting that the variation between plants was greater than
between harvests. Therefore, N-uptake rates between the
two harvests could not be quantified meaningfully.
4.5
Cultivars differed significantly in shoot– as well as in root–dry
matter production at both harvests (Tab. 3, p < 0.001). The
shoot–dry matter production was correlated between the two
harvests (r = 0.64**), suggesting that the shoot–dry matter
production up to the first harvest under high-N treatment influenced the shoot dry matter at the end of the N depletion period at harvest 2. The shoot–dry matter increment between
the two harvests was not related to the shoot dry matter at
harvest 1. Neither the shoot dry matter at harvest 1 or 2 nor
the shoot–dry matter increment between the two harvests
were related to the vegetative dry-matter production under
low-N stress at anthesis in the field experiments in Zimbabwe
and Kenya (results not shown).
Shoot nitrogen uptake also differed significantly between cultivars (Tab. 3) and was highly correlated to shoot dry matter
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
4.0
Grain yield (t ha-1)
3.2 Plant dry matter and N uptake in the nutrientsolution experiment
3.5
3.0
2.5
y = 6.67 - 0.69x
r2 = 0.47**
2.0
1.5
3
4
5
6
7
Leaf senescence score
Figure 1: Relationship between mean grain yield and leaf
senescence score 28 d after anthesis of 16 maize cultivars grown
under low-N stress in field experiments in Zimbabwe in 2003 and
2004. Open symbols represent N-efficient cultivars, while closed
symbols represent N-inefficient cultivars. ** = significant at p < 0.01.
Table 3: Mean shoot dry matter, shoot nitrogen, and root dry matter of 16 maize cultivars grown in nutrient solution with unlimited N supply until
harvest 1 (23 DAG) and then gradually depleted in N until harvest 2 (37 DAG). Cultivars are ranked in order of their N efficiency according to
Worku (2005). LSD: least significant difference.
Cultivar
Harvest 1
Harvest 2
Shoot
Dry matter
(g plant–1)
Root
Shoot
Root
Nitrogen
(mg plant–1)
Dry matter
(g plant–1)
Dry matter
(g plant–1)
Nitrogen
(mg plant–1)
Dry matter
(g plant–1)
C16
7.1
292.4
1.4
27.1
297.6
6.3
C12
8.6
311
2.7
30.5
308.9
6.9
C2
9.4
359.3
2.2
41.5
404.5
6.7
C15
6.9
273.2
1.2
24.4
272.3
4.9
C9
7.2
282.9
1.6
33.5
323.3
4.8
C5
11.8
413.7
2.5
35.6
367.9
5.7
C1
11.8
362.7
2.5
34.1
319.6
5.9
C6
10.2
381.7
1.9
39.7
380.2
5.4
C11
5.7
252.3
0.9
24.9
277.1
4.3
C14
8.4
363.2
1.5
36.4
365.1
6.1
C3
10.6
380.8
1.7
31.3
332.7
5.8
C4
7.9
328.9
1.3
31.3
325.9
4.8
C7
6.9
294.6
1.3
29.5
324.1
5.9
C8
8.6
349.2
1.8
34.1
330.2
6.4
C13
8.8
365.2
1.4
36.6
336.3
4.8
C10
7.1
297.4
1.4
23.9
288.3
5.8
LSD0.05
3.7
134.1
1.3
9.2
94.3
2.2
 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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110
Schulte auf’m Erley, Begum, Worku, Bänziger, Horst
J. Plant Nutr. Soil Sci. 2007, 170, 106–114
3.3 Leaf chlorophyll content
50
50
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
SPAD value
40
30
20
10
0
24
26
28
30
32
34
36
38
Days after germination
Figure 2: SPAD values of leaf 5 of 16 maize cultivars grown in
N-depleted nutrient solution from 25 to 36 DAG. Open symbols
represent N-efficient cultivars while closed symbols represent
N-inefficient cultivars.
The SPAD values at 31 DAG and leaf-senescence score at
anthesis in the field experiments were significantly negatively
correlated (Tab. 4, Fig. 4A). The relationship between SPAD
and leaf-senescence score in the field 14 and 28 d after
anthesis, however, was not significant. Correlations between
SPAD values from the nutrient-solution experiment and grain
Table 4: Simple linear correlations of the SPAD value of leaf 5 of 16
maize cultivars grown in N-depleted nutrient solution 31 DAG with
leaf-senescence score at anthesis, 14 and 28 d after anthesis and
grain yield under low-N stress conditions in the field experiments in
Kenya in 2003 and Zimbabwe in 2003 and 2004. + , * = significant at
p < 0.1 and 0.5, respectively (n = 16).
Date of senescencescore evaluation
Kenya
2003
2003
2004
Anthesis
–0.52*
–0.50*
–0.43+
14 d after anthesis
–0.44+
–0.35
–0.46+
28 d after anthesis
–0.28
–0.21
–0.48+
0.26
0.27
–0.08
Grain yield
Zimbabwe
 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
SPAD value
40
30
20
10
0
0.0
0.1
0.2
0.3
0.4
0.5
-2
Chlorophyll (g m )
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
50
B
y = 8.42 + 27.95x
r2 = 0.55**
40
SPAD value
N deficiency–induced leaf senescence was monitored by
changes in the leaf chlorophyll content as indicated by nondestructive SPAD readings and by photosynthesis-rate
measurements. Values of SPAD of leaf 5 dropped for all cultivars after beginning of the low-N treatment on day 23 (Fig. 2).
Apart from the last evaluation date (36 DAG), cultivar differences were significant on all days (p < 0.01 25 DAG, p <
0.001 later on). At 31 DAG, the mean SPAD value over all
cultivars had dropped to about half of the initial value. The
SPAD values at this date were chosen as a measure for leaf
senescence because cultivar differences in SPAD at this date
reflected most clearly their state of leaf senescence. The
SPAD values were significantly correlated with chlorophyll
(Fig. 3A) and nitrogen (Fig. 3B) contents of the same leaves
over all cultivars. Therefore, the SPAD readings were not corrected for cultivar differences.
A
y = 13.72 + 50.18x
r2 = 0.66***
30
20
10
0
0.0
0.2
0.4
0.6
0.8
1.0
-2
Specific leaf N (g m )
Figure 3: Relationship between SPAD values of 16 maize cultivars
grown in N-depleted nutrient solution and chlorophyll content (A) and
between SPAD values and specific leaf nitrogen content (B) for leaf 8
37 DAG. Open symbols represent N-efficient cultivars, while closed
symbols represent N-inefficient cultivars. **, *** = significant at p <
0.01 and 0.001, respectively.
yield under low-N stress in the field could not be established
(Tab. 4). This was partly due to the fact, that two of the N-efficient cultivars (C1 and C2) were characterized by high leafsenescence scores and thus displayed very low SPAD values
also in nutrient solution (Fig. 4A and B).
3.4 Photosynthesis rates
Photosynthesis rates for leaf 6 declined with time for all cultivars after starting the low-N treatment in the nutrient-solution
experiment. Photosynthesis rates and SPAD values of the
cultivars were significantly positively correlated over the
whole period (r = 0.90***) (Fig. 5). However, calculation of the
correlations for the individual measuring periods revealed
that there was no correlation before the onset of leaf senescence (24 DAG) and that the correlation became increasingly
close with progressing senescence (31 and 36 DAG).
The cultivars differed in their photosynthesis rate before the
onset of leaf senescence 24 DAG (p < 0.05) and more pronounced during leaf senescence 31 and 36 DAG (p < 0.001).
The cultivar differences in photosynthesis rates were not the
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Leaf senescence induced by N deficiency as indicator of N efficiency 111
Photosynthesis rate (µmol m s )
J. Plant Nutr. Soil Sci. 2007, 170, 106–114
-1
40
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
SPAD value
30
25
20
15
10
y = 57.6 - 23.2x
r2 = 0.40**
5
0
0
1
2
-2
A
35
3
Leaf senescence score
40
B
35
24 DAG:
y = 3.19 + 0.51x
r2 = 0.12ns
25
20
31 DAG:
y = -1.14 + 0.48x
r2 = 0.54**
15
10
36 DAG:
y = -0.22 + 0.30x
r2 = 0.77***
5
0
0
10
20
30
40
50
SPAD value
30
SPAD value
25
Figure 5: Relationship between photosynthesis rate of 16 maize
cultivars grown in N-depleted nutrient solution and SPAD values of
leaf 6 24 DAG (circles), 31 DAG (triangles), and 36 DAG (diamonds).
Open symbols represent N-efficient cultivars, while closed symbols
represent N-inefficient cultivars. ns, **, *** = non-significant,
significant at p < 0.01 and 0.001, respectively.
20
15
10
y = 12.7 + 2.1x
2
r = 0.02ns
5
0
0
1
2
3
4
5
-1
Grain yield (t ha )
Figure 4: Relationship between SPAD value of leaf 5 of 16 maize
cultivars grown in N-depleted nutrient solution 31 DAG and mean
leaf-senescence score at anthesis (A) and grain yield (B) under low-N
stress in field experiments in Zimbabwe in 2003 and 2004. Open
symbols represent N-efficient cultivars, while closed symbols
represent N-inefficient cultivars. ns = non-significant, ** = significant
at p < 0.01.
same before and after the onset of leaf senescence. Photosynthesis rates at 24 DAG were not correlated with photosynthesis rates at 31 and 36 DAG while photosynthesis rates
at 31 and 36 DAG were significantly correlated (r = 0.56*).
Photosynthesis rates during leaf senescence from the nutrient-solution experiment showed significant correlations with
Table 5: Simple linear correlations of photosynthesis rate of leaf 6 of
16 maize cultivars grown in N-depleted nutrient solution 31 DAG with
leaf-senescence score at anthesis, 14 and 28 d after anthesis and
grain yield under low-N stress conditions in the field experiments in
Kenya in 2003 and Zimbabwe in 2003 and 2004 for 16 maize
cultivars. + , *, ** = significant at p < 0.1, 0.5 and 0.01, respectively (n = 16).
Date of senescencescore evaluation
Kenya
Zimbabwe
2003
2003
2004
Anthesis
–0.53*
–0.29
–0.50*
14 d after anthesis
–0.70**
–0.52*
–0.50*
28 d after anthesis
–0.51*
–0.41
–0.46+
Grain yield
0.59*
0.57*
0.17
 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the leaf-senescence scores in the field (Tab. 5, Fig. 6A). The
closest relationship was found with the leaf-senescence
score at 14 d after anthesis for all environments, but significant relationships were also found with leaf-senescence
score at anthesis and 28 d after anthesis for some of the
environments (Tab. 5). Most of the N-inefficient cultivars with
a high leaf-senescence score in the field also showed a low
photosynthesis rate in the nutrient solution (Fig. 6A).
Photosynthesis rate during leaf senescence also correlated
significantly with grain yield under low-N stress except for
Zimbabwe in 2004 (Tab. 5). A high photosynthesis rate was
found for most of the N-efficient cultivars (Fig. 6B). However,
some of the N-efficient cultivars (among them C1 and C2)
had low photosynthesis rates.
3.5 Root characteristics
In the nutrient-solution experiment, root length and the kinetic
parameters of N uptake, maximum uptake rate (Imax), and
Michaelis-Menten constant (Km) were determined (Tab. 6).
Significant cultivar differences in root length were found in the
analysis of variance, but mean comparisons did not reveal
higher or lower root lengths for individual cultivars. There
were significant cultivar differences in Imax (p < 0.05) with cultivar C13 having higher Imax values than the cultivars C7 and
C10 (Tab. 6). However, significant differences between N-efficient and N-inefficient cultivars were not found. The determined Imax values were not related to biomass N uptake at
anthesis or maturity under low-N stress. Cultivar differences
in Km could not be found.
www.plant-soil.com
Photosynthesis rate (µ mol m-2 s-1)
112
Schulte auf’m Erley, Begum, Worku, Bänziger, Horst
20
A
18
16
14
12
10
y = 26.1 - 2.91x
2
r = 0.28*
8
0
1
2
3
4
5
6
7
Photosynthesis rate (µmol m-2 s-1)
Leaf senescence score
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
20
B
y = 6.1 + 2.6x
r2 = 0.21+
18
J. Plant Nutr. Soil Sci. 2007, 170, 106–114
Table 6: Mean root length, maximum nitrate influx (Imax), and
Michaelis-Menten constants (Km) of 16 maize cultivars grown in
N-depleted nutrient solution 37 DAG. Cultivars are ranked in order of
their N efficiency according to Worku (2005). LSD: least significant
difference.
Cultivar
Root length
(m plant–1)
Imax
(pmol cm–1 s–1)
Km
(lM)
C16
369
2.47
11.21
C12
391
1.94
16.05
C2
316
2.16
13.82
C15
297
2.52
9.58
C9
291
2.33
8.03
C5
377
2.7
4.5
C1
307
2.21
4.81
C6
358
2.27
5.7
C11
307
2.09
3.59
16
C14
362
2.25
5.58
14
C3
316
2.62
2.85
C4
291
2.66
2.83
C7
467
1.76
10.83
C8
357
2.07
12.35
C13
344
3.22
25.1
C10
444
1.8
LSD0.05
178
1.38
12
10
8
0
1
2
3
4
5
Grain yield (t ha-1)
Figure 6: Relationship between photosynthesis rate of leaf 6 of 16
maize cultivars grown in N-depleted nutrient solution 31 DAG and
mean leaf-senescence score 14 d after anthesis (A) and grain yield
(B) under low-N stress in field experiments in Zimbabwe in 2003 and
2004. Open symbols represent N-efficient cultivars, while closed
symbols represent N-inefficient cultivars. +, * = significant at p < 0.1
and 0.05, respectively.
4 Discussion
The investigated cultivars showed a close relationship between leaf-senescence score after anthesis and grain yield
under low-N stress (Tab. 2). Therefore, a causal positive relationship between delayed leaf senescence and N efficiency
may exist, even though some of the N-efficient cultivars were
able to attain a high grain yield with comparably high leafsenescence scores (Fig. 1). For maize, genetic variation in
leaf senescence exists and has been exploited in crop improvement (Thomas and Smart, 1993). Many recent hybrids
show delayed leaf senescence (stay green), which is connected to high rates of dry-matter accumulation during reproductive growth (Tollenaar, 1991; Tollenaar and Aguilera,
1992), a more active root system, and greater stress tolerance (Tollenaar and Wu, 1999). Drought-tolerant and N-efficient cultivars also showed delayed leaf senescence (Bänziger et al., 2002). A delayed leaf senescence during grain filling enhances the photosynthetic capacity of the plants,
especially when it is limited under stress conditions (Wolfe et
al., 1988). Better assimilate availability is favorable for grain
 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
9.04
ns
formation, but also for root growth, since roots are mainly
supplied by the lower leaves in the canopy (Osaki, 1995), so
that N uptake after anthesis and N supply to the kernels are
also enhanced.
The maize cultivars differed in leaf senescence under low-N
conditions both in the field and under N deficiency in the
short-term nutrient-solution experiment, and those differences were correlated. This suggests that genotypic differences in the susceptibility of leaf senescence induced by N
deficiency exist. No indications were found, that the cultivar
differences in leaf senescence in the nutrient-solution experiment were related to their growth characteristics during the
experiment. Neither SPAD values nor photosynthesis rates of
the cultivars during leaf senescence were significantly correlated to their shoot dry matter, shoot nitrogen, or the dry-matter increment between the two harvests. The molecular
mechanisms of the induction of leaf senescence during N
deficiency, that may explain the cultivar differences, are not
well understood. A higher accumulation of sugars in the
leaves or a lower supply of the leaves with cytokinins and/or
nitrate with the transpiration stream may play a role (Ono et
al., 2001 and references therein). Paponov and Engels
(2003) found higher photosynthesis rates during grain filling
under low-N conditions for an N-efficient compared to an inefficient maize cultivar, which was related to lower leaf contents
of reducing sugars and sucrose. Crafts-Brandner et al.
(1984) investigated maize cultivars differing in leaf senescence and speculated that the nitrate flux to the leaf was a
www.plant-soil.com
J. Plant Nutr. Soil Sci. 2007, 170, 106–114
Leaf senescence induced by N deficiency as indicator of N efficiency 113
factor regulating the differing rates of leaf senescence. Pons
and Bergkotte (1996) suggested that leaf senescence after
shading may be induced by a lower transpiration stream into
the leaf causing lower cytokinin levels. A similar mechanism
may occur under N deficiency, since stomatal conductance of
the leaves and thus also the transpiration stream is reduced
under N stress (Chapin et al., 1988; Broadley et al., 2001).
Leaf chlorophyll contents are usually used as measures of
leaf senescence (Peñarrubia and Moreno, 2002). However,
leaf chlorophyll contents during senescence may not reflect
the maintenance of the photosynthetic capacity of the leaf
(Thomas and Smart, 1993). Therefore, both relative chlorophyll contents (SPAD) and photosynthesis rates were measured to monitor leaf senescence in the nutrient-solution
experiment. Values of SPAD at 31 DAG correlated with leaf
senescence at anthesis in the field, but not with leaf senescence at 14 or 28 d after anthesis except for Zimbabwe 2004
(Tab. 4). Therefore, it seems that leaf-senescence scores at
anthesis and SPAD values in the nutrient solution experiment
mainly reflected genotypic differences at the onset of leaf
senescence induced by N deficiency. Since leaf-senescence
scores after anthesis were not strongly related to SPAD values in the nutrient-solution experiment, the development of
leaf senescence after anthesis was probably influenced by
additional parameters. These parameters may include the
N-uptake rate or the source-to-sink ratio during grain filling
(Rajcan and Tollenaar, 1999a). Leaf senescence at 14 and
28 d after anthesis except for Zimbabwe 2004, however,
were more decisive for N efficiency than leaf senescence at
anthesis (Tab. 2). Therefore, not only the susceptibility of the
cultivars to N deficiency–induced induction of leaf senescence, but also other parameters influencing leaf senescence
seem important for high N efficiency under field conditions.
Photosynthesis rate at 31 DAG was more closely related to
leaf-senescence scores in the field after anthesis than the
SPAD values (Tab. 5). Also significant correlations with grain
yield under low-N conditions in the field could be established,
even though photosynthesis rate did not in all environments
correlate with leaf-senescence scores 28 d after anthesis,
which were most strongly related to grain yield (Tab. 2,
Fig. 1). Differences in the photosynthetic capacity of older
senescing leaves, which are not visible at the early stages of
senescence, may have influenced the further development of
leaf senescence after anthesis. A high photosynthesis rate of
older senescing leaves increases the assimilate availability
for the roots and may thus lead to a high N uptake during
grain filling (Tolley-Henry et al., 1988; Osaki, 1995), which
additionally delays leaf senescence. Leaf-senescence scores
28 d after anthesis may have been also influenced by other
cultivar differences in N uptake or the source capacity of the
cultivars. The impact of delayed leaf senescence on N uptake
may also not be the same for all cultivars.
Genotypic differences in the response of N uptake to assimilate supply or in N-uptake parameters per se may thus also
contribute to N efficiency. Particularly the N-efficient cultivars
C1 and C2, which did not show delayed leaf senescence,
were characterized by a high N uptake between anthesis and
maturity (data not shown). But cultivar differences in physio 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
logical root parameters (Imax and Km), which may explain differences in N uptake and N efficiency were not found in the
nutrient-solution experiment (Tab. 6). However, these parameters are influenced by the energy supply to the roots
(Engels and Marschner, 1995), which may be different from
the energy supply during grain filling under field conditions.
Differences in the genotypic response of the N uptake to
assimilate availability may be due to genotypic differences in
root morphology. For a high N uptake during grain filling, a
high root-length density in the subsoil is particularly important
(Wiesler and Horst, 1994). Besides a high root growth during
later stages of development, also genotypic differences in
root morphology may lead to variations in root-length density
in the subsoil. While no relationship could be established
between N efficiency of maize and root-system size measured by root-capacitance readings (van Beem and Smith,
1997), a positive correlation was found between N efficiency
and root size as determined by vertical-root-pulling resistance
(Kamara et al., 2003), which may better reflect not just the
root-system size but also the rooting depth of the cultivars.
Similar results were found by Worku (2005). A N-inefficient
tropical maize cultivar had a greater total root-system size
than a N-efficient cultivar, but a lower root-length density in
the subsoil, leading to a lower N depletion in the subsoil. In
nutrient-solution experiments, Wang et al. (2004) found that
N-efficient maize cultivars could be characterized by a longer
root system.
In conclusion, leaf senescence seems to be a suitable selection criterion for N-efficient tropical maize cultivars. Rather
than leaf chlorophyll content, the photosynthetic capacity of
senescing leaves correlated with the N efficiency of the cultivars. A more detailed characterization of the photosynthetic
processes during leaf senescence may allow isentification of
more precise physiological or molecular markers for the performance of cultivars under low-N conditions in the field. It
may also be attractive to find additional selection traits
regarding root-morphological parameters for the selection of
N-efficient cultivars, which are not characterized by delayed
leaf senescence.
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