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Discussion
Two AGPase small subunit ( Aps) and four large subunit (Apl) genes were identified from
Arabidopsis and the expression of AtApl3 synergistically regulated by Suc and ABA in
Arabidopsis leaf (Rook et al., 2001). Since it is reported that most of the AGPase activity in the
leaf comes from the AtAps1 and AtApl1 genes (Lin et al., 1988a; Wang et al., 1998; Lin et al.,
1988b; Wang et al., 1997) and an AtApl3 transcript level was approximately 5 and 7 fold lower
than AtAps1 and AtApl1 genes, respectively (Smith et al., 2004), the actual molecular basis and
physiological roles of synergistic regulation system are unknown. In this study, we
demonstrated that OsAGPL3 was also induced by Suc plus ABA within 3 hr after treatment and
starch contents were also increased by Suc plus ABA within 24 hr after treatment . These results
indicated that these regulation systems maybe conserved in other plants and may be involved in
regulation of starch biosynthesis.
In order to get more information about these regulation systems, we used cDNA-amplified
fragment length polymorphism (cDNA-AFLP) technology to identify Suc plus ABA -inducible
genes in rice cultured cells as the reference to levels of OsAGPL3-gene expression . Using TaqI and
AvaII primers carrying two selective nucleotides gives a total of 256 possible nucleotides. With
half of these combinations performed, around 11000 TDFs could be detected (Fig 5). In silico
analysis of 32,127 full-length cDNAs (FL-cDNAs) from rice has shown that 86% of all
transcripts are cut by both AvaII and TaqI restriction enzymes. The 18,377 (about 57% of all
FL-cDNAs) FL-cDNAs are analyzable by 128 primer-pair combinations (only AvaII primer are
labeled with
32
P-ATP) (date not shown). With such a resolution, we monitored the influence of Suc and
ABA on gene expression in rice cells.
It is reported that a lot of genes were up- and down regulated by sugars (Koch KE 1996;
Rolland F et al. 2002) and by ABA (Seki et al. 2002; Rabbani et al. 2003; Yazaki et al. 2003).
We confirmed that 415 and 157 TDFs were up-regulated by ABA and Suc, respectively. These
results indicated that effect of Suc and ABA on genes expression in the rice cells were
successfully
monitored
by
cDNA-AFLP.
Recent
genetic
and
molecular
studies
of
sugar-signalling mutants in Arabidopsis have uncovered many links between sugar and
plant-hormone signaling (Leon and Sheen 2003). Ninety-four TDFs are up-regulated by both
Suc and ABA (or by transfer to flesh medium) (Fig 4). These results indicated that ABA and
Suc signaling in the rice cells are essentially separate but partially cross -talk and converge.
Sixty-three TDFs were synergistically induced by Suc plus ABA and 26 clones were
confirmed as Suc plus ABA-inducible genes with Northern blot analysis (Table 1). Two putative
hexose transporter (HXT) genes were contained in them. Cakir et al (2001) previously reported
that
grape
transcriptional
regulator
(VvMSA)
and
hexose
transporter
(VvHT1)
were
synergistically induced by Suc and ABA, and VvHT1 was regulated by VvMSA vir Suc box3
(Tsukaya et al. 1991) or SURE1(Grierson et al. 1994) in the VvHT1 promoter. We searched for
the 5' sequence of rice two HXTs with those of genomic sequences using DNA databases
(http://www.ncbi.nlm.nih.gov/). There is no such element in each promoter. In addition, we
searched for the promoter sequences of the other Suc plus ABA -inducible genes, two genes
(protein phosphatase 2C-related / PP2C-related and tyrosine specific protein phosphatase
family protein) contained SURE1 in their promoters but other clones did not contained such
element in these promoter. Since Suc plus ABA-inducible genes were categorized into two
groups based on expression patterns (Fig 5), at least two different transcriptional factor(s)
which is synergistically regulated by Suc and ABA may be exit in the rice plants.
Starch biosynthesis
We found that four starch biosynthesis related genes (OsAGPL3, starch branching enzyme 4;
RBE4, Glucose-6-phosphate/phosphate-translocator; GPT, and two HXTs) were induced by Suc
plus ABA treatment. It is generally accepted that AGPase, sucrose synthase (Susy), starch
synthase (SS) and starch branching enzyme (RBE) may play a key role in starch biosynthesis and
over 20 genes were characterized from rice plants. Since these genes except for OsAGPL3 and
RBE4 did not induced by Suc plus ABA treatment, it is possible that OsAGPL3 and RBE4 may
play a direct roll in starch accumulation by Suc plus ABA treatment. It is reported that
exogenous ABA stimulates the uptake of sugars by rice suspension cells (Kashem MA et al.,
1998). Sucrose plus ABA-inducible two HXTs maybe involved in this phenomenon and
transported sugars were maybe used as substrate for starch biosynthesis. GPT maybe promote
the starch accumulate by rise the concentration of hexose in the amyloplast and by activate the
plastidial AGPase vir excretion of organic phosphate which act as an inhibitor of AGPase into
cytoplasm.
Fatty acid biosynthesis
Genes involved in fatty acid biosynthsis stearoyl-acyl-carrier protein desaturase (FAB2),
omega-6-desaturase
(FAD2)
and
S-adenosyl-L-methionine:
phosphoethanolamine
N-methyltransferase (PEAMT) were induced by Suc plus ABA treatment. FAB2 converts stearic
acid (18:0) to oleic acid (18:1) is one of the key steps in the fatty acid biosynthesis pathway
that regulates levels of unsaturated fatty acids in the cell. FAD2 converts oleic acid (18:1) to
palmitic acid (18:2). PEAMT catalyzes the key step in choline (Cho) biosynthesis, the
N-metheylation of phosphoethanolamine. Cho is a vital precursor of phosphatidylch oline,
which is a major membrane lipid in plants, accounting for 40% to 60% of lipids in non-plastid
plant memmbranes (Moore 1990). Arabidopsis fab2 (Lightner et al., 1994) and fad2 (Miquel M
and
Browse
J
1992)
mutants
and
PEAMT
antisense-transgenic
plants
showed
temperature-sensitive phenotype. A general increase in level of polyunsaturated fatty acids is
observed in most plants during growth at low temperatures (Graham and Patterson, 1982).
Fluidity of membranes has been considered to play an important ro le in survival at low
temperatures. A major factor determining the fluidity of the membrane is the degree of
unsaturation of membrane lipids (Nishida and Murata et al., 1996). Since FAB2, FAD2 and
PEAMT induced by Suc plus ABA, it is possible that signals of Suc plus ABA involve in
chilling-resistance. γ-glutamylcyctein synthease (γ-ECS) and ABI3 interacting protein 2 (AIP2)
were also induced by Suc plus ABA treatment. It is reported that γ-ECS catalyzes the first step
in glutathione biosynthesis was induced by chilling stress in Maize (Gomez et al., 2004) and
inhibition of γ-ECS reduced chilling tolerance in transgenic maize(kocsy et al., 2000). AIP2
was increased response to low temperature (Nogueira y et al., 2003). These results support the
possibility of Suc plus ABA signals involve in chilling-resistance.
We demonstrated that Suc + ABA signals are involve in starch and fatty acid biosynthesis in
rice cultured cell. However, it is not clear whether Suc + ABA signal is involve in starch and
fatty acid biosynthesis in rice seed. Suc and hexose contents in the rice seed rapidly increased
after flowering and reached a maximum at 3 DAF and then slowly decreased (Hirose et al.,
2002). On the other hand, ABA contents in the rice grains start to increase at 6 day after
anthesis, and reached a maximum at 15 to 18 day after flowering (DAF) and then slowly
decreased (Yang et al., 2001).Taken together, both Suc and ABA is exitence in developing seed
after 6 DAF. In addition, OsAGPL3, RBE4, FAB2, FAD6 and PEAMT were expressed in
developing seed after 6 DAF(date not shown). These results raise the possibility that Suc plus
ABA signals were involved in starch and fatty acid biosynthesis not only in cultured cell but
also in developing seed. We identified that ABI3 interacting protein2 was induced by Suc plus
ABA. In Arabidopsis thaliana (L.) Heynh, the seed specific transcription factors ABI3 have key
regulatory functions during the development of mature seeds. The Arabidopsis abi3 mutant lack
any significant storage protein or lipid accumulation, but accumulate 3 -6-fold more Suc than
wild type seeds during the seed development (Finkelstein et al., 1990 Ooms et al., 1993). These
results indicated that high Suc is not sufficient to promote reserve accumulation and that ABA
has a central roll in the regulation of seed dev elopment. Therefore, involvement of Suc plus
ABA signals in seed development requires future investigation.
In silico cDNA-AFLP analysis of Oryza sativa
An in silico analysis of the rice 32127 full-length cDNAs was performed to determine the
number of cDNAs in which is analyzable by cDNA-AFLP using 128 primer-pair sets. Based on
in silico analysis, 18377 rice cDNAs (estimated 57 % coverage) can be analyze. Two TDFs are
produced from OsAGPL3 cDNA. However, 8 kinds of TaqI primers which we used cannot
anneal with these TDFs. Thus, OsAGPL3 expression profiles can not be analyzed by
cDNA-AFLP using 128 primers combinations.
However, the actual molecular basis and physiological roles of these regulation mechanisms are
unknown.
In this study, we clarified that the expression of OsAGPL3 was also cooperatively regulated
by Suc and ABA in the rice cells. These results indicated that these regulation systems are
maybe conserved in other plants.
Two AGPase small subunit ( Aps) and four large subunit (Apl) genes were identified from
Arabidopsis.
An AtApl3 transcript level was approximately 5 and 7 fold lower than AtAps1 and AtApl1
genes, respectively (Smith et al., 2004). Mutational analysis showed that most of the AGPase
activity in the leaf comes from the AtAps1 and AtApl1 genes (Lin et al., 1988a; Wang et al.,
1998; Lin et al., 1988b; Wang et al., 1997).
In this study, cDNA-AFLP was used to isolate Suc plus ABA-inducible genes. 63 Suc plus
ABA-inducible TDFs were isolated and 26 TDFs are confirmed as Suc plus ABA-inducible
gene by Northern-blot analysis. This is a first report about global transcriptional monitoring
and isolation of Suc and ABA-inducible genes.
Although the number of TDFs and genes is usually disagree, our results indicated that ABA
and Suc signaling in the rice cultured cell are essentially separate but partially cross -talk and
converge.
Cakir et al (2001) previously reported grape transcriptional regulator (VvMSA) and hexose
transporter (VvHT1)were synergistically induced by Suc and ABA. VvHT1 was regulated by
VvHT1 vir Suc box3 (Tsukaya et al. 1991) or SURE1(Grierson et al. 1994) in the VvHT1
promoter. We identified that two rice putative hexose transporters were also induced by Suc
plus ABA. We search for the 5' sequence data of these genes with those of genomic sequences
of rice using Genome Project databases. Two putative hexose transporter genes do not have Suc
box 3 and SURE1 in their promoters. Same analysis was performed for 23 Suc plus
ABA-inducible rice full-length cDNA clones. Two genes (protein phosphatase 2C-related /
PP2C-related and
tyrosine specific protein phosphatase family protein) contained SURE1 in
their promoters but 21 clones did not contain. These dates imply that Suc box 3 or SURE1
independent Suc plus ABA co-regulation systems are exit in rice cultured cell.
The expression of Arabidopsis Sbe2, one of the starch branching enzyme gene, was also
cooperatively regulated by Suc and ABA in Arabidopsis leaf (Rook et al., 2001).
Because
and these regulation systems maybe involved in regulation of starch biosynthesis.
Although expression of genes related to starch biosynthesis was also cooperatively regulated by
Suc and ABA was identified, whether these regulation systems regulate starch contents or not is
not clear. In this study, we characterized starch contents in the rice cells were cooperatively
regulated by Suc and ABA. These results indicated that these regulation systems are regulate
not only expression of genes related to starch biosynthesis but also starch contents.
In this study, transcriptional factor(s) were not identified by cDNA -AFLP. ABA によ っ て 誘
導 さ れ る 転 写 因子 の 発 現 時 期 は と ても 早 い た め に 単 離 でき な か っ た も の と 思わ れ る 。
In this reports, we demonstrated that the expression of OsAGPL3 was synergistically regulated
by Suc and ABA in the rice cells.
Since These results indicated that these regulation systems are maybe conserved in other plants
and may be involved in regulation of starch biosynthesis .
ABA に よ っ て 誘 導 さ れ る こ と が 知 ら れ て い る 遺 伝 子 が 単 離 さ れ て い な い こ と も 実 験 が
う ま く 行 っ て いる 理 由 と し て 考 え られ た 。
Arabidopsis SBE, one of the starch branching enzyme genes and Apl3,one of AGPase large
subunits genes and Vitis vinifera Hexose transporter (VvHT1) were also synergistically induced
by Suc plus ABA in leaves was previously reported (Rook et al. 2001). These results indicated
that these regulation systems are also conserved in plant species.
Arabidopsis SBE are also synergistically induced by Suc plus ABA in leaves are previously
reported (Rook et al. 2001).These results indicated that these regulation systems are conserved
in plant species.However, it is not clear whether Suc plus ABA treatment increase starch
contents. We demonstrated that starch contents in the rice cultured cell were increased by Suc
plus ABA(Fig
). These results strongly indicated that Suc plus ABA signal involved in the
regulation of starch biosynthesis in the rice cultured cell. We propose a model to explain how
Suc plus ABA signal enhanced starch accumulation.
sugar
transporter
proteins
(two
Suc plus ABA treatment increased three
hexose
transporter
and
glucose-6-phosphate/phosphate-translocator; GPT) and two starch biosynthesis genes (RBE4
and OsAGPL3). In rice cultured cells, Suc was cleaved extracellularly by a cell wall -associated
invertase prior to uptake (Amino S and Tazawa M. 1988). Suc plus ABA treatment may promote
the hexose import vir Suc plus ABA inducible hexose transporters. Imported hexose is
converting
to Glucose-6-Phosphate(Glc6P), and then import into amyloplast via GPT.
Imported G-6-P is converting to Glucose-1-Phospahte (G1P) catalyzed by phosphoglucomutase.
Glc1P is converting to ADP-glucose catalyzed by AGPase. AGPase is exquisitely sensitive to
allosteric regulation, with glycerate-3-phosphate acting as an activator and Pi as an inhibitor
(Sowokins et al., 1982). GPT is glucose-6-phosphate/phosphate antiporter. Suc plus ABA
inducible GPT may act not only supply the substrate for starch synthesis but also activate t he
AGPase activity vir excretion of inhibitor.
Arabidopsis fab2 and fad2 mutant caused increased levels of stearic acid (18:0) (Lightner et al.,
1994) and oleic acid (18:1) (Miquel M and Browse J 1992), respectively. Both mutant show
temperature-sensitive phenotypes. These results indicated that Suc plus ABA signal involved in
control fatty acid biosynthesis.