Download Differential Expression of Genes of the Calvin–Benson Cycle and its

Differential Expression of Genes of the Calvin–Benson Cycle
and its Related Genes During Leaf Development in Rice
Chihiro Yamaoka1, Yuji Suzuki1,* and Amane Makino1,2
1
*Corresponding author: E-mail, [email protected]; Fax, +81-22-717-8765.
(Received September 15, 2015; Accepted November 1, 2015)
To understand how the machinery for photosynthetic
carbon assimilation is formed and maintained during leaf
development, changes in the mRNA levels of the Calvin–
Benson cycle enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase and two key enzymes for
sucrose synthesis were determined in rice (Oryza sativa L.).
According to the patterns of changes in the mRNA levels,
these genes were categorized into three groups. Group 1
included most of the genes involved in the carboxylation
and reduction phases of the Calvin–Benson cycle, as well
as three genes in the regeneration phase. The mRNA levels
increased and reached maxima during leaf expansion and
then rapidly declined, although there were some variations
in the residual mRNA levels in senescent leaves. Group 2
included a number of genes involved in the regeneration
phase, one gene in the reduction phase of the Calvin–
Benson cycle and one gene in sucrose synthesis. The
mRNA levels increased and almost reached maxima before
full expansion and then gradually declined. Group 3
included Rubisco activase, one gene involved in the regeneration phase and one gene in sucrose synthesis. The overall
pattern was similar to that in group 2 genes except that the
mRNA levels reached maxima after the stage of full expansion. Thus, genes of the Calvin–Benson cycle and its related
genes were differentially expressed during leaf development
in rice, suggesting that such differential gene expression is
necessary for formation and maintenance of the machinery
of photosynthetic carbon assimilation.
Keywords: Calvin–Benson cycle Gene expression Leaf
development Rice Sucrose synthesis.
Abbreviations: cFBP, cytosolic fructose-1,6-bisphosphatase;
cpFBP, chloroplastic fructose-1,6-bisphosphatase; FBA, fructose-1,6-biosphosphate aldolase; GAPA, A subunit of glyceraldehyde-3-phosphate dehydrogenase; GAPB, B subunit of
glyceraldehyde-3-phosphate dehydrogenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 3-PGA, 3-phosphoglycerate;
PGK,
3-phosphoglycerate
kinase;
PRK,
phosphoribulokinase; qRT–PCR, quantitative reverse transcription–PCR; RBCS, small subunit of ribulose-1,5-bisphosphate
carboxylase/oxygenase; RCA, ribulose-1,5-bisphosphate carboxylase/oxygenase activase; RPE, ribulose-5-phosphate 3-epimerase; RPI, ribose-5-phosphate isomerase; Rubisco,
ribulose-1,5-bisphosphate
carboxylase/oxygenase;
RuBP,
ribulose 1,5-bisphoshate; SBP, sedoheptulose-1,7-bisphosphatase; SPS, sucrose-phosphate synthase; TKL, transketolase;
TPI, triose-phosphate isomerase.
Introduction
Carbon assimilation is carried out by the Calvin–Benson cycle,
which consists of 11 enzymes and produces carbohydrates from
atmospheric CO2 using NADPH and ATP supplied by photochemical reactions (Calvin 1989, Benson 2002, Heldt and
Piechulla 2011). The Calvin–Benson cycle is comprised of
three phases: (i) carboxylation of ribulose 1,5-bisphoshate
(RuBP) by ribulose-1,5-bisphosphate carboxylase/oxygenase
(Rubisco) to produce 3-phosphoglycerate (3-PGA); (ii) reduction of 3-PGA to produce glyceraldehyde 3-phosphate; and (iii)
regeneration of RuBP. In addition, some other enzymes are
closely related to the function of the Calvin–Benson cycle.
Rubisco activase (RCA) regulates Rubisco activity in vivo (for
reviews, see Portis et al. 2003, 2008, Parry et al. 2013). Cytosolic
fructose-1,6-bisphosphatase (cFBP) and sucrose-phosphate
synthase (SPS) are the key enzymes for sucrose synthesis (for
reviews, see Daie 1993, Huber and Huber 1996, Sharkey et al.
2004, Serrato et al. 2009), whose product, inorganic phosphate,
is used for chloroplastic ATP synthesis required to drive the
Calvin–Benson cycle (Sharkey 1985, Sharkey et al. 1986, Sharkey
et al. 1988, Walters et al. 2004, Sun et al. 2011).
Photosynthetic activity changes during leaf development
and senescence. The rate of CO2 assimilation increases during
leaf expansion, reaches a maximum around full expansion and
declines gradually during senescence. Such a change is accompanied by changes in the activities and/or amounts of the
Calvin–Benson cycle and its related enzymes. For example,
Rubisco, 3-phosphoglycerate kinase (PGK), glyceraldehyde-3phosphate dehydrogenase (GAPDH) and phosphoribulokinase
(PRK) activities change in parallel with the rate of CO2 assimilation in rice (Oryza sativa L.) (Makino et al. 1983, Hidema et al.
1991). Similar tendencies have been observed in wheat
(Triticum aestivum L.) (Suzuki et al. 1987). However, it has
also been reported that changes in the activities and/or
amounts of the Calvin–Benson cycle and its related enzymes
are not uniform. In general, Rubisco is one of the enzymes
whose activities decline at a faster rate during leaf senescence
(Batt and Woolhouse 1975, Wada et al. 1993, Nakano et al.
Plant Cell Physiol. 57(1): 115–124 (2016) doi:10.1093/pcp/pcv183, Advance Access publication on 27 November 2015,
available online at www.pcp.oxfordjournals.org
! The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Regular Paper
Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Sendai, 981-8555 Japan
CREST, JST, Gobancho, Chiyoda-ku, Tokyo, 102-0076 Japan
2
C. Yamaoka et al. | Expression of the Calvin–Benson cycle genes in rice
1995, Fukayama et al. 1996, He et al. 1997, Crafts-Brandner et al.
1998, Ishizuka et al. 2004). In rice, the activities and/or amounts
of chloroplastic fructose-1,6-bisphosphatase (cpFBP), RCA and
SPS were found to decline more slowly than that of Rubisco
(Wada et al. 1993, Nakano et al. 1995, Fukayama et al. 1996,
Ishizuka et al. 2004). On the other hand, the activity of cFBP
declined more rapidly than the amount of Rubisco (Wada et al.
1993). Proteome analysis on senescent flag leaves in rice (Zhang
et al. 2010) provided further information on the Calvin–Benson
cycle and its related enzymes. However, it did not necessarily
coincide with the results by the above-mentioned studies that
directly determined the activities and/or amounts of the enzymes. For example, the amount of cpFBP tended to decrease as
rapidly as that of Rubisco, whereas those of PGK, PRK and cFBP
tended to decline more slowly than that of Rubisco.
It is of interest how genes of the Calvin–Benson cycle and its
related genes are regulated for formation and maintenance of
the carbon assimilation machinery during leaf development.
The mRNA levels of the Rubisco small subunit gene (RBCS)
reach a maximum during leaf expansion but decrease rapidly
with leaf age (Nikolau and Klessig 1987, Loza-Tavera et al. 1990,
Glick et al. 1995, Miller et al. 2000, Suzuki et al. 2001, Suzuki et al.
2010). The genes of the A and B subunits of GAPDH (GAPA and
GAPB) were found to show a similar trend, although their declines were slightly slower (Glick et al. 1995). The decreases in
the mRNA of PGK, PRK, RCA and the sedoheptulose-1,7-bisphosphatase gene (SBP) were also found to be slower during leaf
senescence (Nie et al. 1995, Crafts-Brandner et al. 1998). The
mRNA level of SPS has been found to be even higher in senescent leaves in rice (Okamura et al. 2011). In addition, differences
in expression patterns of a number of genes involved in carbon
metabolism have been observed during senescence of flag
leaves in rice (Zhang et al. 2010). These results suggest that
expression of the Calvin–Benson cycle and its related genes is
not uniformly regulated. However, expression patterns of these
genes have not been examined from leaf emergence to
senescence.
Although there have been a number of transcriptome studies in relation to leaf development, their experimental designs
were similar to the previous gene expression studies in most
cases. There have been some studies focused on leaf senescence
in wheat (Gregersen and Holm 2007), rice (Liu et al. 2008) and
maize (Zea mays L.) (Sekhon et al. 2012), while others have
examined early leaf developmental stages or the periods until
leaf maturation in Arabidopsis (Arabidopsis thaliana L.) (LópezJuez et al. 2008, Skirycz et al. 2010) and maize (Li et al. 2010,
Sekhon et al. 2011). On the other hand, Breeze et al. (2011) have
examined detailed gene expression profiles from leaf expansion
to senescence in Arabidopsis. It was found that the mRNA
levels of the Calvin–Benson cycle and its related genes except
SPS gradually declined during leaf development. These results
are different from those expected from the above-mentioned
gene expression studies. In Arabidopsis, expression patterns of
photosynthetic genes may be different from those in other
plant species. For example, decreases in the mRNA levels of
RBCS were slow during senescence and were similar to those
in the amounts of Rubisco (Hensel et al. 1993, Cheng et al. 1998,
116
Breeze et al. 2011). Palmer et al. (2015) have examined gene
expression profiles from just after full expansion through to
senescence in switchgrass (Panicum virgatum L.), including
both the period of formation of the photosynthetic machinery
and the course of senescence. Expression patterns of the
Calvin–Benson cycle genes were also relatively uniform. In
this study, differential expression was probably not detected
since intervals between the measurements were too long.
Therefore, it is necessary to investigate further whether differential gene expression occurs in formation and maintenance of
the machinery of photosynthetic carbon assimilation during
leaf development and senescence.
In the present study, changes in the mRNA levels of the
genes of the Calvin–Benson cycle enzymes, RCA and two key
enzymes of sucrose synthesis were examined from leaf emergence to senescence in rice. These genes were found to be
differentially expressed during the life span of leaves and
could be divided into three groups based on the patterns of
expression. This categorization was discussed in relation to its
possible physiological significance for the formation and maintenance of the photosynthetic carbon assimilation machinery.
Changes in the mRNA levels were expressed on a tissue weight
basis to compare with those of the activities and/or amounts of
the enzymes in the literature.
Results
The experiments were started on the day of the emergence of
the 12th leaves, the 11th and the 10th leaves at this stage being
on the 8th and 15th day after emergence, respectively. The 11th
leaves had just fully expanded at that time, while the full expansion of the 12th leaves was on the 6th day after their emergence. In the figures, data are plotted against the days after leaf
emergence irrespective of leaf position.
The amounts of total N, Chl and total RNA were determined
(Fig. 1). The amounts of total N and Chl increased with expansion of the 12th leaves and were maximal on the 15th day after
emergence (Fig. 1A, B). These amounts did not change greatly
in the 11th leaves and slightly declined in the 10th leaves.
Although the amount of total RNA did not change greatly
throughout the experiment, it tended to decline slightly with
leaf age, as previously observed in rice (Fig. 1C; Suzuki et al.
2001, Ishizuka et al. 2004, Imai et al. 2008).
Fig. 2 shows changes in the amounts of Rubisco, transketolase (TKL) and RCA. The amount of Rubisco also rapidly
increased with expansion and became almost maximal at the
full expansion of the 12th leaves. The amount tended to decrease gradually in the 11th and the 10th leaves. Their declines
were faster than those of total N and Chl contents as previously
observed in rice (Fig. 2A; Makino et al. 1983, Hidema et al. 1991,
Suzuki et al. 2001, Ishizuka et al. 2004). The amount of TKL
showed a tendency similar to that of Rubisco, although it
decreased only slightly after it reached a maximum (Fig. 2B).
On the other hand, the amount of RCA increased after full
expansion and reached a maximum on the 15th day after emergence of the 12th leaves (Fig. 2C). The declines of RCA with leaf
Plant Cell Physiol. 57(1): 115–124 (2016) doi:10.1093/pcp/pcv183
Fig. 1 Changes in the amounts of total N (A), Chl (B) and total RNA
(C) during development of the 12th, 11th and 10th leaves of rice.
Measurements were started when the 12th leaves had just emerged.
At that time, the 11th leaves were at the 8th day after their emergence
and at the stage of full expansion, while the 10th leaves were at the
15th day after their emergence. The 12th leaves were fully expanded
on the 6th day after their emergence. Circles, triangles and squares
represent the 12th, the 11th and the 10th leaves, respectively. Data are
presented as means ± SE (n = 3). Statistical analysis was carried out by
ANOVA with a post-hoc Tukey HSD test. Columns with the same
letter were not significantly different (P < 0.05).
age were slightly slower than those of Rubisco. These results
agree with previous observations in rice (Fukayama et al. 1996).
Changes in the mRNA levels of the genes of the Calvin–
Benson cycle and its related genes were examined. Fig. 3
shows the results for the RBCS multigene family and RCA.
Four out of five RBCS genes, namely RBCS2, 3, 4 and 5, are
actively expressed in rice leaves (Suzuki et al. 2009b) and contribute to the synthesis of a large amount of Rubisco protein
(Ogawa et al. 2012). These four genes and their sum showed
trends similar to each other and to the previous reports in rice;
the mRNA levels rapidly increased with leaf expansion, reached
maxima and then rapidly decreased (Fig. 3A–E; Suzuki et al.
2001). On the other hand, the pattern of changes in the mRNA
Fig. 2 Changes in the amounts of Rubisco (A), TKL (B) and RCA (C)
during development of the 12th, 11th and 10th leaves of rice. The
developmental stage of each leaf and symbols are the same as in Fig. 1.
Data are presented as means ± SE (n = 3). In (A–C), statistical analysis
was carried out by ANOVA with a post-hoc Tukey HSD test. Columns
with the same letter were not significantly different (P < 0.05).
level of RCA was greatly different from that of RBCS. In the 12th
leaves, the mRNA level of RCA increased and reached a maximum at the 9th day after emergence. The mRNA level in the
11th and 10th leaves remained above 40% of its maximum in
the 12th leaves (Fig. 3F).
Fig. 4 shows the results for PGK, GAPA and GAPB, which are
responsible for the reduction phase of the Calvin–Benson cycle.
PGK showed a trend similar to those of RBCS genes although
the decrease was slightly slower (Fig. 4A). Patterns of changes in
the mRNA levels of the genes of the GAPDH subunits GAPA
and GAPB were different from each other. GAPA showed a
pattern similar to that of PGK except that the mRNA level of
PGK was slightly higher in the 11th leaves (Fig. 4B). On the
other hand, the mRNA level of GAPB in the 12th leaves was
maintained after it reached a maximum during expansion,
whereas the average mRNA levels in the 11th leaf and in the
10th leaf were about 55% of the maximal mRNA level in the
12th leaves (Fig. 4C).
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C. Yamaoka et al. | Expression of the Calvin–Benson cycle genes in rice
Fig. 3 Changes in the mRNA levels of RBCS multigene family (A–D), total RBCS (E) and RCA (F) during development of the 12th, 11th and 10th
leaves of rice. (A–D) The results for RBCS2 to RBCS5, respectively. The developmental stage of each leaf and symbols are the same as in Fig. 1.
Data are presented as means ± SE (n = 3). Statistical analysis was carried out by ANOVA with a post-hoc Tukey HSD test. Columns with the same
letter were not significantly different (P < 0.05).
Fig. 5 shows the results for the eight genes responsible for
the regeneration phase of the Calvin–Benson cycle. Among
these genes, the triose-phosphate isomerase gene (TPI), TKL
and the ribose-5-phosphate isomerase gene (RPI) showed
trends similar to that of GAPA, although the declines of the
latter two were somewhat moderate (Fig. 5A, D, F).
Surprisingly, the pattern of changes in the mRNA level of the
fructose-1,6-bisphosphate aldolase gene (FBA) was almost the
same as that of RCA (Fig. 5B). Changes in the mRNA levels of
SBP, the ribulose-5-phosphate 3-epimerase gene (RPE), PRK and
cpFBP showed trends similar to that of GAPB, although the
mRNA levels of cpFBP in the 10th leaves were lower than for
other genes (Fig. 5C, E, G, H).
Fig. 6 shows the results for cFBP and SPS. Although both
genes encode key enzymes for sucrose synthesis, their changes
in mRNA levels were different from each other. The pattern of
changes in the mRNA level of cFBP was similar to those of SBP,
RPE and PRK, whereas that of SPS was similar to those of RCA
and FBA, although it has been reported that the mRNA level of
SPS was higher in a senescent leaf (Okamura et al. 2011).
The maximal absolute level of mRNA of RCA was 4,600 pmol
kg–1 FW, and this value was the highest among the examined
genes, being even 2.6-fold higher than the second highest, i.e.
the sum of the four RBCS genes. The mRNA levels of FBA, GAPA,
GAPB and PRK were 35–50% of the total RBCS mRNA, whereas
that of TPI was 25%. PGK, SBP and cpFBP had mRNA levels
around 10% of total RBCS. RPI, TKL and RPE, which are involved
in the metabolism of pentose phosphates, had the lowest
mRNA levels among the Calvin–Benson cycle genes that corresponded to <5% of total RBCS. The mRNA levels of cFBP and
SPS were even lower, being <2% of total RBCS. These results
clearly show that there was a large variation in the strength of
expression of the genes of the Calvin–Benson cycle and its
related genes in rice.
In order to examine whether there were similarities among
the expression patterns of the examined genes, changes in the
118
mRNA levels were subjected to hierarchical cluster analysis
(Eisen et al. 1998) (Fig. 7). Based on the results, these genes
were categorized into three groups. Group 1 included RBCS, TPI,
GAPA, TK, PGK and RPI. The mRNA levels became maximal on
the third day after the emergence of the 12th leaves, followed
by their declines. The mRNA levels in the 11th and 10th leaves
tended to remain low. There were some variations among these
genes, decreases in the RBCS mRNA levels being the fastest.
Group 2 included GAPB, cpFBP, cFBP, RPE, PRK and SBP. In
contrast to the group 1 genes, the mRNA levels were high
from the third to the 15th day after the emergence of the
12th leaves. The mRNA levels in the 11th and 10th leaves
were lower, although there were also some variations. Group
3 included FBA, RCA and SPS, whose mRNA levels became
almost maximal on the ninth day after the emergence of the
12th leaves and tended to be lower in the 11th and 10th leaves.
These results are summarized in Fig. 8 as a diagram of the
Calvin–Benson cycle and its related enzymes.
Discussion
The expression of genes of the Calvin–Benson
cycle and its related genes was categorized
into three groups
The present study clearly showed that the expression of genes
of the Calvin–Benson cycle and its related genes could be categorized into three groups according to their expression patterns
during leaf development in rice (Figs. 7, 8). It is suggested from
these results that different gene-regulatory mechanisms are operative for each group of genes. There have been a number of
reports on transcription factors that regulate expression of
photosynthetic genes. Among the genes responsible for
carbon assimilation, RBCS has been extensively studied. A
number of transcription factors that bind to light-responsive
elements in the promoter region have been reported to
Plant Cell Physiol. 57(1): 115–124 (2016) doi:10.1093/pcp/pcv183
genes and it related genes including GAPA, GAPB, PGK, SBP and
PRK and RCA were slower than that of RBCS (Glick et al. 1995,
Nie et al. 1995, Crafts-Brandner et al. 1998), the patterns of the
changes in the mRNA levels were greatly different from those in
the previous work by Zhang et al. (2010) on senescent flag
leaves of rice. They reported that the mRNA levels of FBA,
RPI and RPE decreased faster than those of RBCS, cpFBP, SBP
and PRK. The mRNA levels of PGK and TPI did not change
greatly, whereas those of TKL and GAPDH increased during
leaf senescence. Although the reason for the difference from
our results is unclear, it may be caused by a difference in the
growth conditions. Zhang et al. (2010) used paddy field-grown
rice, whereas we grew rice by water culture in a glasshouse.
Possible physiological significance of the
differential expression of genes of the
Calvin–Benson cycle and its related genes
Fig. 4 Changes in the mRNA levels of genes responsible for the reduction phase of the Calvin–Benson cycle during development of the
12th, 11th and 10th leaves of rice. (A–C) The results for PGK, GAPA
and GAPB, respectively. The developmental stage of each leaf and
symbols are the same as in Fig. 1. Data are presented as means ± SE
(n = 3). Statistical analysis was carried out by ANOVA with a post-hoc
Tukey HSD test. Columns with the same letter were not significantly
different (P < 0.05).
regulate its transcription (for reviews, see Tyagi and Gaur 2003,
Gangappa et al. 2013). It has also been suggested that a single
transcription factor regulates expression of multiple photosynthetic genes. A genome-wide chromatin immunoprecipitation
study suggested that in Arabidopsis, a b-ZIP transcription
factor, ELONGATED HYPOCOTYL 5, binds to the promoter
regions of TPI, FBA, cpFBP and TKL as well as that of RBCS1A
(Lee et al. 2007). In wheat, overexpression of nuclear factor YB
led to an increase in the mRNA levels of cpFBP and SBP as well
as those of a number of photosynthetic components other than
the Calvin–Benson cycle enzymes (Stephenson et al. 2011).
However, transcription factors for leaf age-dependent expression of genes of the Calvin–Benson cycle and its related genes
have not been identified.
Although our results agree with the previous observations
that decreases in the mRNA levels of the Calvin–Benson cycle
It is of interest why genes of the Calvin–Benson cycle and its
related genes were differentially expressed during leaf development in rice. The categorization of the genes may be related to
their respective roles in photosynthetic carbon assimilation.
Group 1 included the genes responsible for a series of reactions
from carbon fixation to isomerization of triose phosphate,
except GAPB (Figs. 3, 4), almost corresponding to the carboxylation and reduction phases of the Calvin–Benson cycle. Group
2 included a number of genes responsible for the regeneration
phase, i.e. cpFBP, SBP, RPE and PRK (Fig. 5C, G, E, H). However,
TKL and RPI, which are also involved in the regeneration phase,
belonged to group 1 (Fig. 5D, F). The key genes for sucrose
synthesis, cFBP and SPS, showed a different expression pattern
(Fig. 6). This was also the case for RBCS and RCA (Fig. 3),
which encode Rubisco and its in vivo regulator, respectively.
Thus, the categorization of the genes by their expression patterns did not necessarily correspond to their functional
categorization.
Otherwise, comparison between the expression patterns
and information on their proteins in the previous studies can
help explain the need for the differential gene expression. The
mRNA levels of the group 1 genes reached maxima during expansion and then declined. The decrease in RBCS mRNA levels
was the sharpest, followed by those of PGK, GAPA and TPI, and
then TKL and RPI (Figs. 3A–E, 4A, B, 5A, D, F). It is known that
Rubisco is a very stable protein. Its synthesis is almost completed during expansion, whereas the amount of Rubisco is
mainly regulated at the level of its protein degradation during
senescence (Mae et al. 1983, Makino et al. 1984, Suzuki et al.
2001). As a result, changes in the mRNA levels and the amounts
of protein show patterns different from each other (Suzuki et al.
2001; Figs. 2A, 3A–E). Changes in the activities of PGK and
GAPDH have been found to be similar to those in the activity
and/or amount of Rubisco (Makino et al. 1983, Hidema et al.
1991). These observations suggest the possibility that PGK is as
stable as Rubisco. This seems to be the case for GAPDH if its
synthesis in senescent leaves was limited by the mRNA level of
GAPA, which was lower than that of GAPB (Fig. 4B, C). In the
case of TKL, the decrease in its amount was slower than that of
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C. Yamaoka et al. | Expression of the Calvin–Benson cycle genes in rice
Fig. 5 Changes in the mRNA levels of genes responsible for the regeneration phase of the Calvin–Benson cycle during development of the 12th,
11th and 10th leaves of rice. (A–H) The results for TPI, FBA, cpFBP, TKL, SBP, RPI, RPE and PRK, respectively. The developmental stage of each leaf
and symbols are the same as in Fig. 1. Data are presented as means ± SE (n = 3). Statistical analysis was carried out by ANOVA with a post-hoc
Tukey HSD test. Columns with the same letter were not significantly different (P < 0.05).
Fig. 6 Changes in the mRNA levels of cFBP (A) and SPS (B) during
development of the 12th, 11th and 10th leaves of rice. The developmental stage of each leaf and symbols are the same as in Fig. 1. Data
are presented as means ± SE (n = 3). Statistical analysis was carried out
by ANOVA with a post-hoc Tukey HSD test. Columns with the same
letter were not significantly different (P < 0.05).
120
Fig. 7 Hierarchical cluster analysis of the genes of the Calvin–Benson
cycle and its related genes based on their expression patterns during
leaf development in rice. Data for each gene presented in Figs. 3–6
were converted to relative values when the maximal level during the
experimental period was defined as 1, the relative values being subjected to the analysis. Pearson correlation was used as the distance
measure for complete linkage clustering. The numbers in the right side
of the figure represent the group of the genes generated by the cluster
analysis.
Plant Cell Physiol. 57(1): 115–124 (2016) doi:10.1093/pcp/pcv183
Fig. 8 Summary of the present study. In the left panel, categorization of genes of the Calvin–Benson cycle and its related genes is mapped onto
the metabolic diagram. The blue, red and orange boxes denote that the genes belong to groups 1, 2 and 3, respectively. In the right panel, changes
in the relative mRNA level in each group are shown. Data are presented as means ± SD (n = 3–9). Abbreviations for the metabolites: RuBP,
ribulose 1,5-bisphosphate; 3-PGA, 3-phosphoglycerate; 1,3-PGA, 1,3-bisphosphoglycerate; G3P, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; F1,6BP, fructose 1,6-bisphosphate; F6P, fructose 6-phosphate; E4P, erythrose 4-phosphate; S1,7BP, sedoheptulose 1,7-bisphosphate; S7P, sedoheptulose 7-phosphate; R5P, ribose 5-phosphate; X5P, xylulose 5-phosphate; Ru5P, ribulose 5-phosphate.
Rubisco during senescence (Fig. 2A, C). Possibly, such a maintained protein content is due to the following reasons: TKL
protein was also stable and its synthesis continued to some
extent during senescence, as suggested from its mRNA level
(Fig. 5D).
In contrast to these genes, the mRNA levels of the group 2
and 3 genes gradually declined after they reached maxima.
These groups included cpFBP, RCA and SPS, in which the activities and/or amounts are known to decrease more slowly than
those of Rubisco (Wada et al. 1993, Nakano et al. 1995,
Fukayama et al. 1996, Ishizuka et al. 2004). On the other
hand, the activity of PRK has been observed to decrease as
rapidly as that of Rubisco (Makino et al. 1983), while that of
cFBP decreased even more rapidly (Wada et al. 1993). These
results indicate that a slow decline in the mRNA levels during
senescence does not always lead to a maintained protein content. One possible explanation for the expression patterns of
the genes in groups 2 and 3 seems to be the instability of protein, as can be deduced from relationships between the mRNA
level and the amount of protein. For example, changes in the
mRNA level of RCA were similar to those in the amount of RCA
protein (Figs. 2C, 3F), which was different from the relationship
between the mRNA and the protein levels in Rubisco (Figs. 2A,
3A–E). This means that the amount of RCA protein is mainly
determined by its synthesis during leaf development, suggesting
that RCA is rapidly degraded. This agrees with previous observations that RCA is more susceptible to proteolysis than
Rubisco under physiological conditions (Fukayama et al.
2010). The low amount of RCA protein despite the high
mRNA level (Figs. 2C, 3F) also supports this assumption. It is
also possible that patterns of changes in the mRNA and protein
levels of PRK and cFBP tended to be similar to each other
(Makino et al. 1983, Wada et al. 1993; Figs. 5H, 6A). This implies
that PRK and cFBP are also continuously expressed to compensate the instability of the proteins.
Therefore, it is possible that the group 1 genes encode stable
proteins, whereas the group 2 and 3 genes tend to encode
relatively unstable proteins that are actively synthesized
during leaf development. Thus, it is likely that expression patterns of genes of the Calvin–Benson cycle and its related genes
are dependent on the stability of protein to yield sufficient
amounts of enzymes required for the machinery of photosynthetic carbon assimilation. In order to examine these assumptions quantitatively, information on the amount and the
turnover rate of each enzyme is required. Recently, protein
turnover rates in their steady state were estimated by proteome
analysis combined with 15N labeling in barley (Hordeum vulgare
L.) (Nelson et al. 2014), whereas their non-steady-state rates
such as those during leaf senescence remain to be studied. It
has been shown that the rates of synthesis and degradation of
bulk soluble protein drastically change during leaf development
in rice (Mae et al. 1983, Makino et al. 1984).
In addition, the difference in the expression pattern may also
be related to differences in translational efficiency of the
mRNAs. It is possible that genes whose translational efficiency
declined during leaf senescence were continuously expressed.
As an index for translational efficiency, the ratio of protein
synthesis to the mRNA level and rate of polysome association
of mRNA can be used. We examined changes in Rubisco synthesis, the mRNA levels of Rubisco genes and their polysome
association in rice leaves at different ages (Suzuki and Makino
2013). It was found that the ratio of RBCS synthesis to the
mRNA level of RBCS tended to be marginally higher in
mature and senescent leaves than that in young, expanding
leaves, and that rates of polysome association of the mRNAs
of RBCS genes were almost the same irrespective of leaf age.
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C. Yamaoka et al. | Expression of the Calvin–Benson cycle genes in rice
These results suggest that the translational efficiency of RBCS
did not change greatly in relation to leaf age in rice. On the
other hand, a recent comprehensive study in Arabidopsis leaves
at different ages suggested that rates of polysome association
changed with leaf age and differed among genes (Yamasaki et al.
2015). As for the individual genes of the Calvin–Benson cycle
and its related enzymes, it was shown that rates of polysome
association of GAPB and FBA1 mRNAs and GAPA and FBA2
mRNAs declined to <80% and 70% of those in young leaves,
respectively, whereas those of other genes were relatively small.
Polysome association analysis of genes of the Calvin–Benson
cycle and its related genes would help the understanding of the
relationship between the differential expression patterns and
translational efficiencies during leaf development and senescence in rice.
Conclusion
In the present study, it was shown that changes in the expression of genes of the Calvin–Benson cycle and its related genes
during leaf development can be categorized into three groups
for formation and maintenance of the machinery of photosynthetic carbon assimilation during leaf development in rice. This
categorization was related to the stabilities of protein rather
than their functions in photosynthetic carbon assimilation.
Further study is needed on the interesting question of what
determines such differential expression of these genes.
Materials and Methods
Plant culture and sampling
Rice (Oryza sativa L. cv. Notohikari) seeds were soaked in tap water, and the
seedlings were then grown on a net floating on tap water for 21 d in an environmentally controlled growth chamber as described in Sudo et al. (2014). Six
seedlings each were transplanted to 3.5 liter plastic pots containing a nutrient
solution, as described by Makino et al. (1988), and grown in a greenhouse. The
nutrient solution was renewed once a week. From the day of the emergence of
the 12th leaves on the main culms, the 12th, 11th and 10th leaf blades were
collected between 10:00 and 14:00 h every 3 d for 15 d. All samples were
weighed, immediately frozen in liquid N2 and stored at –80 C until analysis.
The collected 12th leaf blades included the unexpanded parts that were surrounded by the 11th leaf sheaths, if any. From the beginning of sampling, the
plants were fed with a nutrient solution without N every 6 d. They were fed with
NH4NO3 at a rate of 0.292 mmol per plant every 3 d. Renewal of the nutrient
solution and N feeding were carried out after each sampling since a drastic
change in N concentration immediately affected the mRNA levels of RBCS and
RBCL in rice (Imai et al. 2008).
Biochemical assays
Frozen leaves were homogenized in Na-phosphate buffer, and N contents were
determined with Nessler’s reagent after Kjeldahl digestion as described in
Suzuki et al. (2007). Chl contents were determined according to the method
of Arnon (1949) as described in Makino and Osmond (1991). Rubisco content
was determined by formamide extraction of Coomassie Brilliant Blue R-250stained bands corresponding to the large and small subunits of Rubisco separated by SDS–PAGE using calibration curves made with purified rice Rubisco
(Makino et al. 1985). TKL and RCA contents were determined as follows. An
aliquot of supernatant of the leaf homogenate was combined with an equal
volume of SDS sample buffer [200 mM Tris–HCl containing 2% (v/v) SDS, 20%
(v/v) glycerol and 5% (v/v) 2-mercaptoethanol], boiled for 2 min and stored at
–30 C until analysis. An aliquot of the sample at a volume corresponding to 7
122
mg of total N was subjected to SDS–PAGE using a gel, 16 cm 16 cm in size,
containing acrylamide at 8% (w/v) in the separation gel. Calibration curves were
made with bovine serum albumin. The gel was stained with One-Step
Coomassie Brilliant Blue staining solution (BioCraft), destained with distilled
water and scanned. The intensities of the bands corresponding to TKL and the
small isoform of RCA, which is the major isoform in rice (Zhang and Komatsu
2000, Fukayama et al. 2012), were analyzed with MultiGauge ver. 3.0 (FUJIFILM).
The bands for RCA and TKL were confirmed by Western blotting using a
commercially available RCA antibody (Agrisera) and an antiserum against the
fragment of the putative TKL [residues 72–86 (ETLEGQAATGALLEK) of
AAO33154.1 (GenBank)] raised in rabbit (Sigma Aldrich Japan)
(Supplementary Fig. S1). This putative TKL is considered to be a chloroplastic
isoform since it is highly homologous to the experimentally examined cpTKLs
(for alignments, see Bi et al. 2013, Rocha et al. 2014) and has been found to exist
in rice plastids (Kleffmann et al. 2007). In the 12th leaves just after emergence,
RCA and TKL contents were determined by Western blotting since their bands
were not distinguishable after Coomassie Brilliant Blue staining. An aliquot at a
volume corresponding to 0.15 and 1.5 mg of total N was subjected to SDS–PAGE
for RCA and TKL, respectively. Samples prepared from the 12th leaves on the
6th day after their emergence were loaded as a control. Western blotting was
carried out with a TGX FastCast acrylamide kit with 12% (w/v) acrylamide (BioRad), a semi-dry blotting apparatus (Trans-Blot Turbo Transfer System; BioRad), a polyvinyldifluoridene (PVDF) membrane (Trans-Blot Turbo RTA
Transfer Kit, Mini, PVDF; Bio-Rad), a chemiluminescence detection kit
(SuperSignal West Dura Extended Duration Substrate; Life Technologies
Japan) and an image analyzer (LAS-4000 and MaltiGauge ver. 3.0; FUJIFILM).
RNA analysis
Total RNA was extracted according to the method of Suzuki et al. (2004) with
slight modifications (Suzuki et al. 2009a). The mRNA levels were determined by
quantitative reverse transcription–PCR (qRT–PCR) as described in Ogawa et al.
(2012). The list of the examined genes and the primer pairs is presented in
Supplementary Table S1. PCR amplicons generated with the primer pairs were
cloned with the Mighty TA-cloning Kit for PrimeSTAR (TAKARA) and used to
make calibration curves. Homologs have been searched with the SALAD database (Mihara et al. 2010) and are listed in Izawa et al. (2011). Highly expressed
genes among the homologs were selected by RT–PCR. For genes with low or
undetectable expression levels, genomic PCR was carried out as control. The
lists of examined genes, primer pairs and predicted size of the amplicons are
presented in Supplementary Table S2. Results of RT–PCR are presented in
Supplementary Fig. S2. SPS1 was selected as a highly expressed SPS gene as
previously reported (Okamura et al. 2011). There are two RCA genes,
Os04g0658300 and Os11g0707000, in rice according to the SALAD database
(Mihara et al. 2010). The latter was selected since its cDNA sequence is
almost identical to that of the RCA gene cloned from a cDNA library constructed from leaf total RNA (Zhang and Komatsu 2000).
Statistical treatments
Tukey’s HSD test was performed with JMP11 (SAS Institute Inc.). Hierarchical
cluster analysis (Eisen et al. 1998) was carried out based on relative changes in
the mRNA levels with MultiExperiment Viewer (Saeed et al. 2003). For each
gene, the mRNA level at a given time point was divided by the maximal level
observed during the experimental period to calculate relative mRNA levels.
Pearson correlation was used as the distance measure for complete linkage
clustering.
Supplementary data
Supplementary data are available at PCP online.
Funding
This study was supported the Ministry of Education, Culture,
Sports, Science and Technology [a GRENE NC-CARP project
Plant Cell Physiol. 57(1): 115–124 (2016) doi:10.1093/pcp/pcv183
(to A.M.)]; the Japan Society for the Promotion of Science
[Grants-in-Aid for Scientific Research (No. 26450074 to Y.S.)].
Disclosures
The authors have no conflicts of interest to declare.
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