Download Genetic manipulation and quantitative

Journal of Experimental Botany, Vol. 53, No. 370,
Inorganic Nitrogen Assimilation Special Issue, pp. 917–925, April 2002
Genetic manipulation and quantitative-trait loci mapping
for nitrogen recycling in rice
Tomoyuki Yamaya1,2,4, Mitsuhiro Obara1, Hiroyuki Nakajima1, Shohei Sasaki1,
Toshihiko Hayakawa1 and Tadashi Sato3
1
Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi,
Sendai 981-8555, Japan
2
Plant Science Center, Riken, 2-1 Hirosawa, Wako 351-0198, Japan
3
Graduate School of Life Science, Tohoku University, 2-1-1 Katahira, Sendai 980-8577, Japan
Received 18 July 2001; Accepted 12 October 2001
Abstract
Immunocytological studies in this laboratory have
suggested that NADH-dependent glutamate synthase
(NADH-GOGAT; EC 1.4.1.14) in developing organs of
rice (Oryza sativa L. cv. Sasanishiki) is involved in the
utilization of glutamine remobilized from senescing
organs through the phloem. Because most of the
indica cultivars contained less NADH-GOGAT in their
sink organs than japonica cultivars, over-expression
of NADH-GOGAT gene from japonica rice was
investigated using Kasalath, an indica cultivar.
Several T0 transgenic Kasalath lines over-producing
NADH-GOGAT under the control of a NADH-GOGAT
promoter of Sasanishiki, a japonica rice, showed an
increase in grain weight (80% as a maximum), indicating that NADH-GOGAT is indeed a key step for
nitrogen utilization and grain filling in rice. A genetic
approach using 98 backcross-inbred lines (BC1F6)
developed between Nipponbare (a japonica rice) and
Kasalath were employed to detect putative quantitative trait loci (QTLs) associated with the contents
of cytosolic glutamine synthetase (GS1; EC 6.3.1.2),
which is probably involved in the export of nitrogen
from senescing organs and those of NADH-GOGAT.
Immunoblotting analyses showed transgressive
segregations toward lower or greater contents
of these enzyme proteins in these BC1F6. Seven
chromosomal QTL regions were detected for GS1
protein content and six for NADH-GOGAT. Some
of these QTLs were located in QTL regions for
various biochemical and agronomic traits affected
4
by nitrogen recycling. The relationships between the
genetic variability of complex agronomic traits and
traits for these two enzymes are discussed.
Key words: Glutamate synthase, nitrogen recycling, overexpression, QTL, rice.
Introduction
In japonica rice (Oryza sativa L. cv. Sasanishiki) plants,
approximately 80% of total nitrogen in the ear is
remobilized through the phloem from senescing organs
(Mae and Ohira, 1981). The major forms of nitrogen in
the phloem sap are glutamine and asparagine (Hayashi
and Chino, 1990) that is probably synthesized from
glutamine (Ireland and Lea, 1999). Thus, the synthesis of
glutamine in senescing organs as well as the utilization
of glutamine in developing organs is the key step for
nitrogen recycling in rice plants. Glutamine synthetase
(GS; EC. 6.3.1.2) and glutamate synthase (GOGAT)
could be involved in the process of nitrogen recycling. GS
catalyses the ATP-dependent condensation of NH3 with
glutamate to produce glutamine. Subsequently, GOGAT
catalyses the reductive transfer of the amide group of
glutamine to 2-oxoglutarate to form two glutamate
molecules (Ireland and Lea, 1999). The immunocytological studies suggest that cytosolic GS (GS1) is
necessary for the export of nitrogen from senescing leaves
of rice plants, because the GS1 protein was detected in
companion cells which are important for the phloem
To whom correspondence should be addressed. Fax: q81 22 717 8787. E-mail: [email protected]
ß Society for Experimental Biology 2002
918
Yamaya et al.
loading of solutes, and in vascular parenchyma cells
(Sakurai et al., 1996; Obara et al., 2000). This is in
contrast to the localization of chloroplastic GS (GS2) that
is detected in mesophyll cells (Sakurai et al., 1996; Obara
et al., 2000) and plays the major role in the photorespiratory nitrogen metabolism (Ireland and Lea, 1999).
There are two GOGAT molecular species in rice plants.
One is the ferredoxin (Fd)-dependent GOGAT (EC
1.4.7.1) and the other, NADH-dependent (EC 1.4.1.14).
Although Fd-GOGAT is known to be involved in
photorespiration (Ireland and Lea, 1999), current understanding of the physiological functions of NADHGOGAT in plants is limited. In rice, NADH-GOGAT
is active in developing organs, such as unexpanded nongreen leaves and developing grains (Yamaya et al., 1992).
It is hypothesized that NADH-GOGAT is probably
involved in the utilization of remobilized nitrogen,
because this protein is located in the specific cell types
which are important for solute transport from the phloem
and xylem elements (Hayakawa et al., 1994). NADHGOGAT occurs as a single gene in rice (Goto et al., 1998).
Cell-type-specific and age-dependent expression of the
NADH-GOGAT gene was confirmed by promoter
analysis in transgenic rice (Kojima et al., 2000). Because
no mutant lacking NADH-GOGAT or GS1 has been
found in the plant kingdom, the gene manipulation
technology of either over-expression or reduction of these
target enzymes was used to obtain more direct evidence
for their physiological functions.
Oryza sativa is widespread and there are three types or
subspecies identified for cultivated rice plants, i.e.
japonica, indica, and javanica. Indica and javanica
cultivars are different morphologically compared with
japonica, including plant heights, sizes of leaf blade and
grain, numbers of tillers, as well as their yield and
biomass production (Takahashi, 1984). In general, total
biomass production of indica cultivars is greater than that
of japonica cultivars, whereas grain yield is less. For
example, the mean value of one-spikelet weight on
the main stem of Kasalath (indica) and Nipponbare
( japonica) was 18.0 and 26.9 mg (n ¼ 10), respectively,
when these cultivars were grown to maturity in a
greenhouse at Sendai, Japan, a temperate area. When
various cultivars were tested to compare the contents of
protein for NADH-GOGAT in the non-green portion of
the expanding leaf blade at the reproductive stage, most
of the indica cultivars, including Kasalath, contained less
NADH-GOGAT protein than japonica and javanica
cultivars based on total leaf nitrogen (Obara et al.,
2000). On the other hand, some of the indica cultivars
contained GS1 protein in senescing leaves twice as high as
other cultivars. If the hypothesis was true, indica cultivars
could have a less efficient system in sink organs to utilize
glutamine that is exported from senescing organs than
japonica and javanica cultivars. In other words, overexpression of the NADH-GOGAT gene in sink organs
of indica cultivars in an age- and tissue-specific manner
possibly causes an increase in spikelet weight. As
summarized in Table 1, there are some examples of
over-expression of the GS1 gene in Lotus corniculatus
(Vincent et al., 1997) and alfalfa (Ortega et al., 2001) and
the GS2 gene in tobacco (Migge et al., 2000) and rice
Table 1. Analysis of transgenic plants expressing sense or antisense RNA for either GS or GOGAT
Host plant
Over-expression
Vincent et al. (1997)
L. corniculatus
Ortega et al. (2001)
Alfalfa
Migge et al. (2000)
Tobacco
Hoshida et al. (2000)
Rice
This study (2001)
Rice
Antisense inhibition
Temple et al. (1998)
Alfalfa
Brugière et al. (1999)
Tobacco
Migge and Becker (2000)
Tobacco
Schoenbeck et al. (2000)
Alfalfa
a
Promoter
Transgene
Characteristics
CaM35Sa
Soybean GS1
Accelerate senescence
CaM35S
Alfalfa GS1
No increase in GS1 activity
RubiscoSSUb
Tobacco GS2
Stimulate the growth rate
CaMV35S
Rice GS2
Enhance photorespiration and salt tolerance
Rice NADH-GOGAT
Rice NADH-GOGAT
Enhance grain filling
Alfalfa GS1
Reduce the transcript level
Phloem specific CuuZn SOD
Tobacco GS1
Regulate proline production
RubiscoSSU
Tobacco GS2
Photorespiration
Alfalfa NADH-GOGAT
Assimilate symbiotically fixed nitrogen
CaMV35S
c
Nodule AspAT
Cauliflower Mosaic Virus 35S promoter.
Rubisco small subunit promoter.
Cu/Zn superoxide dismutase promoter.
d
Aspartate aminotransferase promoter.
b
c
d
NADH-GOGAT in rice
(Hoshida et al., 2000). To understand physiological functions, antisense inhibition for GS1 (Temple et al., 1998;
Brugiere et al., 1999), GS2 (Migge and Becker, 2000), or
NADH-GOGAT in nodules of alfalfa (Schoenbeck et al.,
2000) has recently been investigated. However, overexpression of either GOGAT gene has not been reported
in plants, including rice. In the first chapter of this
review, recent transgenic work expressing sense RNA
for NADH-GOGAT in an indica rice is described.
Conventional approaches that involve whole plant
physiology, biochemistry, and molecular physiology are
limited in that they only allow the role of a single or
limited number of enzymes or regulatory elements to be
identified. Agronomic traits, such as the size of seeds
and fruits, time for headings, and nitrogen use efficiency,
could be determined by multiple gene functions.
Molecular cloning of all genes is one way to understand
phenotypic characteristics. This upward method is, however, not easy to cover all genes, because the individual
effect of each gene on the phenotype could be relatively
small. DNA markers and their linkage map on chromosomes are efficient tools and methods for mapping
individual ‘quantitative trait loci’ (QTLs) (Tanksley,
1993; Paterson, 1995), as well as the recent progress
of DNA array and proteome analyses as post-genomics
tools in Arabidopsis and green algae. A number of QTL
analyses using interspecific crosses have been conducted
to identify the loci anduor genes controlling physiological
traits in various crop plants including tomato (Frary et al.,
2000) and maize (Hirel et al., 2001; Wang et al., 2001). In
rice, QTL analysis with DNA markers, based on a wellsaturated genetic linkage map, has been employed to
detect genomic regions associated with several traits
exhibiting complex inheritance (Yano and Sasaki, 1997).
Genes which are functioning in the regulation of time to
heading have recently been identified from QTL analysis
(Takahashi et al., 2001; Yano et al., 2000). As far as is
known, however, there have been no trials to combine
genetic traits of rice and biochemicalumolecular biological
characteristics in QTL analysis. Such downward analyses
could provide valuable information on many genes
related to the regulation of target steps for specific traits.
In addition, the recent development of genome sequencing projects in a number of model plant species, including rice, will give valuable information in the near future
for the identification of the precise location of key genes
in QTL regions. The detection of QTLs associated with
the contents of GS1 and NADH-GOGAT in rice leaves
together with the agronomic traits are also described.
Transgenic analysis for NADH-GOGAT in rice
The NADH-GOGAT gene is 17.1 kb long (Goto et al.,
1998) which is probably too long to transform rice,
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so that construction of a chimeric gene containing the
NADH-GOGAT promoter region and NADH-GOGAT
cDNA was designed. Identification of cis-element(s)
related to the tissue-specific expression of NADHGOGAT gene has not yet been completed in rice
(Kojima et al., 2000). There are, however, several
instances that indicate the importance of the presence of
the first intron and 39-UTR in addition to the 59-UTR
as a promoter for tissue-specific gene expression in
Arabidopsis thaliana (Sieburth and Meyerowitz, 1997;
Larkin et al., 1993) and potato (Fu et al., 1995).
Therefore, the region from –2 352 in 59-UTR (q1 as
the transcriptional start) to q1 322 in the 3rd exon of the
NADH-GOGAT gene (AB001916; Goto et al., 1998)
from Sasanishiki ( japonica) was fused with the NADHGOGAT cDNA (AB008845; Goto et al., 1998), from
which had been deleted the overlapped region, containing
the poly(A) tail (262 to 6762 in AB008845). This chimeric
NADH-GOGAT gene was inserted at the XbaI site
between NTPII and HTP in the uidA gene (encoding
GUS) deleted Ti plasmid pBI101Hm (Kojima et al.,
2000). Agrobacterium tumefaciens-mediated transformation of Kasalath (indica) was carried out as described
previously (Kojima et al., 2000). Transformation of
Kasalath using pBI101Hm was also adopted as a control.
Twenty-two lines of the NADH-GOGAT transformant (T0) and 11 lines of the control transformant (TC)
were obtained at a frequency of 3.1% and 3.6%, respectively. The occurrence of the chimeric gene (1–6 copies for
the NADH-GOGAT chimeric gene and 3–4 copies of hpt
for the control) in each line was confirmed by DNA gel
blot analysis (data not shown). These regenerated lines as
well as the wild-type Kasalath were grown hydroponically
(Hayakawa et al., 1994) in an isolated greenhouse until
maturity. Molecular biological and biochemical analyses
were performed as described previously (Hayakawa et al.,
1994; Kojima et al., 2000; Obara et al., 2000). As shown
in Fig. 1, there were large variation in the NADHGOGAT protein contents, when unexpanded non-green
leaf blades of three productive tillers from each line and
from wild-type plants were analysed with immunoblotting. The NADH-GOGAT protein content increased
in several lines. Line 34 contained the protein approximately 1.8-fold higher than the control line or wild-type
plant. On the other hand, some lines severely reduced the
content of NADH-GOGAT protein, which is probably
caused by so-called ‘co-suppression’ or ‘homologydependent gene silencing’ (Iyer et al., 2000; Matzke and
Matzke, 1995). This phenomenon is sometimes seen when
a gene or cDNA from a plant is introduced into the same
plants in a sense-orientation (Voinnet and Baulcombe,
1997). There was also some variation in the content of
Fd-GOGAT protein and that of soluble protein in those
lines. The changes in the contents of Fd-GOGAT protein
and soluble protein were apparently independent of that
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Yamaya et al.
Fig. 1. Contents of NADH-GOGAT protein (A), Fd-GOGAT protein
(B), and soluble proteins (C) in unexpanded non-green leaf blades of
various lines of Kasalath at T0 generation introduced with the chimeric
NADH-GOGAT gene at the sense orientation. Wild-type Kasalath (W)
and transformants with pBI101Hm (TC) were used as a control. Both
GOGAT proteins were determined with immunoblotting methods. Error
bars represent SE of at least three independent plants.
of NADH-GOGAT protein. The variation seen in the
soluble protein content could not be caused by the efficiency of protein extraction from different lines, since the
values of standard error were small.
Three lines were chosen from either the over-expressed
or co-suppressed T0-transformants, and NADHGOGAT activity in unexpanded non-green leaf blades
of three productive tillers from each line and agronomic
characteristics at a particular stage of harvest were
investigated (Fig. 2). These lines are associated with the
higher content of NADH-GOGAT protein seen in Fig. 1
and also showed higher NADH-GOGAT activity. Plant
height and the number of spikelets on the main stem
were identical among these transformants and wild-type
plants. It should be noted, however, that the mean value
Fig. 2. Morphological and biochemical characteristics of representative
‘over-expressed’ lines and ‘co-suppressed’ lines compared to those
of wild-type Kasalath (W) and transformed control with pBI101Hm
(TC). Vertical bars represent SE of at least five independent plants.
(A) NADH-GOGAT activity in unexpanded non-green leaf blades; (B)
plant height; (C) weight of a spikelet; (D) spikelet number on the main
stem; (E) panicle number per plant; and (F) panicle weight per plant.
of one spikelet weight of the over-expressed lines was
higher than that of the wild-type or control transformants, although the rate of increase in the NADHGOGAT protein in unexpanded non-green leaf blades
NADH-GOGAT in rice
was not correlated with that in the one-spikelet weight
among these three lines. This correlation should be evaluated in future by estimating the NADH-GOGAT protein
contents and its activity in spikelets using progenies
of the T0-transformants. Co-suppressed lines showed a
much lower one-spikelet weight. Although these results
were obtained from the T0 generation having multiple
trans-genes, other results with a homozygous T3-progeny
from transgenic Sasanishiki ( japonica) expressing an
antisense RNA for NADH-GOGAT under the control
of cauliflower mosaic virus 35S promoter also showed a
severe reduction in the one-spikelet weight (K. Ishiyama
et al., unpublished results). In this case, total nitrogen
percentage (about 1.4%) in the dry weight of spikelets
was identical in the antisense progeny and the wild type,
so that a reduction of NADH-GOGAT caused a reduction in the entire process of grain filling. Isolation of a
progeny of the over-expressed transgenic Kasalath having
homozygous and fewer sense genes is now in progress.
QTL analysis for contents of GS1 and
NADH-GOGAT protein in rice
In rice, there are two cytosolic GS isozymes encoded by
distinct genes, i.e. GS1 (mainly expressed in leaves) and
GSr (mainly expressed in roots) (Sakamoto et al., 1989).
By contrast, NADH-GOGAT occurs as a single gene
(Goto et al., 1998). Because nitrogen recycling largely
occurs in leaves and spikelets, the focus was on GS1 and
NADH-GOGAT in the QTL analyses. Plant phenotype,
including the content of target enzyme protein as well as
agronomic and physiological traits, is determined by a
number of gene activities in which some genes contribute
positively and other genes work negatively (Goldman
et al., 1993). There is a large variation in the content of
GS1 protein in senescing leaf blades and that of NADHGOGAT protein in the unexpanded young leaf blades
among rice cultivars (Obara et al., 2000). As the first step
towards identifying some of the many genes related to the
regulation of nitrogen recycling, the locus of GS1 was
mapped and the QTLs associated with GS1 protein
content in the senescing leaf blade and NADH-GOGAT
protein content in the developing leaf blade were detected.
A genetic approach using 98 backcross-inbred lines
(BIL: BC1F6) developed between Nipponbare (japonica)
and Kasalath (indica) was employed to detect putative
QTLs associated with the contents of GS1 and NADHGOGAT in leaves. Although a given QTL region would
still contain many genes, some of them could be tightly
related to the physiological and biochemical traits. The
GS1 protein content in senescing leaf blades of the
BILs showed a continuous distribution with one peak
ranging from 2.55 to 16.18 mg g1 FW, and demonstrated
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transgressive segregation in both directions. Also, the
NADH-GOGAT protein content in developing leaf
blades of the BILs was continuously distributed with one
peak ranging from 1.27 to 4.48 mg g1 FW, the mean
value of the BILs being within the range of the parents.
Thus, the contents of GS1 and NADH-GOGAT proteins
in rice plants are considered to be quantitative traits.
Details of analytical methods and results have been
recently published (Obara et al., 2001).
Seven chromosomal regions were detected having
QTLs associated with GS1 protein content in the senescing leaf blade and six with NADH-GOGAT protein
content in the developing leaf blade. Three putative QTLs
for GS1 protein content including the major QTL (linked
to the marker S2260: positive allele from Kasalath) on
chromosome 11 were detected in the vicinity of QTL
regions for physiological traits (Fig. 3). The GS1 gene
was mapped between C560 and C1408 on chromosome 2
(Fig. 3). The genetic distance was 10.8 cM from C560 and
0.8 cM from C1408, where the GS1 gene was detected to
overlap with a putative QTL for one-spikelet weight.
Alleles from Nipponbare and Kasalath that were identified with a higher GS1 protein content were also associated with a higher rate of leaf discoloration (rate of leaf
senescence) in the case of QTLs linked with the markers
C950 (positive allele from Nipponbare) and R728
(positive allele from Kasalath) on chromosome 11. Therefore, the alleles that increase the GS1 protein content
apparently promote the rate of leaf senescence. These
results suggest that variation of the GS1 protein content
in this population might change the rate of export of
glutamine from senescing organs to developing organs
and then lead to changes in these physiological traits. This
possibility should be confirmed in future experiments by
identifying genes.
Similarly, two putative QTLs for NADH-GOGAT
protein content on chromosome 2 were detected in the
vicinity of QTLs for physiological traits (Fig. 3). The
major QTL linked to the marker R712 (positive allele
from Nipponbare) was located close to the QTL regions
for soluble protein content in developing leaves, spikelet
number per panicle on the main stem, and rate of leaf
discoloration. In this case, the alleles from Nipponbare
contributed to increases in NADH-GOGAT protein
content and spikelet number whilst also promoting the
rate of discoloration. One QTL for spikelet number on
the main stem linked to marker G227 (positive allele
from Kasalath) was located close to the marker R712.
Therefore, the alleles from Nipponbare in this region
negatively contributed to an increase in spikelet number
on the main stem. Another QTL for NADH-GOGAT
protein content linked to the marker C1221 (positive
allele from Kasalath) on chromosome 2 was located in
QTL regions for spikelet number on the main stem. In this
case, the alleles from Kasalath contributed to increases in
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Yamaya et al.
Fig. 3. The regions of putative QTLs for GS1 protein content in senescing leaf blades NADH-GOGAT protein content in developing leaf blades, and
physiologicaluagronomic traits. The chromosomes in which QTLs were examined for GS1 and NADH-GOGAT protein contents are shown. The QTLs
for content of GS1 (black) and NADH-GOGAT (red) are shown on the left of the chromosome arms, those for physiological traits on the right. PC
(brown), soluble protein contents from developing leaf blades; SPN (pink), spikelet number on the main stem; PNW (yellow), panicle weight on the
main stem; SPW (blue), one-spikelet weight; RFD (green), rate for full-discoloration. The arrows on the left of the chromosome arms show the locus of
the structural gene for GS1 (in this study) and NADH-GOGAT (Sasaki et al., 1994). Black and red arrowheads on the left of the chromosome arms
indicate the co-localization of QTLs for GS1 or NADH-GOGAT and other physiological traits, respectively. Genetic distance, in cM, is based on
linkage analysis of 98 BILs (Lin et al., 1998). A probability of less than 0.05 was used to define the borders of the confidence intervals for QTLs
(modified from Obara et al., 2001).
NADH-GOGAT protein content and spikelet number on
the main stem. One putative QTL for one-spikelet weight
linked to marker R3393 (positive allele from Nipponbare)
was located close to the marker C1221 on chromosome 2.
This suggests that alleles from Kasalath in this
QTL region negatively contributed to an increase in
one-spikelet weight. It should be noted that a major QTL
linked to the marker R728 (positive allele from Kasalath)
on chromosome 11 was located in the QTL region for
one-spikelet weight. A QTL for spikelet number on
chromosome 1 linked to the marker R886 (positive allele
from Nipponbare) indicates that the allele from Kasalath
NADH-GOGAT in rice
923
in this region negatively contributes spikelet number on
the main stem.
As described previously (Obara et al., 2001), onespikelet weight in Kasalath was 33% lower than that
in Nipponbare. Over-expression of NADH-GOGAT in
transgenic Kasalath showed an increase in panicle weight
and in spikelet weight, but had no effect on the spikelet
number on the main stem, as mentioned in the previous
chapter. On the other hand, the existence of QTL regions
for NADH-GOGAT content linked to the markers
C1221uR3393 and R712uG227 indicates that alleles from
Kasalath in these regions negatively contribute to onespikelet weight and promote the increase in spikelet
number, respectively. Although this seems inconsistent
with what was observed in the over-expressed transgenic
Kasalath, it is suggested that other effective QTLs, such
as a QTL linked to the marker R728 (positive allele from
Kasalath) on chromosome 11, could overcome the QTLs
linked to R712, resulting in an increase in one-spikelet
weight. Similarly, a QTL linked to the marker R886
(positive allele from Nipponbare) on chromosome 1 may
overcome the QTLs linked to G227 and C499 (positive
alleles from Kasalath) on chromosome 2, resulting in a
small difference in spikelet number on the main stem.
Furthermore, putative QTLs for NADH-GOGAT protein content on chromosome 1 were located in the QTL
region for soluble protein content in developing leaves.
At these QTLs, the alleles from Kasalath were identified
with an increase of NADH-GOGAT protein content
and soluble protein content in developing leaves. These
findings suggested that the variation in NADH-GOGAT
protein content in this population is related to changes in
the rate of regeneration of glutamic acid from glutamine
transported via the phloem and xylem in developing
organs leading to changes in protein content. A QTL
region for NADH-GOGAT protein content was detected
at the position mapped for the NADH-GOGAT structural gene on chromosome 1. A QTL region for soluble
protein content in developing leaves was also detected in
this region. Although fine mapping is required to identify
individual genes, QTL analysis could be a useful postgenomic tool to study the gene functions for regulation of
nitrogen recycling in rice.
thickness of leaves and stem, development of root system,
etc. all affect the productivity of rice. The findings of the
over-expression of NADH-GOGAT in sink organs of
Kasalath as well as the co-suppression or the antisense
inhibition in Sasanishiki (Ishiyama et al., 1999) strongly
supported the hypothesis that NADH-GOGAT in spikelets at the early stage of ripening is important to re-utilize
glutamine. Not only that, but also the grain-filling process
is co-regulated by the status of the re-utilization in which
the precise mechanisms are largely unknown. This is a
good example of the limitation of upward analysis by
manipulating a limited number of target genes.
QTL analysis provides multiple independent loci
expected to have regulatory functions on the gene
products, although fine mapping is needed to identify
these genes. QTLs for GS1 protein and rate of leaf senescence detected on chromosome 11 could be a good candidate gene that can, at least partially, explain variations in
nitrogen export from senescing leaf blades. Two QTLs for
NADH-GOGAT on chromosome 2 could be related to
grain yield. From QTL analysis using 99 recombinant
inbred lines of maize, leaf nitrate accumulation and reactions catalysed by nitrate reductase and GS are recently
suggested to be co-regulated and represent key elements
controlling nitrogen use efficiency (Hirel et al., 2001).
Prioul et al. showed that QTLs for the activities of
sucrose-phosphate-synthase and acid-soluble invertase
were detected in the regions where each structural gene
was mapped in maize (Prioul et al., 1999). Furthermore,
these QTLs were located close to QTLs for the contents of
products and substrates of each enzyme. In these experiments, two out of seven QTLs for GS1 protein content
and three out of six QTLs for NADH-GOGAT protein
content were detected in different regions from other
biological and physiological traits (Fig. 3). To identify the
candidate genes from the QTL analysis more precisely,
it is necessary to investigate lines that are near isogenic
in the QTL region. Elucidation of the genetic nature
regulating GS1 and NADH-GOGAT protein contents as
a whole could aid in the efficient breeding of highperformance rice cultivars. A combination of genetic
manipulation and QTL analysis should be useful for the
elucidation of the nitrogen recycling mechanism.
Conclusion
Acknowledgements
Nitrogen recycling and grain-filling in rice are complex
traits that depend on many factors such as rate of senescence to provide nitrogen source, photosynthesis and
respiration to provide carbon skeleton and energy for
amino acid biosynthesis and transport, re-utilization of
precursors in sink organs for storage of protein and
starch, and so on. Morphological traits, such as size of
leaves and spikelets, the number of spikelets and panicles,
We thank Miss Aiko Shudo for technical assistance and
Dr Masahiro Yano, National Institute of Agrobiological
Resources, Tsukuba, Japan, for providing seeds of the backcross
inbred lines. We also thank to two referees for helpful comments
and critical reading of the manuscript. This work was supported
in part by a program of CREST of JST (Japan Science and
Technology) and in part by Grant-in-Aid for Scientific Research
(Nos 10556075 and 12460029) from the Ministry of Education,
Science, Sport, Technology, and Culture of Japan.
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Yamaya et al.
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