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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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The expression patterns of Ib-RCA genes in a photoperiod. a Expression patterns of the Ib-RCA genes by relative quantitative real-time PCR as shown in graphs. RNA samples were collected at 2 h interval from 0 to 22 h. b Expression patterns of the Ib-RCA genes by semi-quantitative PCR as shown in gels. The β-actin was used as a control. Inside graph is the change of temperature in a photoperiod. Data (mean ± SE) are typical results from three independent experiments performed
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Fig4: The expression patterns of Ib-RCA genes in a photoperiod. a Expression patterns of the Ib-RCA genes by relative quantitative real-time PCR as shown in graphs. RNA samples were collected at 2 h interval from 0 to 22 h. b Expression patterns of the Ib-RCA genes by semi-quantitative PCR as shown in gels. The β-actin was used as a control. Inside graph is the change of temperature in a photoperiod. Data (mean ± SE) are typical results from three independent experiments performed

Mentions: To study whether mRNA’s accumulation of Ib-RCA in leaf tissue is regulated by light, we determined the mRNA and protein levels of Ib-RCAs/Ib-RCAl by q-PCR and Western-blot. Before quantitative analysis, serial dilutions for each cDNA (10−1–10−5) were used to make a quantitative PCR standard curve to calculate the PCR efficiencies of Ib-RCAs, Ib-RCAl and β-actin. The standard curves demonstrated that the amplification efficiencies for Ib-RCAs, Ib-RCAl, β-actin of the real-time PCR were 98.2, 100.8, 98.8 %, respectively, and the values of R2 (correlation coefficient′s square) were 0.994, 0.966, 0.991, respectively (Supplementary Fig. S2). The relative quantitative PCR results of Ib-RCAs and Ib-RCAl showed that the maximal mRNA level of Ib-RCAs was detected at 10:00, 4 h after the beginning of the light period. The expression level decreased moderately at 12:00 compared with that at 10:00. However, the mRNA level of Ib-RCAs recovered slightly after 12:00, and then decreased gradually to the minimum level at midnight (Fig. 4). Compared with Ib-RCAs, the expression level of Ib-RCAl began to rise at 12:00 and reached the peak at 14:00, then decreased quickly (Fig. 4). Protein levels of these two Ib-RCA isoforms in the leaves of sweet potato shared similar characteristics at the mRNA levels. From 10:00, the Ib-RCAs accumulated quickly, declined gradually after 16:00, and then maintained at a comparatively low level at night. After 14:00, the accumulation of Ib-RCAl began to increase, and then declined after 16:00 (Fig. 5a, b).Fig. 4


Characterization and expression of Rubisco activase genes in Ipomoea batatas.

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

The expression patterns of Ib-RCA genes in a photoperiod. a Expression patterns of the Ib-RCA genes by relative quantitative real-time PCR as shown in graphs. RNA samples were collected at 2 h interval from 0 to 22 h. b Expression patterns of the Ib-RCA genes by semi-quantitative PCR as shown in gels. The β-actin was used as a control. Inside graph is the change of temperature in a photoperiod. Data (mean ± SE) are typical results from three independent experiments performed
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: The expression patterns of Ib-RCA genes in a photoperiod. a Expression patterns of the Ib-RCA genes by relative quantitative real-time PCR as shown in graphs. RNA samples were collected at 2 h interval from 0 to 22 h. b Expression patterns of the Ib-RCA genes by semi-quantitative PCR as shown in gels. The β-actin was used as a control. Inside graph is the change of temperature in a photoperiod. Data (mean ± SE) are typical results from three independent experiments performed
Mentions: To study whether mRNA’s accumulation of Ib-RCA in leaf tissue is regulated by light, we determined the mRNA and protein levels of Ib-RCAs/Ib-RCAl by q-PCR and Western-blot. Before quantitative analysis, serial dilutions for each cDNA (10−1–10−5) were used to make a quantitative PCR standard curve to calculate the PCR efficiencies of Ib-RCAs, Ib-RCAl and β-actin. The standard curves demonstrated that the amplification efficiencies for Ib-RCAs, Ib-RCAl, β-actin of the real-time PCR were 98.2, 100.8, 98.8 %, respectively, and the values of R2 (correlation coefficient′s square) were 0.994, 0.966, 0.991, respectively (Supplementary Fig. S2). The relative quantitative PCR results of Ib-RCAs and Ib-RCAl showed that the maximal mRNA level of Ib-RCAs was detected at 10:00, 4 h after the beginning of the light period. The expression level decreased moderately at 12:00 compared with that at 10:00. However, the mRNA level of Ib-RCAs recovered slightly after 12:00, and then decreased gradually to the minimum level at midnight (Fig. 4). Compared with Ib-RCAs, the expression level of Ib-RCAl began to rise at 12:00 and reached the peak at 14:00, then decreased quickly (Fig. 4). Protein levels of these two Ib-RCA isoforms in the leaves of sweet potato shared similar characteristics at the mRNA levels. From 10:00, the Ib-RCAs accumulated quickly, declined gradually after 16:00, and then maintained at a comparatively low level at night. After 14:00, the accumulation of Ib-RCAl began to increase, and then declined after 16:00 (Fig. 5a, b).Fig. 4

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