Cloning and expression of rice (Oryza sativa) sucrose synthase 1 (RSs1) in developing seed endosperm

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Plant Science I I3 (1996) 67-78

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Cloning and expression of rice (Oryza sativa) sucrose synthase (RSsl) in developing seed endosperm

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William Odegard, Jing Jie Liu, Benito 0. de Lumen* Department of Nutritional Sciences, 231 Morgan Hall, University of Calfornia, Berkelev. CA 94720, USA

Received 21 September 1995; accepted 6 November 1995

Abstract

A previously cloned cDNA to rice (Oryza saliva L.) sucrose synthase 1 (R&l) was used to determine spatial expression of the gene in rice tissues and temporal expression in developing rice endosperm. RSsl was expressed predominantly in the endosperm of milky stage rice seeds with maximum expression at 3-5 days after pollination which were &IO-fold over leaf levels. The peak in RSsl transcript levels preceded the peak of enzyme activity which occured 9-l 1 days after pollination. RSsl transcript and activity levels were analyzed in two starch deficient mutants of rice to determine if the lesion in these mutants resides at the locus for RSsl. No correlation between sucrose synthase activity and starch biosynthesis was seen in these mutants, although slight elevations of RSsl transcript levels were observed. Keywords:

Sucrose synthase; Rice; Endosperm; Starch biosynthesis

1, Introduction The enzyme sucrose synthase

(SS) (UDP-G~c:D-

Fru 2-glucosyl-transferase, EC 2.4.1.13) is proposed to be important as a source of glucosyl units for glycolysis, cell wall and starch biosynthesis [l-4], and has been used as a determinant of sink strength for storage organs actively importing sucrose [5-71. SS catalyzes the reversible reaction ’ Abbreviations:

ADPG, ADP-glucose; AGP, ADP-glucose

pyrophosphorylase; DAP, days after pollination; ORF, open reading frame; SS, sucrose synthase. l Corresponding author, Tel.: 510 642 8144; Fax: 510 642 0535.

UDP-glucose + fructose CI sucrose + UDP. In the developing endosperm of maize (Zea mays), the activity of the enzyme lies in the direction of sucrose degradation [8]. Although known- to be abundant in many plant tissues under different conditions, the exact role of the enzyme in endosperm development, particularly in starch biosynthesis, remains controversial [9]. Starch is the major reserve carbohydrate in higher plants, the single largest source of calories in the human diet and the major determinant of yield in grain. The biochemical pathway of starch biosynthesis and its control points are well established for chloroplasts, but less completely

0168.9452/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved SSDI 0168-9452(95)04288-6

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understood for amyloplasts of developing storage organs such as tubers, rhizomes, seeds and fruits. During starch biosynthesis, starch synthase, found in the amyloplasts of endosperm cells, utilizes ADP-glucose (ADPG) as the principal glucosyl donor for polymerization into the growing starch chain [l]. The enzyme ADP-glucose pyrophosphorylase (AGP) is currently believed to be the primary source of ADPG for starch synthesis [lo]. This enzyme is also located in the amyloplast and converts glucose- 1-P imported from the cytosol to ADPG. Considerable evidence exists that AGP is essential for starch biosynthesis. Starch deficient mutants of maize, pea and A. rhaliana also have sharply reduced AGP activity [ 1l-151. The production of transgenic potato plants with antisense genes for AGP led to an almost complete elimination of starch synthesis in the tubers of these plants [ 161. The strongest evidence that sucrose synthase contributes to starch biosynthesis comes from Shl mutants of maize. These mutants have a 90-95% reduction of sucrose synthase activity and contain -40% less starch in mature seeds than wild type maize [2,17]. The lesion in these mutants is unique to the Sh locus encoding SS 1. SS could participate either sequentially or parallel to the AGP pathway. For the latter to be true, either the UDP-glucose produced must be converted to ADPG or the enzyme must be capable of using ADP. Some investigators have described ADP-dependent SS [18,19], but the existence of one of these [18] remains in question [20]. In addition to the above conditions, ADPG must enter the amyloplast. Akazawa and coworkers have demonstrated that the adenylate translocator, located in the membranes of mitochondria and amyloplasts in cultured cells of sycamore (Acer pseudoplatanus), can transport ADP-glucose in addition to ATP, ADP and AMP [21,22]. Alternatively, SS could participate sequentially where UDPG is converted to glucose- IP, imported into the amyloplast and then converted to ADPG by AGP. SS has been studied in a number of plant species since its activity was first described [23]. By far the most extensive studies have been performed on the

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maize enzyme encoded by the Sh gene. The enzyme is a tetramer of subunits -87 kD in size. In maize endosperm the enzyme exists only as a homotetramer composed of SSl subunits due to spatially separate expression of the Sh gene, but is present as a heterotetramer of SSl and the Ss2 encoded monomer, SS2, in other tissues [24]. Such differential expression is indicative of differential in vivo roles for the two SS isoforms. Additional evidence suggests that SS exhibits posttranslational regulation. In maize roots, transcription of both Sh and Ss2 increases in response to anaerobiosis, but translation does not [25] whereas in rice seedlings, anaerobiosis induces both transcription and translation [26]. Sh was the first sucrose synthase gene cloned [17]. Since then, SS genes and/or cDNAs have been cloned from several species including potato [27], broad bean [28], rice 1291, barley [30,31], wheat [32], carrot [33] and Arabidopsis [34,35]. The monocots, maize and barley, carry two distinct genes for the enzyme and there is evidence for a family of three SS genes in rice [36], however, a full length gene for RSs3 has not yet been cloned. Genomic clones or cDNAs for RSsl and RSs2 have been isolated [29,37,38], however their patterns of expression have not been demonstrated. We are interested in the biochemical role of sucrose synthase in rice endosperm development. A complete understanding of this process is essential to improve the yield and nutritional characteristics of this grain using molecular methods. Using a cDNA isolated in our lab (371, we have determined the temporal and spatial pattern of RSsI expression in the IR36 cultivar of rice and in two IR36 based starch deficient mutants EM20 (shrunken) and 82GF (sugary). 2. Materials and methods 2.1. Plant material

Greenhouse-grown cultivars of Oryza sativa, IR36 (wild type), EM20 (shrunken) and 82GF (sugary) were used as a source of material for DNA and RNA isolation. Leaves, stems and seeds were removed, frozen in liquid nitrogen and stored at -80°C. Root tissue was collected in the same

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way from plants of the same cultivars grown hydroponically in a l/Zconcentrated solution of Hoagland’s salts. 2.2. cDNA Library and Sh 1 cDNA A XgtlO cDNA library made from milky stage rice seeds was the generous gift of Dr. Susan Wessler. A full length cDNA to the maize Sh gene encoding Ssl was provided by Dr. P.S. Chourey [39]. This cDNA was transferred into pBluescript@ (pBSKS+, Stratagene, La Jolla, CA) and isolated as an EcoRI fragment from 1% agarose tris-acetate gels with geneclean@ (Bio 101, La Jolla, CA) for use as a template to produce radiolabelled hybridization probes. 2.3. Preparation probes

of radiolabelled

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and hybridized overnight at 42°C. Hybridized filters were washed twice in 2 x SSC for 5 min at room temperature followed by 2 washes in 0.1 x SSC/O.l% SDS at 52°C and exposed to XAR-5 film from Eastman Kodak Co. (Rochester, NY). 2.5. cDNA subcloning and sequencing Plaques hybridizing to the Sh probe were purified, plate lysates were prepared and used for phage DNA isolation according to [40]. Inserts from h clones were gel purified, extracted from 1% agarose tris-acetate gels with geneclean@ (Bio 101) and ligated into the EcoRI site of pBSKS+ (Stratagene). Inserts were completely sequenced according to Sanger et al. [41] using United States Biochemical’s (Cleveland, OH) Sequenase@ kit.

hybridization

Radiolabelled hybridization probes were prepared by random primed labelling (BoeringerManheim, Indianapolis, IN) using 25 ng of template DNA and [ar32P]dCTP (DuPontMEN, Boston, MA). Unincorporated nucleotides were removed with nut-trap@ push columns (Stratagene) in TE buffer (10 mM Tris-HCl pH 7.5, 1.0 mM EDTA). The various RSsl restriction fragments used as templates in particular experiments are indicated in the Results section (Fig. 1). 2.4. Library screening The rice seed cDNA library was plated at densities of 102-lo3 plaque-forming units on lo-cm Luria Broth plates. Plaques were lifted in duplicate onto BA-85 nitrocellulose filters (Shleicher and Schuell, Keene, NH), denatured by laying on 0.5 N NaOWl.5 M NaCl, neutralized on 0.5 M Tris-HCl, pH 7.5/1.5 M NaCl, rinsed in 2 x SSC, dried and fixed by baking at 80°C for 2 h under vacuum. Filters were prehybridized (50% formamide, 10x Denhardt’s (2% each BSA, ficoll, polyvinylpyrrolidone), 50 mM Tris-HCl pH 7.5, 5% dextran sulfate, 1.0 M NaCl, 1% SDS, 0.1% Na-pyrophosphate, 0.1 mg/ml sonicated herring sperm DNA) l-2 h at 42°C. Radiolabelled probes prepared using the maize Sh cDNA as a template were heated to 100°C for 5 min, quenched on ice, added directly to filters in prehybridization fluid

2.6. DNA isolation Leaf tissue (0.2 g) was ground to a powder in liquid nitrogen, placed in an Eppendorf tube with 700 ~1 DNA extraction buffer (7 M urea, 0.35 M NaCl, 0.5 M Tris-HCl pH 8.0, 20mM EDTA, 1% w/v sarkosyl) and vortexed. Phenol:CHC13 (600 ~1 of 1:1) was added and the mixture rocked for l-2 h. Samples were centrifuged 15 min in a microfuge at full speed. The aqueous phase was transferred to a new tube and extracted once with an equal volume of CHC13. The DNA was precipitated with l/10 volume of 4.4 M ammonium acetate (pH 5.2) and 2.5 volumes of 100% EtOH at -20°C overnight or on dry ice for 20 min. DNA was recovered by centrifugation, resuspended in H20 and reprecipitated with l/10 volume 3.0 M NaAc, 2.5 volumes of 100% EtOH and a 20 min dry ice incubation. DNA was recovered by centrifugation as before, resuspended in 105 ~1 H20 and the concentration determined spectrophotometrically. 2.7. Southern blotting and hybridization DNA (10 pg) was incubated with 20 units of various restriction enzymes in the appropriate buffer with the addition of 0.1 mg/ml acetylated BSA and 4 mM spermidine. After l-2 h at 37°C another 20 units of enzyme were added and the samples incubated another 4 h to overnight. Samples were run on 1.0% agarose Tris-acetate gels for 2-4 h, stained in ethidium bromide (0.5 &ml) and

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photographed. Gels were transferred to GeneScreen Plus’s’ membranes (DuPont/NEN) and hybridized, according to the manufacturers specifications, to radiolabelled probes indicated in the Results section. Blots were visualized by autoradiography with Hyperlilm-MP (Amersham, Arlington Heights, Ill). 2.8. RNA isolation Total RNA was extracted from 0.3-0.5 g of rice seeds at various stages or 2 g of leaf, root or stem tissue. Samples were ground to a powder in liquid nitrogen, placed in a 15 ml Corning polyallomer tube with 5.0 ml LiCl extraction buffer (150 mM LiCl, 50 mM Tris-base, 5 mM EDTA, 1% w/v SDS, 0.1 mg/ml proteinase K) and rocked gently for 30 min at room temperature (RT) followed by two extractions with 1:l phenol:CHCls and once with CHCls. Organic phases were back-extracted with 1.0 ml extraction buffer and the aqueous phases were pooled and brought to 2.0 M LiCl with 2.0 ml 8.0 M LiCl. RNA was precipitated overnight at 4°C and centrifuged at 9000 rev./min for 15 min at 4°C. Pellet was washed with 2.0 M LiCl and resuspended in 300 ~1 DEPC HZO. RNA was precipitated by adding 30 ~1 3.0 M NaAc, 2.5 vol. 100% EtOH and storing overnight at -20°C followed by centrifugation in a microfuge 15 min, 4°C at full speed. Pellet was rinsed with cold 70% EtOH, dried, resuspended in 105 ~1 DEPC Hz0 and the RNA concentration determined spectrophotometrically. 2.9. Northern blotting and hybridization Total RNA (20 pg) was diluted to 9 ~1 with DEPC Hz0 and 3 1 ~1 of RNA premix (100 ~1 10x MAE buffer (50 mM NaAc, 200 mM 3-(4morpholino)-propane sulfonic acid (MOPS), 10 mM EDTA, pH 7.0), 175 ~1 37% formaldehyde, 500 ~1 formamide) was added. After adding 9 ~1 of loading dye (0.25% bromophenol blue, 0.25% xylene cyanol, 50% glycerol, 0.4 pg/ml ethidium bromide), samples were vortexed, incubated at 65°C for 15 min, on ice for 5 min then loaded on 1% agarose gels (3 g agarose dissolved in 261 ml DEPC HzO, 30 ml 10 x MAE buffer, 9 ml 37% formaldehyde) and electrophoresed for 3 h at 110 V. RNA was electroblotted to Genescreen Plus@

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membranes (DuPontNEN) in 25 mM NaP04 for 1 h at 1.85 A, 4°C. After transfer, blots were rinsed in 25 mM NaP04 and baked l-2 h at 80°C. Blots were wetted in 2 x SSC, placed in roller tubes with 10 ml hybridization solution (250 mM Na2HP04, 7% SDS, 1 mg/ml BSA) and prehybridized 1 h at 65°C. After prehybridization, at least lo5 counts/mm/ml of radiolabelled probe was added to roller tubes followed by overnight hybridization at 65°C. After hybridization, blots were washed in 75 mM Na2HP04, 1% SDS twice for 15 min at 65°C (moderate stringency) followed by washing in a 1:2 dilution of the first wash (high stringency) twice for 15 min at 65°C. Blots were exposed to Hyperfilm-MP (Amersham) and quantitated using a model 400s PhosphoImager and ImageQuantTM software from Molecular Dynamics (Sunnyvale, CA). Where indicated, data was normalized for equal loading based on fluorescence from ethidium bromide staining of gels using the Gel DoclOOO UV Gel Documentation System from Bio-Rad (Richmond, CA). 2.10. Protein extraction and enzyme assays Seeds (50- 100 mg) at various stages of development were homogenized in 400 ~1extraction buffer (50 mM Tris-HCl, pH 7.5; 1.0 mM DTT; 1.0 mM EDTA and 2 mM PMSF) and kept at 4°C. Ammonium sulfate fractions (30-50% w/v) were precipitated then resuspended in dialysis buffer (50 mM Tris-HCl, pH 8.0, 5 mM MgSO,, 5 mM 2mercaptoethanol) and dialyzed overnight at 4°C. Protein concentrations were determined by the Bradford method (Bio-Rad) and 40 mg protein were used per assay. SS assays were performed using a modified method of Ricard et al. [26] in 20 mM MES pH 6.4,20OmM sucrose and 4 mM UDP for 15 min at 30°C. Reactions were stopped by boiling for 2 min followed by measurement of fructose by the Nelson-Somogyi method [42]. Control tubes lacked UDP. 3. Results 3.1. Identification, subcloning and sequence of cDNA clones for RSsl

The cDNA we used to generate probes for ex-

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pression studies [37] was constructed from two incomplete cDNAs Fl and Gla. Fl contains the 3 ’ 1847 bp of an open reading frame (ORF) encoding SS and 196 bp of 3 ’ flanking sequences including the translation stop and polyadenylation sites. Gla contains the 5’ 2379 bp of an SS ORF including the translation start site and 9 bp of the 5’ untranslated region. These cDNAs share a 100% homologous overlap of 1799 bp corresponding to bp 1- 1799 of F 1 and bp 590-2388 of G 1a. Beyond this point Fl continues to code for an SS protein while Gla contains the first 69 bp of intron 15 of the rice sucrose synthase 1 (RSsl) gene described by Wang et al. [29]. By fusing the 5 ’ 701 bp of Gla to the 3 ’ 1931 bp of F 1 at a Sac1 site located at bp 697 in Gl a and bp 108 of Fl, a complete cDNA of 2632 bp was generated containing an open reading frame of 2427 bp encoding a protein of 808 amino acids. A graphic map of this cDNA, including regions used to generate radiolabelled probes for experiments described below, is presented in Fig. 1. Interestingly, the 3’ end of Gla corresponding to intron 15 of the RSsl gene contains an open reading frame for an alternative C-terminus to the peptide and may indicate an alternatively spliced mRNA. Translation of the 3 ’ end of such an mRNA would result in a change of the expected 14 C-terminal amino acids for 10 with a considerably

Rice

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SSl

Fig. 2. Phylogenetic tree of SS peptides recorded in the GenBanke and EMBL databases, Tree was produced using the Higgins algorithm with the MacDNASISo Sequence Analysis Software from Hitachi Software (San Bruno. CA).

X

A

6

B Kb -

23.1

-9.4

-

2.3

-

2.0

-0.5

Fig. 1.Graphic representation of the RSsl cDNA including location of important restriction sites, translation start and stop codons and restriction fragments used to generate various hybridization probes. Restriction enzyme sites are EcoRI (E), EcoRV (EV), PsrI (P), Sac1 (S) and Nsil (N). The location of the Sac1 site fusing the two cDNAs, Gla and Fl, is indicated with an asterisk.

A

B

Fig. 3. Southern analysis of sucrose genome. Genomic DNA (IO *g) cut or XbaI (X) was loaded in each lane. either the 1.2 kb EcoRV fragment 183 bp fragment

synthase genes in the rice with BarnHI (B), Apal (A), Blots were hybridized with (A) or the 3’ untranslated

(B) of RSsl (Fig. 1).

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Fig. 4. Northern analysis of RSsl expression in developing seeds and other tissues from lR36 (A) including the ethidium bromidestained gel showing approximately equal amounts of RNA in each lane. Panel B shows a histogram of steady state mRNA levels from this blot. Expression levels are in arbitrary units based on a value of leaf expression equal to I unit. Total RNA (20 pg) was loaded in each lane and blots were hybridized with the I83 bp, 3’ untranslated fragment of RSsl (Fig. 1). Data was normalized for equal loading based on fluorescence from ethidium bromide-staining of gels using the Gel DoclOOOUV Gel Documentation System from Bio-Rad (Richmond, CA).

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B

l-l EM20 L-J82GF -I

T

T

r

ii 7-9

Seeds-Days

Root

Stem

After Pollination

Fig. 5. Northern analysis comparing RSsl expression in seeds at 1-3, 3-5, 5-7 and 7-9 DAP seeds, leaf. root and stem of IR36 (wild type), EM20 (shrunken) and 82GF (sugary) rice cultivars (A) including the ethidium bromide-stained gel showing approximately equal amounts of RNA in each lane (lower panel). Panel B shows a histogram of steady state mRNA levels from this blot. Expression levels are in arbitrary units based on a value of leaf expression in IR36 equal to I unit. Total RNA (20 rg) was loaded in each lane and blots were hybridized with the 183 bp, 3’ untranslated fragment of RSsl (Fig. I). Data were normalized for equal loading based on fluorescence from ethidium bromide staining of gels using the Gel DoclOOO UV Gel Documentation System from Bio-Rad (Richmond, CA).

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different sequence sharing no homology with the C-termini of any SS peptides known. Homology searches of the GenBank@ and EMBL databases with this cDNA showed it to be identical to the coding regions of the RSsl gene [29] and share 87% homology with the maize Sh gene. The peptide sequence encoded by this cDNA had 94% homology with the endosperm specific maize enzyme SSl. A phylogenetic tree of several SS peptide sequences currently in the GenBank@ and EMBL databases is presented in Fig. 2. 3.2. Southern hybridization Results from Southern blot hybridization of IR36 genomic DNA (Fig. 3) identified three bands hybidizing to probes derived from the 1.2 kb EcoRV fragment of RSsl (Fig. I), but only a single band is seen in blots hybridized with probes derived from the 3’ untranslated region (Fig. 1). This confirms that probes derived from the 3 ’ untranslated region of our RSsl cDNA are specific to a single gene in the rice SS gene family while probes derived from the coding region hybridize with three related genes. 3.3. Expression of RSsl in developing seeds and other tissues of wild type, shrunken and sugary varieties of rice Northern hybridization was performed on blots containing total RNA from a series of developing rice seeds and other tissues from the IR36 cultivar of rice (Fig. 4A). Blots were probed with the 3 ’ untranslated 183 bp fragment of RSsl which is specific to RSsl. Data was normalized for equal loading using Bio-Rad’s Gel DoclOOO UV Gel Documentation System. Probes hybridized to an mRNA of -3.0 kb under high stringency conditions. This mRNA is of sufficient size to encode an 808 amino acid peptide. The steady state level of this mRNA reached a maximum at 3-5 days after pollination (DAP) diminishing to below leaf levels at 9- 11 DAP and remaining low through maturity at 2 1+ DAP. The peak of expression was 3.6-fold higher than initial levels of the RSsl mRNA in endosperm (l-3 DAP) and almost 8-fold over RSsl levels in leaves. Expression in both stem and root tissue was about 3.8-fold over leaf levels on average. A histogram of RSsl transcript levels is presented in Fig. 4B.

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Fig. 6. Histogram of sucrose synthase activity in a developmental series of seeds from IR36 (wild type), EM20 (shrunken) and 82GF (sugary) rice cultivars. Assays were performed in 20 mM MES pH 6.4, 200 mM sucrose and 4 mM UDP for 15 min at 3O”C, stopped by boiling for 2 min followed by measurement of fructose by the Nelson-Somogyi method (Somogyi, 1952). Control tubes lacked UDP. Values are averages of duplicate samples.

Two mutants, EM20 (shrunken) and 82GF (sugary) have been shown to be deficient in starch accumulation [43]. Northern blots containing total RNA from 1-3, 3-5, 5-7 and 7-9 DAP seeds, leaf, root and stem of these mutants and IR36 (Fig. 5A) showed similar expression patterns. The level of the RSsl transcript reached a maximum level at 3-5 DAP in all three varieties, however, RSsl mRNA levels in EM20 and 82GF were 1.4and 1.3-fold higher, respectively than in IR36 at 3-5 DAP. Expression in roots was lower in the mutants, but in 82GF stem expression was higher. This data was also normalized for equal loading using Bio-Rad’s Gel DoclOOO UV Gel Documentation System. A histogram of data comparing these mutants with IR36 is presented in Fig. 5B. 3.4. Sucrose synthase activity assays Proteins were extracted from seeds of IR36, EM20 and 82GF and the 30-50% (w/v) ammonium sulfate fractions were assayed for sucrose synthase activity. Although mRNA levels for RSsl are higher, SS activity was essentially the same for the two starch mutants EM20 and 82GF as in IR36. Results from these assays are presented in Fig. 6. 4. Discussion A cDNA to RSsl previously isolated in our laboratory [37] was used to generate radiolabelled

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probes to investigate spatial and temporal patterns of RSsl expression in rice. This cDNA was constructed from two incomplete but homologous cDNAs Fl and Gla (Fig. 1) which were isolated from a Xgt10 library prepared from milky stage seeds. Several other cDNAs were isolated, but all were either identical to the two chosen by restriction mapping, or too short to contain a full length ORF for sucrose synthase. Inserts of the h clones Fl and Gla also terminated in endogenous EcoRI restriction sites suggesting that the cDNAs used to prepare the library were incompletely methylated prior to cloning into the XgtlO vector. Of particular interest was the difference in the 3 ’ ends of the two cDNAs. Fl encoded a peptide highly homologous to the maize SSl peptide, however, the 3’ end of Gla contained the first 69 bp of intron 15 of the rice sucrose synthase 1 (RSsl) gene described by Wang et al. [29]. This portion of intron 15 contains an open reading frame for an alternative C-terminus to the peptide. Although Gla may have resulted from a pre-mRNA for R&l which was incompletely spliced prior to cDNA sythesis, the possibility that it represents an alternatively spliced mRNA for RSsZ is intriguing and warrants further study. Alternative gene splicing has been shown to occur in essentially all higher eucaryotes and is an important mechanism in the control of gene expression [43]. Although the mechanisms of determining splice sites have been well-studied, conditions which induce alternative splicing are not well-known. Developmental controls affecting sex determination are known to occur in drosophila, but alternative splicing of important mRNAs in response to environmental conditions or stress has not been demonstrated. Several questions arise from the possibility that the RSsl pre-mRNA is alternatively spliced. Could alternative splicing be a response to some environmental condition to which the plants were exposed or is it developmentally controlled? Does a change in the C-terminus affect targeting or localization of the peptide to a particular location in the endosperm? Could such a change at the Cterminus have an effect on enzymatic properties which make this form of the peptide more suitable to the needs of the endosperm? A phylogenetic tree of SS peptides recorded in the GenBank” and EMBL databases (Fig. 2B)

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reveals two main families of genes consistent with the botanical classification of the species they represent. The first, belonging to monocots, is subdivided into two subfamilies composed of SS 1 and SS2. The second family, belonging to the dicots, are divided into two subgroups containing members of the solanaceae, tomato and potato, with D. currota included as a distant relative. The second subgroup contains two beans with the inclusion of A. thaliana. It is interesting that all monocots analysed for SS contain at least two genes for this enzyme forming two groups with higher homology between species than between the two groups. This implies that if the two genes resulted from a gene duplication, such a duplication occured prior to the differentiation of the separate species. The SSl isoform from maize and rice both show predominant expression in the endosperm. Plants which produce endospermous seeds may have evolved separate SS isoforms more suited to the needs of the developing endostarch than more sperm which contain cotyledonous seeds. Northern blots in the IR36 wild type (WT) cultivar (Fig. 5A) reveal an mRNA of -3.0 kb hybridizing to the 3 ’ untranslated region of our RSsl cDNA. Like Sh in maize, RSsl encodes a form of the enzyme expressed predominantly in seed endospetm, although it is expressed at lower levels in leaf, root and stem. The steady state level of the RSsl mRNA reaches a peak between 3 and 5 DAP in developing seeds, 8-lo-fold higher than expression in leaf (Fig. SB). This corresponds to the milky stage of rice seed development characterized by a liquid endosperm. High levels of RSsl expression would be expected here as both starch and cell wall biosynthesis are very active during the grain filling period. SS enzyme assays on the same developmental series of IR36 show a peak in SS activity at - 10 DAP (Fig. 6). A lag of about 6 days exists between the peaks in transcript level and enzyme activity. This could indicate that post-translational modifications are occurring. Two mutants, EM20 (shrunken) and 82GF (sugary) have been shown to be deficient in starch accumulation [44]. Developed at the International Rice Research Institute, these mutants contain 59.2% and 49.5% of the starch found in wild type

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IR36, respectively. Although the lesion in these mutants is not known, we found transcript levels of RSsl were - 1.5-fold higher in the shrunken and sugary seeds than wild type seeds at 3-5 DAP (Figs. 6 and 7). This is particularly interesting considering the results of sucrose synthase assays (Fig. 6) on seeds from the three cultivars which showed SS activity was essentially the same in the two mutants as in IR36. The presence of a feedback control element in the promoter of the maize Sh gene has been demonstrated by Maas et al. [45]. The authors of this study propose that Sh gene transcription is repressed by high UDP glucose concentrations and induced by low UDPG levels during cell wall biosynthesis in maize protoplasts. An alignment of the transcription start site of RSsl with that of the maize Sh gene showed strong homology with regions of Sh proposed to contain this feedback control element near the translation start site. If the R&l genes in the Sh and Su rice mutants produce an enzyme with depressed rates for sucrose cleavage, UDPG would remain at low levels causing an increase in RSsl transcription and translation in an attempt to compensate. This suggests that there is a particular level of SS activity required by developing seeds. If the activity of the RSsl protein is impaired in these mutants, one would expect protein levels to be higher than in IR36. The fact that SS activity levels in the mutants were similar to IR36 levels indicates that disruption of the SS pathway is not responsible for the diminished starch content in the endosperm of these mutants. Several genes for SS have been cloned from a number of species and the details of SS transcription, translation, activity and responses to environmental changes are being elucidated. We have established the spatial expression of RSsl in rice and found it to be expressed predominantly in the seed endosperm. Temporal expression analysis revealed maximum expression of seed development at 3-5 DAP. Activity of the enzyme in seeds reached a maximum at 9-l 1 DAP. Two uncharacterized starch deficient mutants of rice showed similar levels of RSsl expression and activity suggesting no connection between RSsl expression and starch accumulation in these

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mutants. Additional work must be done to determine the role of R&l in rice endosperm development, particularly its role in starch synthesis. Acknowledgements We would like to thank Dr. Susan Wessler for the rice seed cDNA, Dr. P.S. Chourey for the maize Sh cDNA, Dr. Linda H. Chen for assistance with the Gel DoclOOO UV Gel Documentation System and Drs. R. Fischer and Stan Goldman for critical reading of the manuscript. This work was supported in part by USDA NRICGP (Grant no. 93-03045), Hasselblad Foundation (Sweden), California Dry Bean Board and Nebraska Dry Bean Commission. References 111J.F. Turner and D.H. Turner, The regulation ofcarbohydrate metabolism. Ann. Rev. Plant Physiol., 26 (1975) 159-186. 121P.S. Chourey and O.E. Nelson, The enzymatic deficiency conditioned by the shrunken-i mutations in maize. B&hem. Genet., 14 (1976) 1041-1055. 131 S.C. Huber and T. Akazawa, A novel sucrose synthase pathway for sucrose degradation in cultured sycamore cells. Plant Physiol., 81 (1986) 1008-1013. 141 P.S. Chourey, E.W. Taliercio and E.J. Kane, Tissuespecific expression and anaerobically induced posttranscriptional modulation of sucrose synthase genes in Sorghum bicolor M. Plant Physiol., 96 (1991) 485-490. 151D.P. Xu, S.S. Sung and CC. Black, Sucrose metabolism in lima bean seeds. Plant Physiol., 89 (1989) 1106-I116. 161S.S. Sung, D.P. Xu and C.C. Black, Identification of actively filling sucrose sinks. Plant Physiol., 89 (1989) 1117-1121. I71 F. Wang, A. Sanz, M.L. Brenner and A. Smith, Sucrose synthase, starch accumulation, and tomato fruit sink strength. Plant Physiol., 101 (1993) 321-327. 181G.B. Cobb and L.C. Hannah, Shrunken-l encoded sucrose synthase is not required for sucrose synthesis in the maize endosperm. Plant Physiol.. 88 (1988) 1219-1221. 191 T.W. Okita, Is there an alternative pathway for starch biosynthesis? Plant Physiol., 100 (1992) 560-564. IlO1 L.A. KIeczkowski, Plant ADP-glucose pyrophosphorylase - recent advances and biotechnological perspectives (A review). Z. Naturforsch, 46~ (1991) 605. 1111 C.Y. Tsai and O.E. Nelson, Starch deficient maize mutants lacking adenosine diphosphate glucose pyrophosphorylase activity. Science, 151 (1966) 341-343. WI D.B. Dickenson and J. Preiss, Presence of ADP-glucose

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