Oral Immunization with Soybean Storage Protein Containing Amyloid-ß 4-10 Prevents Spatial Learning Decline.

Amyloid-ß (Aß) plays a central role in the pathogenesis of Alzheimer's disease (AD). Because AD pathologies begin two decades before the onset of dementia, prevention of Aß amyloidosis has been proposed as a mean to block the pathological cascade. Here, we generate a transgenic plant-based vaccine, a soybean storage protein containing Aß4-10, named Aß+, for oral Aß immunization. One mg of Aß+ or control protein (Aß-) was administered to TgCRND8 mice once a week from 9 weeks up to 58 weeks. Aß+ immunization raised both anti-Aß antibodies and cellular immune responses. Spatial learning decline was prevented in the Aß+ immunized group in an extended reference memory version of Morris water maze test from 21 to 57 weeks. In Tris-buffered saline (TBS), sodium dodecyl sulfate (SDS), and formic acid (FA) serial extractions, all sets of Aß species from Aß monomer, low to high molecular weight Aß oligomers, and Aß smears had different solubility in TgCRND8 brains. Aß oligomers decreased in TBS fractions, corresponding to an increase in high molecular weight Aß oligomers in SDS extracts and Aß smears in FA fraction of the Aß+ treated group. There was significant inhibition of histological Aß burden, especially in diffuse plaques, and suppression of microglial inflammation. Processing of amyloid-ß protein precursor was not different between Aß+ and Aß- groups. No evidence of amyloid-related inflammatory angiopathy was observed. Thus, Aß+ oral immunization could be a promising, cheap, and long-term safe disease-modifying therapy to prevent the pathological process in AD.

Characterization of very long chain fatty acid synthesis inhibition by ipfencarbazone.

Ipfencarbazone exhibits excellent herbicidal activity against Echinochloa spp. and is safe for rice. The effects of ipfencarbazone on very long chain fatty acid (VLCFA) elongation in rice and late watergrass and its inhibitory mechanism were investigated in this study. Although ipfencarbazone inhibited VLCFA elongation in the microsomes prepared from late watergrass and rice at low concentrations, the inhibitory effect was higher in late watergrass than in rice. These results suggested that the primary site of action of ipfencarbazone is VLCFA elongase (VLCFAE) and ipfencarbazone has a differential affinity between the VLCFAEs of the plants. The inhibitory activity of ipfencarbazone became higher in proportion to pre-incubation period with the VLCFAE. The degree of inhibition did not decrease by dilution of the VLCFAE-ipfencarbazone complex. These results suggested that ipfencarbazone binds to the VLCFAE irreversibly.

A vacuolar sorting receptor-independent sorting mechanism for storage vacuoles in soybean seeds.

The seed storage proteins of soybean (Glycine max) are composed mainly of glycinin (11S globulin) and ß-conglycinin (7S globulin). The subunits of glycinin (A1aB1b, A1bB2, A2B1a, A3B4, and A5A4B3) are synthesized as a single polypeptide precursor. These precursors are assembled into trimers with a random combination of subunits in the endoplasmic reticulum, and are sorted to the protein storage vacuoles. Proteins destined for transport to protein storage vacuoles possess a vacuolar sorting determinant, and in this regard, the A1aB1b subunit contains a C-terminal peptide that is sufficient for its sorting to protein storage vacuoles. The A3B4 subunit, however, lacks a corresponding C-terminal sorting determinant. In this study, we found that, unlike the A1aB1b subunit, the A3B4 subunit does not bind to previously reported vacuolar sorting receptors. Despite this difference, we observed that the A3B4 subunit is sorted to protein storage vacuoles in a transgenic soybean line expressing the A3B4 subunit of glycinin. These results indicate that a protein storage vacuolar sorting mechanism that functions independently of the known vacuolar sorting receptors in seeds might be present in soybean seeds.

Stable accumulation of seed storage proteins containing vaccine peptides in transgenic soybean seeds.

There has been a significant increase in the use of transgenic plants for the large-scale production of pharmaceuticals and industrial proteins. Here, we report the stable accumulation of seed storage proteins containing disease vaccine peptides in transgenic soybean seeds. To synthesize vaccine peptides in soybean seeds, we used seed storage proteins as a carrier and a soybean breeding line lacking major seed storage proteins as a host. Vaccine peptides were inserted into the flexible disordered regions in the A1aB1b subunit three-dimensional structure. The A1aB1b subunit containing vaccine peptides in the disordered regions were sorted to the protein storage vacuoles where vaccine peptides are partially cleaved by proteases. In contrast, the endoplasmic reticulum (ER)-retention type of the A1aB1b subunit containing vaccine peptides accumulated in compartments that originated from the ER as an intact pro-form. These results indicate that the ER may be an organelle suitable for the stable accumulation of bioactive peptides using seed storage proteins as carriers.

The Sg-1 glycosyltransferase locus regulates structural diversity of triterpenoid saponins of soybean.

Triterpene saponins are a diverse group of biologically functional products in plants. Saponins usually are glycosylated, which gives rise to a wide diversity of structures and functions. In the group A saponins of soybean (Glycine max), differences in the terminal sugar species located on the C-22 sugar chain of an aglycone core, soyasapogenol A, were observed to be under genetic control. Further genetic analyses and mapping revealed that the structural diversity of glycosylation was determined by multiple alleles of a single locus, Sg-1, and led to identification of a UDP-sugar-dependent glycosyltransferase gene (Glyma07g38460). Although their sequences are highly similar and both glycosylate the nonacetylated saponin A0-αg, the Sg-1(a) allele encodes the xylosyltransferase UGT73F4, whereas Sg-1(b) encodes the glucosyltransferase UGT73F2. Homology models and site-directed mutagenesis analyses showed that Ser-138 in Sg-1(a) and Gly-138 in Sg-1(b) proteins are crucial residues for their respective sugar donor specificities. Transgenic complementation tests followed by recombinant enzyme assays in vitro demonstrated that sg-1(0) is a loss-of-function allele of Sg-1. Considering that the terminal sugar species in the group A saponins are responsible for the strong bitterness and astringent aftertastes of soybean seeds, our findings herein provide useful tools to improve commercial properties of soybean products.

Metabolic engineering for the production of prenylated polyphenols in transgenic legume plants using bacterial and plant prenyltransferases.

Prenylated polyphenols are secondary metabolites beneficial for human health because of their various biological activities. Metabolic engineering was performed using Streptomyces and Sophora flavescens prenyltransferase genes to produce prenylated polyphenols in transgenic legume plants. Three Streptomyces genes, NphB, SCO7190, and NovQ, whose gene products have broad substrate specificity, were overexpressed in a model legume, Lotus japonicus, in the cytosol, plastids or mitochondria with modification to induce the protein localization. Two plant genes, N8DT and G6DT, from Sophora flavescens whose gene products show narrow substrate specificity were also overexpressed in Lotus japonicus. Prenylated polyphenols were undetectable in these plants; however, supplementation of a flavonoid substrate resulted in the production of prenylated polyphenols such as 7-O-geranylgenistein, 6-dimethylallylnaringenin, 6-dimethylallylgenistein, 8-dimethylallynaringenin, and 6-dimethylallylgenistein in transgenic plants. Although transformants with the native NovQ did not produce prenylated polyphenols, modification of its codon usage led to the production of 6-dimethylallylnaringenin and 6-dimethylallylgenistein in transformants following naringenin supplementation. Prenylated polyphenols were not produced in mitochondrial-targeted transformants even under substrate feeding. SCO7190 was also expressed in soybean, and dimethylallylapigenin and dimethylallyldaidzein were produced by supplementing naringenin. This study demonstrated the potential for the production of novel prenylated polyphenols in transgenic plants. In particular, the enzymatic properties of prenyltransferases seemed to be altered in transgenic plants in a host species-dependent manner.

Novel bacterial N-acetyltransferase gene for herbicide detoxification in land plants and selection maker in plant transformation.

Phosphinothricin (PPT) is the active ingredient in bialaphos, which specifically inhibits glutamine synthetase in land plants. We isolated a novel PPT-resistant gene from a soil bacterium, Nocardia sp., and characterized it. The encoded protein, consisting of 177 amino acids, showed significant similarity to bacterial N-acetyltransferases, and we originally designated the gene MAT (methionine sulfone N-acetyltransferase). The recombinant MAT protein exhibited functions as a methionine sulfone and PPT N-acetyltransferase in vitro. The PPT N-acetyltransferase activity reached the maximum at pH 8-8.5, indicating that the protein might optimally function in chloroplasts. We therefore constructed a MAT gene, encoding the enzyme with a chloroplast-localizing signal in its amino-terminus. Plant transformation with the construct resulted in the generation of PPT-resistant rice and Arabidopsis. Furthermore, the transformed Arabidopsis was selectable in a synthetic medium containing PPT. The MAT gene thus facilitated establishment of herbicide-resistant plants, and as a new selectable gene marker.

High-level tryptophan accumulation in seeds of transgenic rice and its limited effects on agronomic traits and seed metabolite profile.

Metabolic manipulation of plants to improve their nutritional quality is an important goal of plant biotechnology. Expression in rice (Oryza sativa L.) of a transgene (OASA1D) encoding a feedback-insensitive alpha subunit of rice anthranilate synthase results in the accumulation of tryptophan (Trp) in calli and leaves. It is shown here that the amount of free Trp in the seeds of such plants is increased by about two orders of magnitude compared with that in the seeds of wild-type plants. The total Trp content in the seeds of the transgenic plants was also increased. Two homozygous lines, HW1 and HW5, of OASA1D transgenic rice were generated for characterization of agronomic traits and aromatic metabolite profiling of seeds. The marked overproduction of Trp was stable in these lines under field conditions, although spikelet fertility and yield, as well as seed germination ability, were reduced compared with the wild type. These differences in agronomic traits were small, however, in HW5. In spite of the high Trp content in the seeds of the HW lines, metabolic profiling revealed no substantial changes in the amounts of other phenolic compounds. The amount of indole acetic acid was increased about 2-fold in the seeds of the transgenic lines. The establishment and characterization of these OASA1D transgenic lines have thus demonstrated the feasibility of increasing the Trp content in the seeds of rice (or of other crops) as a means of improving its nutritional value for human consumption or animal feed.