Folie 1

1
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
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 18C,
night 16C. Temperature logged using mobile
temperature logger at frequency 1/30 min, to
obtain temperature sum (base temperature 1C).
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), x0half-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.
Nitrogen levels 2, 8, 12 mM
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)
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").
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
b

• 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.

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. 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 1,58SPADLeaf4_35d 1,20 SPADLeaf5_35d
1,32SPADLeaf5_42d 1,79LenLeaf2_35d (R2
80,0) GSleaf2 - 88,4 1,33SPADLeaf5_35d
1,67 LenLeaf4_35d 1,75SPADLeaf3_35d -
0,826LenLeaf3_42d (R2 62,6) GSleaf4 102
1,04SPADLeaf2_42d - 2,54LenLeaf1_28d
0,828SPADLeaf1_42d 5,71FWStem_35d - 2,03
SPADLeaf1_21d (R2 81,4)

• Fig. 4 (below) Meristem length.
• Apical meristems of DOM were shorter than
  those of REC, throughout all N treatments and at
  all pheno-
• logical stages.
• Meristem length increased with N
  concentration, particularly visible after 28 days.

Fig. 2 (below) Sigmoid curve with three
parameters (a, b, x0) fitted to blade length
extension data (see MM).

• Summary
• 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.

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, GN 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, GN
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).
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.