Journal of Experimental Botany, Vol. 52, No. 362, pp. 1827–1833, September 2001
Are contents of Rubisco, soluble protein and nitrogen
in flag leaves of rice controlled by the same genetics?
Ken Ishimaru1,3, Noriko Kobayashi 2, Kiyomi Ono1, Masahiro Yano1 and Ryu Ohsugi1
1
National Institute of Agrobiological Sciences, 2-1-2, Kannondai, Tsukuba, Ibaraki 305-8602, Japan
Rice Genome Research Program, National Institute of Agrobiological SciencesuInstitute of
Society for Techno-Innovation of Agriculture, Forestry, and Fisheries, 2-1-2, Kannondai, Tsukuba,
Ibaraki 305-8602, Japan
2
Received 2 February 2001; Accepted 11 June 2001
Abstract
Introduction
Genetic relations among the contents of Rubisco,
soluble protein and total leaf nitrogen (N) in leaves of
rice (Or yza sativa L.) were studied by quantitative trait
loci (QTL) analysis with a population of backcross
inbred lines (BILs) of japonica Nipponbare 3 indica
Kasalath. The ratio of Rubisco to total leaf N in
leaves is the main target in improving photosynthetic
N-use efficiency in plants. QTLs controlling Rubisco
content were not detected near QTLs for total leaf N
content. These results indicate that contents of
Rubisco and total leaf N are controlled by different
genetics. QTLs that controlled the ratio of Rubisco
to total leaf N (CORNs) were detected. These results
suggest that some mechanism(s) may be involved
in determining this ratio, while the contents of
Rubisco and total leaf N are controlled in other
ways. In elite BILs, the ratios of Rubisco to total leaf
N were higher than those of both parents. These
results suggest a good possibility of improving N-use
efficiency by CORNs in cultivated rice. A QTL controlling Rubisco content was mapped near a QTL
for soluble protein content on chromosome 8 at 5 d
after heading and on chromosome 9 at 25 d. In each
chromosome region, the peaks of both QTLs overlapped accurately, giving a high possibility of pleiotropic effects by the same genes. Different QTLs
controlling soluble protein or Rubisco were detected
from those detected at 5 d or 25 d after heading. This
suggests that these traits are genetically controlled
depending on the growth stages of leaves.
Rubisco, the key enzyme in C3 photosynthesis, is the
most abundant protein in leaves (Evans, 1989). The
kinetics of Rubisco and the ratio of Rubisco to total
leaf nitrogen (N) are the two main factors determining
N-use efficiency, indicating the rate of CO2 assimilation
per unit amount of N per unit leaf area (for review see
Mae, 1997). Rubisco from higher plants consists of eight
large subunits (rbcL) encoded in the chloroplast genome
and eight small subunits (rbcS) encoded in the nuclear
genome (Spreitzer, 1999). It is difficult to change the
kinetic character of Rubisco (Mae, 1997). Therefore, the
ratio of Rubisco to total leaf N is the main target for
the improvement of N-use efficiency (Mae, 1997). The
relationship between Rubisco and N content is studied
with respect to changes in environmental factors. Regardless of irradiance, temperature or high CO2 conditioning
during growth, the ratio of Rubisco to total N in a rice
leaf appears to be determined only by the amount of N. In
other words, Rubisco content is thought to be due to
total leaf N content (Mae, 1997; Makino et al., 1994,
1997; Nakano et al., 1997).
Recent progress in the generation of a well-saturated
molecular genetic map and markers for rice has made it
possible to map individual genes associated with complex
traits called quantitative trait loci (QTL) (Yano and
Sasaki, 1997; Ishimaru et al., 2001a, b). Identifying the
location of QTLs for traits clarifies the linkage relationships and whether a gene with pleiotropic effects controls
plural traits or not. There are several reports of the map
locations of QTLs and major genes affecting the same
trait (Beavis et al., 1994; Edwards et al., 1992; Veldboom
and Lee, 1994). These reports support Robertson’s
hypothesis that QTLs allelic with major genes affect the
Key words: Nitrogen, Oryza sativa (rice), quantitative trait loci
(QTL), Rubisco.
3
To whom correspondence should be addressed. Fax: q81 298 38 8347. E-mail: [email protected]
ß Society for Experimental Biology 2001
1828
Ishimaru et al.
same trait (Robertson, 1985). A comparison of locations
between structural genes and QTLs can be available for
the determination of the most likely candidate among
various genes that comprehensively control a trait.
Edwards et al. called this the candidate gene strategy
(Edwards et al., 1992). By this method, Sh1 was thought
to be the most important candidate gene controlling
sucrose content in maize leaves (Prioul et al., 1997). QTL
analysis can now evaluate the existence of unknown
genes. Xiao et al. identified new genes at QTLs affecting
the yield from wild rice, Oryza rufipogon (Xiao et al.,
1996a).
As Koornneef et al. and the authors of this paper
have already mentioned (Koornneef et al., 1997; Ishimaru
et al., 2001b), genetic analysis by the QTL approach is a
powerful tool for plant physiologists. However, as far as
is known, there is no report about the genetic relationships among the contents of Rubisco, soluble protein and
total leaf N. In this study, an attempt has been made
to elucidate these genetic relationships in rice leaves by
QTL analysis, and the relationships between QTLs and
candidate genes have been studied.
Materials and methods
Plant materials
A rice japonica variety, Nipponbare, was crossed with an indica
variety, Kasalath. A resultant F1 plant was crossed with
Nipponbare to produce seeds of backcross inbred lines
(BILsF1). Ninety-eight BILsF7 (BILs) were developed from the
resultant BILsF1 plants by the single-seed descent method.
Genotypes of 245 RFLP markers were determined (Harushima
et al., 1998; Lin et al., 1998). The 98 BILsF7 and their two
parental lines were sown on 6 May 1997. For evaluation of their
traits, their seedlings were transplanted on 5 June and were
grown under natural conditions in Tsukuba, Japan (latitude
388 N), in a random design to reduce the effects of environmental factors. Forty seedlings per line were grown under
natural conditions in a randomized complete design with two
replicates. At 5 d or 25 d after heading, flag leaves were
harvested at around noon (about 7 h after sunrise) on a sunny
day. Leaves were frozen immediately in liquid nitrogen and
stored at 80 8C before use. The frozen leaves were divided into
several parts and used for several measurements.
Biochemical assays
The contents of soluble protein and Rubisco in leaves at 5 d and
25 d after heading were analysed. The amounts of soluble
protein and Rubisco were determined according to the methods
of Makino et al. (Makino et al., 1985) with some modifications.
Frozen leaves were ground in liquid nitrogen to a fine powder
and suspended in a buffer containing 100 mM NaH2PO4u
Na2HPO4 (pH 7.0), 1 mM phenylmethylsulphonylfluoride,
1% (wuv) insoluble polyvinyl polypyrrolidone, and 1% (vuv)
b-mercaptoethanol, and then centrifuged for 5 min at 9800 g.
The soluble protein concentration in the supernatant was
determined using BSA as a standard (Bradford, 1976). Soluble
proteins (2 mg) were subjected to SDS-PAGE on 12.5% (wuv)
gels containing 0.1% (wuv) SDS; the gels were stained with
Coomassie brilliant blue. The intensity of the band corresponding to the large subunit of Rubisco was measured with an image
analyser (Umax Technologies, Inc., CA, USA). A calibration
curve with a high correlation (r)0.99) was obtained for Rubisco
purified from rice leaves.
Content of total leaf N
Samples were oven-dried at 80 8C for 3 d. Total leaf N contents
were determined with a CN-Corder (Yanaco Co., Tokyo,
Japan).
QTL and statistical analysis
Chromosomal locations of putative QTLs were determined by
a single-point analysis with the general linear model procedure
of QGENE (Nelson, 1997) according to the method of Ishimaru
et al. (Ishimaru et al., 2001b). A probability level of 0.01 was
used as the threshold for detecting significant differences
in mean values of the two genotypic classes, homozygous
for Nipponbare and Kasalath alleles (Ishimaru et al., 2001b).
To represent a QTL on the map, the chromosome region
corresponding to a log of the likelihood odds ratio (LOD)
greater than the maximum LOD minus 1 was selected, called an
LOD–1 interval (Hirel et al., 2001). The location of the nitrate
reductase (NR) gene came from a rice linkage map (Kurata
et al., 1994), and that of rbcS from the database of the Rice
Genome Project (http:uuwww.dna.affrc.go.jp:82u). The means
of 10 replications per line were used in the data analysis for
each trait. Means and descriptive statistics were generated with
SAS (SAS Institute, 1988). The QTL controlling Rubisco
content at 5 d after heading has been reported (Ishimaru et al.,
2001b).
Results
Variation in the contents of soluble protein, Rubisco,
and total leaf N in flag leaves
BILs showed continuous variations in contents of soluble
protein, Rubisco and total leaf N, and in the ratio of
Rubisco to leaf N; all traits were inherited quantitatively
(Fig. 1). Transgressive segregants were observed in all
traits. The contents of soluble protein and Rubisco in
leaves of Nipponbare were higher than those of Kasalath
at 5 d and 25 d after heading (Fig. 1A, B). The contents
of soluble protein and Rubisco decreased markedly
at 25 d compared with those at 5 d (Fig. 1A–D). The
contents of total leaf N were similar in both Nipponbare
and Kasalath, at about 70 mmol m2, whereas those in
BILs ranged from 50–130 mmol m2 (Fig. 1E). The ratios
of Rubisco to total leaf N were almost the same in
Nipponbare and Kasalath; 17% of BILs outperformed
their parents (Fig. 1F). In BILs there was a high correlation among the contents of soluble protein and
Rubisco at 5 d (r ¼ 0.554; P-0.01), and a low correlation
at 25 d (r ¼ 0.248; P-0.05) after heading (Table 1). Leaf
N contents were positively correlated with the contents
of soluble protein (r ¼ 0.456; P-0.01) and Rubisco
(r ¼ 0.367; P-0.05).
Genetic relationships in rice
1829
Fig. 1. Frequency distributions of BILs for the contents of soluble protein and Rubisco at 5 d or 25 d after heading, total leaf N content, and the ratio
of Rubisco to total leaf N in flag leaves of rice at 5 d after heading. Soluble protein content at (A) 5 d and (B) 25 d after heading. Rubisco content at
(C) 5 d and (D) 25 d after heading. (E) Contents of total leaf N at 5 d after heading. (F) Ratio of Rubisco to total leaf N. Phenotypes of parents
(Nipponbare and Kasalath) are shown by arrows. The data are the means of 10 individual experiments.
Table 1. Correlation coefficients among traits of Rubisco and
soluble protein content at 5 d and 25 d after heading, and total
leaf N content at 5 d after heading
Traits
Protein 5 d Protein 25 d Rubisco 5 d Rubisco 25 d
Protein 25 d
Rubisco 5 d
Rubisco 25 d
Total
leaf N 5 d
0.299*
0.554**
N.S.
0.456**
–
N.S.
0.248*
N.S.
–
0.367*
N.S.
*, ** indicate significant differences at P-0.05 and P-0.01,
respectively. N.S. indicate no significant correlation. For traits see
legend of Fig. 1.
QTLs for traits
QTL controlling total leaf N content was not mapped
near other QTLs for Rubisco content or soluble protein
content (Fig. 2). Three CORNs (QTLs controlling the
ratio of Rubisco to leaf N) were detected. Nipponbare
alleles gave positive effects at CORN1 or CORN5;
Kasalath alleles did so at CORN12. QTLs controlling
soluble protein were detected differently depending on
the growth stage of leaves (5 d or 25 d after heading)
(Fig. 2). The QTL for Rubisco at 5 d was mapped to the
same region of chromosome 8 as a QTL associated with
soluble protein content at 5 d (Fig. 2). The peak of LOD
of Rubisco at 5 d coincided with that of soluble protein
content (Fig. 3A). The effect of the allele contributed by
Kasalath was positive for both phenotypes. At 25 d after
heading, the QTL controlling Rubisco was closely linked
to the QTL controlling soluble protein on chromosome 9,
and the Nipponbare allele positively affected both
(Fig. 2). The peaks of their QTLs overlapped accurately
(Fig. 3B).
1830
Ishimaru et al.
Fig. 2. Linkage map of BILs showing the putative location of QTLs associated with the contents of soluble protein and Rubisco at 5 d or 25 d after
heading. Putative QTLs were determined by QGENE, and the chromosome region corresponding to the LOD of a QTL greater than the maximum
LOD minus 1 was selected. Markers significant at the 0.01 probability level or higher (ANOVA) are shown: ** and * indicate significant differences at
P-0.001 and P-0.005, respectively. K means the direction of phenotypic effect by Kasalath. The QTL controlling Rubisco content at 5 d after
heading has been reported (Ishimaru et al., 2001b).
Comparison between QTLs and candidate genes
Discussion
The QTL for soluble protein content at 25 d was positioned between markers C1370 and C86 on chromosome
1 (Fig. 2). The peak of LOD of this QTL was close to that
of C813 and corresponded to a position of 4.4 cm from
NR. rbcS did not detect the region of the QTL controlling
Rubisco content at 25 d that was mapped on chromosome 12 (Fig. 2). The maximum LOD of CORN12 lay
between C443 and G2140 on chromosome 12. There was
a difference of 5.6 cm between the peak of LOD of
CORN12 and the location of rbcS.
The ratio of Rubisco to total leaf N content is not
influenced by environmental changes (Mae, 1997; Makino
et al., 1994, 1997; Nakano et al., 1997). However, QTLs
controlling the Rubisco content of flag leaves were not
mapped near those controlling total leaf N content. These
results suggest that Rubisco and total leaf N content are
controlled by different genetics (Fig. 2). The detection
of CORNs suggests that there may be some mechanisms
that determine the ratio of Rubisco to total leaf N content. A few reports showed that when N content was
Genetic relationships in rice
1831
Fig. 3. Likelihood odds ratio score of the QTL controlling Rubisco and soluble protein contents on chromosome 8 at 5 d (A) or on chromosome 9
at 25 d (B).
limiting to growth, the content of Rubisco declined in
proportion to the content of total leaf N under long-term
CO2 enrichment (Sage et al., 1989; Rowland-Bamford
et al., 1991; Rogers et al., 1996). In these results, 17%
of BILs outperformed their parents (Nipponbare and
Kasalath) in this ratio (Fig. 1F). These results suggest
that the ratio of Rubisco to total leaf N can be controlled
by genetic methods. For example, CORN12, with positive
effects by the Kasalath allele, may improve the N-use
efficiency of cultivar Nipponbare, even though Nipponbare and Kasalath had almost the same ratio. The
combination of high-yielding rice cultivars and the high
use of N fertilizer since 1960 have been credited for the
increased yields of the Green Revolution, but the high
input of N-fertilizer has reduced the fertility of the soil
(Mann, 1999). As mentioned in the Introduction, the
ratio of Rubisco to total leaf N content is the main target
for improving N-use efficiency in rice (Mae, 1997). Therefore, controlling N-use efficiency with CORNs could be
valuable for maintaining high yields with reduced inputs
of N fertilizer.
BILs showed continuous variations in all traits
(Fig. 1); these variations could be caused by transgressive
segregants. Xiao et al. found transgressive segregants in
BILs where there was no significant difference between
parents (Xiao et al., 1996b). Prioul et al. reported that
transgressive segregants seemed to result from alleles
affecting a trait that may be dispersed between parents
(Prioul et al., 1997).
The overlap between QTLs controlling Rubisco and
soluble protein contents was detected on chromosome
8 at 5 d after heading or on chromosome 9 at 25 d
(Figs 2, 3). There are two possible explanations for the
occurrence of QTLs associated with different traits at
the same locus. One is that the QTLs are closely linked
genetically but unrelated phenotypically. The second is
that multiple traits are controlled by a single locus, and a
gene may have two functions, or the expression of one
trait may, in part, cause the expression of another trait.
The peaks of the LODs of the QTLs for Rubisco and
soluble protein contents overlapped exactly in each
region of the chromosome (Fig. 3). This suggests a strong
possibility of pleiotropic effects by the same genes on
these chromosome regions. In contrast, a few QTLs for
soluble protein content that did not overlap QTLs for
Rubisco content were detected (Fig. 2). These results
suggest that the Rubisco and soluble protein contents
may be partly controlled by the same genetic regulation,
and that these QTLs may be related to the contents of
other soluble proteins besides Rubisco.
Because a trait is controlled by different expression
dynamics of QTLs during development, QTL analysis by
plural observations gives better information in genetics
than analysis by only 1 observation (Wu et al., 1999). The
coincidence of QTLs between Rubisco and soluble
protein contents differed between 5 d and 25 d after
heading, and some QTLs for these traits were detected at
either 5 d or 25 d (Fig. 2; Table 2). These results suggest
that these traits might be genetically controlled depending on the growth stage of leaves. In rice plants, N is
reallocated from leaves to new sinks (panicles) during
senescence (Mae, 1997). Thus, QTLs controlling Rubisco
and soluble protein contents at 25 d after heading (the
senescence period) could be related to this reallocation.
Nitrate taken up from the soil by higher plants is
reduced to ammonium by two enzymes, NR and nitrite
reductase. Reduction of nitrate to nitrite catalysed by
NR is a rate-limiting step in the nitrate assimilation
pathway in higher plants (Miflin and Lea, 1977). The
amount of Rubisco is reduced in rbcS antisense C3 and C4
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Ishimaru et al.
Table 2. QTL traits of Rubisco and soluble protein contents
at 5 d and 25 d after heading and total leaf N content and the ratio
of Rubisco to total leaf N content at 5 d after heading
Traits
Number
of QTLs
Protein 5 d
4
Protein 25 d
4
Rubisco 5 db
Rubisco 25 d
1
2
Total leaf N 5 d
RubiscouN 5 d
1
3
Chromosome
number
1
3
8
1
4
6
9
8
9
12
2
1 (CORN1)
5 (CORN5)
12(CORN12)
Nearest
marker
DPEa
LOD
C112
R1927
G1073
R2662
C813
C335
R674
C570
G1073
C570
G2140
R2510
R2635
R3166
C443
N
N
K
K
N
N
N
N
K
N
K
K
N
N
K
2.00
1.55
2.00
2.00
1.48
2.10
2.00
2.23
2.00
3.30
2.15
1.48
1.41
2.18
2.00
a
DPE; direction of phenotypic effect. N and K indicate Nipponbare
and Kasalath, respectively.
b
QTL for Rubisco 5 d (Ishimaru et al., 2001b).
plants (Furbank et al., 1996; Makino et al., 1997). At the
level of translation, rbcS may directly control the expression of rbcL (Rodermel et al., 1996). NR and rbcS were
mapped near QTLs controlling soluble protein content
at 25 d after heading and CORN12, respectively (Fig. 2).
However, neither NR nor rbcS coincided with the peak of
the LOD score for their QTLs. These results suggest that
the contents of soluble protein, Rubisco or total leaf N
may not be directly related to the structural genes, NR
and rbcS, in this environment and with the materials
used in this study.
The degradation of Rubisco in rice leaves occurs
independently of the synthesis of Rubisco (Makino et al.,
1984), and the total leaf N content is determined by the
contents of nitrate and ammonium but not protein.
CORNs might relate to the degradation of Rubisco or to
the contents of nitrate and ammonium, or both. Near
isogenic lines have been developed with a specific
chromosome region in Nipponbare replaced by that of
Kasalath (Yano and Sasaki, 1997). Such lines with
Kasalath CORN replacing the Nipponbare alleles can
be used for investigating how the ratio of Rubisco to total
leaf N is determined.
Acknowledgements
We thank the staff of the Farm Management Division (National
Institute of Agrobiological Sciences, Japan) for their care of
the plants used in these experiments, and Mr H Sakai (National
Institute of Agro-Environmental Sciences) for the operation
of the CN-Corder. This work was supported in part by a Grantin-Aid (Bio Cosmos Program) from the Ministry of Agriculture,
Forestry and Fisheries, Japan.
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