Overexpression of Rubisco subunits with RAF1 increases Rubisco content in maize


Rubisco catalyses a rate-limiting step in photosynthesis and has long been a target for improvement due to its slow turnover rate. An alternative to modifying catalytic properties of Rubisco is to increase its abundance within C4 plant chloroplasts, which might increase activity and confer a higher carbon assimilation rate. Here, we overexpress the Rubisco large (LS) and small (SS) subunits with the Rubisco assembly chaperone RUBISCO ASSEMBLY FACTOR 1 (RAF1). While overexpression of LS and/or SS had no discernable impact on Rubisco content, addition of RAF1 overexpression resulted in a >30% increase in Rubisco content. Gas exchange showed a 15% increase in CO2 assimilation (ASAT) in UBI-LSSS-RAF1 transgenic plants, which correlated with increased fresh weight and in vitro Vcmax calculations. The divergence of Rubisco content and assimilation could be accounted for by the Rubisco activation state, which decreased up to 23%, suggesting that Rubisco activase may be limiting Vcmax, and impinging on the realization of photosynthetic potential from increased Rubisco content.

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Fig. 1: Maize transformation constructs and analysis of Rubisco content, cell type expression and assembly status.
Fig. 2: Rubisco activity and activation state, and relationship to Rubisco content.
Fig. 3: Photosynthetic performance of maize lines.
Fig. 4: Growth analysis.
Fig. 5: Relationships between in vitro and in vivo C4 and C3 cycle photosynthetic parameters.

Data availability

The data generated and analysed during this study are available from the corresponding author on reasonable request. Raw data would include photosynthesis and enzyme activity analyses.


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We acknowledge T. Clemente and S. Sato (University of Nebraska–Lincoln) for assembling the final transformation constructs, performing maize transformations, and providing seed from T0 lines. T. Nelson is acknowledged for sharing ME antibody. This research was supported by the Agriculture and Food Research Initiative from the National Institute of Food and Agriculture, US Department of Agriculture, under award number 2016-67013-24464. Travel to the Australian National University was supported by the Mario Einaudi Center for International Studies, International Research Travel Grant at Cornell University. We thank S. Long (University of Illinois) for helpful discussions and manuscript suggestions. R.E.S. is funded by the ARC Centre of Excellence for Translational Photosynthesis (CE140100015) and ARC DECRA (DE13010760).

Author information

C.S. participated in all experiments and drafted the manuscript. R.S. participated in experiments shown in Figs. 1, 2, 3, 5 and Table 1. F.A.B. participated in experiments shown in Fig. 3 and Table 1. J.K. participated in some experiments presented in Fig. 5 and Table 1. V.B. participated in experiments shown in Supplementary Figs. 3 and 4. D.S. was responsible for project management, finalization of data analysis and manuscript preparation.

Correspondence to David B. Stern.

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