JXB Advance Access originally published online on January 10, 2005
Journal of Experimental Botany 2005 56(412):623-632; doi:10.1093/jxb/eri065
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RESEARCH PAPER |
Evolution and expression analysis of starch synthase III and IV in rice
State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310029, PR China
To whom correspondence should be addressed. Fax: +86 571 86971323. E-mail: clspwu{at}zju.edu.cn
Received 23 August 2004; Accepted 25 October 2004
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Plants contain at least five subfamilies of starch synthases, granule bound starch synthase (GBSS) and starch synthases I, II, III, and IV (SSI, SSII, SSIII, SSIV). In this work, two members of SSIII and SSIV, respectively, were cloned and designated OsSSIII-1/-2 and OsSSIV-1/-2 in rice. Together with six other previously reported genes, the SS gene family in rice therefore is known to be duplicated and to comprise ten SS genes distributed among the five subfamilies. The starch synthase activity of each SS was confirmed by expression and enzyme activity assay in E. coli. Expression profile analysis with reverse transcription-PCR, western blotting and zymogram, indicates that OsSSIII-2 and OsSSIV-1 are mainly expressed in endosperm, while OsSSIII-1 and OsSSIV-2 are mainly expressed in the leaves. With a similar pattern of genes encoding other enzymes for starch synthesis, (such as GBSS, SSII, ADP-glucose pyrophosphorylases, and branching enzymes), it is suggested that two divergent groups of these genes should be classified in rice. Group I genes are preferentially expressed in the endosperm and function on storage starch synthesis. Group II genes are mainly expressed in leaves and some of them in the early developing endosperm, and function on transient starch synthesis in rice.
Key words: Gene duplication, gene expression, Oryza sativa L, starch biosynthesis, starch synthase
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Plant starches are comprised of two classes of glucose homopolymers. Firstly, there is amylose, which is a lightly branched linear molecule with a degree of polymerization of 1000 to 5000 Glc units. Secondly, amylopectin, which has a much larger polymer unit (with a degree of polymerization of 105–106 Glc units) and contains frequent
-1,6 branch linkages. In higher plants, biosynthesis of starch occurs in plastids with the involvement of a series of biosynthetic enzymes, including ADP-Glc pyrophosphorylase (AGPase), starch synthase (SS), starch branching enzyme (BE), and debranching enzyme (DBE) (Smith et al., 1997
; Myers et al., 2000; James et al., 2003).
The -glucan chains of both types of polymers are elongated by starch synthase (ADP-Glc: -1,4 glucan -4-glucosyl-transferase; EC 2. 4. 1. 21). Starch synthase catalyses the transfer of -D-glucose from ADP-Glc to the non-reducing end of the chain by an -1,4-linkage. Five subfamilies of starch synthases have been identified in higher plants, including granule-bound starch synthase (GBSS), starch synthase I (SSI), starch synthase II (SSII), starch synthase III (SSIII), and starch synthase IV (SSIV). GBSS is essential for amylose synthesis and is exclusively bound to the starch granule. SSI, SSII, SSIII, and SSIV (named as SSV in dicots) are responsible for amylopectin chain elongation with their distribution between the granular and soluble fractions (Ball and Morell, 2003; Li et al., 2003). Each class of SS genes plays a distinct role in the synthesis of amylopectin. Analysis of a rice mutant with a retrotransposon inserted into a gene that encodes for SSI indicates that amylopectin is depleted in chains of DP8-12 and enriched in chains of DP6 and 7 in the mutant. This suggests a distinct capacity of SSI for the synthesis of chains with DP8-12 from DP6-7 (Nakamura, 2002). SSII has a specific role in the synthesis of intermediate-length chains (B2 and B3 chains) (Fontaine et al., 1993; Craig et al., 1998; Edwards et al., 1999; Morell et al., 2003). Mutations eliminating SSIII activity are known in maize as du1 (Gao et al., 1998) and antisense suppression of SSIII has been reported in the potato (Edwards et al., 1999). The suppression of SSIII activity reduced amylopectin synthesis and therefore the length of development of the amylopectin long chains (DP25-35). This suggests that SSIII preferentially synthesizes long B1 and B2 chains.
The origin of these five starch synthase subfamilies is clearly ancient, given the conservation of the orthologous genes from Chlamydomonas through to the dicots and monocots (Ball and Morell, 2003). Evidence is increasing from a range of species that two members of GBSS (Fujita and Taira, 1998; Vrinten and Nakamura, 2000; Edwards et al., 2002; Dian et al., 2003), and two classes of SSII (Harn et al., 1998; Jiang et al., 2004) are present and functionally divergent with one member expressed in seeds and another member expressed in other tissues.
In rice, six distinct genes encoding starch synthases have been reported (Wang et al., 1990; Baba et al., 1993; Dian et al., 2003; Jiang et al., 2004). The identification of two genes encoding SSIII and two genes encoding SSIV in rice is reported here. The gene structure, expression patterns, genomic distribution, and evolution of the starch synthase genes have also been investigated and discussed. The expression pattern of the genes for starch synthesis and their role in starch accumulation in cereal seeds is also a key focus.
In the present paper, the two-letter prefix, Os, is used to indicate rice (Oryza sativa L.). The genes were designated OsSSIIIa, OsSSIIIb, OsSSIVa, and OsSSIVb, in chronological order of the submission to GenBank.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Plant material
The indica rice (Oryza sativa L.) variety Zhe733 and japonica rice variety Zhenongda104 (for zymogram analysis) were grown in a greenhouse at 22/28 °C (night/day) and 80% relative humidity. The grains were harvested several times during maturation while the leaves and roots were taken from 4-leaf-stage seedlings.
For the sugar effect test, the 4-leaf-stage seedlings were cultured for 2 d under dark condition to deplete endogenous sugars before treatments with sugars. After 2 d, the second and third leaves were excised from the plants (now about 1 cm in length) and then transferred and incubated in rice nutrition solution (Yoshida et al., 1976) with different sugar or sugar analogues in the dark at 28 °C. Excised leaves were also incubated in a rice solution without sugar as a control. The leaf samples were harvested after 12 h of sugar treatments.
cDNA cloning of OsSSIII and OsSSIV
The TBLASTN searching of the GenBank (http://www.ncbi.nlm.nih.gov) databases with the conserved C-terminal amino acids of rice starch synthases yielded four highly homologous rice genomic clones: AL606645
[GenBank]
(OSJN00079), AP005441
[GenBank]
, AP003292
[GenBank]
, and AC121365
[GenBank]
. The predicted polypeptides encoding by AL606645
[GenBank]
and AP005441
[GenBank]
bear an analogy to maize SSIII/DU1, named as OsSSIII-1 and OsSSIII-2, while those of AP003292
[GenBank]
and AC121365
[GenBank]
bear an analogy to wheat SSIV, named as OsSSIV-1 and OsSSIV-2, respectively. The OsSSIII-1 cDNA clone containing the complete coding domain sequence (CDS) and an OsSSIII-1 cDNA fragment (lack 560 bp at the 5' region of the putative ORF) were isolated by screening the immature seed cDNA library (Jiang et al., 2004). For sequencing, the positive clones were converted to pTriplEx2. The plasmid was prepared using GFXTM Micro Plasmid Prep Kit (Amersham Pharmacia Biotech), and then the cDNA fragments were subcloned into the pBsSK vector and sequenced (MegaBACETM 1000, Amersham Pharmacia Biotech). To clone the two putative SSIV cDNA, two primer pairs were designed for each gene. S411 [5'-AGT GCC CCT CCT CGC CCG CTT G (–24 to –3 bp, A –ATG is No. 1)] and S412 [5'-GTC GTA TTT GGG AAG AAC AAT CTC (+1540 to +1563 bp)], S413 [5'-AAA AAA GGT ACC GGA AAC AGT TCT GGC TTG CAC ATT GTC-3' (+1414 to +1440 bp)] and S414 [5'-AAA AAA GAG CTC ATG TTT GAG CCG CTC CCC TTG CCT G-3' (+2893 to +2917 bp)]; S421 [5'-TTC CCC AGC CTC CGC ATC CGA TTC (–26 to –3 bp)] and S422 [5'-TCA CAT CCG CCA AAC CAC CAA CC (+1308 to +1330 bp), S423 [5'-AAA AAA GGT ACC ACT AGT TCT GGC TTG CAC ATC ATC C (+1252 to +1276 bp)] and S424 [5'-AAA AAA GAG CTC TGC TGC TGC CCT CGC TCG AGC CAC TG (+2720 to +2745 bp)], respectively. The amplified fragments were cloned in the pUCm-T vector and sequenced (MegaBACETM 1000, Amersham Pharmacia Biotech). GenBank accession numbers of these four cDNA clones were AF432915 (OsSSIII-1), AY100469 (OsSSIII-2), AY373257 (OsSSIV-1), and AY373258 (OsSSIV-2), respectively.
Expression of OsSSIII and OsSSIV in E. coli
The following primers were used for PCR modification of the N-termini of OsSSIIIa, OsSSIIIb, OsSSIVa, and OsSSIVb: pS311 [5'-AAA TTG CAT ATG ATG GAC TTT GTT TTC TCT-3' (+2194 to +2214)] and pS312 [5'-AAA TTG GAT ATC TCA GTT CTT GCG AGC GGA ATG G-3' (+3630 to +3651)]; pS321 [5'-AAA TTG CAT ATG ATG GAC TTT GTT TTC TCT-3' (+3908 to +3930)] and pS322 [5'-AAA AAA GAG CTC TCA AAA TTT gGTG AGC TGA ATG-3' (+5346 to +5367)]; S413 [5'-AAA AAA GGT ACC GGA AAC AGT TCT GGC TTG CAC ATT GTC-3' (+1414 to +1440)] and S414 [5'-AAA AAA GAG CTC ATG TTT GAG CCG CTC CCC TTG CCT G-3' (+2893 to +2917)]; S423 [5'-AAA AAA GGT ACC ACT AGT TCT GGC TTG CAC ATC ATC C (+1252 to +1276 bp)] and S424 [5'-AAA AAA GAG CTC TGC TGC TGC CCT CGC TCG AGC CAC TG (+2720 to +2745 bp)]. These primers were used to introduce NdeI/EcoRV, NdeI/ SacI, KpnI/SacI, and KpnI/SacI restriction sites at the C-termini of OsSSIII-1, OsSSIII-2, OsSSIV-1, and OsSSIV-2, respectively. The reconstructed sequences were subcloned into pET-29b (Novagen), named pSSIII-1, pSSIII-2, pSSIV-1, and pSSIV-2, respectively. The SDS-PAGE analysis of the expressed proteins and enzyme assays were carried out as described previously (Dian et al., 2003).
Semiquantitative RT-PCR analysis
Total RNA from leaves, roots, and grains of plants were isolated with Trizol (GIBICOL). PCR amplifications were performed on first cDNA strand corresponding to 2 µg of total RNA, using eight specific primer sets (Table 1). All specific primers were designed to span intron sequences, thus allowing the signals resulting from genomic DNA contamination to be accounted for. Primers (Table 1) that amplify Actin (Genbank accession number X16280) from rice were used as a control. The amplified fragments were cloned in the pUCm-T vector and sequenced (MegaBACETM 1000, Amersham Pharmacia Biotech). Thermocycling time and temperature were as follows: 95 °C for 5 min, followed by 30 cycles (or as indicated) of 95 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min, and a final extension period 72 °C for 7 min. PCR products were separated on 1% agarose gels, and stained with ethidium bromide.
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Gel electrophoresis and immunoblot analysis
Native PAGE was carried out using 7.5% (resolving gel) and 2.5% (stacking gel) polyacrylamide and activity staining was performed as previously described (Jiang et al., 2003
). Each lane was loaded with 50 µg of the crude extract from leaves (L) or endosperms. Starch synthase activity was detected after incubation of the gel in 50 mM Tricine-NaOH (pH 8.5), 0.5 M sodium citrate, 25 mM potassium acetate, 2 mM EDTA, 2 mM DTT, 0.1% (w/v) potato amylopectin, and the indicated content of ADP-glucose at the indicated temperature for 10 h.
In immunoblot analysis, the following antisera were used: antisera (Anti-ZmSSIII), raised against the N-terminal region of the maize SSIII protein, were the generous gift of Dr Alan M Myers (Iowa State University, Ames) and are described by Cao et al. (1999). The rabbit polyclonal antisera (designated Anti-OsSSI and Anti-OsSSIII) were raised against the deduced mature OsSSI or the C-terminus of OsSSIII-1 expressed in E. coli (Jiang et al., 2004). The Anti-OsSSII-2 and Anti-OsSSII-3 are described by Jiang et al. (2004). Protein extracts were separated by 7.5% Native-PAGE or 7.5% SDS–PAGE, transferred to polyvinylidenedifluoride (PVDF) membranes (AMRESCO), and visualized using rabbit anti-SS antiserum followed by alkaline phosphatase–conjugated goat anti-rabbit serum (Santa Cruz, USA).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Cloning and characterization of OsSSIII and OsSSIV genes
BLAST analysis of the rice genome (http://www.ncbi.nlm.nih.gov/BLAST/) using the conserved C-terminus of starch synthase amino acid sequences, revealed four new homologous genes. According to the putative amino acid sequences, two genes were homologous to maize SSIII/DU1, designated OsSSIII-1 and OsSSIII-2, and the other two genes homologous to wheat SSIV, designated OsSSIV-1 and OsSSIV-2. The cDNA of the four genes were cloned by screening a cDNA library or RT-PCR. According to the cDNA sequences, rice SSIII genes contain 16 exons separated by 15 introns as in wheat. The sequences of exon3 are the variable repeat region (Li et al., 2000
), and vary between OsSSIIIa and OsSSIIIb in length. The structures of the SSIV genes contain 16 exons separated by 15 introns, the first three exons in the 5' region of the genes are variable in length.
Duplication and distribution of SS gene families in rice
The four genes of SSIII-1/-2 and SSIV-1/-2 cloned in this case together with the other six genes cloned previously (Wang et al., 1990; Baba et al., 1993; Dian et al., 2003; Jiang et al., 2004), compose 10 members of the SS gene family in rice with five different subfamilies distributed over eight rice chromosomes. SSIII-1, SSIII-2, SSIV-3, and SSIV-4 were located on chromosomes 4, 8, 1, and 5, respectively. However, many genes clustered around the loci of SSIII-1 and SSIII-2 and the loci of the SSIV-1 and SSIV-2 were identical (Fig. 1). This observation indicated that the duplications of SSIII and SSIV were associated with the duplication of a large chromosome segment during evolution. In addition to the 10 genes, a truncated SSI gene was identified on chromosome 10 and a truncated SSII-3 gene (Accession number BAC16084) on chromosome 7. The putative sequence of amino acids of the truncated SSI shared high identity with C-terminus of OsSSI (Fig. 2).
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Domain organization of OsSSIII and OsSSIV and expression of OsSSIII and OsSSIV in E. coli
As is the case in the SSIII protein in maize and wheat (Gao et al., 1998
; Li et al., 2000), rice SSIII proteins also contain four distinct regions. These are a putative transit peptide region (47/49 amino acids for OsSSIII-1/-2) (identified using the ChloroP neural network analysis of the 100 amino acids at the N-terminus of each sequence), a variable repeat region, a SSIII specific region, and a C-terminal region that contains the catalytic domain (Fig. 3A). The SSIII specific region in OsSSIII-1/-2 was composed by a set of three repeated units of amino acid similarity in each gene. Thirteen amino acids were conserved in these repeat units (Fig. 3B). Rice SSIV proteins contain three distinct regions: a putative transit peptide region (78/33 amino acids for SSIV-1/-2), a region homologous to Smc (COG1196)/myosin tail 1 (pfam01576) and a C-terminal catalytic domain region (Fig. 3A).
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The deduced amino acid sequences of the C-terminal catalytic domain region from the ten OsSS proteins were aligned with one prokaryotic glucogen synthase (E. coli glucogen synthase, EcGS) using the Clustal W program. Nine conserved regions were identified. The invariant residues were listed (Fig. 3C). The first homologous region contains the consensus motif KXGGL, which is believed to be the ADP-Glc binding site of starch synthase (Furukawa et al. 1990
). To determine whether the OsSSIII and OsSSIV genes encode authentic starch synthases, expression (Fig. 4) of OsSSIII-1, OsSSIII-2, OsSSIV-1 and OsSSIV-2 genes in E. coli (BL21) were tested. The activities of starch synthase were increased by 4.1-fold, 4.8-fold, 2.7-fold, and 2.4-fold above baseline activity level of E. coli glycogen synthase, for each of the four genes, respectively. This indicated that the OsSSIII and OsSSIV genes encoded functional starch synthase enzymes in rice.
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Organ expression profile of OsSSIII and OsSSIV genes
The spatial expression of OsSSIII-1, OsSSIII-2, OsSSIV-1, and OsSSIV-2 was examined in storage and non-storage organs of rice by RT-PCR (Fig. 5A). The result shows that OsSSIII-1 and OsSSIV-2 were expressed mainly in leaves and weakly in endosperms, while OsSSIII-2 and OsSSIV-1 mainly in endosperms and weakly in leaves. Total proteins extracted from rice leaves and endosperms were electrophoresed in SDS–PAGE gels using antibodies to maize SSIII polypeptide (N-terminus) (anti-ZmSSIII) and anti OsSSIII-1 polypeptide (C-terminus) (anti-OsSSIII-1). Both antibodies were bound to 230 kDa proteins from rice endosperms and 170 kDa proteins from rice leaves and endosperms (Fig. 5B). The calculated molecular mass of OsSSIII-1 and OsSSIII-2 was 138 kDa and 201 kDa, respectively. The apparent molecular mass of SS as estimated on SDS-PAGE, was often noted to be larger than the calculated molecular mass (Knight et al., 1998
), suggesting that the 230 kDa protein should be OsSSIII-1 and the 170 kDa protein should be OsSSIII-1. This result is consistent with the expression patterns of OsSSIII-1 and OsSSIII-2b transcripts. OsSSIII-1 was expressed at the early developing stage of endosperm, while OsSSIII-2 reached maximum levels at the middle age. The transcription of OsSSIV-1 reached maximum levels in endosperm at the late developmental stage (Fig. 5C). Western blotting also indicated that OsSSIII-2 protein maintains higher levels at the middle developmental stage of rice endosperms (Fig. 5D).
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Zymogram analysis for SS activities in rice endosperm and leaf extracts
The zymogram experiment was employed to identify further the expression of different SS genes in rice endosperms and leaves. Figure 6A shows that at least four SS activity bands were detected in rice leaves and five SS activity bands in rice endosperms. Immunoblotting showed that the lowest mobility activity band in endosperms should be that of OsSSIII-2, and in leaves should be that of OsSSIII-1 (Fig. 6B). Rice SSIII-2 showed temperature-sensitive activity, with notably lower starch synthase activity at 37 °C than in 25 °C. Figure 6B also shows that the expression of OsSSI was not tissue-specific, whilst OsSSII-2 was leaf-specific and OsSSII-3 was endosperm-specific. The mobility of OsSSII-2 in leaves and OsSSII-3 in endosperms is seen as equal. SSI and SSII showed an identified band in many rice cultivars such as Zhenongda104, Nipponbare and Kasalath (the data of last two cultivars is not shown).
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Influence of sugar level on SS gene expression in rice leaves
It has been demonstrated that the gene expression of OsGBSSII is regulated by sugar level in excised leaves (Dian et al., 2003
). The effects of sugars on the other SS genes that expressed in rice leaves were tested in this case. Excised leaves were treated with 175 mM of mannitol (mal), sucrose (suc), 3-O-methyl-glucose (3-OMG), and 2-deoxyglucose (2-DOG), respectively. RT-PCR analysis indicated that accumulation of OsSSI, OsSSII-2, and OsSSIII-1 transcripts were up-regulated by sucrose, but not for OsSSIII-2 transcripts. Glucosamine eliminated the accumulation of these transcripts induced by sucrose (Fig. 7). Sequence analysis has revealed the sugar response elements in the promoter regions (up to 2000 bp from the initiation codon of ATG) of the three SS genes: OsSSIIIa, SP8BFIBSP8BIB (TACTATT) (Ishiguro and Nakamura, 1992), –1431, SURE2STPAT21 (AATACTAAT) (Grierson et al., 1994), –1758; OsSS1, SURE1STPAT21 (AATAGAAAA) (Grierson et al., 1994), –830; OsSSII-2, SURE1STPAT21, –362. This suggested that the sugar regulation of these SS genes in rice leaves is consistent and the sugar response elements are active in rice.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Characterization of SSIII and SSIV genes in rice
Five subfamilies of starch synthase genes have been reported in higher plants (Ball and Morell, 2003
). Among them, three subfamilies have been characterized in rice, including GBSS (Wang et al., 1990; Dian et al., 2003), SSI (Baba et al., 1993; Tanaka et al., 1995), and SSII (Jiang et al., 2004). In this paper the genes of other two subfamilies, SSIII and SSIV, in rice were cloned and characterized. The gene structure analysis indicates that the arrangements of exon and intron of SSIII and SSIV genes are highly conserved in rice, wheat, and Arabidopsis, respectively, whereas they differ from those of GBSS, SSI, and SSII genes (Li et al., 2003; Dian et al., 2003; Jiang et al., 2004). SSIII proteins contain a SSIII-specific region with a set of three repeated units, while SSIV proteins contain a region homologous to the Smc motif. According to these observations, it is clear that the separation of GBSS, SSI, SSII, SSIII, and SSIV predates the separation of monocotyledonous and dicotyledonous plants. The proteins of OsSSIII-1/-2 and OsSSIV-1/-2 contain conserved domains characteristic of GlgA (glycogen synthase) at the C-termini. Each class of GlgA domain can catalyse starch synthesis when expressed in E. coli. This result indicates that the SSIII and SSIV genes encode functional starch synthase enzymes in rice.
Evolution of the SS gene families in rice
It has been reported that GBSS and SSII subfamilies have diverged through gene duplication events in cereals (Harn et al., 1998; Vrinten and Nakamura, 2000; Dian et al., 2003; Jiang et al., 2004). The present observation indicated that other two subfamilies, SSIII and SSIV, are also duplicated and diverged in rice. The homologous ESTs of SSIII and SSIV genes are detected in other cereals in GenBank and Tiger EST databases (data not shown), which suggests the duplication and divergence of SSIII and SSIV presented in other Gramineae. Although the duplicated SSI genes have been destroyed, gene duplication has also been identified (Fig. 2). This is the first report that the duplication event occurred in all the five subfamilies genes for starch synthase in Gramineae.
Subfunctionalization of the duplicated genes for starch synthesis in rice
The expression patterns of the five SS gene subfamilies were investigated as a first step toward understanding their respective functions. Previous observations indicated that the transcripts and proteins of the OsSSII-2 and OsGBSSII genes were detected mainly in leaves, while OsSSII-3 and GBSSI (Wx) primarily in endosperms (Harn et al., 1998; Vrinten and Nakamura, 2000; Dian et al., 2003; Jiang et al., 2004). The present observation with reverse transcription-PCR, western blotting or zymogram, indicate that OsSSIII-2 and OsSSIV-1 are mainly expressed in endosperm, while OsSSIII-1 and OsSSIV-2 are mainly expressed in leaves (Figs 5, 7). In rice endosperms, OsSSIII-1 was expressed at an early developing stage, while OsSSIII-2 reached maximum levels at the middle stage. In rice leaves, as for the OsGBSSII, the expression of OsSSI, OsSSII-2, and OsSSIIIa is regulated by sugars, suggesting that sugar regulation of these SS genes converged during rice evolution. The consistent regulation of these OsSS genes allows starch synthesis to be modulated in response to the accumulation of sugars in rice leaves under conditions such as high light and nitrogen or phosphate starvation.
In cereals, evidence is increasing that the different subfamilies of genes for starch synthesis may be partitioned into two gene groups, with one group expressed in the endosperm and a second mainly expressed in other tissues. For instance, BEIIb (RBE3) is specifically expressed in endosperms, while BEIIa (RBE4) is mainly expressed in vegetative tissues and in the early developing endosperms in cereals (Yamanouchi and Nakamura, 1992; Gao et al., 1996; Morell et al., 1997; Sun et al., 1998; Jiang et al., 2003; Mutisya et al., 2003). Transcripts of AgpL1 and AgpS1a genes were specifically detected in endosperms, while AgpL2, AgpS1b, and AgpS2 are mainly expressed in leaves of maize, wheat, barley, and rice (AgpS1a and AgpS1b are encoded by a single gene in wheat, barley, and rice). AgpL2, AgpS1b, and AgpS2 are likewise expressed in the early developing endosperm in wheat, barley, and rice (Burton et al., 2002; Johnson et al., 2003). Thus, in cereals, the duplicated starch synthesis genes diverged into two groups: Group I, preferentially expressed in endosperms, including AgpL1, AgpS1a, GBSSI, SSII-3 (SSIIa), SSIII-2 (DU1), SSIV-1 (may be including SSIV-2), and BEIIb (RBE3), and Group II, mainly expressed in other tissues, and some in the early developing endosperms, including ApgL2, AgpS1b, AgpS2, GBSSII, SSII-2 (SSIIb), SSIII-1, SSIV-2 (may be including SSIV-1), and BEIIa (RBE4). The single member subfamily genes of SSI and BEI (RBE1) are expressed constitutively and function in both groups (Fig. 8). Thus the preservation of these duplicate genes is likely to be by subfunctionalization (Lynch and Force, 2000), with the primary function of the Group II genes on transient starch synthesis, and Group I genes on storage starch synthesis in rice.
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Expression profiles of the corresponding genes suggest a two-step model for starch biosynthesis in rice
It is already known that angiosperm seeds accumulate storage protein, oil, and carbohydrate during seed filling and degrade them to support early seedling growth upon germination. The relative abundance of storage reserves varies among seeds of different species. Many oilseeds produce 50–70% oil, some legumes contain 40% protein, whereas most cereal seeds contain 70–85% of the seed dry weight as starch (Baud et al., 2002
). In developing legume seeds, the model of detailed gene expression regarding the control of starch and protein synthesis has been established through the proteomics research on Medicago truncatula seed development (Gallardo et al., 2003). Contrapuntal networks of gene expression during the Arabidopsis seed-filling period reveal that the genes involved in carbohydrate metabolism peaks are expressed in early seed development, those required for oil synthesis are expressed later, and the genes for oil-body and storage-protein are expressed later still (Ruuska et al., 2002). Although the cytosolic localization of AGPase in cereal endosperms has been suggested to have functional significance for partitioning large amounts of carbon into starch when sucrose is plentiful (Beckles et al., 2001), the accumulation of storage components requires the co-ordination of many genes that encode the enzymes of the corresponding pathways (Ruuska et al., 2002). In rice, the expression of Group II starch synthesis genes peaks early in seed development, similar to that in the developing seeds of Arabidopsis (Fig. 8B), whilst the expressed pattern of Group I starch synthesis genes was consistent with the age of the rapid accumulation of starch in the endosperms. This suggests that this is one of the primary factors in the synthesis and storage of starch in rice endosperms (Fig. 8B).
The expression profiles of these genes are likely to suggest a two-step model for starch biosynthesis in rice endosperms. Group II genes synthesize the early transient starch mainly in leaves and Group I genes synthesize the storage starch in rice endosperms. This result is also in support of the hypothesis of subfunctionalization regarding the preservation of the duplicated genes in the genome (Lynch and Force, 2000). Therefore this can help in understanding the co-evolution of the relevant genes regarding the functional divergences in duplicated genes as revealed in gene expression changes in plants.
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Acknowledgements |
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The authors thank Dr Alan M Myers for the generous donation of antibodies to the maize DU1 protein. This project was supported by the Natural Science Foundation of China, the Natural Science Foundation of Zhejiang Province (Grant No. ZA0106), and the China Postdoctoral Science Foundation (Grant No. 2003034068).
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Footnotes |
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* These authors contributed equally to this study.
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References |
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Baba T, Nishihara M, Mizuno K, Kawasaki T, Shimada H, Kobayashi E, Ohnishi S, Tanaka K, Arai Y. 1993. Identification, cDNA cloning, and gene expression of soluble starch synthase in rice (Oryza sativa L.) immature seeds. Plant Physiology 103, 565–573.[Abstract]
Ball SG, Morell MK. 2003. From bacterial glycogen to starch: understanding the biogenesis of the plant starch granule. Annual Review of Plant Biology 54, 207–233.[CrossRef][Medline]
Baud S, Boutin J, Miquel M, Lepiniec L, Rochat C. 2002, An integrated overview of seed development in Arabidopsis thaliana ecotype WS. Plant Physiology and Biochemistry 40, 151–160.[CrossRef][ISI]
Beckles DM, Smith AM, Rees T. 2001. A cytosolic ADP-glucose pyrophosphorylase is a feature of graminaceous endosperms, but not of other starch-storing organs. Plant Physiolgy 125, 818–827.
Burton RA, Jenner H, Carrangis L, et al. 2002. Starch granule initiation and growth are altered in barley mutants that lack isoamylase activity. The Plant Journal 31, 97–112.[CrossRef][ISI][Medline]
Cao H, Imparl-Radosevich J, Guan H, Keeling PL, James MG, Myers AM. 1999. Identification of the soluble starch synthase activities of maize endosperm. Plant Physiology 120, 205–215.
Craig J, Lloyd JR, Tomlinson K, Barber L, Edwards A, Wang TL, Martin C, Hedley CL, Smith AM. 1998. Mutations in the gene encoding starch synthase II profoundly alter amylopectin structure in pea embryos. The Plant Cell 10, 413–426.
Dian WM, Jiang HW, Chen QS, Liu FY, Wu P. 2003. Cloning and characterization of the granule-bound starch synthase II gene in rice: gene expression is regulated by the nitrogen level, sugar and circadian rhythm. Planta 218, 261–268.[CrossRef][ISI][Medline]
Edwards A, Borthakur A, Bornemann S, Venail J, Denyer K, Waite D, Fulton D, Smith A, Martin C. 1999. Specificity of starch synthase isoforms from potato. European Journal of Biochemistry 266, 724–736.[ISI][Medline]
Edwards A, Vincken JP, Suurs LC, Visser RG, Zeeman S, Smith A, Martin C. 2002. Discrete forms of amylose are synthesized by isoforms of GBSSI in pea. The Plant Cell 14, 1767–1785.
Fontaine T, D'Hulst C, Maddelein ML, Routier F, Pepin TM, Decq A, Wieruszeski JM, Delrue B, Van den Koornhuyse N, Bossu JP. 1993. Toward an understanding of the biogenesis of the starch granule. Evidence that Chlamydomonas soluble starch synthase II controls the synthesis of intermediate size glucans of amylopectin. Journal of Biological Chemistry 268, 16223–16230.
Fujita N, Taira T. 1998. A 56 kda protein is a novel granule-bound starch synthase existing in the pericarps, aleurone layers, and embryos of immature seed in diploid wheat (Triticum monococcum L.). Planta 207, 125–132.[CrossRef][ISI][Medline]
Furukawa K, Tagaya M, Inouye M, Preiss J, Fukui T. 1990. Identification of lysine 15 at the active site in Escherichia coli glycogen synthase. Conservation of a lys-X-gly-gly sequence in the bacterial and mammalian enzymes. Journal of Biological Chemistry 265, 2086–2090.
Gallardo K, Le Signor C, Vandekerckhove J, Thompson RD, Burstin J. 2003. Proteomics of Medicago truncatula seed development establishes the time frame of diverse metabolic processes related to reserve accumulation. Plant Physiology 133, 664–682.
Gao M, Fisher DK, Kim KN, Shannon JC, Guiltinan MJ. 1996. Evolutionary conservation and expression patterns of maize starch branching enzyme I and IIb genes suggests isoform specialization. Plant Molecular Biology 30, 1223–1232.[CrossRef][ISI][Medline]
Gao M, Wanat J, Stinard PS, James MG, Myers AM. 1998. Characterization of dull1, a maize gene coding for a novel starch synthase. The Plant Cell 10, 399–412.
Grierson C, Du JS, Zabala MT, Beggs K, Smith C, Holdsworth M, Bevan M. 1994. Separate cis sequences and trans factors direct metabolic and developmental regulation of a potato tuber storage protein gene. The Plant Journal 5, 815–826.[CrossRef][ISI][Medline]
Harn C, Knight M, Ramakrishnan A, Guan H, Keeling PL, Wasserman BP. 1998. Isolation and characterization of the zSSIIa and zSSIIb starch synthase cDNA clones from maize endosperm. Plant Molecular Biology 37, 639–649.[CrossRef][ISI][Medline]
Ishiguro S, Nakamura K. 1992. The nuclear factor SP8BF binds to the 5'-upstream regions of three different genes coding for major proteins of sweet potato tuberous roots. Plant Molecular Biology 18, 97–108.[CrossRef][ISI][Medline]
James MG, Denyer K, Myers AM. 2003. Starch synthesis in the cereal endosperm. Current Opinion in Plant Biology 6, 215–222.[CrossRef][ISI][Medline]
Jiang HW, Dian WM, Liu FY, Wu P. 2004. Molecular cloning and expression analysis of three genes encoding starch synthase II in rice. Planta 218, 1062–1070.[CrossRef][ISI][Medline]
Jiang HW, Dian WM, Wu P. 2003. Effect of high temperature on fine structure of amylopectin in rice endosperm by reducing the activity of the starch branching enzyme. Phytochemistry 63, 53–59.[CrossRef][ISI][Medline]
Johnson PE, Patron NJ, Bottrill AR, Dinges JR, Fahy BF, Parker ML, Waite DN, Denyer K. 2003. A low-starch barley mutant, riso 16, lacking the cytosolic small subunit of ADP-glucose pyrophosphorylase, reveals the importance of the cytosolic isoform and the identity of the plastidial small subunit. Plant Physiology 131, 684–696.
Knight ME, Harn C, Lilley CE, Guan H, Singletary GW, MuForster C, Wasserman BP, Keeling PL. 1998. Molecular cloning of starch synthase I from maize (W64) endosperm and expression in Escherichia coli. The Plant Journal 14, 613–622.[CrossRef][ISI][Medline]
Li Z, Mouille G, Kosar-Hashemi B, Rahman S, Clarke B, Gale KR, Appels R, Morell MK. 2000. The structure and expression of the wheat starch synthase III gene. Motifs in the expressed gene define the lineage of the starch synthase III gene family. Plant Physiology 123, 613–624.
Li Z, Sun F, Xu S, Chu X, et al. 2003. The structural organisation of the gene encoding class II starch synthase of wheat and barley and the evolution of the genes encoding starch synthases in plants. Functional and Integrative Genomics 3, 76–85.
Lynch M, Force A. 2000. The probability of duplicate gene preservation by subfunctionalization. Genetics 154, 459–473.
Morell MK, Blennow A, Kosar-Hashemi B, Samuel MS. 1997. Differential expression and properties of starch-branching enzyme isoforms in developing wheat endosperm. Plant Physiology 113, 201–208.[Abstract]
Morell MK, Kosar-Hashemi B, Cmiel M, Samuel MS, Chandler P, Rahman S, Buleon A, Batey IL, Li Z. 2003. Barley sex6 mutants lack starch synthase IIa activity and contain a starch with novel properties. The Plant Journal 34, 173–185.[CrossRef][Medline]
Mutisya J, Sathish P, Sun C, Andersson L, Ahlandsberg S, Baguma Y, Palmqvist S, Odhiambo B, Aman P, Jansson C. 2003. Starch-branching enzymes in sorghum (Sorghum bicolor) and barley (Hordeum vulgare): comparative analyses of enzyme structure and gene expression. Journal of Plant Physiology 160, 921–930.[CrossRef][ISI][Medline]
Myers AM, Morell MK, James MG, Ball SG. 2000. Recent progress toward understanding biosynthesis of the amylopectin crystal. Plant Physiology 122, 989–997.
Nakamura Y. 2002. Towards a better understanding of the metabolic system for amylopectin biosynthesis in plants: rice endosperm as a model tissue. Plant Cell Physiology 43, 718–725.
Ruuska SA, Girke T, Benning C, Ohlrogge JB. 2002. Contrapuntal networks of gene expression during Arabidopsis seed filling. The Plant Cell 14, 1191–1206.
Smith AM, Denyer K, Martin C. 1997. The synthesis of the starch granule. Annual Review of Plant Physiology and Plant Molecular Biology 48, 67–87.[CrossRef]
Sun C, Sathish P, Ahlandsberg S, Jansson C. 1998. The two genes encoding starch-branching enzymes IIa and IIb are differentially expressed in barley. Plant Physiology 118, 37–49.
Tanaka K, Ohnishi S, Kishimoto N, Kawasaki T, Baba T. 1995. Structure, organization, and chromosomal location of the gene encoding a form of rice soluble starch synthase. Plant Physiology 108, 677–683.[Abstract]
Vrinten PL, Nakamura T. 2000. Wheat granule-bound starch synthase I and II are encoded by separate genes that are expressed in different tissues. Plant Physiology 122, 255–264.
Wang ZY, Wu ZL, Xing YY, Zheng FG, Guo XL, Zhang WG, Hong MM. 1990. Nucleotide sequence of rice waxy gene. Nucleic Acids Research 18, 5898.
Yamanouchi H, Nakamura Y. 1992. Organ specificity of isoforms of starch branching enzyme (Q-enzyme) in rice. Plant Cell Physiology 33, 985–991.
Yoshida S, Forno DA, Cock JH, Gomez KA. 1976. Laboratory manual for physiological studies of rice, 3rd edn. Manila, The Philippines: International Rice Research Institute, 61–64.
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