Rice Seed Storage Protein Research Articles

Native and Artificial Reticuloplasmins Co-Accumulate in Distinct Domains of the Endoplasmic Reticulum and in Post-Endoplasmic Reticulum Compartments

We compared the subcellular distribution of native and artificial reticuloplasmins in endosperm, callus, and leaf tissues of transgenic rice (Oryza sativa) to determine the distribution of these proteins among endoplasmic reticulum (ER) and post-ER compartments. The native reticuloplasmin was calreticulin. The artificial reticuloplasmin was a recombinant single-chain antibody (scFv), expressed with an N-terminal signal peptide and the C-terminal KDEL sequence for retrieval to the ER (scFvT84.66-KDEL). We found that both molecules were distributed in the same manner. In endosperm, each accumulated in ER-derived prolamine protein bodies, but also in glutelin protein storage vacuoles, even though glutelins are known to pass through the Golgi apparatus en route to these organelles. This finding may suggest that similar mechanisms are involved in the sorting of reticuloplasmins and rice seed storage proteins. However, the presence of reticuloplasmins in protein storage vacuoles could also be due to simple dispersal into these compartments during protein storage vacuole biogenesis, before glutelin deposition. In callus and leaf mesophyll cells, both reticuloplasmins accumulated in ribosome-coated vesicles probably derived directly from the rough ER.

Messenger RNA targeting of rice seed storage proteins to specific ER subdomains.

Rice seeds, a rich reserve of starch and protein, are a major food source in many countries. Unlike the seeds of other plants, which typically accumulate one major type of storage protein, rice seeds use two major classes, prolamines and globulin-like glutelins. Both storage proteins are synthesized on the endoplasmic reticulum (ER) and translocated to the ER lumen, but are then sorted into separate intracellular compartments. Prolamines are retained in the ER lumen as protein bodies whereas glutelins are transported and stored in protein storage vacuoles. Mechanisms responsible for the retention of prolamines within the ER lumen and their assembly into intracisternal inclusion granules are unknown, but the involvement of RNA localization has been suggested. Here we show that the storage protein RNAs are localized to distinct ER membranes and that prolamine RNAs are targeted to the prolamine protein bodies by a mechanism based on RNA signal(s), a process that also requires a translation initiation codon. Our results indicate that the ER may be composed of subdomains that specialize in the synthesis of proteins directed to different compartments of the plant endomembrane system.

Quantitative nature of the Prolamin‐box, ACGT and AACA motifs in a rice glutelin gene promoter: minimal cis‐element requirements for endosperm‐specific gene expression

The -197 bp promoter of the rice seed storage protein gene, GluB-1, is capable of conferring endosperm-specific gene expression. This proximal 5' flanking region contains four motifs, GCN4, AACA, ACGT and Prolamin-box, which are conserved in many seed storage protein genes. We previously showed that multiple copies of GCN4 conferred endosperm expression pattern when fused to the -46 core promoter of CaMV 35S. In this paper we demonstrate, using a similar approach, that tandem repeated copies of any of the other three motifs are unable to direct expression in seeds as well as other tissues of transgenic rice plants. Mutational analysis of individual motifs in the -197 bp promoter resulted in remarkable reductions in promoter activity. These results indicate that the GCN4 motif acts as an essential element determining endosperm-specific expression and that the AACA, ACGT and Prolamin-box are involved in quantitative regulation of the GluB-1 gene. A set of gain-of-function experiments using transgenic rice showed that either the Prolamin-box or AACA, although often coupled with GCN4 in many genes, is insufficient to form a functional promoter unit with GCN4, whereas a combination of GCN4, AACA and ACGT motifs was found sufficient to confer a detectable level of endosperm expression. Taken together, our results provide direct insight into the importance of combinatorial interplay between cis-elements in regulating the expression of seed storage protein genes.

Development of biopharmaceuticals in plant expression systems: cloning, expression and immunological reactivity of human cytomegalovirus glycoprotein B (UL55) in seeds of transgenic tobacco

Plant seeds offer unique opportunities for the production and delivery of oral subunit vaccines. We have used the immunodominant glycoprotein B complex of human cytomegalovirus (HCMV), introduced into tobacco plants, as a model system for studying the merit of this promising approach. Given the advantages of expressing proteins in seeds, a novel expression vector was developed incorporating regulatory sequences of glutelin, the major rice seed storage protein, to direct synthesis of recombinant glycoprotein B. Analysis of genomic DNA of 28 selected tobacco transformants by PCR amplification showed that 71% harboured the gB cDNA, a finding further documented by Southern blotting. Specific immunoassays of protein extracts from seeds of positive plants showed that all were producing antigenic glycoprotein B at levels ranging from 70–146 ng/mg extracted protein. In addition, similarity with native glycoprotein B produced in HCMV-infected cells was also demonstrated by inhibition of immunofluorescence on HCMV-infected human fibroblasts. These data are the first to report the expression of an immunodominant antigen of HCMV in plant tissues, indicating the fidelity with which this very large heterologous viral glycoprotein can be synthesized in this model system.

Amino Acid Sequencing and cDNA Cloning of Rice Seed Storage Proteins, the 13kDa Prolamins, Extracted from Type I Protein Bodies.

The 13kDa prolamins, one of the rice storage proteins, consist of complex mixtures of polypeptides encoded by multigene family that show heterogeneity both in size and solubility. Although many researchers have isolated prolamin cDNA clones, it has not been possible to correlate most of these cDNA clones with individual 13kDa prolamin mature polypeptides. We isolated three new prolamin cDNA clones, λRM1, λRM4 and λRM9. Further more, we purified six 13kDa prolamin polypeptides from rice type I protein bodies, and determined these amino acid sequences. Here we demonstrate a classification for the 13kDa prolamin polypeptides which can be divided four classes, 13-I, 13-IIa, 13-IIb and 13-III. Cysteine labeling of the prolamin polypeptides indicated that 13-I contains cysteine residues, but 13-IIa or 13-IIb have no Cysteine residues. The 13-I polypeptide was soluble in nonreducing solution when their cysteine residues form inteamolecular disulfide bonds, but not soluble at intermolecular bonding.

Promoters of Rice Seed Storage Protein Genes Direct Endosperm-Specific Gene Expression in Transgenic Rice

Chuan-Yin Wu, Takahiro Adachi, Tetsuya Hatano, Haruhiko Washida-, Akihiro Suzuki and Fumio Takaiwa 1 Department of Biotechnology, National Institute of Agrobiological Resources, Tsukuba, Ibaraki, 305-0856 Japan 2 Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-Ku, Tokyo, 113-0034 Japan 3 Faculty of Science, Shimane University, Matsue, Shimane, 690-0823 Japan 4 Faculty of Horticulture, Chiba University, Matsudo, Chiba, 271-0092 Japan

The GCN4 motif in a rice glutelin gene is essential for endosperm-specific gene expression and is activated by Opaque-2 in transgenic rice plants.

The GCN4 motif is conserved in a number of seed storage protein genes, and promoter fragments containing this motif have been shown to be involved in controlling seed-specific expression of the genes studied. All genes encoding the rice seed storage protein glutelin contain the GCN4 motif at similar sites in their 5' flanking regions. Using a stable homologous transgenic system, we have analysed the promoter of the rice glutelin gene GluB-1 and demonstrated that the GCN4 motif functions as an essential cis-element for endosperm-specific gene expression. Moreover, a 21 bp GluB-1 promoter fragment spanning the GCN4 motif, as a multimer, directed GUS gene expression in endosperm of transgenic rice plants, when fused directly to the core promoter (-46) of CaMV 35S. In transiently transfected rice protoplasts, over a hundred-fold transactivation was observed from the 21 bp sequence by the bZIP type transcriptional activator Opaque-2 (O2) co-expressed under a CaMV 35S promoter. The transactivation was also evident in transgenic plants containing both O2 and the 21 bp sequence/GUS fusion. The O2-mediated activation requires binding of O2 to an intact GCN4 motif. Our results suggest that a bZIP protein functionally similar to O2 may exist in rice and participate in controlling the endosperm-specific expression of GluB-1 through the GCN4 motif.

The pea seed storage protein legumin was synthesized, processed, and accumulated stably in transgenic rice endosperm

To improve the amino acid composition of rice grain, the pea seed storage protein legumin was expressed in the endosperm of transgenic rice plants. Genomic or cDNA coding sequence of the pea legumin gene (LegA, including the 3′-downstream region) was placed under transcriptional control of either a 1.8 or 5.1 kb promoter of the rice seed storage protein glutelin gene (Gt1). The 5.1 kb promoter conferred a higher level of legumin accumulation in mature seeds than the 1.8 kb promoter, while the choice of genomic or cDNA coding sequence did not affect expression level significantly. Segregation analysis of RO seeds suggests a monogenic Mendelian inheritance, indicating a single locus or closely linked loci of gene insertion. Western blot analysis indicated that the legumin precursor is cleaved endoproteolytically into the acidic and basic subunits in the rice endosperm as is in its native pea cotyledon. We expect a significant improvement in the nutritional quality of rice seed generated by a high level expression of legumin.

A distal promoter region of the rice seed storage protein glutelin gene enhanced quantitative gene expression

We have demonstrated previously that a distal promoter region of the rice seed storage protein glutelin (Gt1) gene plays a role in increasing the quantitative level of gene expression. To determine the approximate location of cis-acting elements, if any, for the increased promoter activity, 18 progressive 5′- or internal deletions were introduced into the distal promoter region between −5.1 k and −507 relative to the transcription initiation site. The deleted promoters were fused to the coding sequence of β-glucuronidase (GUS) gene, and the fusion genes were introduced via protoplasts into homologous rice plants. GUS activities were assayed in developing seeds and vegetative organs of mature plants containing wild-type and mutant constructs. The results showed that the 4.6 kb-long wild-type distal promoter enhanced the quantitative level of GUS reporter gene by nearly 100-fold and also increased the frequency of plants showing high levels of expression. Any deletions from the 4.6 kb distal promoter region reduced the GUS activity but no distinct element was evident within the region by this analysis. We suggest that the distal promoter region may form a favorable chromatin structure to maximize the quantitative level of the glutelin gene expression. This finding also has an important practical implication in using the glutelin gene promoter to achieve a high expression level of introduced gene products in transgenic plants.

The same nuclear proteins bind to the 5'-flanking regions of genes for the rice seed storage protein: 16 kDa albumin, 13 kDa prolamin and type II glutelin.

Expression of rice seed storage-protein genes is dramatically regulated over a short period of seed maturation. To characterize the expression mechanism of the rice seed storage protein genes, their expression of major storage protein genes (16 kDa albumin, 13 kDa prolamin and type II glutelin) were compared by RNA blot analysis. Their coordinate expression suggests that the transcriptional regulatory machinery is shared among the glutelin, prolamin and albumin-genes. We isolated two novel genomic genes for prolamins (PG5a and PG5b) and obtained the promoter region of the glutelin gene by PCR. The 5'-flanking regions of these three rice seed storage protein genes were found to contain some similar conserved sequences. Nuclear extract partially purified from maturing rice seeds was used for the gel shift assay of the 5' region of the RA gene. We identified two DNA sequences of RA gene which were recognized by independent DNA-binding proteins. The complexes of these DNA sequences and DNA-binding proteins were inhibited by the fragments containing the 5' regions of the prolamin and glutelin genes, suggesting that these three genes share transcription factors.

The Bean Seed Storage Protein [beta]-Phaseolin Is Synthesized, Processed, and Accumulated in the Vacuolar Type-II Protein Bodies of Transgenic Rice Endosperm.

The seed storage protein [beta]-phaseolin of the common bean (Phaseolus vulgaris L.) was expressed in the endosperm of transgenic rice (Oryza sativa L.) plants. The 5.1- or 1.8-kb promoter fragment of the rice seed storage protein glutelin Gt1 gene was fused transcriptionally to either the genomic or cDNA coding sequence of the [beta]-phaseolin gene. The highest quantity of phaseolin estimated by enzyme-linked immunosorbent assay was 4.0% of the total endosperm protein in the transgenic rice seeds. The phaseolin trait was segregated as a single dominant trait with a positive gene dosage effect and was stably inherited through three successive generations. Both phaseolin genomic and cDNA coding sequences were used to synthesize four isoforms of mature phaseolin protein with apparent molecular masses of 51, 48, 47, and 45 kD. Enzyme deglycosylation experiments indicated that the 51-kD form contains high-mannose N-glycans; the 48- and 47-kD forms have further modified N-glycans; and the 45-kD form is a nonglycosylated protein. Immunolabeling studies using light and electron microscopy demonstrated that phaseolin accumulates primarily in the vacuolar type-II protein bodies located at the periphery of the endosperm near the aleurone layer. We discuss the implications of these results on nutritional improvement of rice grains.

5' distal and proximal cis-acting regulator elements are required for developmental control of a rice seed storage protein glutelin gene.

Using a homologous transgenic rice system it is demonstrated that 5' distal and proximal cis-acting transcriptional regulatory elements are required for developmental control of a rice seed storage protein glutelin gene. Analyses of gene expression of nine progressively truncated 5' promoter sequences in developing endosperm indicated the existence of at least one major positive element located from the -5.1 to -1.8 kb region. The functional importance of proximal elements in the context of 1.8 kb promoter was demonstrated by single substitution mutations in the TATA box (-28/-23), AACA motif (-73/-61), and protein-binding boxes I (-103/-86), II (-124/-110), III (-175/-158) and IV (-200/-217). A simultaneous mutation of five protein-binding sites (-410/-86) essentially eliminated the activity of the 1.8 kb promoter. Although temporal control of the Gt1 gene during endosperm development was retained in plants of constructs from -5.1 kb to -155 bp, spatial control of the glutelin gene was altered when the 5.1 kb promoter was deleted to -507 bp or -154 bp as the reporter gene activities of these constructs were detected in phloem of leaves, and in stems, sheaths and roots of plants.

Rice seed storage proteins: A structural insight

Rice seed storage proteins, albumin, prolamin and glutelin, ( Oryzae sativa, cv. Basmati 370) were characterized by measurement of their CD and fluorescence spectra in native and denaturing conditions to gain some information about their secondary structure. While rice albumin showed a CD spectrum typical to protein containing β-sheet and β-bends (reverse turns), rice prolamin is predominantly α-helical in nature, which transforms to β-helical nature with the decrease in hydrophobicity. Rice glutelin exhibits a random coil structure with the presence of a small percentage of α-helix secondary structure. During denaturation with guanidine hydrochloride (Gdn.HCl), the relative fluorescence intensity of all three storage proteins shows a decrease at 274 nm and 295 nm indicating unfolding of the protein molecule as is expected. When the fluorescence spectra of prolamin are examined at 274 nm and 295 nm, with a decrease in ethanol concentration (from 70% to 40%, v/v), there is a sudden increase in the relative fluorescence at 50% ethanol concentration, indicating a further folding of the protein molecule before it unfolds at 40% ethanol concentration. The fluorescence data thus substantiates the CD results. This is the first time that the three rice seed storage proteins are examined from the secondary structure point of view.