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. 2001 May;13(5):1165-78.
doi: 10.1105/tpc.13.5.1165.

Cosuppression of the alpha subunits of beta-conglycinin in transgenic soybean seeds induces the formation of endoplasmic reticulum-derived protein bodies

A J Kinney  1 , R Jung, E M Herman
Affiliations

Cosuppression of the alpha subunits of beta-conglycinin in transgenic soybean seeds induces the formation of endoplasmic reticulum-derived protein bodies

A J Kinney et al. Plant Cell. 2001 May.

Abstract

The expression of the alpha and alpha' subunits of beta-conglycinin was suppressed by sequence-mediated gene silencing in transgenic soybean seed. The resulting seeds had similar total oil and protein content and ratio compared with the parent line. The decrease in beta-conglycinin protein was apparently compensated by an increased accumulation of glycinin. In addition, proglycinin, the precursor of glycinin, was detected as a prominent polypeptide band in the protein profile of the transgenic seed extract. Electron microscopic analysis and immunocytochemistry of maturing transgenic soybean seeds indicated that the process of storage protein accumulation was altered in the transgenic line. In normal soybeans, the storage proteins are deposited in pre-existing vacuoles by Golgi-derived vesicles. In contrast, in transgenic seed with reduced beta-conglycinin levels, endoplasmic reticulum (ER)-derived vesicles were observed that resembled precursor accumulating-vesicles of pumpkin seeds and the protein bodies accumulated by cereal seeds. Their ER-derived membrane of the novel vesicles did not contain the protein storage vacuole tonoplast-specific protein alpha-TIP, and the sequestered polypeptides did not contain complex glycans, indicating a preGolgi and nonvacuolar nature. Glycinin was identified as a major component of these novel protein bodies and its diversion from normal storage protein trafficking appears to be related to the proglycinin buildup in the transgenic seed. The stable accumulation of proteins in a protein body compartment instead of vacuolar accumulation of proteins may provide an alternative intracellular site to sequester proteins when soybeans are used as protein factories.

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Figures

Figure 1.
Figure 1.
RNA Gel Blot Shows Conglycinin Cosuppression. RNA gel blots of mRNA purified from developing seed of transgenic line G19 cosuppressed in conglycinin (G19) and of nontransgenic control (C) are shown. The blots were probed with conglycinin and Fad 2 cDNA probes. Note the almost complete absence of conglycinin mRNA in the G19 line compared with the abundant conglycinin mRNA in the control. Fad 3 (fatty acid desaturase 3) was used as a loading control.
Figure 2.
Figure 2.
Immunoblots Show the Absence of Conglycinin and the Accumulation of ProGY and P34 precursor. Extracts of fully mature G19 and control (C) seeds were fractionated by SDS-PAGE and transferred to nitrocellulose membranes. The blot was probed with antibodies against β-conglycinin, GY, and P34. This blot shows that the G19 line contains very little conglycinin compared with the abundant level of the protein in the control. In contrast to both mature GY and mature P34, polypeptide bands appear similar in both the G19 and control samples, indicating that neither of these proteins are reduced. The G19 line contains abundant proGY (arrowhead) and proP34 (arrowhead) compared with the control.
Figure 3.
Figure 3.
Vacuolar Processing Enzyme (VPE) Is Not Suppressed in Conglycinin-Cosuppressing Seed. (A) RNA gel blot shows that the G19 and control (C) seed contain very similar amounts of VPE message. Fad 3 labeling was used as a loading control as in Figure 1. (B) SDS-PAGE immunoblots probed with VPE2 and VPE1 isoform-specific antibodies show that both forms accumulate in G19 and control seed. The mature VPE 33-kD bands are similar in both G19 and control seeds for both VPE2 and VPE1 isoforms. The G19 seed contain abundant higher Mr precursors of both VPE2 and VPE1 isoforms that are not present in the control seed.
Figure 4.
Figure 4.
Assembly of GY Subunits into 11S Hexamers Is Impeded in Transgenic G19 Seed. Sedimentation analysis of soybean seed protein fractions. Sucrose gradient fractions of protein isolated from transgenic developing G19 seed (top section), protein isolated from mature G19 seed (middle section), and protein isolated from nontransgenic mature soybean seed (bottom section). Indicated fractions were separated by SDS-PAGE (top blot in each set of sections) and probed by immunoblotting using GY-specific antibodies (middle blot in each set of sections) and Kunitz trypsin inhibitor (KTI)–specific antibodies (bottom blot in each set of sections). Fraction numbers are given at the top of the lanes; the approximate sedimentation coefficients of fractions are indicated at the bottom. In mature transgenic seed, GY chains (acidic and basic) are present in all gradient fractions, indicating only partial assembly of GY subunits into 11S hexamers.
Figure 5.
Figure 5.
Conglycinin-Suppressing Seed Contain Abundant PBs. (A) Examination of maturing G19 seed by conventional electron microscopy with osmium postfixation demonstrates that the cytoplasm contains numerous electron-dense vesicles. (B) Higher magnification images of the electron-dense vesicles show that they possess an electron-dense core surrounded by a rough ER-derived membrane that is characteristic of seed PBs. N, nucleus; OB, oil body; PB, protein body; PSV, protein storage vacuole.
Figure 6.
Figure 6.
PBs in Maturing Conglycinin-Cosuppressing Soybeans Contain GY and P34. Thin sections of G19 soybeans were labeled with specific antibodies against GY and P34 to assay antigen content of the PBs. (A) and (B) Low- and high-magnification images of antiglycinin-labeled sections. The antibody labels both the PSV and PBs with similar gold particle density (B). (C) A monoclonal antibody against P34 also labels the PBs. OB, oil body; PB, protein body; PSV, protein storage vacuole; V, vacuole.
Figure 7.
Figure 7.
PBs Remain in Germinated Seed and Do Not Contain Oil Body, Golgi-processed, or Tonoplast Proteins. The PBs of germinated seed were assayed with antibodies against GY (A), complex glycan (B), soybean oleosin (C), and α-TIP (D). AntiGY antibodies label both the PBs and PSV (A), as in the maturing seed (A) and (B). In contrast, the anticomplex glycan antibodies label the PSV but do not label the PB. Oil bodies, like PBs are ER derived, and labeling with anti-oleosin antibodies shows that the OB proteins are restricted to the OBs (C). Although PBs contain PSV matrix proteins, they do not contain tonoplast proteins, as indicated by the lack of labeling with antibodies against the PSV tonoplast protein α-TIP (D). OB, oil body; PB, protein body; PSV, protein storage vacuole.
Figure 8.
Figure 8.
Diagram of the Formation of PBs. A diagram showing a model for the diversion of PSV matrix proteins into PBs is shown. A portion of the proteins that would otherwise progress through the endomembrane system for transport to the vacuole instead aggregate and are budded from the ER as PBs. The resulting PBs are a stable population of organelles that persist through seed maturation and remain in the dry mature seed. ER, endoplasmic reticulum; PB, protein body.
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