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Effects of proteome rebalancing and sulfur nutrition on the accumulation of methionine rich δ-zein in transgenic soybeans.

Kim WS, Jez JM, Krishnan HB - Front Plant Sci (2014)

Bottom Line: Western blot and 2D-gel analysis revealed that β-conglycinin knockdown line (SAM) failed to accumulate the α', α, and β-subunits of β-conglycinin.The introgression of the 11 kDa δ-zein into β-conglycinin knockdown line did not enhance the accumulation of the 11 kDa δ-zein.However, when the same plants were grown in sulfur-rich medium, we observed 3- to 16-fold increased accumulation of the 11 kDa δ-zein.

View Article: PubMed Central - PubMed

Affiliation: Plant Genetics Research Unit, Agricultural Research Service, U.S. Department of Agriculture, University of Missouri Columbia, MO, USA.

ABSTRACT
Expression of heterologous methionine-rich proteins to increase the overall sulfur amino acid content of soybean seeds has been only marginally successful, presumably due to low accumulation of transgenes in soybeans or due to gene silencing. Proteome rebalancing of seed proteins has been shown to promote the accumulation of foreign proteins. In this study, we have utilized RNAi technology to suppress the expression of the β-conglycinin, the abundant 7S seed storage proteins of soybean. Western blot and 2D-gel analysis revealed that β-conglycinin knockdown line (SAM) failed to accumulate the α', α, and β-subunits of β-conglycinin. The proteome rebalanced SAM retained the overall protein and oil content similar to that of wild-type soybean. We also generated transgenic soybean lines expressing methionine-rich 11 kDa δ-zein under the control of either the glycinin or β-conglycinin promoter. The introgression of the 11 kDa δ-zein into β-conglycinin knockdown line did not enhance the accumulation of the 11 kDa δ-zein. However, when the same plants were grown in sulfur-rich medium, we observed 3- to 16-fold increased accumulation of the 11 kDa δ-zein. Transmission electron microscopy observation revealed that seeds grown in sulfur-rich medium contained numerous endoplasmic reticulum derived protein bodies. Our findings suggest that sulfur availability, not proteome rebalancing, is needed for high-level accumulation of heterologous methionine-rich proteins in soybean seeds.

No MeSH data available.


Related in: MedlinePlus

Suppression of β-conglycinin accumulation in transgenic soybean lines. Total seed proteins from wild-type (lane 1) and from seven independent RNAi transgenic lines (lanes 2–8) were fractionated in duplicate gels by 10% SDS-PAGE and stained with Coomassie Blue (A) or transferred to nitrocellulose membranes. The membrane was probed with the β-conglycinin antibodies (B). Immunoreactive proteins were identified using anti rabbit IgG-horseradish peroxidase conjugate followed by chemiluminescent detection. Note the absence of α′, α, and β-subunits of β-conglycinin in some of the RNAi lines.
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Figure 2: Suppression of β-conglycinin accumulation in transgenic soybean lines. Total seed proteins from wild-type (lane 1) and from seven independent RNAi transgenic lines (lanes 2–8) were fractionated in duplicate gels by 10% SDS-PAGE and stained with Coomassie Blue (A) or transferred to nitrocellulose membranes. The membrane was probed with the β-conglycinin antibodies (B). Immunoreactive proteins were identified using anti rabbit IgG-horseradish peroxidase conjugate followed by chemiluminescent detection. Note the absence of α′, α, and β-subunits of β-conglycinin in some of the RNAi lines.

Mentions: The suppression of the 7S globulins accumulation in soybean seeds was accomplished by RNAi utilizing Agrobacterium-mediated cotyledonary node transformation protocol (Hinchee et al., 1988). Seeds from several independent transgenic lines were screened for their protein composition by SDS-PAGE and immunoblot analysis using antibodies raised against the β-subunit of β-conglycinin (Figure 2). An examination of the Coomassie stained gel revealed that in 50% of the transgenic plants no significant reduction in the accumulation of β-conglycinin had occurred when compared to the wild-type plant (Figure 2A). However, four transgenic RNAi lines showed a complete absence of the β-conglycinin accumulation in their seeds (Figure 2A). The lack of β-conglycinin in these RNAi lines was confirmed by immunoblot analysis using β-conglycinin antibodies (Figure 2B). These four lines were grown for five generations. Twenty individual T5 seeds from each transgenic line were subjected to Western blot analysis. Transgenic plants where all tested seeds failed to accumulate the β-conglycinin were considered to be homozygous plants. These homozygous transgenic RNAi plants are referred as SAM from this point onwards.


Effects of proteome rebalancing and sulfur nutrition on the accumulation of methionine rich δ-zein in transgenic soybeans.

Kim WS, Jez JM, Krishnan HB - Front Plant Sci (2014)

Suppression of β-conglycinin accumulation in transgenic soybean lines. Total seed proteins from wild-type (lane 1) and from seven independent RNAi transgenic lines (lanes 2–8) were fractionated in duplicate gels by 10% SDS-PAGE and stained with Coomassie Blue (A) or transferred to nitrocellulose membranes. The membrane was probed with the β-conglycinin antibodies (B). Immunoreactive proteins were identified using anti rabbit IgG-horseradish peroxidase conjugate followed by chemiluminescent detection. Note the absence of α′, α, and β-subunits of β-conglycinin in some of the RNAi lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4227475&req=5

Figure 2: Suppression of β-conglycinin accumulation in transgenic soybean lines. Total seed proteins from wild-type (lane 1) and from seven independent RNAi transgenic lines (lanes 2–8) were fractionated in duplicate gels by 10% SDS-PAGE and stained with Coomassie Blue (A) or transferred to nitrocellulose membranes. The membrane was probed with the β-conglycinin antibodies (B). Immunoreactive proteins were identified using anti rabbit IgG-horseradish peroxidase conjugate followed by chemiluminescent detection. Note the absence of α′, α, and β-subunits of β-conglycinin in some of the RNAi lines.
Mentions: The suppression of the 7S globulins accumulation in soybean seeds was accomplished by RNAi utilizing Agrobacterium-mediated cotyledonary node transformation protocol (Hinchee et al., 1988). Seeds from several independent transgenic lines were screened for their protein composition by SDS-PAGE and immunoblot analysis using antibodies raised against the β-subunit of β-conglycinin (Figure 2). An examination of the Coomassie stained gel revealed that in 50% of the transgenic plants no significant reduction in the accumulation of β-conglycinin had occurred when compared to the wild-type plant (Figure 2A). However, four transgenic RNAi lines showed a complete absence of the β-conglycinin accumulation in their seeds (Figure 2A). The lack of β-conglycinin in these RNAi lines was confirmed by immunoblot analysis using β-conglycinin antibodies (Figure 2B). These four lines were grown for five generations. Twenty individual T5 seeds from each transgenic line were subjected to Western blot analysis. Transgenic plants where all tested seeds failed to accumulate the β-conglycinin were considered to be homozygous plants. These homozygous transgenic RNAi plants are referred as SAM from this point onwards.

Bottom Line: Western blot and 2D-gel analysis revealed that β-conglycinin knockdown line (SAM) failed to accumulate the α', α, and β-subunits of β-conglycinin.The introgression of the 11 kDa δ-zein into β-conglycinin knockdown line did not enhance the accumulation of the 11 kDa δ-zein.However, when the same plants were grown in sulfur-rich medium, we observed 3- to 16-fold increased accumulation of the 11 kDa δ-zein.

View Article: PubMed Central - PubMed

Affiliation: Plant Genetics Research Unit, Agricultural Research Service, U.S. Department of Agriculture, University of Missouri Columbia, MO, USA.

ABSTRACT
Expression of heterologous methionine-rich proteins to increase the overall sulfur amino acid content of soybean seeds has been only marginally successful, presumably due to low accumulation of transgenes in soybeans or due to gene silencing. Proteome rebalancing of seed proteins has been shown to promote the accumulation of foreign proteins. In this study, we have utilized RNAi technology to suppress the expression of the β-conglycinin, the abundant 7S seed storage proteins of soybean. Western blot and 2D-gel analysis revealed that β-conglycinin knockdown line (SAM) failed to accumulate the α', α, and β-subunits of β-conglycinin. The proteome rebalanced SAM retained the overall protein and oil content similar to that of wild-type soybean. We also generated transgenic soybean lines expressing methionine-rich 11 kDa δ-zein under the control of either the glycinin or β-conglycinin promoter. The introgression of the 11 kDa δ-zein into β-conglycinin knockdown line did not enhance the accumulation of the 11 kDa δ-zein. However, when the same plants were grown in sulfur-rich medium, we observed 3- to 16-fold increased accumulation of the 11 kDa δ-zein. Transmission electron microscopy observation revealed that seeds grown in sulfur-rich medium contained numerous endoplasmic reticulum derived protein bodies. Our findings suggest that sulfur availability, not proteome rebalancing, is needed for high-level accumulation of heterologous methionine-rich proteins in soybean seeds.

No MeSH data available.


Related in: MedlinePlus