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. 2010 Jun;61(11):2939-50.
doi: 10.1093/jxb/erq122. Epub 2010 May 17.

Reversible association of ribulose-1, 5-bisphosphate carboxylase/oxygenase activase with the thylakoid membrane depends upon the ATP level and pH in rice without heat stress

Juan Chen  1 , Peng Wang, Hua-Ling Mi, Gen-Yun Chen, Da-Quan Xu
Affiliations

Reversible association of ribulose-1, 5-bisphosphate carboxylase/oxygenase activase with the thylakoid membrane depends upon the ATP level and pH in rice without heat stress

Juan Chen et al. J Exp Bot. 2010 Jun.

Abstract

Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activase (RCA) in the thylakoid membrane (TM) has been shown to play a role in protection and regulation of photosynthesis under moderate heat stress. However, the physiological significance of RCA bound to the TM (TM-RCA) without heat stress remains unknown. In this study, it is first shown, using experiments in vivo, that the TM-RCA varies in rice leaves at different development stages, under different environmental conditions, and in a rice mutant. Furthermore, it is shown that the amount of TM-RCA always increased when the Rubisco activation state and the pH gradient across the TM (DeltapH) decreased. It was then demonstrated in vitro that the RCA bound dynamically to TM and the amount of TM-RCA increased during Rubisco activation. A high level of ATP and a high pH value promoted the dissociation of RCA from the TM. Both the RCA association with and dissociation from the TM showed conformational changes related to the ATP level or pH as indicated by the changes in fluorescence intensity of 1-anilinonaphthalene-8-sulphonic acid (ANS) binding to RCA. These results suggest that the reversible association of RCA with the TM is ATP and pH (or DeltapH) dependent; it might be involved in the RCA activation of Rubisco, in addition to the previously discovered role in the protection and regulation of photosynthesis under heat stress.

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Figures

Fig. 1.
Fig. 1.
BN-PAGE and western blot analysis of the TM–RCA from rice leaves. (A) BN-PAGE, (B) second dimension electrophoresis, (C) western blot analysis.
Fig. 2.
Fig. 2.
Changes in the amount of Rubisco, total RCA, and TM–RCA in rice leaves at different leaf ages (A) and leaf positions (B). 1w, 2w, and 4w represent 1-, 2-, and 4-week-old flag leaves, respectively, while 1st, 2nd, and 3rd represent the first (flag, 4-week-old) leaf, second leaf (∼6-week-old), and third leaf (∼8-week-old) (counted from the top of the plant), respectively. RCA amounts are all expressed on a leaf area basis. Each value is the mean of more than three independent experiments with the SE expressed as a vertical bar. The amount of total RCA (∼2.93 μg cm−2) and TM–RCA (∼0.58 μg cm−2) in 4-week-old flag leaves in the day was defined as 100%, respectively.
Fig. 3.
Fig. 3.
Changes in the amount of Rubisco, total RCA, and TM–RCA of rice leaves collected at different times of the day (A), treated with high CO2 and low light (B), and in Δlut (C). Day and Night represent the rice leaves collected in the daytime and at night. LH represents the rice leaves treated with high CO2 and low light. Four-week-old flag leaves at the filling stage were used in this figure.
Fig. 4.
Fig. 4.
The intensity of ms-DLE of rice leaves at different leaf ages (A), leaf positions (B), and treated with high CO2 and low light (C). ms-DLE was measured as described in the Materials and methods, and measurements were made at the heading and filling stages. 1w, 2w, 4w, 1st, 2nd, 3rd, and LH are as defined in the legends of Figs 2 and 3.
Fig. 5.
Fig. 5.
Correlation analysis of the TM–RCA/total RCA ratio to photosynthetic parameters (A), Rubisco activity (B), and Chl fluorescence (C). Correlation analysis of the data in Tables 1–3, and Fig.4 was performed using the software SPSS 10.0 (SPSS Inc., USA). R: Pearson correlation coefficients (two tailed).
Fig. 6.
Fig. 6.
The binding of RCA to the TM during activation of Rubisco from different species by rice RCA. A thylakoid sample containing 1–2 μg of Chl was loaded in each line. (A) RCA and TMs from rice leaves were incubated with activated (–RuBP) and deactivated (+RuBP) Rubisco from rice leaves in an assay system containing 1 mM ATP. (B) RCA and TMs from rice were incubated with deactivated Rubisco (+RuBP) from non-Solanaceae plants (rice and spinach) and Solanaceae plants (tobacco and chili pepper). R, S, T, and C represent Rubisco from rice, spinach, tobacco, and chili pepper, respectively.
Fig. 7.
Fig. 7.
Effects of ATP level or pH on the amount of RCA on the TM in vitro. RCA and TMs were incubated with Rubisco (+RuBP) in the assay at different ATP levels (A) and different pH values (B) at 25 °C for 20 min. TMs from fresh rice leaves incubated at different ATP levels (C) or different pH values (D) at 25 °C for 20 min. The amount of TM–RCA was detected by western blot.
Fig. 8.
Fig. 8.
Effects of ATP on the fluorescence of ANS binding to RCA during Rubisco activation. The mixture of Rubisco and RCA in the presence of 2 mM (A) and 25 μM RuBP (B) was incubated with 0, 1, and 4 mM ATP at room temperature for 30 min, then ANS was added and the fluorescence intensity was measured after 15 min in the dark according to the method described in the Materials and methods. The numbers in the figure indicate the ATP concentration (mM).
Fig. 9.
Fig. 9.
Hypothetical model of Rubisco activation by reversible association of RCA with the TM. In the chloroplast stroma, inactive Rubisco (due to binding inhibition or another reason) is surrounded by RCA in a ring structure (RCAn, n=16 or n=8). ATP hydrolysis in the RCA–Rubisco supercomplex releases Pi and causes conformational changes in the Rubisco and RCA ring structure, resulting in activation of inactive Rubisco. Then some of the dissociated RCAs from the complex bind to the TM. Subsequently, the TM–RCA complex leaves the TM at higher ATP concentrations and alkaline pH, and forms the ring structure again on Rubisco (based on the work of Portis, 2003).
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References

    1. Arnon DJ. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiology. 1949;24:1–15. - PMC - PubMed
    1. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry. 1976;72:248–254. - PubMed
    1. Brooks A, Portis AR, Jr, Sharkey T. Effects of irradiance and methyl viologen treatment on ATP, ADP, and activation of ribulose bisphosphate carboxylase in spinach leaves. Plant Physiology. 1988;88:850–853. - PMC - PubMed
    1. Campbell WJ, Ogren WL. A novel role for light in the activation of ribulosebisphosphate carboxylase/oxygenase. Plant Physiology. 1990a;92:110–115. - PMC - PubMed
    1. Campbell WJ, Ogren WL. Electron transport through photosystem I stimulates light activation of ribulose bisphosphate carboxylase/oxygenase (Rubisco) by Rubisco activase. Plant Physiology. 1990b;94:479–484. - PMC - PubMed

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