Genetic Background Negates Improvements in Rice Flour Characteristics and Food Processing Properties Caused by a Mutant Allele of the PDIL1-1 Seed Storage Protein Gene.

Phenotypic differences among breeding lines that introduce the same superior gene allele can be a barrier to effective development of cultivars with desirable traits in some crop species. For example, a deficient mutation of the Protein Disulfide Isomerase Like 1-1 (PDIL1-1) gene can cause accumulation of glutelin seed storage protein precursors in rice endosperm, and improves rice flour characteristics and food processing properties. However, the gene must be expressed to be useful. A deficient mutant allele of PDIL1-1 was introduced into two rice cultivars with different genetic backgrounds (Koshihikari and Oonari). The grain components, agronomic traits, and rice flour and food processing properties of the resulting lines were evaluated. The two breeding lines had similar seed storage protein accumulation, amylose content, and low-molecular-weight metabolites. However, only the Koshihikari breeding line had high flour quality and was highly suitable for rice bread, noodles, and sponge cake, evidence of the formation of high-molecular-weight protein complexes in the endosperm. Transcriptome analysis revealed that mRNA levels of fourteen PDI, Ero1, and BiP genes were increased in the Koshihikari breeding line, whereas this change was not observed in the Oonari breeding line. We elucidated part of the molecular basis of the phenotypic differences between two breeding lines possessing the same mutant allele in different genetic backgrounds. The results suggest that certain genetic backgrounds can negate the beneficial effect of the PDIL1-1 mutant allele. Better understanding of the molecular basis for such interactions may accelerate future breeding of novel rice cultivars to meet the strong demand for gluten-free foods.

Eukaryotic peptide chain release factor 1 participates in translation termination of specific cysteine-poor prolamines in rice endosperm.

Prolamines are alcohol-soluble proteins classified as either cysteine-poor (CysP) or cysteine-rich (CysR) based on whether they can be alcohol-extracted without or with reducing agents, respectively. In rice esp1 mutants, various CysP prolamines exhibit both reduced and normal amounts of isoelectric focusing bands, indicating that the mutation affects only certain prolamine classes. To examine the genetic regulation of CysP prolamine synthesis and accumulation, we constructed a high-resolution genetic linkage map of ESP1. The ESP1 gene was mapped to within a 20 kb region on rice chromosome 7. Sequencing analysis of annotated genes in this region revealed a single-nucleotide polymorphism within eukaryotic peptide chain release factor (eRF1), which participates in stop-codon recognition and nascent-polypeptide release from ribosomes during translation. A subsequent complementation test revealed that ESP1 encodes eRF1. We also identified UAA as the stop codon of CysP prolamines with reduced concentration in esp1 mutants. Recognition assays and microarray analysis confirmed that ESP1/eRF1 recognizes UAA/UAG, but not UGA. Our results provide convincing evidence that ESP1/eRF1 participates in the translation termination of CysP prolamines during seed development.

The Dual Roles of the Golgi Transport 1 (GOT1B): RNA Localization to the Cortical Endoplasmic Reticulum and the Export of Proglutelin and α-Globulin from the Cortical ER to the Golgi.

The rice glup2 lines are characterized by their abnormally high levels of endosperm 57 kDa proglutelins and of the luminal chaperone binding protein (BiP), features characteristic of a defect within the endoplasmic reticulum (ER). To elucidate the underlying genetic basis, the glup2 locus was identified by map based cloning. DNA sequencing of the genomes of three glup2 alleles and wild type demonstrated that the underlying genetic basis was mutations in the Golgi transport 1 (GOT1B) coding sequence. This conclusion was further validated by restoration of normal proglutelin levels in a glup2 line complemented by a GOT1B gene. Microscopic analyses indicated the presence of proglutelin-α-globulin-containing intracisternal granules surrounded by prolamine inclusions within the ER lumen. As assessed by in situ reverse transcriptase polymerase chain reaction (RT-PCR) analysis of developing endosperm sections, prolamine and α-globulin RNAs were found to be mis-targeted from their usual sites on the protein body ER to the cisternal ER, the normal sites of proglutelin synthesis. Our results indicate that GLUP2/GOT1B has a dual role during rice endosperm development. It is required for localization of prolamine and α-globulin RNAs to the protein body ER and for efficient export of proglutelin and α-globulin proteins from the ER to the Golgi apparatus.

Rice OsVAMP714, a membrane-trafficking protein localized to the chloroplast and vacuolar membrane, is involved in resistance to rice blast disease.

Membrane trafficking plays pivotal roles in many cellular processes including plant immunity. Here, we report the characterization of OsVAMP714, an intracellular SNARE protein, focusing on its role in resistance to rice blast disease caused by the fungal pathogen Magnaporthe oryzae. Disease resistance tests using OsVAMP714 knockdown and overexpressing rice plants demonstrated the involvement of OsVAMP714 in blast resistance. The overexpression of OsVAMP7111, whose product is highly homologous to OsVAMP714, did not enhance blast resistance to rice, implying a potential specificity of OsVAMP714 to blast resistance. OsVAMP714 was localized to the chloroplast in mesophyll cells and to the cellular periphery in epidermal cells of transgenic rice plant leaves. We showed that chloroplast localization is critical for the normal OsVAMP714 functioning in blast resistance by analyzing the rice plants overexpressing OsVAMP714 mutants whose products did not localize in the chloroplast. We also found that OsVAMP714 was located in the vacuolar membrane surrounding the invasive hyphae of M. oryzae. Furthermore, we showed that OsVAMP714 overexpression promotes leaf sheath elongation and that the first 19 amino acids, which are highly conserved between animal and plant VAMP7 proteins, are crucial for normal rice plant growths. Our studies imply that the OsVAMP714-mediated trafficking pathway plays an important role in rice blast resistance as well as in the vegetative growth of rice.

Deficiency of Starch Synthase IIIa and IVb Alters Starch Granule Morphology from Polyhedral to Spherical in Rice Endosperm.

Starch granule morphology differs markedly among plant species. However, the mechanisms controlling starch granule morphology have not been elucidated. Rice (Oryza sativa) endosperm produces characteristic compound-type granules containing dozens of polyhedral starch granules within an amyloplast. Some other cereal species produce simple-type granules, in which only one starch granule is present per amyloplast. A double mutant rice deficient in the starch synthase (SS) genes SSIIIa and SSIVb (ss3a ss4b) produced spherical starch granules, whereas the parental single mutants produced polyhedral starch granules similar to the wild type. The ss3a ss4b amyloplasts contained compound-type starch granules during early developmental stages, and spherical granules were separated from each other during subsequent amyloplast development and seed dehydration. Analysis of glucan chain length distribution identified overlapping roles for SSIIIa and SSIVb in amylopectin chain synthesis, with a degree of polymerization of 42 or greater. Confocal fluorescence microscopy and immunoelectron microscopy of wild-type developing rice seeds revealed that the majority of SSIVb was localized between starch granules. Therefore, we propose that SSIIIa and SSIVb have crucial roles in determining starch granule morphology and in maintaining the amyloplast envelope structure. We present a model of spherical starch granule production.

Fusion, rupture, and degeneration: the fate of in vivo-labelled PSVs in developing barley endosperm.

Cereal endosperm is a highly differentiated tissue containing specialized organelles for the accumulation of storage proteins. The endosperm of barley contains hordeins, which are ultimately deposited within protein storage vacuoles (PSVs). These organelles have been characterized predominantly by the histochemical analysis of fixed immature tissue samples. However, little is known about the fate of PSVs during barley endosperm development, and in vivo imaging has not been attempted in order to gain further insight. In this report, young seeds were followed through development to characterize the dynamic morphology of PSVs from aleurone, subaleurone, and central starchy endosperm cells. TIP3-GFP was used as a PSV membrane marker and several fluorescent tracers were used to identify membranes and monitor endomembrane organelles in real time. Whereas the spherical appearance of strongly labelled TIP3-GFP PSVs in the aleurone remained constant, those in the subaleurone and central starchy endosperm underwent substantial morphological changes. Fusion and rupture events were observed in the subaleurone, and internal membranes derived from both the tonoplast and endoplasmic reticulum were identified within these PSVs. TIP3-GFP-labelled PSVs in the starchy endosperm cells underwent a dramatic reduction in size, so that finally the protein bodies were tightly enclosed. Potential desiccation-related membrane-altering processes that may be causally linked to these dynamic endomembrane events in the barley endosperm are discussed.

Amyloplast-localized SUBSTANDARD STARCH GRAIN4 protein influences the size of starch grains in rice endosperm.

Starch is a biologically and commercially important polymer of glucose and is synthesized to form starch grains (SGs) inside amyloplasts. Cereal endosperm accumulates starch to levels that are more than 90% of the total weight, and most of the intracellular space is occupied by SGs. The size of SGs differs depending on the plant species and is one of the most important factors for industrial applications of starch. However, the molecular machinery that regulates the size of SGs is unknown. In this study, we report a novel rice (Oryza sativa) mutant called substandard starch grain4 (ssg4) that develops enlarged SGs in the endosperm. 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 acid residues and an amino-terminal amyloplast-targeted sequence. SSG4 contains a domain of unknown function490 that is conserved from bacteria to higher plants. Domain of unknown function490-containing proteins with lengths greater than 2,000 amino acid residues 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 for future starch breeding and biotechnology.

A guanine nucleotide exchange factor for Rab5 proteins is essential for intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm.

Rice (Oryza sativa) glutelins are synthesized on the endoplasmic reticulum as a precursor, which are then transported via the Golgi to protein storage vacuoles (PSVs), where they are proteolytically processed into acidic and basic subunits. The glutelin precursor mutant6 (glup6) accumulates abnormally large amounts of proglutelin. Map-base cloning studies showed that glup6 was a loss-of-function mutant of guanine nucleotide exchange factor (GEF), which activates Rab GTPase, a key regulator of membrane trafficking. Immunofluorescence studies showed that the transport of proglutelins and α-globulins to PSV was disrupted in glup6 endosperm. Secreted granules of glutelin and α-globulin were readily observed in young glup6 endosperm, followed by the formation of large dilated paramural bodies (PMBs) containing both proteins as the endosperm matures. The PMBs also contained membrane biomarkers for the Golgi and prevacuolar compartment as well as the cell wall component, ß-glucan. Direct evidence was gathered showing that GLUP6/GEF activated in vitro GLUP4/Rab5 as well as several Arabidopsis (Arabidopsis thaliana) Rab5 isoforms to the GTP-bound form. Therefore, loss-of-function mutations in GEF or Rab5 disrupt the normal transport of proglutelin from the Golgi to PSVs, resulting in the initial extracellular secretion of these proteins followed, in turn, by the formation of PMBs. Overall, our results indicate that GLUP6/GEF is the activator of Rab5 GTPase and that the cycling of GTP- and GDP-bound forms of this regulatory protein is essential for the intracellular transport of proglutelin and α-globulin from the Golgi to PSVs and in the maintenance of the general structural organization of the endomembrane system in rice seeds.

P5-type sulfhydryl oxidoreductase promotes the sorting of proteins to protein body I in rice endosperm cells.

In rice (Oryza sativa) endosperm cells, oxidative protein folding is necessary for the sorting of storage proteins to protein bodies, PB-I and PB-II. Here we examined the role of sulfhydryl oxidoreductase PDIL2;3 (a human P5 ortholog) in the endoplasmic reticulum (ER), using GFP-AB, a PB-I marker in which the N-terminal region (AB) of α-globulin is fused to green fluorescent protein (GFP). RNAi knockdown of PDIL2;3 inhibited the accumulation of GFP-AB in PB-I and promoted its exit from the ER. We discuss the role of PDIL2;3 in retaining proteins within the ER and specifying their localization to PB-I through disulfide bond formation.

Oxidative protein folding: selective pressure for prolamin evolution in rice.

During seed development, endosperm cells of highly productive cereals, including rice, synthesize disulfide-rich proteins in large amounts and deposit them into storage organelles. Disulfide bond formation involves electron transfer and generates H(2)O(2) as a by-product. To ensure proper development and maturation of seeds, the endosperm cells must supply large amounts of oxidizing equivalents to dithiols in nascent proteins in a controlled manner. This review compares multiple oxidative protein folding systems in yeast, cultured human cells, and rice endosperm. We discuss possible roles of ERO1, other sulfhydryl oxidases, and the protein disulfide isomerase family in the formation of disulfide bonds in storage proteins and the development of protein bodies. Rice prolamins, encoded by a multigene family, are divided into Cys-rich and Cys-depleted subgroups. We discuss the potential importance of disulfide bond formation in the evolution of the prolamin family in japonica rice.

The small GTPase Rab5a is essential for intracellular transport of proglutelin from the Golgi apparatus to the protein storage vacuole and endosomal membrane organization in developing rice endosperm.

Rice (Oryza sativa) glutelins are synthesized on the endoplasmic reticulum as larger precursors, which are then transported via the Golgi to the protein storage vacuole (PSV), where they are processed into acidic and basic subunits. Three independent glutelin precursor mutant4 (glup4) rice lines, which accumulated elevated levels of proglutelin over the wild type, were identified as loss-of-function mutants of Rab5a, the small GTPase involved in vesicular membrane transport. In addition to the plasma membrane, Rab5a colocalizes with glutelins on the Golgi apparatus, Golgi-derived dense vesicles, and the PSV, suggesting that Rab5a participates in the transport of the proglutelin from the Golgi to the PSV. This spatial distribution pattern was dramatically altered in the glup4 mutants. Numerous smaller protein bodies containing glutelin and α-globulin were evident, and the proteins were secreted extracellularly. Moreover, all three independent glup4 allelic lines displayed the novel appearance of a large dilated, structurally complex paramural body containing proglutelins, α-globulins, membrane biomarkers for the Golgi apparatus, prevacuolar compartment, PSV, and the endoplasmic reticulum luminal chaperones BiP and protein disulfide isomerase as well as ß-glucan. These results indicate that the formation of the paramural bodies in glup4 endosperm was due to a significant disruption of endocytosis and membrane vesicular transport by Rab5a loss of function. Overall, Rab5a is required not only for the intracellular transport of proglutelins from the Golgi to the PSV in rice endosperm but also in the maintenance of the general structural organization of the endomembrane system in developing rice seeds.

Rice debranching enzyme isoamylase3 facilitates starch metabolism and affects plastid morphogenesis.

Debranching enzymes, which hydrolyze α-1 and 6-glucosidic linkages in α-polyglucans, play a dual role in the synthesis and degradation of starch in plants. A transposon-inserted rice mutant of isoamylase3 (isa3) contained an increased amount of starch in the leaf blade at the end of the night, indicating that ISA3 plays a role in the degradation of transitory starch during the night. An epitope-tagged ISA3 expressed in Escherichia coli exhibited hydrolytic activity on ß-limit dextrin and amylopectin. We investigated whether ISA3 plays a role in amyloplast development and starch metabolism in the developing endosperm. ISA3-green fluorescent protein (GFP) fusion protein expressed under the control of the rice ISA3 promoter was targeted to the amyloplast stroma in the endosperm. Overexpression of ISA3 in the sugary1 mutant, which is deficient in ISA1 activity, did not convert water-soluble phytoglycogen to starch granules, indicating that ISA1 and ISA3 are not functionally redundant. Both overexpression and loss of function of ISA3 in the endosperm generated pleomorphic amyloplasts and starch granules. Furthermore, chloroplasts in the leaf blade of isa3 seedlings were large and pleomorphic. These results suggest that ISA3 facilitates starch metabolism and affects morphological characteristics of plastids in rice.

A role for the cysteine-rich 10 kDa prolamin in protein body I formation in rice.

The rice prolamins consist of cysteine-rich 10 kDa (CysR10), 14 kDa (CysR14) and 16 kDa (CysR16) molecular species and a cysteine-poor 13 kDa (CysP13) polypeptide. These storage proteins form protein bodies (PBs) composed of single spherical intracisternal inclusions assembled within the lumen of the rough endoplasmic reticulum. Immunofluorescence and immunoelectron microscopy demonstrated that CysR10 and CysP13 were asymmetrically distributed within the PBs, with the former concentrated at the electron-dense center core region and the latter distributed mainly to the electron-lucent peripheral region. These results together with temporal expression data showed that the formation of prolamin-containing PB-I in the wild-type endosperm was initiated by the accumulation of CysR10 to form the center core. In mutants deficient for cysteine-rich prolamins, the typical PB-I structures containing the electron-dense center core were not observed, and instead were replaced by irregularly shaped, electron-lucent, hypertrophied PBs. Similar, deformed PBs were observed in a CysR10 RNA interference plant line. These results suggest that CysR10, through its formation of the central core and its possible interaction with other cysteine-rich prolamins, is required for tight packaging of the proteins into a compact spherical structure.

Distinct roles of protein disulfide isomerase and P5 sulfhydryl oxidoreductases in multiple pathways for oxidation of structurally diverse storage proteins in rice.

In the rice (Oryza sativa) endosperm, storage proteins are synthesized on the rough endoplasmic reticulum (ER), in which prolamins are sorted to protein bodies (PBs) called type-I PB (PB-I). Protein disulfide isomerase (PDI) family oxidoreductase PDIL2;3, an ortholog of human P5, contains a conserved structural disulfide in the redox-inactive thioredoxin-like (TRX) domain and was efficiently targeted to the surface of PB-I in a redox active site-dependent manner, whereas PDIL1;1, an ortholog of human PDI, was localized in the ER lumen. Complementation analyses using PDIL1;1 knockout esp2 mutant indicated that the a and a' TRX domains of PDIL1;1 exhibited similar redox activities and that PDIL2;3 was unable to perform the PDIL1;1 functions. PDIL2;3 knockdown inhibited the accumulation of Cys-rich 10-kD prolamin (crP10) in the core of PB-I. Conversely, crP10 knockdown dispersed PDIL2;3 into the ER lumen. Glutathione S-transferase-PDIL2;3 formed a stable tetramer when it was expressed in Escherichia coli, and the recombinant PDIL2;3 tetramer facilitated α-globulin(C79F) mutant protein to form nonnative intermolecular disulfide bonds in vitro. These results indicate that PDIL2;3 and PDIL1;1 are not functionally redundant in sulfhydryl oxidations of structurally diverse storage proteins and play distinct roles in PB development. We discuss PDIL2;3-dependent and PDIL2;3-independent oxidation pathways that sustain disulfide bonds of crP10 in PB-I.

Septum formation in amyloplasts produces compound granules in the rice endosperm and is regulated by plastid division proteins.

Storage tissues such as seed endosperm and tubers store starch in the form of granules in the amyloplast. In the rice (Oryza sativa) endosperm, each amyloplast produces compound granules consisting of several dozen polyhedral, sharp-edged and easily separable granules; whereas in other cereals, including wheat (Triticum aestivum), barley (Hordeum vulgare) and maize (Zea mays), each amyloplast synthesizes one granule. Despite extensive studies on mutants of starch synthesis in cereals, the molecular mechanisms involved in compound granule synthesis in rice have remained elusive. In this study, we expressed green fluorescent protein (GFP) fused to rice Brittle1 (BT1), an inner envelope membrane protein, to characterize dividing amyloplasts in the rice endosperm. Confocal microscopic analyses revealed that a septum-like structure, or cross-wall, containing BT1-GFP divides granules in the amyloplast. Plastid division proteins including FtsZ, Min and PDV2 play significant roles not only in amyloplast division, but also in septum synthesis, suggesting that amyloplast division and septum synthesis are related processes that share common factors. We propose that successive septum syntheses which create sections inside the amyloplast and de novo granule synthesis in each section are primarily responsible for the synthesis of compound granules.

The rice alpha-amylase glycoprotein is targeted from the Golgi apparatus through the secretory pathway to the plastids.

The well-characterized secretory glycoprotein, rice (Oryza sativa) alpha-amylase isoform I-1 (AmyI-1), was localized within the plastids and proved to be involved in the degradation of starch granules in the organelles of rice cells. In addition, a large portion of transiently expressed AmyI-1 fused to green fluorescent protein (AmyI-1-GFP) colocalized with a simultaneously expressed fluorescent plastid marker in onion (Allium cepa) epidermal cells. The plastid targeting of AmyI-1 was inhibited by both dominant-negative and constitutively active mutants of Arabidopsis thaliana ARF1 and Arabidopsis SAR1, which arrest endoplasmic reticulum-to-Golgi traffic. In cells expressing fluorescent trans-Golgi and plastid markers, these fluorescent markers frequently colocalized when coexpressed with AmyI-1. Three-dimensional time-lapse imaging and electron microscopy of high-pressure frozen/freeze-substituted cells demonstrated that contact of the Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids occur within the cells. The transient expression of a series of C-terminal-truncated AmyI-1-GFP fusion proteins in the onion cell system showed that the region from Trp-301 to Gln-369 is necessary for plastid targeting of AmyI-1. Furthermore, the results obtained by site-directed mutations of Trp-302 and Gly-354, located on the surface and on opposite sides of the AmyI-1 protein, suggest that multiple surface regions are necessary for plastid targeting. Thus, Golgi-to-plastid traffic appears to be involved in the transport of glycoproteins to plastids and plastid targeting seems to be accomplished in a sorting signal-dependent manner.

ER membrane-localized oxidoreductase Ero1 is required for disulfide bond formation in the rice endosperm.

The developing endosperm of rice (Oryza sativa, Os) synthesizes a large amount of storage proteins on the rough (r)ER. The major storage proteins, glutelins and prolamins, contain either intra or intermolecular disulfide bonds, and oxidative protein folding is necessary for the sorting of the proteins to the protein bodies. Here, we investigated an electron transfer pathway for the formation of protein disulfide bonds in the rER of the rice endosperm, focusing on the roles of the thiol-disulfide oxidoreductase, OsEro1. Confocal microscopic analysis revealed that N-glycosylated OsEro1 is localized to the rER membrane in the subaleurone cells, and that targeting of OsEro1 to the rER membrane depends on the N-terminal region from Met-1 to Ser-55. The RNAi knockdown of OsERO1 inhibited the formation of native disulfide bonds in the glutelin precursors (proglutelins) and promoted aggregation of the proglutelins through nonnative intermolecular disulfide bonds in the rER. Inhibition of the formation of native disulfide bonds was also observed in the seeds of the esp2 mutant, which lacks protein disulfide isomerase-like (PDIL)1;1, but shows enhanced OsEro1 expression. We detected the generation of H(2)O(2) in the rER of the WT subaleurone cells, whereas the rER-derived H(2)O(2) levels decreased markedly in EM49 homozygous mutant seeds, which have fewer sulfhydryl groups than the WT seeds. Together, we propose that the formation of native disulfide bonds in proglutelins depends on an electron transfer pathway involving OsEro1 and OsPDIL.

Amyloplast division progresses simultaneously at multiple sites in the endosperm of rice.

The amyloplast, a form of differentiated plastid, proliferates in sink tissues, where it synthesizes and stores starch granules. Little is known about the molecular mechanism for amyloplast division and development. The rice (Oryza sativa) endosperm provides an excellent model system for studying molecular mechanisms involved in amyloplast division and starch synthesis. We compared amyloplast division processes in the endosperm of wild type and a mutant of ARC5, a member of the dynamin superfamily. Plant growth and fertility of arc5 were not significantly different from the wild type. Unlike binary fission of chloroplast in the leaf, small amyloplasts in the endosperm of wild type divide simultaneously at multiple sites, generating a beads-on-a-string structure. In addition, large amyloplasts divide by budding-type division, giving rise to small amyloplasts attached to their surfaces. ARC5 and FtsZ2-1 fused to fluorescent proteins were targeted to the constriction sites in dividing amyloplasts. Both the loss of function of ARC5 and overexpression of ARC5 fusion proteins in the endosperm did not produce spherical amyloplasts with increased diameter, but produced either fused amyloplasts with thick connections or pleomorphic types, suggesting that proper stoichiometry between ARC5 and other components in the amyloplast division machinery is necessary for the completion of the late stage of amyloplast division. The size distribution of starch granules purified from arc5 was shifted to small and the starch gelatinization peak temperature was significantly higher than for wild-type starch, suggesting that amyloplast division processes have a significant effect on starch synthesis.

Carbohydrate-binding module of a rice endo-beta-1,4-glycanase, OsCel9A, expressed in auxin-induced lateral root primordia, is post-translationally truncated.

We report the cloning of a glycoside hydrolase family (GHF) 9 gene of rice (Oryza sativa L. cv. Sasanishiki), OsCel9A, corresponding to the auxin-induced 51 kDa endo-1,4-beta-glucanase (EGase). This enzyme reveals a broad substrate specificity with respect to sugar backbones (glucose and xylose) in beta-1,4-glycans of type II cell wall. OsCel9A encodes a 640 amino acid polypeptide and is an ortholog of TomCel8, a tomato EGase containing a carbohydrate-binding module (CBM) 2 sequence at its C-terminus. The expression of four rice EGase genes including OsCel9A showed different patterns of organ specificity and responses to auxin. OsCel9A was preferentially expressed during the initiation of lateral roots or subcultured root calli, but was hardly expressed during auxin-induced coleoptile elongation or in seed calli, in contrast to OsCel9D, a KORRIGAN (KOR) homolog. In situ localization of OsCel9A transcripts demonstrated that its expression was specifically up-regulated in lateral root primordia (LRP). Northern blotting analysis showed the presence of a single product of OsCel9A. In contrast, both mass spectrometric analyses of peptide fragments from purified 51 kDa EGase proteins and immunogel blot analysis of EGase proteins in root extracts using two antibodies against internal peptide sequences of OsCel9A revealed that the entire CBM2 region was post-translationally truncated from the 67 kDa nascent protein to generate 51 kDa EGase isoforms. Analyses of auxin concentration and time course dependence of accumulation of two EGase isoforms suggested that the translation and post-translational CBM2 truncation of the OsCel9A gene may participate in lateral root development.

Roles of isoamylase and ADP-glucose pyrophosphorylase in starch granule synthesis in rice endosperm.

Amyloplast-targeted green fluorescent protein (GFP) was used to monitor amyloplast division and starch granule synthesis in the developing endosperm of transgenic rice. Two classical starch mutants, sugary and shrunken, contain reduced activities of isoamylase1 (ISA1) and cytosolic ADP-glucose pyrophosphorylase, respectively. Dividing amyloplasts in the wild-type and shrunken endosperms contained starch granules, whereas those in sugary endosperm did not contain detectable granules, suggesting that ISA1 plays a role in granule synthesis at the initiation step. The transition from phytoglycogen to sugary-amylopectin was gradual in the boundary region between the inner and outer endosperms of sugary. These results suggest that the synthesis of sugary-amylopectin and phytoglycogen involved a stochastic process and that ISA1 activity plays a critical role in the stochastic process in starch synthesis in rice endosperm. The reduction of cytosolic ADP-glucose pyrophosphorylase activity in shrunken endosperm did not inhibit granule initiation but severely restrained the subsequent enlargement of granules. The shrunken endosperm often developed pleomorphic amyloplasts containing a large number of underdeveloped granules or a large cluster of small grains of amyloplasts, each containing a simple-type starch granule. Although constriction-type divisions of amyloplasts were much more frequent, budding-type divisions were also found in the shrunken endosperm. We show that monitoring GFP in developing amyloplasts was an effective means of evaluating the roles of enzymes involved in starch granule synthesis in the rice endosperm.