TP 3
The Oregon Wolfe Barley parental lines as a source for the study of
nitrogen use efficiency in barley.
I. Statistical analysis (Cooperation between subprojects 3 and 9)
Sang-Il Kim1, Steffen Amme2, Gerhard Buck-Sorlin2,3, Hans-Peter Mock2, Winfried Kurth3
1Pyongyang
Crop Genetic Resources Institute, Academy of Agricultural Sciences, Duru-dong, Sadong District, Pyongyang, D.P.R. of Korea.
2Institute of Plant Genetics, Dept. of Molecular Plant Physiology, Corrensstrasse 3, D-06466 Gatersleben, Germany
3Brandenburg Technical University (BTU), Chair for Practical Informatics / Graphical Systems, P.O.Box 101344, D-03013 Cottbus, Germany
Introduction
Nitrogen is an element of central importance for plants. It is essential for growth and development as is signified by its presence in proteins, nucleic acids and
chlorophyll. Nitrogen is assimilated by plant roots mostly in the form of ammonium and nitrate. Crop yield is largely dependent on nitrogen availability.
Nitrogen use efficiency (NUE): the level of nitrogen used by the plant under a given nitrogen input. Under limited nitrogen supply, a plant with a higher NUE is
expected to produce a higher yield than other plants with lower ones.
Barley
DOM / REC
Nitrogen levels
2, 8, 12 mM
Materials and Methods
Plant material: Two contrasting spring barley genotypes: ‘DOM’ (two-rowed) and ‘REC’ (six-rowed): parents of mapping population 'Oregon Wolfe
Barley'.
Growth conditions: Greenhouse (day-length 16 hrs, light intensity 300µmol m-2 s-1, temperature day 18°C,
night 16°C. Temperature logged
using mobile temperature logger at frequency 1/30 min, to obtain temperature sum (base temperature 1°C). Plants irrigated daily with a nutrient solution
(Geiger et al., 1999): solution containing either 2mM, 8mM or 12mM of NH4NO3.
Comparison of leaf growth rates:
No. of plants restricted to 12 plants/treatment. Extension of leaf blades and sheaths usually follows
a sigmoid function (Buck-Sorlin 2002):
where a = asymptote of function (max. organ length), b = slope (extension speed: cm/°Cd), x0=half-value of function
(characterizes timing
of extension).
Lengths of blades and sheaths of main culms of twelve plants were measured daily, repetitively on the
same organs and thus
non-destructively.
Plant and meristem harvest: At 14, 21, 28, 35 and 42 days after sowing, entire plants were harvested. Every leaf of a plant was separated
from the main stem and herbarium specimens prepared. Apical meristems were cut under binocular microscope and preserved in alcohol (100%).
Preserved specimems were stained in astra blue/safranin. Images were taken under a dissecting binocular microscope along with appropriate scale
(1 mm) using a digital camera. Length and basal diameter of meristems determined using image analysis software ("Korn").
Morphological
Study
Biochemical
Study
- Growth rate and phenology
of leaves (reparameterisation
of model)
- Development of apical
meristems
- Leaf biometry (herbarium
specimens)
- Analysis of key enzymes of
C/N-metabolism (different
tissues, developmental stages)
as a function of N-supply
- Western blotting for the study
of expression levels of enzymes.
Testcase NUE:
Calibration and validation of
Integrated Ecophysiological
Barley Crop Model
(contribution to VCMS)
Statistical analysis: ANOVA (incl. Tukey test of pairwise comparison) and multiple regression, were carried out using Minitab v. 12.1 software. Sigmoid models were fitted using Sigmaplot 2001, v. 7.0 (SPSS Inc.)
Western Blotting, Enzyme assay (Glutamine synthetase) : see separate poster of subproject 3
Results:
a
Fig. 2 (below): Sigmoid curve with three
parameters (a, b, x0) fitted to blade length
extension data (see M&M).
b
90
80
GS, leaf 2, 42 DAS
Fresh weight (FW) (Table 1, left):
• Genotype (G) effects: Increasing effect of G
for stem (from 28 DAS), tillers (on 42 DAS)
and leaf 1 (21 to 35 DAS). Decreasing effect
for leaves 2, 5 and 6. Equally significant effect
for leaf 4. No significant effect of G on root
FW.
• Nitrate (N) effects: Increasing effect of N for
stem (28 to 35 DAS), (very strong for) tillers
(28 to 42 DAS), leaves 1, 2, (3, 4), 5, 6.
• G * N interactions: FW of leaves 3 and 6
(both at 35 and 42 DAS). Single significant
interactions for root and main stem FW (14
DAS), tillers (35 DAS), leaves 4 (35 DAS)
and 7. No significant interactions for leaves 1,
2 and 5.
Y = 40,1992 + 1,14422X
R-Sq = 54,7 %
Fig. 1a (above): ANOVA of GS
concentration (amount of protein from
Western Blot analysis) at last harvest
(42 DAS) in leaf 1: significant
influence of low N level (2 mM) in
both genotypes on the GS level. Same
observation for leaves 2 and 4 (though
in rank 4, GS levels were only low in
DOM, 2 mM).
Fig. 4 (below): Meristem length.
• Apical meristems of DOM were shorter than those of
REC, throughout all N treatments and at all phenological stages.
• Meristem length increased with N concentration,
particularly visible after 28 days.
70
60
50
40
30
20
0
10
20
30
SPAD, Leaf 1, 42 DAS
Fig. 1b (above): Linear regression between final (42
DAS) GS level (leaf 2) and SPAD value of leaf 1.
Multiple regressions between GS levels (leaf 1, 2, and
4) and biometric parameters:
GSleaf1 = -146 + 1,58*SPADLeaf4_35d + 1,20 *
SPADLeaf5_35d + 1,32*SPADLeaf5_42d + 1,79*LenLeaf2_35d
(R2 = 80,0%)
GSleaf2 = - 88,4 + 1,33*SPADLeaf5_35d + 1,67 *
LenLeaf4_35d + 1,75*SPADLeaf3_35d - 0,826*LenLeaf3_42d
(R2 = 62,6%)
GSleaf4 = 102 + 1,04*SPADLeaf2_42d - 2,54*LenLeaf1_28d
+ 0,828*SPADLeaf1_42d + 5,71*FWStem_35d - 2,03 *
SPADLeaf1_21d (R2 = 81,4%)
Summary
Leaf blade extension (Fig. 2, 3, Table 2):
a (maximum length): G effect in ranks 2, 4, 5 and 6, N effects in all but rank 2, G*N interaction in all ranks. Longest
blades in DOM-8mM (rank 2), DOM-2 (ranks 3, 4), REC-12 (ranks 5, 6, 7).
b (extension speed): G effects in all ranks but rank 2, N effects in ranks 4, 6 and 7, G*N interactions in all ranks but rank
2. Fastest blade extension in REC-12 (rank 2), REC-2 (ranks 3, 6, 7), DOM-12 (ranks 4, 5).
x0 (extension time): Very strong G effect in rank 2, decreasing up to rank 4. Significant N effects in ranks 2, 4, 6 and 7.
(Weak) G * N interaction only in rank 3. Earliest leaf blade extensions in REC-2 (all ranks)  low N levels caused
earliness of extension (also tendency in DOM).
Fig. 3 (below): Time series of average data points for each genotype-treatment combination (blades and sheaths),
illustrating the ANOVA (Table 2).
REC 2 mM: leaf blades and sheaths
35
5
6
4
blade
sheath
20
2
8
15
10
7
6
5
43
2 8
5
0
0
100
200
300
400
500
600
700
800
3
2
6
7
8
76
20
10
85
4
23
900
100
200
300
400
500
600
700
800
sheath
7
7
8
8
10
sheath
0
600
700
800
900
20
7
6
5
42
3
100
200
300
400
500
600
700
Temperature sum (°C d)
500
600
700
800
900
800
5
34
sheath
20
10
0
400
blade
0
500
300
25
2
7
6
400
200
DOM 12 mM: leaf blades and sheaths
6
5
4
3
blade
5
3,24
300
100
Temperature sum (°C d)
30
Length (cm)
20
Temperature sum (°C d)
0
900
DOM 8 mM: leaf blades and sheaths
4
5
3
6
2
blade
200
7
10
Temperature sum (°C d)
30
100
8
0
0
DOM 2 mM: leaf blades and sheaths
0
7
2
20
6
5
32
8 4
0
Temperature sum (°C d)
Length (cm)
3
sheath
5
4
6
blade
sheath
30
Length (cm)
Length (cm)
blade
7
3
25
4
5
30
Length (cm)
30
REC 8 mM: leaf blades and sheaths
Length (cm)
REC 12 mM: leaf blades and sheaths
21d
900
6
2
15
76
7, 8
8
10
5
5
42
3
0
0
100
200
300
400
500
600
700
800
900
28d
• The overall objective of the research project
‘Morphological, phenological, physiological and
genetical characterization of contrasting genotypes
of barley (Hordeum vulgare L.)’ is the
establishment – within the frame of the research
group "Virtual Crops" – of an ecophysiological crop
growth model integrating a number of correlated
data sets with expert knowledge of plant physiology
and morphology.
• The influence of nitrogen on two contrasting
spring barley genotypes, the two-rowed ‘DOM’,
and the six-rowed ‘REC’, at different
developmental stages was tested, by determining
fresh weights, chlorophyll content, extension
phenology of leaf blades and sheaths, lengths and
growth stage of apical meristems, as well as protein
expression of glutamine synthetase by western
blotting.
• Treatment with a nutrient solution containing
different levels of NH4NO3 lead to phenological
differences between the two contrasting spring
barley genotypes. Higher nitrogen levels increased
fresh weight of tillers, length of meristems and
protein content of glutamine synthetase.
• The two contrasting genotypes exhibited
different reaction patterns to nitrogen supply.
Overall, good correlations between morphological,
phenological and physiological parameters were
found.
Temperature sum (°C d)
References: Buck-Sorlin, G.H. (2002): L-System Model of the Vegetative Growth of Winter Barley (Hordeum vulgare L.). In: D.Polani, J.Kim, T.Martinetz (Eds.): Fifth German Workshop on Artificial Life. March 18-20, 2002, Lübeck, Germany. pp.
53-64. Akademische Verlagsgesellschaft Aka GmbH, Berlin
Geiger, M., Haake, V., Ludewig, F., Sonnewald, U., Stitt, M. (1999): The nitrate and ammonium nitrate supply have a major influence on the response of photosynthesis, carbon metabolism, nitrogen metabolism and growth to elevated carbon dioxide in
tobacco. Plant Cell Environment 22: 1177-1199.
Acknowledgments: Sang-Il Kim was kindly supported by InWent programme AB044 during his stay at IPK Gatersleben. Technical support by Petra Linow is gratefully acknowledged. The third author thanks IPK, in particular Dr. Patrick
Schweizer, for providing office facilities while being a guest researcher at the institute.