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Sucrose phosphate synthase and sucrose phosphate phosphatase interact in planta and promote plant growth and biomass accumulation.

Maloney VJ, Park JY, Unda F, Mansfield SD - J. Exp. Bot. (2015)

Bottom Line: Herein, the formation of an enzyme complex between SPS and SPP was examined, and the results from yeast two-hybrid experiments suggest that there is indeed an association between these proteins.The findings clearly demonstrated that SPS interacts with SPP and that this interaction impacts soluble carbohydrate pools and affects carbon partitioning to starch.Moreover, a fusion construct between the two genes promotes plant growth in both transgenic Arabidopsis and hybrid poplar.

View Article: PubMed Central - PubMed

Affiliation: Department of Wood Science, University of British Columbia, 2424 Main Mall, Vancouver, BC, V6T 1Z4, Canada.

No MeSH data available.


Related in: MedlinePlus

Protein interaction of Arabidopsis AtSPS (At5g20280) and AtSPP (At2g35750) using a yeast two-hybrid assay. (A) SC/-Leu/-Trp plate confirming the introduction of both plasmids into the cells. (B) Prototrophic growth of the yeast two-hybrid strain MaV203 on media depleted in histidine+10mM 3AT indicates positive interactions. (C) Growth inhibition of cells containing interacting proteins results from induction of URA3 reporter genes on medium containing 0.2% 5-fluoroorotic acid (5FOA). (D) Induction of the lacZ gene with X-gal resulted in a blue colour, indicating that a positive protein interaction between AtSPS and AtSPP was produced.
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Figure 1: Protein interaction of Arabidopsis AtSPS (At5g20280) and AtSPP (At2g35750) using a yeast two-hybrid assay. (A) SC/-Leu/-Trp plate confirming the introduction of both plasmids into the cells. (B) Prototrophic growth of the yeast two-hybrid strain MaV203 on media depleted in histidine+10mM 3AT indicates positive interactions. (C) Growth inhibition of cells containing interacting proteins results from induction of URA3 reporter genes on medium containing 0.2% 5-fluoroorotic acid (5FOA). (D) Induction of the lacZ gene with X-gal resulted in a blue colour, indicating that a positive protein interaction between AtSPS and AtSPP was produced.

Mentions: In an attempt to assess the putative protein–protein interaction between AtSPS and AtSPP, the full-length AtSPS cDNA fused in-frame with the DNA-binding domain of GAL4 and the AtSPP fused in-frame with the GAL4 activation domain were used for co-transformation in a direct yeast two-hybrid assay system. Transformed colonies were first selected on medium lacking leucine and tryptophan, confirming the introduction of both plasmids into cells (Fig. 1A), while positive interactions were detected by selection on plates lacking the auxotrophic markers histidine or uracil. These cells were then grown on selective medium lacking histidine, which included 10mM 3AT as a HIS3 inhibitor (Fig. 1B). In order to monitor the putative positive interaction further, the cells were grown on media with 0.2% 5FOA. The positive yeast cell lines, pDEST™ 32/SPS and pDEST™ 22/SPP, produce the two-hybrid-dependent induction of URA3, which results in the conversion of 5FOA to 5-fluorouracil, which is toxic. Hence, the AtSPS and AtSPP proteins in the cells containing both plasmids must be interacting, as indicated by their inability to grow on medium containing 5FOA (Fig. 1C). Furthermore, induction of the lacZ gene with X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) using the colorimetric assays for β-galactosidase resulted in a blue colour, indicating that a positive protein interaction between AtSPS and AtSPP was produced. The combination of pDEST32-SPS and the empty vector pDEST22 served as a negative interaction control and, as expected, showed no interaction (Fig. 1D). The combination of empty vectors pDEST32 and pDEST22 served as additional negative controls on each selective medium.


Sucrose phosphate synthase and sucrose phosphate phosphatase interact in planta and promote plant growth and biomass accumulation.

Maloney VJ, Park JY, Unda F, Mansfield SD - J. Exp. Bot. (2015)

Protein interaction of Arabidopsis AtSPS (At5g20280) and AtSPP (At2g35750) using a yeast two-hybrid assay. (A) SC/-Leu/-Trp plate confirming the introduction of both plasmids into the cells. (B) Prototrophic growth of the yeast two-hybrid strain MaV203 on media depleted in histidine+10mM 3AT indicates positive interactions. (C) Growth inhibition of cells containing interacting proteins results from induction of URA3 reporter genes on medium containing 0.2% 5-fluoroorotic acid (5FOA). (D) Induction of the lacZ gene with X-gal resulted in a blue colour, indicating that a positive protein interaction between AtSPS and AtSPP was produced.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4493782&req=5

Figure 1: Protein interaction of Arabidopsis AtSPS (At5g20280) and AtSPP (At2g35750) using a yeast two-hybrid assay. (A) SC/-Leu/-Trp plate confirming the introduction of both plasmids into the cells. (B) Prototrophic growth of the yeast two-hybrid strain MaV203 on media depleted in histidine+10mM 3AT indicates positive interactions. (C) Growth inhibition of cells containing interacting proteins results from induction of URA3 reporter genes on medium containing 0.2% 5-fluoroorotic acid (5FOA). (D) Induction of the lacZ gene with X-gal resulted in a blue colour, indicating that a positive protein interaction between AtSPS and AtSPP was produced.
Mentions: In an attempt to assess the putative protein–protein interaction between AtSPS and AtSPP, the full-length AtSPS cDNA fused in-frame with the DNA-binding domain of GAL4 and the AtSPP fused in-frame with the GAL4 activation domain were used for co-transformation in a direct yeast two-hybrid assay system. Transformed colonies were first selected on medium lacking leucine and tryptophan, confirming the introduction of both plasmids into cells (Fig. 1A), while positive interactions were detected by selection on plates lacking the auxotrophic markers histidine or uracil. These cells were then grown on selective medium lacking histidine, which included 10mM 3AT as a HIS3 inhibitor (Fig. 1B). In order to monitor the putative positive interaction further, the cells were grown on media with 0.2% 5FOA. The positive yeast cell lines, pDEST™ 32/SPS and pDEST™ 22/SPP, produce the two-hybrid-dependent induction of URA3, which results in the conversion of 5FOA to 5-fluorouracil, which is toxic. Hence, the AtSPS and AtSPP proteins in the cells containing both plasmids must be interacting, as indicated by their inability to grow on medium containing 5FOA (Fig. 1C). Furthermore, induction of the lacZ gene with X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) using the colorimetric assays for β-galactosidase resulted in a blue colour, indicating that a positive protein interaction between AtSPS and AtSPP was produced. The combination of pDEST32-SPS and the empty vector pDEST22 served as a negative interaction control and, as expected, showed no interaction (Fig. 1D). The combination of empty vectors pDEST32 and pDEST22 served as additional negative controls on each selective medium.

Bottom Line: Herein, the formation of an enzyme complex between SPS and SPP was examined, and the results from yeast two-hybrid experiments suggest that there is indeed an association between these proteins.The findings clearly demonstrated that SPS interacts with SPP and that this interaction impacts soluble carbohydrate pools and affects carbon partitioning to starch.Moreover, a fusion construct between the two genes promotes plant growth in both transgenic Arabidopsis and hybrid poplar.

View Article: PubMed Central - PubMed

Affiliation: Department of Wood Science, University of British Columbia, 2424 Main Mall, Vancouver, BC, V6T 1Z4, Canada.

No MeSH data available.


Related in: MedlinePlus