The composition of newly synthesized proteins in the endoplasmic reticulum determines the transport pathways of soybean seed storage proteins

Glycinin (11S) and beta-conglycinin (7S) are major storage proteins in soybean (Glycine max L.) seeds and accumulate in the protein storage vacuole (PSV). These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the PSV by vesicles. Electron microscopic analysis of developing soybean cotyledons of the wild type and mutants with storage protein composition different from that of the wild type showed that there are two transport pathways: one is via the Golgi and the other bypasses it. Golgi-derived vesicles were observed in all lines used in this study and formed smooth dense bodies with a diameter of 0.5 to several micrometers. ER-derived protein bodies (PBs) with a diameter of 0.3-0.5 microm were observed at high frequency in the mutants containing higher amount of 11S group I subunit than the wild type, whereas they were hardly observed in the mutants lacking 11S group I subunit. These indicate that pro11S group I may affect the formation of PBs. Thus, the composition of newly synthesized proteins in the ER is important in the selection of the transport pathways.     

Arabidopsis vacuolar sorting mutants (green fluorescent seed) can be identified efficiently by secretion of vacuole-targeted green fluorescent protein in their seeds

Two Arabidopsis thaliana genes have been shown to function in vacuolar sorting of seed storage proteins: a vacuolar sorting receptor, VSR1/ATELP1, and a retromer component, MAIGO1 (MAG1)/VPS29. Here, we show an efficient and simple method for isolating vacuolar sorting mutants of Arabidopsis. The method was based on two findings in this study. First, VSR1 functioned as a sorting receptor for beta-conglycinin by recognizing the vacuolar targeting signal. Second, when green fluorescent protein (GFP) fusion with the signal (GFP-CT24) was expressed in vsr1, mag1/vps29, and wild-type seeds, both vsr1and mag1/vps29 gave strongly fluorescent seeds but the wild type did not, suggesting that a defect in vacuolar sorting provided fluorescent seeds by the secretion of GFP-CT24 out of the cells. We mutagenized transformant seeds expressing GFP-CT24. From approximately 3,000,000 lines of M2 seeds, we obtained >100 fluorescent seeds and designated them green fluorescent seed (gfs) mutants. We report 10 gfs mutants, all of which caused missorting of storage proteins. We mapped gfs1 to VSR1, gfs2 to KAM2/GRV2, gfs10 to the At4g35870 gene encoding a novel membrane protein, and the others to different loci. This method should provide valuable insights into the complex molecular mechanisms underlying vacuolar sorting of storage proteins.     

Anti-hypertensive activity of genetically modified soybean seeds accumulating novokinin

Novokinin (Arg-Pro-Leu-Lys-Pro-Trp), which has been designed based on the structure of ovokinin (2-7), significantly reduces the systolic blood pressure at a dose of 100 microg/kg after oral administration in spontaneously hypertensive rats (SHRs). In this study, we generated a transgenic soybean which accumulates novokinin. A vector encoding a modified beta-conglycinin alpha' subunit (4novokinin-alpha') in which four novokinin sequences have been incorporated by site-directed mutagenesis was introduced into somatic embryos by whisker-mediated gene transformation to produce a transgenic soybean. The 4novokinin-alpha' occupied 0.5% of total soluble protein and 5% of the beta-conglycinin alpha' subunit in the transgenic soybean seeds. Protein extracted from the transgenic soybean reduced systolic blood pressure after single oral administration in SHRs at a dose of 0.15 g/kg. Defatted flour from the transgenic soybean also reduced the systolic blood pressure at a dose of 0.25 g/kg. Thus, the 4novokinin-alpha' produced in soybean exhibited an anti-hypertensive activity in SHRs after oral administration.     

Transcriptome-wide effects of expansin gene manipulation in etiolated Arabidopsis seedling

Journal of Plant Research - Expansin is a non-enzymatic protein which plays a pivotal role in cell wall loosening by inducing stress relaxation and extension in the plant cell wall. Previous...     

A red fluorescent protein, DsRed2, as a visual reporter for transient expression and stable transformation in soybean

Fluorescent proteins such as green fluorescent protein (GFP) from Aequorea victoria are often used as markers for transient expression and stable transformation in plants, given that their detection does not require a substrate and they can be monitored in a nondestructive manner. We have now evaluated the red fluorescent protein DsRed2 (a mutant form of DsRed from Discosoma sp.) for its suitability as a visual marker in combination with antibiotic selection for genetic transformation of soybean [Glycine max (L.) Merrill]. Transient and stable expression of DsRed2 in somatic embryos was readily detected by fluorescence microscopy, allowing easy confirmation of gene introduction. We obtained several fertile transgenic lines, including homozygous lines, that grew and produced seeds in an apparently normal manner. The red fluorescence of DsRed2 was detected by fluorescence microscopy without background fluorescence in both leaves and seeds of the transgenic plants. Furthermore, in contrast to seeds expressing GFP, those expressing DsRed2 were readily identifiable even under white light by the color conferred by the transgene product. The protein composition of seeds was not affected by the introduction of DsRed2, with the exception of the accumulation of DsRed2 itself, which was detectable as an additional band on electrophoresis. These results indicate that DsRed2 is a suitable reporter (even more suitable than GFP) for genetic transformation of soybean.     

Identification of the 23-kDa peptide derived from the precursor of Gly m Bd 28K, a major soybean allergen, as a new allergen

One of the major soybean allergens, Gly m Bd 28K, is suggested to be biosynthesized as a preproprotein form, which would be composed of a signal peptide, Gly m Bd 28K and the C-terminal peptide (the 23-kDa peptide). However, the 23-kDa peptide has never been characterized. In the present study, we prepared a monoclonal antibody (mAb) against a recombinant 23-kDa peptide expressed in Escherichia coli to detect the 23-kDa peptide in soybean. Several proteins were detected by immunoblotting with the mAb. All of the proteins were shown to have the identical N-terminal amino acid sequence, suggesting that the proteins correspond to the C-terminal part of the Gly m Bd 28K precursor. Furthermore, Gly m Bd 28K and the 23-kDa peptide were observed to come out at the 21st day after flowering and to locate in the crystalloid part of protein storage vacuoles in growing cotyledons. Some of the 23-kDa peptides were shown to be glycoproteins with an N-linked glycan moiety and exhibited the binding to IgE antibodies in the sera of patients sensitive to soybean. The binding of the peptides to IgE antibodies was suggested to be predominantly dependent on their glycan moiety. This study proves the occurrence of the 23-kDa peptide in soybean and that it is a new allergen.     

Regulated DNA rearrangement during sporulation in Bacillus weihenstephanensis KBAB4

Temperate phages can integrate their genomes into a specific region of a host chromosome to produce lysogens (prophage). During genome insertion, prophages may interrupt the gene coding sequence. In Bacillus subtilis, the sigma factor gene sigK is interrupted by a 48 kb prophage‐like element. sigK is a composite coding sequence from two partial genes during sporulation. For over two decades, however, no further examples of DNA element‐mediated gene reconstitution other than sigK have been identified in spore formers. Here we report that the gene for dipicolinic acid (DPA) synthetase β subunit spoVFB in B. weihenstephanensis KBAB4 is interrupted by a prophage‐like element named vfbin. DPA is synthesized in the mother cell and required for maintaining spore dormancy. We found that spoVFB was a composite coding sequence generated in the mother cell via chromosomal rearrangement that excised vfbin. Furthermore, vfbin caused excision after phage‐inducer treatment, but vfbin appeared to be defective as a prophage. We also found various spore‐forming bacteria in which sporulation‐related genes were disrupted by prophage‐like DNA elements. These results demonstrate the first example of a similar mechanism that affects a sporulation gene other than sigK and suggest that this prophage‐mediated DNA rearrangement is a common phenomenon in spore‐forming bacteria.