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Characterization and expression of Rubisco activase genes in Ipomoea batatas.

Jiang Y, Wang J, Tao X, Zhang Y - Mol. Biol. Rep. (2013)

Bottom Line: The results indicated that these two RCA isoforms may play different roles in regulating photosynthesis and they may be regulated by light, heat or both.In addition, there were interactions between Rubisco large subunit (RBCl) and short isoform RCA (RCAs) as well as RCAs and long isoform RCA (RCAl), but no interaction between RBCl and RCAl, implying they might form a sandwich-like structure (RBCl-RCAs-RCAl), at least in yeast cells.These results provided new information on the modulation of RCA genes in sweet potato, which could be useful in improving photosynthesis and plant growth in sweet potato.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Resource Biology and Eco-environment of Ministry of Education, Sichuan Key Laboratory of Molecular Biology and Biotechnology, College of Life Science, Sichuan University, Chengdu, 610064, People's Republic of China.

ABSTRACT
Two-dimensional electrophoresis, coupled with MALDI-TOF-MS, was used to identify differentially expressed proteins between young and mature leaves of sweet potato [Ipomoea batatas (L.) Lam]. The results showed that there were 25 differential proteins between young and mature leaves. The Rubisco activase (RCA) that catalyzes the activation of Rubisco in vivo and plays a crucial role in photosynthesis was among these 25 proteins. So far, little was known about the molecular biology of RCA in sweet potato. Here, this research reports the cloning and characterization of two genes encoding the short isoform and the long isoform of sweet potato RCAs. Analysis of DNA sequences of RCA suggested that the corresponding mRNAs were transcribed from two different genes. To study the roles of these two RCA isoforms in photosynthesis, we investigated the expression patterns of these RCA genes at the mRNA and protein levels every 2 h in a photoperiod and under different temperatures conditions. The results indicated that these two RCA isoforms may play different roles in regulating photosynthesis and they may be regulated by light, heat or both. In addition, there were interactions between Rubisco large subunit (RBCl) and short isoform RCA (RCAs) as well as RCAs and long isoform RCA (RCAl), but no interaction between RBCl and RCAl, implying they might form a sandwich-like structure (RBCl-RCAs-RCAl), at least in yeast cells. These results provided new information on the modulation of RCA genes in sweet potato, which could be useful in improving photosynthesis and plant growth in sweet potato.

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Yeast two-hybrid assay for detecting protein–protein interactions among Rubisco large subunit and two RCA isoforms. a The interaction of Rubisco large subunit with Ib-RCAs.b The interaction of Rubisco large subunit with Ib-RCAl. c The interaction of Ib-RCAl with Ib-RCAs. Yeast cells transformed with plasmids expressing murine p53 and SV40 large T-antigen were used as positive controls. Yeast cells transformed with various empty vectors (no insert) were used negative controls
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Fig6: Yeast two-hybrid assay for detecting protein–protein interactions among Rubisco large subunit and two RCA isoforms. a The interaction of Rubisco large subunit with Ib-RCAs.b The interaction of Rubisco large subunit with Ib-RCAl. c The interaction of Ib-RCAl with Ib-RCAs. Yeast cells transformed with plasmids expressing murine p53 and SV40 large T-antigen were used as positive controls. Yeast cells transformed with various empty vectors (no insert) were used negative controls

Mentions: To investigate whether both Ib-RCAs and Ib-RCAl could activate Rubisco, we studied the interactions of Ib-RBCl with these two isoform Ib-RCA. The yeast strain AH109 was co-transformed with bait plasmid pGBKT7-RCAs and prey plasmid pGADT7-RCAl, or co-transformed with bait plasmid pGBKT7-RBCl and prey plasmid pGADT7-RCAs, both formed blue colonies on filter paper by colony-lift filter assay with X-β-gal (data not shown) and the corresponding colonies grew normally on Quadruple Drop Out medium (Fig. 6). However, yeast AH109 strains co-transformed with bait plasmid pGBKT7-RBCl and prey plasmid pGADT7-RCAl could only grow on the plate of SD/-leu-Trp, and no blue yeast colonies appeared on filter paper by colony-lift filter assay (data not shown). Yeast cells co-transformed with various empty vectors (no insert) lacked any visible growth on Quadruple Drop Out medium (SD/-Leu/-Trp/-His/-Ade, high stringency selection), providing clear evidence that expression of Ib-RBCl, Ib-RCAs or Ib-RCAl alone did not lead to expression of the reporter genes in yeast. These results indicated that at least in yeast cell, there was interaction between Ib-RBCl and Ib-RCAs as well as Ib-RCAs and Ib-RCAl, and no interaction between Ib-RBCl and Ib-RCAl.Fig. 6


Characterization and expression of Rubisco activase genes in Ipomoea batatas.

Jiang Y, Wang J, Tao X, Zhang Y - Mol. Biol. Rep. (2013)

Yeast two-hybrid assay for detecting protein–protein interactions among Rubisco large subunit and two RCA isoforms. a The interaction of Rubisco large subunit with Ib-RCAs.b The interaction of Rubisco large subunit with Ib-RCAl. c The interaction of Ib-RCAl with Ib-RCAs. Yeast cells transformed with plasmids expressing murine p53 and SV40 large T-antigen were used as positive controls. Yeast cells transformed with various empty vectors (no insert) were used negative controls
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3824211&req=5

Fig6: Yeast two-hybrid assay for detecting protein–protein interactions among Rubisco large subunit and two RCA isoforms. a The interaction of Rubisco large subunit with Ib-RCAs.b The interaction of Rubisco large subunit with Ib-RCAl. c The interaction of Ib-RCAl with Ib-RCAs. Yeast cells transformed with plasmids expressing murine p53 and SV40 large T-antigen were used as positive controls. Yeast cells transformed with various empty vectors (no insert) were used negative controls
Mentions: To investigate whether both Ib-RCAs and Ib-RCAl could activate Rubisco, we studied the interactions of Ib-RBCl with these two isoform Ib-RCA. The yeast strain AH109 was co-transformed with bait plasmid pGBKT7-RCAs and prey plasmid pGADT7-RCAl, or co-transformed with bait plasmid pGBKT7-RBCl and prey plasmid pGADT7-RCAs, both formed blue colonies on filter paper by colony-lift filter assay with X-β-gal (data not shown) and the corresponding colonies grew normally on Quadruple Drop Out medium (Fig. 6). However, yeast AH109 strains co-transformed with bait plasmid pGBKT7-RBCl and prey plasmid pGADT7-RCAl could only grow on the plate of SD/-leu-Trp, and no blue yeast colonies appeared on filter paper by colony-lift filter assay (data not shown). Yeast cells co-transformed with various empty vectors (no insert) lacked any visible growth on Quadruple Drop Out medium (SD/-Leu/-Trp/-His/-Ade, high stringency selection), providing clear evidence that expression of Ib-RBCl, Ib-RCAs or Ib-RCAl alone did not lead to expression of the reporter genes in yeast. These results indicated that at least in yeast cell, there was interaction between Ib-RBCl and Ib-RCAs as well as Ib-RCAs and Ib-RCAl, and no interaction between Ib-RBCl and Ib-RCAl.Fig. 6

Bottom Line: The results indicated that these two RCA isoforms may play different roles in regulating photosynthesis and they may be regulated by light, heat or both.In addition, there were interactions between Rubisco large subunit (RBCl) and short isoform RCA (RCAs) as well as RCAs and long isoform RCA (RCAl), but no interaction between RBCl and RCAl, implying they might form a sandwich-like structure (RBCl-RCAs-RCAl), at least in yeast cells.These results provided new information on the modulation of RCA genes in sweet potato, which could be useful in improving photosynthesis and plant growth in sweet potato.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Resource Biology and Eco-environment of Ministry of Education, Sichuan Key Laboratory of Molecular Biology and Biotechnology, College of Life Science, Sichuan University, Chengdu, 610064, People's Republic of China.

ABSTRACT
Two-dimensional electrophoresis, coupled with MALDI-TOF-MS, was used to identify differentially expressed proteins between young and mature leaves of sweet potato [Ipomoea batatas (L.) Lam]. The results showed that there were 25 differential proteins between young and mature leaves. The Rubisco activase (RCA) that catalyzes the activation of Rubisco in vivo and plays a crucial role in photosynthesis was among these 25 proteins. So far, little was known about the molecular biology of RCA in sweet potato. Here, this research reports the cloning and characterization of two genes encoding the short isoform and the long isoform of sweet potato RCAs. Analysis of DNA sequences of RCA suggested that the corresponding mRNAs were transcribed from two different genes. To study the roles of these two RCA isoforms in photosynthesis, we investigated the expression patterns of these RCA genes at the mRNA and protein levels every 2 h in a photoperiod and under different temperatures conditions. The results indicated that these two RCA isoforms may play different roles in regulating photosynthesis and they may be regulated by light, heat or both. In addition, there were interactions between Rubisco large subunit (RBCl) and short isoform RCA (RCAs) as well as RCAs and long isoform RCA (RCAl), but no interaction between RBCl and RCAl, implying they might form a sandwich-like structure (RBCl-RCAs-RCAl), at least in yeast cells. These results provided new information on the modulation of RCA genes in sweet potato, which could be useful in improving photosynthesis and plant growth in sweet potato.

Show MeSH
Related in: MedlinePlus