Toshihiro Kumamaru

The Rice Mutant esp2 Greatly Accumulates the Glutelin Precursor and Deletes the Protein Disulfide Isomerase

Rice (Oryza sativa) accumulates prolamins and glutelins as storage proteins. The latter storage protein is synthesized on the endoplasmic reticulum (ER) as a 57-kD proglutelin precursor, which is then processed into acidic and basic subunits in the protein storage vacuole. Three esp2mutants, CM1787, EM44, and EM747, contain larger amounts of the 57-kD polypeptide and corresponding lower levels of acidic and basic glutelin subunits than normal. Electron microscopic observation revealed thatesp2 contained normal-appearing glutelin-containing protein bodies (PB-II), but lacked the normal prolamin-containing PB (PB-I). Instead, numerous small ER-derived PBs of uniform size (0.5 μm in diameter) and low electron density were readily observed. Immunoblot analysis of purified subcellular fractions and immunocytochemistry at the electron microscopy level showed that these new PBs contained the 57-kD proglutelin precursor and prolamin polypeptides. The 57-kD proglutelin was extracted with 1% (v/v) lactic acid solution only after removal of cysteine-rich prolamin polypeptides, suggesting that these proteins form glutelin-prolamin aggregates via interchain disulfide bonds within the ER lumen. The endosperm of esp2 mutants contains the lumenal chaperones, binding protein and calnexin, but lacks protein disulfide isomerase (PDI) at the protein and RNA levels. The transcript of PDI was expressed in the seed only during the early stage of seed development in the wild type. These results suggest that PDI plays an essential role in the segregation of proglutelin and prolamin polypeptides within the ER lumen.

Mutants for rice storage proteins : 1. Screening of mutants for rice storage proteins of protein bodies in the starchy endosperm.

SummaryTo obtain genetic materials to breed qualitatively improved rice storage proteins, we screened about 3,000 mutant lines induced by the treatment of rice fertilized egg cell with N-methyl-N-nitrosourea (MNU). The screening was performed by comparing the profiles of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with that of the original variety, Kinmaze, especially focussing on the changes in polypeptides present in two kinds of protein bodies, PB-I and PB-II. We selected 17 mutant lines and classified them into 4 types on the basis of variations of the relative contents of the polypeptides. Determination of extracted protein in the starchy endosperm of the mutants revealed changes in the content of prolamin and glutelin but not globulin. In some mutants there was marked accumulation of 57 kDa polypeptide concomitant with the remarkable reduction of glutelin subunits. Treatment of the fertilized egg cell with MNU was found to be an effective method to induce mutations for storage proteins in protein bodies of rice.

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 H2O2 in the rER of the WT subaleurone cells, whereas the rER-derived H2O2 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.

Distinct Roles of Protein Disulfide Isomerase and P5 Sulfhydryl Oxidoreductases in Multiple Pathways for Oxidation of Structurally Diverse Storage Proteins in Rice

This work examines the localization and functions of two protein disulfide isomerase family oxidoreductases in formation of protein storage bodies in rice endosperm, finding that the two have nonoverlapping localizations, activities, and biological functions. 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.

Targeting of Proteins to Endoplasmic Reticulum-Derived Compartments in Plants. The Importance of RNA Localization

The targeting of proteins to the endoplasmic reticulum (ER) is a topic of considerable interest since this organelle serves as an entry point for proteins destined for other organelles, as well as for the ER itself. A unique feature of plants is that they are able to store proteins in the ER in

TAWAWA1, a regulator of rice inflorescence architecture, functions through the suppression of meristem phase transition

Inflorescence structures result from the activities of meristems, which coordinate both the renewal of stem cells in the center and organ formation at the periphery. The fate of a meristem is specified at its initiation and changes as the plant develops. During rice inflorescence development, newly formed meristems acquire a branch meristem (BM) identity, and can generate further meristems or terminate as spikelets. Thus, the form of rice inflorescence is determined by a reiterative pattern of decisions made at the meristems. In the dominant gain-of-function mutant tawawa1-D, the activity of the inflorescence meristem (IM) is extended and spikelet specification is delayed, resulting in prolonged branch formation and increased numbers of spikelets. In contrast, reductions in TAWAWA1 (TAW1) activity cause precocious IM abortion and spikelet formation, resulting in the generation of small inflorescences. TAW1 encodes a nuclear protein of unknown function and shows high levels of expression in the shoot apical meristem, the IM, and the BMs. TAW1 expression disappears from incipient spikelet meristems (SMs). We also demonstrate that members of the SHORT VEGETATIVE PHASE subfamily of MADS-box genes function downstream of TAW1. We thus propose that TAW1 is a unique regulator of meristem activity in rice and regulates inflorescence development through the promotion of IM activity and suppression of the phase change to SM identity.

Vacuolar Processing Enzyme plays an Essential Role in the Crystalline Structure of Glutelin in Rice Seed

To identify the function of genes that regulate the processing of proglutelin, we performed an analysis of glup3 mutants, which accumulates excess amounts of proglutelin and lack the vacuolar processing enzyme (VPE). VPE activity in developing seeds from glup3 lines was reduced remarkably compared with the wild type. DNA sequencing of the VPE gene in glup3 mutants revealed either amino acid substitutions or the appearance of a stop codon within the coding region. Microscopic observations showed that alpha-globulin and proglutelin were distributed homogeneously within glup3 protein storage vacuoles (PSVs), and that glup3 PSVs lacked the crystalline lattice structure typical of wild-type PSVs. This suggests that the processing of proglutelin by VPE in rice is essential for proper PSV structure and compartmentalization of storage proteins. Growth retardation in glup3 seedlings was also observed, indicating that the processing of proglutelin influences early seedling development. These findings indicate that storage of glutelin in its mature form as a crystalline structure in PSVs is required for the rapid use of glutelin as a source of amino acids during early seedling development. In conclusion, VPE plays an important role in the formation of protein crystalline structures in PSVs.

Protein disulfide isomerase like 1-1 participates in the maturation of proglutelin within the endoplasmic reticulum in rice endosperm

The rice esp2 mutation was previously characterized by the abnormal accumulation of elevated levels of proglutelin and the absence of an endosperm-specific protein disulfide isomerase like (PDIL1-1). Here we show that Esp2 is the structural gene for PDIL1-1 and that this lumenal chaperone is asymmetrically distributed within the cortical endoplasmic reticulum (ER) and largely restricted to the cisternal ER. Temporal studies indicate that PDIL1-1 is essential for the maturation of proglutelin only when its rate of synthesis significantly exceeds its export from the ER, a condition resulting in its build up in the ER lumen and the induction of ER quality control processes which lower glutelin levels as well as those of the other storage proteins. As proglutelin is initially synthesized on the cisternal ER, its deposition within prolamine protein bodies in esp2 suggests that PDIL1-1 helps retain proglutelin in the cisternal ER lumen until it attains competence for ER export and, thereby, indirectly preventing heterotypic interactions with prolamine polypeptides.

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.

Axillary Meristem Formation in Rice Requires the WUSCHEL Ortholog TILLERS ABSENT1

A WOX gene, TAB1/WUS, plays a critical role in the initiation of the axillary meristem development in rice. Axillary shoot formation is a key determinant of plant architecture. Formation of the axillary shoot is regulated by initiation of the axillary meristem or outgrowth of the axillary bud. Here, we show that rice (Oryza sativa) TILLERS ABSENT1 (TAB1; also known as Os WUS), an ortholog of Arabidopsis thaliana WUS, is required to initiate axillary meristem development. We found that formation of the axillary meristem in rice proceeds via a transient state, which we term the premeristem, characterized by the expression of OSH1, a marker of indeterminate cells in the shoot apical meristem. In the tab1-1 (wus-1) mutant, however, formation of the axillary meristem is arrested at various stages of the premeristem zone, and OSH1 expression is highly reduced. TAB1/WUS is expressed in the premeristem zone, where it shows a partially overlapping pattern with OSH1. It is likely, therefore, that TAB1 plays an important role in maintaining the premeristem zone and in promoting the formation of the axillary meristem by promoting OSH1 expression. Temporal expression patterns of WUSCHEL-RELATED HOMEOBOX4 (WOX4) indicate that WOX4 is likely to regulate meristem maintenance instead of TAB1 after establishment of the axillary meristem. Lastly, we show that the prophyll, the first leaf in the secondary axis, is formed from the premeristem zone and not from the axillary meristem.