amyloplast-localized substandard starch grain4 protein ... · amyloplast-localized substandard...

Amyloplast-Localized SUBSTANDARD STARCHGRAIN4 Protein Influences the Size of Starch Grains inRice Endosperm1[W]

Ryo Matsushima*, Masahiko Maekawa, Miyako Kusano, Hideki Kondo, Naoko Fujita, Yasushi Kawagoe2,and Wataru Sakamoto

Institute of Plant Science and Resources, Okayama University, Kurashiki 710–0046, Japan (R.M., M.M., H.K.,W.S.); RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa 230–0045, Japan (M.K.);Department of Biological Production, Akita Prefectural University, Akita 010–0195, Japan (N.F.); and Divisionof Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba 305–8602, Japan (Y.K.)

Starch is a biologically and commercially important polymer of glucose and is synthesized to form starch grains (SGs) insideamyloplasts. Cereal endosperm accumulates starch to levels that are more than 90% of the total weight, and most of theintracellular space is occupied by SGs. The size of SGs differs depending on the plant species and is one of the most importantfactors for industrial applications of starch. However, the molecular machinery that regulates the size of SGs is unknown. In thisstudy, we report a novel rice (Oryza sativa) mutant called substandard starch grain4 (ssg4) that develops enlarged SGs in theendosperm. Enlargement of SGs in ssg4 was also observed in other starch-accumulating tissues such as pollen grains, root caps,and young pericarps. The SSG4 gene was identified by map-based cloning. SSG4 encodes a protein that contains 2,135 amino acidresidues and an amino-terminal amyloplast-targeted sequence. SSG4 contains a domain of unknown function490 that is conservedfrom bacteria to higher plants. Domain of unknown function490-containing proteins with lengths greater than 2,000 amino acidresidues are predominant in photosynthetic organisms such as cyanobacteria and higher plants but are minor in proteobacteria.The results of this study suggest that SSG4 is a novel protein that influences the size of SGs. SSG4 will be a useful molecular tool forfuture starch breeding and biotechnology.

Plastids originated from the endosymbiosis of cya-nobacteria and can differentiate into several formsdepending on their intracellular functions during theplant life cycle (Sakamoto et al., 2008). The amyloplast isa terminally differentiated plastid responsible for starchsynthesis and storage. Starch forms insoluble particles inamyloplasts, referred to as starch grains (SGs). SGs areeasily visualized by staining with iodine solution, andthey can be observed using a light microscope. SGs areobserved in storage organs such as seed endosperm,

potato (Solanum tuberosum) tubers, and pollen grains.Nonstorage tissues such as endodermis and root capsalso contain SGs (Morita, 2010).

Cereal endosperm accumulates high levels of starch inamyloplasts. The volume of SGs is approximately thesame as the volume of amyloplasts that fill most of theintracellular space. SGs in rice (Oryza sativa) endospermare normally 10 to 20 mm in diameter (Matsushima et al.,2010). One amyloplast contains a single SG that is as-sembled of several dozen smaller starch granules. Eachstarch granule is a sharp-edged polyhedron with atypical diameter of 3 to 8 mm. This type of SG is called acompound SG (Tateoka, 1962). For compound SGs,starch granules are assembled (but not fused) to form asingle SG, which is easily separated by conventionalpurification procedures. By contrast, simple SGs containa single starch granule. Simple SGs are produced inseveral important crops, such as maize (Zea mays), sor-ghum (Sorghum bicolor), barley (Hordeum vulgare), andwheat (Triticum aestivum; Tateoka, 1962; Matsushimaet al., 2010, 2013).

The size of SGs in cereal endosperm is diverse. Maizeand sorghum SGs have a uniform size distribution ofapproximately 10 mm in diameter (Jane et al., 1994;Matsushima et al., 2010; Ai et al., 2011). In barley andwheat, SGs of two discrete size classes (approximately15225 mm and less than 10 mm) coexist in the same cells(Evers, 1973; French, 1984; Jane et al., 1994; Matsushimaet al., 2010). In Bromus species, intrageneric size

1 This work was supported by the Ministry of Education, Culture,Sports, Science, and Technology (Grant-in-Aid for Scientific Researchno. 23770046 to R.M.), by the Program for the Promotion of Basic andApplied Researches for Innovations in Bio-oriented Industry (to N.F.),by the Japan Advanced Plant Science Network, and by the followingfoundations: the Iijima Memorial Foundation for the Promotion ofFood Science and Technology, the Japan Prize Foundation, the ShoraiFoundation for Science and Technology, the Wesco Scientific Promo-tion Foundation, the Towa Foundation for Food Research, the Foun-dation of the Skylark Food Science Institute, and the OoharaFoundation.

2 Deceased.* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Ryo Matsushima ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.113.229591

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variations of SGs are observed in which even phyloge-netic neighbors develop distinctly sized SGs (Matsushimaet al., 2013). The size of SGs can be controlled by ma-nipulating the activity of starch synthetic enzymesusing transgenic plants or genetic mutants (Gutiérrezet al., 2002; Bustos et al., 2004; Ji et al., 2004; Stahl et al.,2004; Matsushima et al., 2010). However, the molecularmechanism that controls the interspecific size variationsof SGs has not been resolved.

The SG occupies most of the amyloplast interior, be-cause the SG is approximately the same size as theamyloplast. The size of amyloplasts may affect the sizeof SGs, or vice versa. Amyloplasts and chloroplasts bothdevelop from proplastids. The size of chloroplasts iscontrolled by the chloroplast binary fission divisionmachinery, especially by the ring structures that form atthe division sites (Miyagishima, 2011). Proteins involvedin the ring structures have been isolated, includingFilamenting temperature-sensitive mutantZ (FtsZ),Minicell locusD (MinD), MinE, and ACCUMULATIONAND REPLICATIONS OF CHLOROPLAST5 (ARC5).Arabidopsis (Arabidopsis thaliana) mutants that are de-fective in these proteins have defects in chloroplastdivision and contain enlarged and dumbbell-shapedchloroplasts. In contrast to the binary fission of chloro-plasts, amyloplasts divide at multiple sites and generatea beads-on-a-string structure (Yun and Kawagoe, 2009).The inhibition of the chloroplast division machinerydoes not result in enlarged amyloplasts (Yun andKawagoe, 2009).

We recently developed a rapid method to prepare thinsections of endosperm (Matsushima et al., 2010). Usingthis method, SGs in mature endosperm can be easily andclearly observed. We performed genetic screening for ricemutants defective in SG morphology and size. One of theisolated mutants, substandard starch grain4 (ssg4), developsenlarged SGs in its endosperm. In this study, we char-acterized ssg4 phenotypes and identified the responsiblegene. SSG4 encodes a protein containing 2,135 amino acidresidues and an N-terminal plastid-targeted sequence.The domain of unknown function 490 (DUF490) wasfound at the C terminus of SSG4, where the ssg4mutationwas located. This suggests that SSG4 is a novel factor thatinfluences the size of SGs and has potential as a moleculartool for starch breeding and biotechnology.

RESULTS

Enlarged SGs in ssg4 Mutant Endosperm

The chalkiness of seeds was a distinguishing pheno-type of ssg4 grains when compared with wild-typegrains of cv Nipponbare (Fig. 1, A–D). Seed size wasslightly smaller in ssg4 than in cv Nipponbare, especiallywith respect to seed width and depth (Fig. 1E). Theiodine-stained thin sections of mature endosperm clearlyshowed enlarged SGs in ssg4 endosperms (Fig. 1, F–I).Quantification of the areas occupied by SGs in the thinsections showed that SGs were approximately 6-foldlarger in ssg4 than in cv Nipponbare (Fig. 1J).

The endosperm is a triploid tissue generated by thefusion of sperm and the binucleate central cell of thefemale gametophyte (Li and Berger, 2012). Therefore,endosperm has four possible genotypes at one genelocus:AAA, AAa, Aaa, and aaa. We performed reciprocalcrosses to obtain two distinct heterozygous seeds ofSSG4SSG4ssg4 and SSG4ssg4ssg4. Chalkiness was notobserved in the endosperm of either heterozygote(Supplemental Fig. S1). The SG sizes of SSG4SSG4ssg4and cv Nipponbare seeds did not significantly differ,whereas the SG sizes of the SSG4ssg4ssg4 seeds wereslightly larger than those of cv Nipponbare (Fig. 1J). Thisindicated that two wild-type alleles supplied from thefemale gametophyte were sufficient for the formation ofnormal-sized SGs, whereas one copy of the SSG4 allelesupplied by the sperm was functional but not sufficientfor the formation of normal-sized SGs. We next examinedstarch accumulation in ssg4 grains. The total amount ofstarch was lower in ssg4 seeds than in wild-type seeds(Fig. 1K). No significant difference in the gelatinizationproperties of ssg4 starch compared with wild-type starchwas observed; therefore, the structural properties ofstarch were similar in ssg4 and cv Nipponbare (Table I).This result was consistent with previous work showingthat the amylopectin chain-length distribution of ssg4starch is normal (Matsushima et al., 2010).

The Arabidopsis phosphoglucomutase (pgm) mutantcontains small amounts of starch in leaves but exhibitshigh levels of accumulation of soluble sugars, such asSuc, D-Glc, and D-Fru (Bläsing et al., 2005). This isexplained by the defective conversion of photosynthateinto starch in pgm1 leaves. Less starch accumulation inssg4 seeds also might cause the abnormal level of sugaraccumulation. We analyzed the soluble sugars in ssg4and cv Nipponbare seeds by using gas chromatography-time of flight-mass spectrometry. Levels of Suc andD-Glc were much higher in ssg4 seeds than in cv Nip-ponbare seeds (Supplemental Fig. S2, A and B), whilethe D-Fru level was less abundant in ssg4 seeds than incv Nipponbare seeds (Supplemental Fig. S2C).

Rice grains require more than 1 month for full rip-ening after flowering. During this period, a large num-ber of SGs are developed and fill the endosperm. Toinvestigate when the enlarged SGs were developed inthe ssg4 mutant, we focused on early-developing seedsat 3, 5, and 7 d after flowering (DAF). Seed enlargementfrom 3 to 7 DAF in cv Nipponbare and ssg4 was es-sentially the same (Fig. 2, A–F). By contrast, the sizesand numbers of SGs from 3 to 7 DAF in cv Nipponbareand ssg4 were different (Fig. 2, G–L). At 3 DAF, mostSGs in the ssg4 endosperm were larger than those in cvNipponbare (Fig. 2, G and J) and occupied an area thatwas more than 3-fold larger than that occupied by SGsin cv Nipponbare (Fig. 2M). At 7 DAF, the area occupiedby SGs was more than 5-fold larger in ssg4 than in cvNipponbare. When the SGs were assumed to be spher-ical, the volume of SGs at 7 DAF was approximately10-fold larger in ssg4 than in cv Nipponbare. Thenumber of SGs showed the opposite pattern to the sizesof SGs (Fig. 2N) and was lower in ssg4 than in cv

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Nipponbare at all days tested. At 3 to 7 DAF, thenumber of SGs in ssg4 was one-third less than thenumber in cv Nipponbare (Fig. 2N).We also investigated ssg4 endosperms at 5 DAF by

transmission electron microscopy (TEM; SupplementalFig. S3). Morphologies of ssg4 SGs in TEM imageswere spherical, like the iodine-stained SGs in Figure 2.

SG Morphologies in Other Tissues

Endosperm tissue accumulates the highest levels ofstarch in rice plants. Other tissues also accumulate SGs,

including pollen grains, root caps, and pericarps. Weexamined SG morphologies in these tissues in ssg4mutants. Pollen grains were immersed in iodine solutionto stain SGs, and many rod-like SGs were visualized incv Nipponbare pollen grains (Fig. 3A). By contrast, ssg4SGs in pollen were more spherically shaped (Fig. 3B). Inboth cases, pollen SGs displayed different morphologiesfrom those of endosperm SGs. When pollen grains weresquashed under coverslips, SGs were released and themorphologies were clearer (Fig. 3, C and D). Scanningelectron micrographs of the released SGs also showedthat the SG morphologies were different in cv Nippon-bare and ssg4 (Fig. 3, C and D, insets). Most SGs in

Figure 1. Enlarged SGs of mature endosperm in the ssg4 mutant. A and B, Grains of cv Nipponbare, front and side view images,respectively. Bars = 1 mm. C and D, ssg4 grains, front and side view images, respectively. Bars = 1 mm. E, Quantification of cvNipponbare and ssg4 seed sizes (n = 30 each). F and G, Iodine-stained thin sections of cv Nipponbare endosperm at low and highmagnification, respectively. Bars = 10 mm. H and I, Iodine-stained thin sections of ssg4 endosperm at low and high magnification,respectively. Bars = 10 mm. J, Quantification of the areas occupied by SGs in sections of different genotypes (n = 6 each).K, Quantification of the starch amount in mature seeds expressed as the percentage of weight (n = 3 each). Data are given as means6 SD.Statistical comparisons were performed using Welch’s t test; all data were compared with cv Nipponbare (*P , 0.05, **P , 0.01).

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A Rice Mutant with Enlarged Starch Grains





pollen grains of both cv Nipponbare and ssg4 appear tobe simple SGs. SGs were slightly larger in ssg4 pollengrains than in cv Nipponbare pollen grains (Fig. 3E).Root caps developed many SGs that were the com-pound type (Fig. 3, F–I). The pericarp is the wall of the

mature ovary, and it surrounds the entire seed. In early-developing rice seeds, many compound SGs developedin the pericarp (Fig. 3, K–N). The SGs in the ssg4pericarps were more spherical than those in the cvNipponbare pericarps (Fig. 3, K–N). In root caps and

Table I. Effects of the ssg4 mutation on the gelatinization properties of starch in endosperm determined by differ-ential scanning calorimetry

Gelatinization properties of the starch in ssg4 seeds were analyzed by a differential scanning calorimeter. Valuesare means 6 SE of three independent determinations. TO, TP, and TC are onset, peak, and conclusion temperatures,respectively. DH is gelatinization enthalpy of starch.

Plant TO TP TC DH

˚C mJ mg21

cv Nipponbare 53.2 6 2.6 63.2 6 0.8 69.2 6 0.5 5.3 6 0.8ssg4 51.6 6 2.1 61.6 6 0.4 67.6 6 0.8 5.8 6 0.5

Figure 2. SGs in maturing endo-sperm. A to C, Developing seeds ofcv Nipponbare (NP) at 3, 5, and7 DAF, respectively. Bars = 1 mm.D to F, Developing seeds of ssg4at 3, 5, and 7 DAF, respectively.Bars = 1 mm. G to I, Iodine-stainedthin sections of cv Nipponbare endo-sperm at 3, 5, and 7 DAF, res-pectively. Bars = 20 mm. J to L,Iodine-stained thin sections of ssg4endosperm at 3, 5, and 7 DAF,respectively. Bars = 20 mm.M, Quantification of the areasoccupied by SGs in sections at 3, 5,and 7 DAF (n = 20 each). N, Quan-tification of the numbers of SGs per10,000 mm2 at 3, 5, and 7 DAF. Dataare given as means 6 SD. Statisticalcomparisons were performed byWelch’s t test; all data werecompared with cv Nipponbare(**P , 0.01).


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pericarps, ssg4 SGs were more than 2-fold largerthan cv Nipponbare SGs (Fig. 3, J and O). All theseresults suggest that the ssg4 mutation affects the sizeof SGs in pollen grains, root caps, pericarps, andendosperm.The third leaves of ssg4mutants showed a variegated

phenotype (Fig. 4, A and B). We speculated that chlo-roplasts might also be affected by the ssg4 mutation. Tovisualize chloroplasts, thin sections of third leaves fromyoung seedlings were double stained with methyleneblue and basic fuchsin (Fig. 4, C–F). The cv Nipponbarechloroplasts displayed elongated, lens-like shapes,whereas those of ssg4were more spherical (Fig. 4, E andF). The areas of chloroplasts were approximately 2-foldlarger in ssg4 than in cv Nipponbare (Fig. 4G). Theseresults indicate that the ssg4 mutation affects the size ofchloroplasts and amyloplasts. We also investigated ssg4

chloroplasts by TEM to observe the chloroplastic ul-trastructures, such as starch granules, grana stacks, andenvelope membranes. The size of starch granules in ssg4chloroplasts were similar to those in cv Nipponbarechloroplasts (Fig. 4, H and I). Grana stacks and enve-lope membranes were not affected in ssg4 chloroplasts(Fig. 4, J and K). In contrast to the third leaves, ssg4 flagleaves did not show the variegated phenotype(Supplemental Fig. S4, A and B). The shapes of chlo-roplasts in the flag leaves did not show much differ-ence between cv Nipponbare and ssg4 (SupplementalFig. S4, C–F). Areas of chloroplasts in the ssg4 flagleaves were a little larger than those in cv Nipponbare,but not to the degree in the third leaves (SupplementalFig. S4G). TEM showed that the chloroplastic ultra-structures were not affected in the ssg4 flag leaves(Supplemental Fig. S4, H–K).

Figure 3. SG morphologies in pollen grains, root caps,and pericarps. A and B, Iodine-stained pollen grains ofcv Nipponbare and ssg4, respectively. Bars = 10 mm.C and D, Released SGs from squashed pollen grains ofcv Nipponbare and ssg4, respectively. Bars = 10 mm.Insets show scanning electron micrographs of the re-leased SGs. Bars = 1 mm. E, Quantification of theareas occupied by SGs in pollen grains (n = 30 each).F and G, Iodine-stained thin sections of root caps of cvNipponbare and ssg4, respectively. Bars = 20 mm.H and I, Magnified images of F and G. Bars = 20 mm.J, Quantification of the areas occupied by SGs in rootcaps (n = 24 each). K and L, Iodine-stained thin sec-tions of pericarps in 3-DAF seeds of cv Nipponbareand ssg4, respectively. Bars = 10 mm. M and N,Magnified images of K and L. Bars = 10 mm.O, Quantification of the areas occupied by SGs inpericarps (n = 12 each). Data are given as means6 SD.Statistical comparisons were performed by Welch’st test; all data were compared with cv Nipponbare(**P , 0.01).











Genetic Analysis and Map-Based Cloning of theSSG4 Gene

When ssg4 was crossed with cv Nipponbare, ap-proximately half of the pollen grains of the F1 plants hadssg4 phenotypes (Table II). This indicates that ssg4 be-haves in a gametophytic manner in pollen grains. Thessg4 phenotype in endosperm tissue was completelypenetrant in ssg4 selfed progeny and segregated as asingle recessive allele in F2 progeny (Table III). Weidentified the SSG4 gene using conventional map-basedcloning. We mapped the ssg4 mutation within a 62-kbregion on chromosome 1 (Fig. 5A). Ten open readingframes are expected in this region according to the RiceAnnotation Project Database (http://rapdb.dna.affrc.go.jp/). We identified a base change in the Os1g0179400gene of the ssg4mutant. The ssg4mutant carries a G-to-A

transition at position of 4,139,234 (The InternationalRice Genome Sequencing Project [IRGSP] 1.0-basedposition) on chromosome 1. The G-to-A transition isconsistent with an ethyl methanesulfonate-inducedmutation. A previously isolated complementary DNA(cDNA) clone of Os1g0179400 (AK063507) encodes aprotein containing 1,022 amino acid residues with aDUF490, according to the Pfam database (Punta et al.,2012). SSG4 is similar to the EMBRYO DEFECTIVE2410(EMB2410) protein in Arabidopsis. Although AK063507was registered as a full-length cDNA, all other homol-ogous proteins from Arabidopsis, Brachypodium dis-tachyon, and maize contain more than 1,000 additionalamino acids at their N termini, compared with theOs1g0179400 protein predicted from AK063507. Thisraises the possibility that the reported 59 terminus ofAK063507 is incorrect and that a longer protein is

Figure 4. Chloroplast morphologies in ssg4 third leaves. A and B, Third leaves of cv Nipponbare and ssg4, respectively.Bars = 1 mm. C and D, Thin sections of the third leaves were double stained with methylene blue and basic fuchsin in cv Nipponbareand ssg4, respectively. Bars = 10 mm. E and F, Magnified images of C and D. Bars = 10 mm. G, Quantification of the areasoccupied by chloroplasts in third leaves (n = 12 each). Data are given as means6 SD. Statistical comparisons were performed byWelch’s t test; all data were compared with cv Nipponbare (**P, 0.01). H and I, TEM images of chloroplasts of cv Nipponbareand ssg4, respectively. Bars = 1 mm. J and K, TEM images of thylakoid and envelope membranes of cv Nipponbare and ssg4,respectively. Bars = 200 nm.

Table II. Segregation of ssg4 pollen grains of F1 plants

Mature anthers from the F1 hybrid between ssg4 and cv Nipponbare were disrupted with forceps in the diluted Lugol solution on a glass slide toobtain the iodine-stained mature pollen grains. The released pollen grains were subsequently examined with the microscope.

Parental Genotype No. of Wild-Type Pollen Grains No. of ssg4 Pollen Grains Total Percentage of ssg4 Pollen Grains

ssg42/+ 59 58 117 50.4


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http://rapdb.dna.affrc.go.jp/

http://rapdb.dna.affrc.go.jp/


encoded by the real Os1g0179400 full-length cDNA. Toinvestigate this possibility, we performed a 59 RACEexperiment to determine the 59 end of Os1g0179400.The RACE experiment showed that the 59 end ofOs1g0179400 is far longer than that of AK063507. Thenew full-length cDNA of Os1g0179400 is derived from23 exons and the 59 untranslated region at the 59 ter-minus (Fig. 5B). The deduced protein had 2,135 aminoacid residues and contains a putative plastid-targetingsequence at the N terminus. For the complementationtest, we cloned the genomic sequence of 14,263 nu-cleotides, starting from the putative first ATG to 1,299nucleotides downstream of the stop codon of theOs01g0179400 gene. We could not clone the promotersequence of SSG4 because it was unstable and causeddeletions during plasmid construction. Therefore, weused the maize UBIQUITIN1 promoter to express theOs01g0179400 genomic clone (Himmelbach et al.,2007). The genomic clone was introduced into the ssg4mutant, and the transgenic plants that were homo-zygous for the transgene were isolated and namedUbi:SSG4genomic/ssg4. The sizes and morphologies ofSGs in transgenicUbi:SSG4genomic/ssg4 endosperm andpollen grains were very similar to those in cv Nippon-bare (Fig. 6). This indicates that the SG phenotypes inendosperm and pollen grains were completely rescuedby the transgene. We conclude that Os1g0179400 is thegene responsible for the ssg4 mutation.SSG4 had a putative plastid-targeting sequence in its

N-terminal region (Fig. 5B). Other than the plastid-targeting sequence and DUF490, no other functionaldomains were identified in the SSG4 protein. Phylo-genic analysis showed that DUF490s from photosyn-thetic organisms form a different group separate fromproteobacterial DUF490s (Fig. 5C). The ssg4 mutationsubstitutes the Gly residue at position 1,924, which is lo-cated within DUF490, with a Ser residue. This Gly residueis conserved from proteobacteria to higher plants, whichsuggests that it is important for the function of DUF490(Fig. 5D).

Expression Patterns of the SSG4 Gene

The expression patterns of SSG4 in various tissues ofdifferent developmental stages were investigated usingreal-time quantitative PCR by three different sets ofprimers (Supplemental Fig. S5). P1, P2, and P3 primersets were used to detect the first, middle, and last exonsof the SSG4 gene, respectively (Fig. 5B). All tissues ex-cept for third leaves were sampled from plants grown

in a paddy field. To obtain third leaves, plants weregrown in a greenhouse. Real-time quantitative PCRshowed that SSG4 was expressed in all tissues exam-ined in both cv Nipponbare and ssg4 (SupplementalFig. S5A). This suggests that SSG4 is needed at all devel-opmental stages. During early seed development, SSG4transcripts started to accumulate at 4 DAF in cv Nip-ponbare, but the accumulation was delayed in ssg4. At 5to 7 DAF, the expression of SSG4 continued to increasein both cv Nipponbare and ssg4, reaching a high level.In young plants, the third leaves in cv Nipponbare hada high level of SSG4 expression, which was approxi-mately twice as high in ssg4. The higher expression ofSSG4 in third leaves compared with the flag leaves incv Nipponbare may reflect the greater requirements ofSSG4 in third leaves. This is consistent with the severeenlargement of chloroplasts in the third leaves com-pared with the flag leaves in ssg4 (Fig. 4; SupplementalFig. S4). The expression patterns obtained using the P2and P3 primer sets were approximately the same as thatobtained using the P1 primers (Supplemental Fig. S5, Band C). This indicates that all three primer sets ampli-fied the same cDNA species. Therefore, the long full-length SSG4 cDNA determined in this study should bethe dominant cDNA species.

Subcellular Localization of the SSG4 Protein

The target prediction programs TargetP (Emanuelssonet al., 2007) and WoLF PSORT (Nakai and Horton, 2007)predicted that the SSG4 protein is targeted to chlo-roplasts and has a putative transit peptide at theN terminus. To confirm the chloroplast localization ofSSG4, we attempted to construct the SSG4 gene fusedwithGFP. We used the N-terminal coding region (639 bp)of the SSG4 cDNA instead of the full-length cDNA be-cause the full-length cDNA sequence strongly inhibitedbacterial growth and was difficult for plasmid construc-tion. The plasmid construct containing the N terminus ofSSG4 fused to GFP was designated SSG4N-GFP. WhenSSG4N-GFP was transiently expressed in Nicotianabenthamiana leaves, the SSG4N-GFP signals were detectedinside chloroplasts, and the patterns were very similar tothe stroma-localized GFP (Supplemental Fig. S6). Thisresult indicates that SSG4N-GFP was mainly localized instroma of chloroplasts.

We constructed stable transgenic rice plants expressingthe SSG4N-GFP gene under the control of the maizeUBIQUITIN1 promoter. In SSG4N-GFP plants, SSG4N-GFPfluorescence was detected in pollen grains, endosperm, and

Table III. Segregation of ssg4 seeds in the F2 population

F2 seeds were obtained from the cross between ssg4 and cv Nipponbare. Endosperm thin sections were preparedfrom 100 F2 seeds. The size of starch grains was examined with a microscope.

Parental Genotype No. of ssg4 Seeds No. of Wild-Type Seeds Total x2 Value (P) for 1:3 Segregation

ssg42/+ 26 74 100 0.053 (0.82)











Figure 5. Map-based cloning of the SSG4 gene. A, Fine-mapping of the SSG4 locus on chromosome 1. A total of 229 F2progeny (458 chromosomes) with homozygous ssg4 alleles were analyzed. The numbers of recombinations that occurredbetween SSG4 and the molecular markers are indicated. The SSG4 locus was mapped to a 62-kb region between two molecularmarkers (Marker1169 and Marker13025). This region contains 10 open reading frames (boxes). The ssg4 mutant has a mutation


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pericarps (Fig. 7). In pollen grains, SSG4N-GFP fluo-rescence was observed as a ring-like structure (Fig. 7,A–F). Differential interference contrast images of pollenshowed that the ring-like GFP fluorescence surroundedrod-shaped structures (Fig. 7, E and F), which are likelyto be SGs, as their morphologies are consistent with theiodine-stained SGs shown in Figure 3. In developingendosperm and pericarps, SSG4N-GFP colocalized withthe amyloplasts, whose interiors contained compoundSGs (Fig. 7, G–I). SSG4N-GFP was excluded from theSGs and accumulated in nonstarch areas (Fig. 7, J–L). Inendosperm SGs, each starch granule is compactly as-sembled, which might prevent the SSG4N-GFP proteinfrom entering the intergranule space. SSG4N-GFP ac-cumulated in the spaces between SGs and amyloplastmembranes (Fig. 7, J–L, arrowheads). This space willcorrespond to the stroma in endosperm amyloplasts. Bycontrast, SSG4N-GFP fluorescence accumulated in thespace between the starch granules in pericarp SGs(Fig. 7, M–O). This suggests that starch granules in peri-carp SGs are loosely assembled, which allows SSG4N-GFPto enter the intergranule space. Taken together, these datashow that SSG4N-GFP is localized in the amyloplasts ofvarious tissues and suggest that SSG4 is an amyloplast-localized protein with an N-terminal plastid-targetingsignal.

Accumulation of Proteins Involved in ChloroplastDivision in ssg4 Seeds

In rice, the arc5 mutant is the only mutant reported tobe defective in chloroplast division. However, the arc5endosperm does not produce spherical amyloplast withincreased diameter, as ssg4 does (Yun and Kawagoe,2009). The proteins involved in chloroplast division(FtsZ1, FtsZ2, MinD, and MinE) accumulated at the samelevel in ssg4 and cv Nipponbare (Supplemental Fig. S7).

Therefore, we speculate that SSG4 is not directly involvedin the regulation of plastid division.

Protein Length Diversity of DUF490-Containing Proteins

In the InterPro protein sequence analysis and clas-sification database (Hunter et al., 2012), 4,546 DUF490-containing proteins are registered. Proteins containingDUF490 are found from bacteria to higher plants butnot in animals. TamB (for translocation and assemblymodule B) is a well-characterized DUF490-containingprotein in proteobacteria and is responsible for the in-sertion and assembly of outer membrane proteins(Selkrig et al., 2012). Out of the 4,546 DUF490-containingproteins, proteobacterial proteins predominate and in-clude 3,566 proteins, whereas 166 proteins are registeredfor cyanobacteria and 41 proteins are registered forViridiplantae (green algae and land plants). A com-parison of the lengths of these DUF490-containingproteins showed that proteins from cyanobacteria andViridiplantae are clearly longer than those from proteo-bacteria (Supplemental Fig. S8, A–C). The lengths of mostproteobacterial proteins are approximately 1,300 aminoacid residues. For example, TamBs from Citrobacterrodentium, Salmonella enterica, and Escherichia coli are all1,259 amino acids residues (Selkrig et al., 2012), whilethe majority of cyanobacterial and Viridiplantae proteinsare around 2,000 amino acid residues. The differences inprotein length distributions among proteobacteria, cya-nobacteria, and Viridiplantae are statistically significant(Steel-Dwass analysis: proteobacteria and cyanobacteria,P , 0.001; proteobacteria and Viridiplantae, P = 0.005;cyanobacteria and Viridiplantae, P = 0.542). SeveralDUF490-containing proteins of Viridiplantae with around2,000 amino acid residues are predicted to target plastids.Therefore, the longer DUF490-containing proteins may beneeded for photosynthetic organisms and organelles.

Figure 5. (Continued.)in Os1g0179400 (gray box). The position (4,133,70324,140,631) is based on IRGSP 1.0. B, Schematic representation of theexon and intron organization of Os01g0179400 and its cDNA obtained from RACE analysis. The deduced protein structure isalso shown. The numbers in parentheses are the positions of chromosome 1 based on IRGSP 1.0. The ssg4 mutant has a base-pair change (G to A) at position 4,139,234. The positions of primers (P12P3) that were used for real-time PCR are indicated. Thepreviously isolated cDNA clone (AK063507) covered approximately half of the full-length cDNA. SSG4 encodes a proteincontaining 2,135 amino acid residues with a DUF490. Putative transit peptides (amino acids 1242) and DUF490 (amino acids1,73022,119) are indicated by red and yellow boxes, respectively. The base-pair change in ssg4 causes an amino acid sub-stitution at position 1,924, indicated by the red arrow. C, Phylogenic relationships of DUF490 sequences from bacteria to higherplants. Sequences are named by the GenBank/EMBL/DDBJ database or UniProt Knowledgebase identifications. SSG4 (rice),BRADI2G05017 (B. distachyon), and DAA53165 (maize) are monocot proteins. XP_002281904 (grape [Vitis vinifera]),AT2G25660 (Arabidopsis), and XP_003545508 (soybean [Glycine max]) are dicot proteins. XP_002966241 (Selaginellamoellendorffii) is from a pteridophyte; XP_001779881 (Physcomitrella patens) is from a bryophyte; CCO16912 (Bathycoccusprasinos) and Q016Y8 (Ostreococcus tauri) are from green algae; BAB74129 (Anabaena sp. PCC 7120) and P73551 (Syn-echocystis sp. PCC 6803) are cyanobacterial proteins; and E1WAU5 (Salmonella enterica), P39321 (Escherichia coli), andD2TN57 (Citrobacter rodentium) are proteobacterial proteins. Bootstrap values from 1,000 trials are indicated. The 0.2 scaleshows substitution distance. D, Multiple amino acid sequence alignments of DUF490-containing proteins near the ssg4 mu-tation site. The alignment was produced with ClustalW using default parameters and was refined manually. Highly andmoderately conserved residues are highlighted with green and yellow backgrounds, respectively. Different groups are shown bycolored lines to the left of the protein names: red, monocot; blue, dicot; brown, pteridophyte; orange, bryophyte; green, greenalgae; violet, cyanobacteria; and black, proteobacteria. The Gly residue that was substituted with Ser in the ssg4 mutant isindicated by the black arrowhead.







DISCUSSION

Regulation of SG Sizes by SSG4

The size of an SG is one of the most important char-acteristics of starch for industrial applications (Lindeboomet al., 2004). Small starch granules are used to replacefat in food applications, because aqueous dispersionsof small starch granules exhibit fat-mimetic properties(Malinski et al., 2003). In maize and cassava (Manihot

esculenta) crops, larger starch granules are desirablebecause they improve the final yield after wet-millingpurification (Gutiérrez et al., 2002).

In this study, we characterized ssg4 phenotypes thatshow enlarged SGs in endosperm, pollen grains, rootcaps, and pericarps (Figs. 1–3). SSG4 was identified asthe gene that influences SG size (Fig. 5). An amino acidsubstitution from Gly to Ser in DUF490 of SSG4 in-creased the size of SGs. However, the enlargement ofSGs did not result in the direct expansion of starchgranules in ssg4, because SGs in ssg4 endosperm are thecompound SG type. The information obtained in thisstudy will be applicable to other crops for the produc-tion of larger starch granules. For simple SGs, the size of

Figure 6. Complementation of the ssg4 mutant with the genomicclone of the SSG4 gene. The genomic fragment including the SSG4gene was expressed under the control of the maize UBIQUITIN1promoter in the ssg4 mutant background. Two independent transgenicplants (Ubi:SSG4genomic/ssg4 #5 and #8) were examined using thefollowing tissues: mature endosperm (A–D) and pollen grains (E–H).A and B are low magnification; C and D are high magnification; E and Fare images of whole-mount pollen grains; G and H show SGs that werereleased from squashed pollen grains. Bars = 10 mm.

Figure 7. Amyloplast localizations of SSG4N-GFP of various tissues.Confocal and differential interference contrast (DIC) images of SSG4N-GFP transgenic plants are shown. A to C, Whole-mount images ofpollen grains. Bar = 10 mm. D to F, Higher magnification images ofamyloplasts in pollen grains. Bar = 5 mm. G to I, Endosperm sectionsobtained by vibratome sectioning. Bar = 10 mm. J to L, Higher mag-nification images of endosperm sections. Arrowheads indicate thenonstarch regions in which GFPs accumulated. Bar = 5 mm. M to O,Pericarp sections obtained by vibratome sectioning. Bar = 10 mm.


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the SG is consistent with the size of starch granules.Therefore, the enlargement of SGs will directly generatelarger starch granules. Barley, maize, and sorghumdevelop simple SGs, and all these species have homo-logs of SSG4. These homologs have a conserved Glyresidue at the mutation site of ssg4 reported in thisstudy, like the wild-type rice cv Nipponbare. Therefore,introduction of this same mutation into these crops ordown-regulation of homologs of SSG4 will producelarger starch granules.A number of mutants defective in starch biosynthetic

enzymes of endosperms have been isolated in severalplant species (Walker andMerritt, 1969; Jarvi and Eslick,1975; Satoh and Omura, 1981; Yano et al., 1984; Satohet al., 2003a, 2003b, 2008; Kang et al., 2005; Fujita et al.,2007, 2009). Some of these mutants exhibit distinct SGmorphologies in endosperms compared with those ofwild-type plants. Mutations in amylopectin-branchingenzyme IIb reduce the size of SGs in rice and maizeendosperm (Yano et al., 1985; Li et al., 2007; Matsushimaet al., 2010), while the Arabidopsis mutant of starchsynthase IV forms one huge starch granule per chlo-roplast in leaves (Roldán et al., 2007). Starch synthaseIV is suggested to be involved in the process of initi-ation of the starch granule and in the priming of starchsynthesis (Szydlowski et al., 2009; D’Hulst and Mérida,2010). However, the role of starch synthase IV in cerealendosperms has remained unknown so far.

Subcellular Localization of SSG4

The SSG4 N-terminal sequence targeted GFP to thestroma of chloroplasts and amyloplast in various tissues(Fig. 7; Supplemental Fig. S6), while proteomic analysisof cyanobacteria showed that SSG4 cyanobacterial ho-mologs are localized in the outer membrane (Moslavacet al., 2005). The Prediction of Transmembrane Regionsand Orientation program (http://www.ch.embnet.org/software/TMPRED_form.html) predicted three trans-membrane regions in the SSG4 sequence (amino acids1042127, 5172540, and 1,46321,485). The first regionhad the highest confidence interval. However, the firstregion should not be a transmembrane domain becauseit is included in SSG4N-GFP. The latter two regionsmay be important for the intraplastidic localization ofSSG4 and may target SSG4 to membranes.

Possible Functions of the SSG4 Protein

To date, SSG4 homologs of photosynthetic organismshave not been functionally characterized. Transfer DNAknockout mutations of the Arabidopsis homolog ofSSG4 (At2g25660), denoted as emb2410, arrest embryodevelopment at the globular stage (Meinke et al., 2008).Many embryo-defective mutants with the disruption ofplastid-targeted proteins have been shown to exhibit theimpaired plastid development (Hsu et al., 2010; Bryantet al., 2011). In the case of the Arabidopsis arc1 mutant,

the weak allele mutant has smaller, more numerouschloroplasts than the wild type, while the strongtransfer DNA insertion allele causes embryo lethality(Kadirjan-Kalbach et al., 2012). ARC1 likely functionsin an essential process of plastid development thatmay be coupled with plastid division. In a similar way,the essential function of At2g25660 for embryogenesisraises the possibility that the function of SSG4 is moreinvolved in plastid development than any direct rolefor SG size control. The pleiotropic effect of the ssg4mutation on chloroplast organization in third leavesand the conservation of DUF490-containing proteins incyanobacteria that do not develop SGs support thisidea (Figs. 4 and 5).

Septum-like structures have been suggested to existbetween starch granules during the formation of com-pound SGs (Yun and Kawagoe, 2010). The successivesynthesis of septa during plastid division promotes theformation of compound SGs. SGs in endosperms, rootcaps, and pericarps were the compound type, while SGsin pollen grain were the simple type (Figs. 1–3). Theenlargement of SGs by the ssg4mutation was extreme incompound SGs compared with simple SGs (Fig. 3).Therefore, SSG4 may be related to septum formationand may have some specific roles in the compound na-ture of dividing plastids. The amino acid substitution inssg4 is the first nonlethal mutation in DUF490-containingproteins of photosynthetic organisms. Future studies ofSSG4 will reveal more detailed information about thefunction of DUF490 in higher plants.

MATERIALS AND METHODS

Plant Materials and Growth Condition

Rice (Oryza sativa subspecies japonica ‘Nipponbare’ and subspecies indica‘Kasalath’) were used as wild-type plants. The ssg4mutant was previously isolatedfrom an ethyl methanesulfonate-treated cv NipponbareM2 population (Matsushimaet al., 2010). We backcrossed the ssg4 mutant with cv Nipponbare, and theirprogeny with ssg4 phenotypes were used in this study. Rice plants were grownat an experimental paddy field at the Institute of Plant Science and Resources,Okayama University, under natural conditions or at 28°C in a greenhouse.

Characterization of Grain Appearance, Size, andStarch Amount

Mature dry seeds and maturing young seeds were photographed witha macromicroscope (MVX10; Olympus) and a digital camera (DP72; Olympus).The sizes of grains weremeasured by vernier caliper. The total amount of starchwasmeasured by enzymaticmethods using the Total StarchAssayKit (MegazymeInternational).

Thermal Properties of Starch

Dried rice grain was dehulled, crushed with pliers, and hand homogenizedusing a motor and pestle. The weighed starch (3 mg) was placed in a silversample cup (560-003; Seiko Instruments), mixed with 9 mL of distilled water,and sealed. Gelatinization properties of the starch were analyzed by a dif-ferential scanning calorimeter (DSC-6100; Seiko Instruments). The heating ratewas 3°C min–1 over a temperature range of 5°C to 90°C.

Semiquantification of D-Glc, D-Fru, and Suc Levels

Extracts from mature seeds of cv Nipponbare and the ssg4 mutant(equivalent to 5 mg) were subjected to gas chromatography-time of flight-mass





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spectrometry as described previously (Kusano et al., 2007). Peaks of D-Glc, D-Fru,and Suc in each analyte were identified by comparing retention indices and themass spectra of the corresponding authentic standards.

Thin Sections of Technovit 7100 Resin of Endospermand Staining

For mature dry seeds, approximately 1-mm3 blocks were cut from the centerregion of the endosperm and fixed in solution containing 5% (v/v) formalin, 5%(v/v) acetic acid, and 50% (v/v) ethanol for at least 12 h at room temperature. Formaturing endosperm, approximately 1-mm blocks were cut out from the maturingendosperm at 3, 5, and 7 DAF and fixed in 3% (v/v) glutaraldehyde in 20 mM

cacodylate buffer (pH 7.4) for at least 24 h at 4°C. To observe root caps, the seminalroot tips (1 mm) were cut out and fixed in the same buffer as for the maturingendosperm. To observe chloroplasts, the middle region of leaves was sampled.After fixation, samples were subsequently dehydrated and then embedded inTechnovit 7100 resin (Kulzer) as described previously (Matsushima et al., 2010).The embedded samples were cut in 1-mm sections with an ultramicrotome (EMUC7; Leica Microsystems) and diamond knives and then dried on coverslips. Tostain SGs, thin sections were stained with 403 diluted Lugol solution (iodine/potassium iodine solution; MP Biomedicals) in deionized water for at least 5 s andsubsequently examined with a microscope (AX70; Olympus). Quantifications ofamyloplast areas were analyzed with ImageJ 1.46r software (http://rsbweb.nih.gov/ij/). To stain the chloroplast in leaves, the Technovit sections were doublestained with 0.026% (w/v) methylene blue and basic fuchsin.

TEM Observation

The middle region of third leaves of seedlings and 5-DAF endosperms werefixed in 2% (w/v) paraformaldehyde and 2% (v/v) glutaraldehyde in 50 mM

cacodylate buffer (pH 7.4) at 4°C overnight followed by postfixation with2% (w/v) osmium tetroxide at 4°C for 3 h. Samples were subsequently dehy-drated using a graded ethanol series and infiltrated with propylene oxide. Thesamples were then embedded in Quetol-651 resin (Nisshin EM). The embeddedsamples were ultrathin sectioned at 70 nm with a diamond knife, and sectionswere placed on copper grids. They were stained with 2% (w/v) uranyl acetatefor 15 min, and then they were secondary stained with lead stain solution for3 min. The grids were observed by a transmission electron microscope (JEM-1400Plus; JEOL) at an acceleration voltage of 80 kV. Digital images were takenwith a CCD camera (VELETA; Olympus Soft Imaging Solutions).

Observation of Pollen Grains

To obtain the iodine-stained mature pollen grains, anthers just before anthesiswere disrupted with forceps in the diluted Lugol solution on a glass slide, and thereleased pollen grains were subsequently examined with the AX70 microscope.Furthermore, pollen grains were squashed by putting gentle pressure on a cov-erslip to release SGs from pollen vegetative cells. The released SGs were alsoobserved with a scanning electron microscope (Quanta 250; FEI).

Map-Based Cloning of the SSG4 Gene

For mapping the SSG4 gene, we constructed an F2 population derived froma cross between the ssg4 mutant and cv Kasalath. To select ssg4 mutant seedsfrom the F2 populations, endosperm thin sections of each F2 seed were ex-amined by a rapid method that was developed previously (Matsushima et al.,2010). The genomic DNA of these ssg4 mutants was individually isolated andanalyzed using simple sequence-length polymorphism markers to determinethe molecular markers linked to the ssg4 phenotype (Temnykh et al., 2000;McCouch et al., 2002). Primers used for the molecular markers were as follows:Marker1169, 59-TAAGACTGAACTTAAATGATTGTGT-39 and 59-AAAAACA-TATAATCCAAAACGTTAG-39; Marker13025, 59-TAGGAGAGGAGAGAAGT-TTGTG-39 and 59-GTTTAGGATCGCTACCAAATAG-39.

The SSG4 locus was mapped in the 62-kb region between Marker1169 andMaker13025 on the short arm of chromosome 1. We determined the nucleotidesequences of several candidate genes in this region and identified a single-basesubstitution in a candidate Os01g0179400 gene.

59 RACE Experiment

The multiple alignments between Os01g0179400 and other plant homologousproteins indicated that the cDNA clone (AK063507) for the Os01g0179400 gene

should be missing the 59 end of the predicted open reading frame. Therefore, themissing part was obtained using a 59 RACE experiment using the SMARTerRACE cDNA amplification kit (Clontech Laboratories), according to the man-ufacturer’s instructions. The first-strand cDNA was synthesized from total RNAof DAF-5 developing seeds or 7-d-old young seedlings of cv Nipponbare. Thefollowing primer was used as a gene-specific primer: 59-TGACAACAGC-CTTCCGTCTTGAATGG-39. The sequence of the obtained PCR fragment wasused as a template for direct sequencing using the Big Dye Terminator Version3.1 Cycle Sequencing Kit (Applied Biosystems) and a genetic analyzer (AppliedBiosystems).

Plasmid Construction

For complementation of the ssg4 mutant, a genomic fragment containingthe Os01g0179400 gene was cloned into pIPKb002, a binary vector for thetransformation of cereals (Himmelbach et al., 2007). The genomic fragment is14,263 nucleotides, starting from the putative first ATG up to 1,299 nucleotidesdownstream of the stop codon of Os01g0179400 (chromosome 1, 4,127,368–4,141,631). The genomic fragment was amplified separately as two fragmentsto construct the plasmid. First, the 39 half of the genomic region(4,131,703–4,141,631) was amplified using the following primers: 59-TCAGTC-GACTGGATCCAATGGGCGGTGGTTTATCTCAAAA-39 and 59-GTGCGGCCG-CGAATTGGGAAATGGAAAGAACCTAGATTGG-39.

The fragment was cloned into the BamHI and EcoRI sites of the pENTR2Bentry vector (Invitrogen) using the In-Fusion Cloning Kit (Clontech). Theresulting plasmid is called pENTR-latter. The remaining 59 half of the geno-mic region (4,131,703–4,133,132) was amplified using the following primers:59-AACCAATTCAGTCGAATGTCCCACTGCCTCCGGGCGTCGC-39 and59-TAGTCTCTCCATTGAGCTCTCCAATTT-39 (the SacI site is underlined). Theamplified fragment was inserted into the SalI and SacI sites of pENTR-latter. TheSalI site is located in the vector-derived region, and the SacI site is in the middleof the genomic region. The resulting plasmid, pENTR-full, was used for the LRrecombination reaction with the destination vector pIPKb002 using the Gatewaysystem (Invitrogen). The resulting plasmid was then introduced into the ssg4mutant using an Agrobacterium tumefaciens-mediated method (Hiei et al., 1994).To construct the transgenic plants expressing the SSG4 protein fused with GFP,cDNA encoding the SSG4 N-terminal fragment (amino acids 1–213) was am-plified and cloned into the pENTR2B entry vector (Invitrogen) together with theGFP gene. The SSG4 N-terminal coding region (SSG4N; 639 bp) was amplifiedby PCR amplification using the full-length cDNA as a template and the follow-ing primers: 59-AACCAATTCAGTCGAATGTCCCACTGCCTCCGGGCGTCGC-39and 59-GCCCTTGCTCACCATCTCGGACAGCACGGCGTCGACGACG-39. TheGFP gene was amplified from LAT52-GFPN plasmids (Matsushima et al.,2008) using the following primers: 59-ATGGTGAGCAAGGGCGAGGAGCT-39and 59-AAGCTGGGTCTAGATTTACTTGTACAGCTCGTCCATGC-39. Bothfragments were connected and inserted into the SalI and EcoRV sites of thepENTR2B vector using the In-Fusion Cloning Kit. In the resulting plasmid,SSG4N was connected to the GFP gene (SSG4N-GFP). The resulting plasmid(pENTRSSG4N-GFP) was used for the LR reaction with the destination vectorpIPKb002. The resulting plasmid (pK02SSG4NTM-GFP) was then introducedinto cv Nipponbare. pENTRSSG4N-GFP was also used for the LR reactionwith the destination vector pGWB2 (Nakagawa et al., 2007) to introduceSSG4N-GFP under the control of the cauliflower mosaic virus 35S promoter.The resulting plasmid (pG2SSG4N-GFP) was used for the A. tumefaciens-mediatedtransient transformation of Nicotiana benthamiana.

Phylogenetic Analysis

Sequences containing DUF490 were searched through BLAST in theGenBank/EMBL/DNA Data Bank of Japan (DDBJ) databases and the MunichInformation Center for Protein Sequences database (http://mips.helmholtz-muenchen.de/plant/index.jsp). Sequences were aligned with ClustalW (http://www.genome.jp/tools/clustalw/), followed by manual alignment. Trees wereconstructed on conserved positions of the alignment by clustered protein se-quences from plants with the neighbor-joining algorithm as implemented inMEGA 5.2 with pairwise deletion for gap filling (Tamura et al., 2011). To testinferred phylogeny, we used bootstraps with 1,000 bootstrap replicates.

Expression Pattern of the SSG4 Gene

Different tissues, including developing seeds, anthers, pistils, young pan-icles, and third and flag leaf blades were sampled. Except for third leaf blades,


Matsushima et al.


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all tissues were obtained from plants grown in the paddy field frommidMay tothe end of September. To obtain the third leaf blades, plants were grown in agreenhouse. For developing seeds, maturing seeds were sampled at 1, 2, 3, 4, 5,and 7 DAF. The anthers and pistils were sampled from spikelets on the pri-mary rachis branches when the distance between auricles of the last two leaveswas 12 cm. Young panicles were sampled when their length was 3.5 to 4.5 cm.All samples were frozen in liquid nitrogen for RNA extraction. Extraction oftotal RNA was done using the RNeasy plant mini kit (Qiagen). One hundredsixty nanograms of total RNA was used for first-strand cDNA synthesis usinga ReverTra Ace qPCR RT Master Mix with gDNA Remover kit (Toyobo).The expression was determined with the Thunderbird SYBR qPCR Mix kit(Toyobo) on Light Cycler 2.0 (Roche Diagnostics).

The PCR conditions were as follows: 95°C for 30 s, and 40 cycles of 95°C for5 s and 55°C for 30 s. LightCycler Software (version 4.0; Roche Diagnostics) wasused to quantify PCR results. Histone H3 was used as an internal control (Sasakiet al., 2011). Data shown are averages and SD of three biological replicates. Theprimers used were as follows: P1, 59-CATATTTTCAAAACGAGTGTAGTG-39and 59-AATCCATAGATGTTGTCTCAGAGT-39; P2, 59-TGCTGATTTATAT-GGCATTAGAG-39 and 59-GACAAATTAATATCCATAGCAGAAT-39; P3,59-ACAATATATCTTTTGCTACTGAGGT-39 and 59-GTTAGCTGATATATG-AGTGACCA-39; Histone H3, 59-GGTCAACTTGTTGATTCCCCTCT-39 and59-AACCGCAAAATCCAAAGAACG-39.

A. tumefaciens-Mediated Transient Transformation ofN. benthamiana

Suspensions of transformed A. tumefaciens GV3101 bacteria were ad-justed to an optical density at 600 nm of 0.6 in MES buffer (10 mM MgCl2 and10 mM MES, pH 5.6), and acetosyringone was added to a final concentrationof 20 mM. Bacterial suspensions were then maintained at room temperaturefor 2 to 3 h. Infiltrations were conducted by gently pressing a 1-mL dis-posable syringe to the abaxial surface of fully expanded leaves that wereapproximately 3.5 cm wide and slowly depressing the plunger. A sufficientamount of bacterial suspension was used to completely infiltrate the leavesand give a water-soaked appearance. Following the infiltration, plants weremaintained in a growth chamber at 25°C with a 12-h/12-h light/darkphotoperiod. Leaves were examined by microscopy between 50 and 90 hpost infiltration. For chloroplast stroma-localized GFP, plasmid (pL12-GFP)expressing GFP fused to transit peptide of chloroplast ribosomal protein L12was used (Arimura et al., 1999). For chloroplast envelope-localized GFP,plasmid (pCor413im1-GFP) expressing GFP fused to Cor413 chloroplastinner envelope membrane protein1 was used (Okawa et al., 2008). GFPsignals were detected using a laser scanning confocal microscope (FV1000;Olympus).

Detection of GFP Signals in Endosperms and Pericarps ofSSG4N-GFP Transgenic Plants

Developing seeds (3 DAF) without husks were embedded in 5% (w/v)agarose and cross sectioned through the middle portion of the seed in150-mm-thick sections with a Vibrating Blade Microtome (VT-1200S; LeicaMicrosystems). The sections were incubated in phosphate-buffered saline, andthe samples were examined using the laser scanning confocal microscope(FV1000).

Total Protein Extraction and Immunoblotting

For protein extraction from developing seeds, samples were homogenizedin the extraction buffer (10 mL mg21) consisting of 50 mM Tris-HCl (pH 6.8), 8 M

urea, 4% (w/v) SDS, 20% (v/v) glycerol, and 5% (v/v) b-mercaptoethanol(35 mL mg21) using a plastic homogenizer. After centrifugation at 12,000g for5 min, proteins were separated by SDS-PAGE on precast 10% to 20% poly-acrylamide gels (ATTO). The gels were blotted onto a polyvinylidene fluoridemembrane for immunoblotting with antibodies and enhanced chemilumi-nescence (GE Healthcare). Antibodies were prepared previously (Yun andKawagoe, 2009).

The accession number for the SSG4-coding DNA sequence is AB856288in GenBank/EMBL/DDBJ. All other sequence data used in this article canbe found in the GenBank/EMBL/DDBJ databases and the UniProtKnowledgebase.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Grain chalkiness of ssg4 and heterozygous mu-tant seeds.

Supplemental Figure S2. Semiquantification of Suc, D-Glc, and D-Fru levelsin cv Nipponbare and ssg4.

Supplemental Figure S3. TEM images of SGs at 5 DAF.

Supplemental Figure S4. Chloroplast morphologies in ssg4 flag leaves.

Supplemental Figure S5. SSG4 expression patterns in various tissues.

Supplemental Figure S6. Stroma localization of SSG4N-GFP inchloroplasts.

Supplemental Figure S7. Accumulation of proteins involved in chloroplastdivision in ssg4 seeds at 7 DAF.

Supplemental Figure S8. Histograms showing the protein length distribu-tions of DUF490-containing proteins.

ACKNOWLEDGMENTS

We thank Dr. Shin-ichi Arimura (University of Tokyo) and Dr. TakehitoInaba (Miyazaki University) for providing us the plasmids, pL12-GFP andpCor413im1-GFP, respectively, Makoto Kobayashi (RIKEN) and Kazuki Saito(RIKEN) for metabolite profiling analysis, and Rie Hijiya (Okayama Univer-sity) and the Biotechnology Center of Akita Prefectural University for theirtechnical assistance.

Received October 7, 2013; accepted December 13, 2013; published December13, 2013.

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