Over the past few years, three photorespiratory bypasses have been introduced into plants, two of which led to observable increases in photosynthesis and biomass yield. However, most of the experiments were carried out using Arabidopsis under controlled environmental conditions, and the increases were only observed under low-light and short-day conditions. In this study, we designed a new photorespiratory bypass (called GOC bypass), characterized by no reducing equivalents being produced during a complete oxidation of glycolate into CO 2 catalyzed by three rice-self-originating enzymes, i.e., glycolate oxidase, oxalate oxidase, and catalase. We successfully established this bypass in rice chloroplasts using a multi-gene assembly and transformation system. Transgenic rice plants carrying GOC bypass (GOC plants) showed significant increases in photosynthesis efficiency, biomass yield, and nitrogen content, as well as several other CO 2 -enriched phenotypes under both greenhouse and field conditions. Grain yield of GOC plants varied depending on seeding season and was increased significantly in the spring. We further demonstrated that GOC plants had significant advantages under high-light conditions and that the improvements in GOC plants resulted primarily from a photosynthetic CO 2 -concentrating effect rather than from improved energy balance. Taken together, our results reveal that engineering a newly designed chloroplastic photorespiratory bypass could increase photosynthetic efficiency and yield of rice plants grown in field conditions, particularly under high light.

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Various chloroplast transit peptides (CTP) have been used to successfully target some foreign proteins into chloroplasts, but for other proteins these same CTPs have reduced localization efficiencies or fail completely. The underlying cause of the failures remains an open question, and more effective CTPs are needed. In this study, we initially observed that two E.coli enzymes, EcTSR and EcGCL, failed to be targeted into rice chloroplasts by the commonly-used rice rbcS transit peptide (rCTP) and were subsequently degraded. Further analyses revealed that the N-terminal unfolded region of cargo proteins is critical for their localization capability, and that a length of about 20 amino acids is required to attain the maximum localization efficiency. We considered that the unfolded region may alleviate the steric hindrance produced by the cargo protein, by functioning as a spacer to which cytosolic translocators can bind. Based on this inference, an optimized CTP, named RC2, was constructed. Analyses showed that RC2 can more effectively target diverse proteins, including EcTSR and EcGCL, into rice chloroplasts. Collectively, our results provide further insight into the mechanism of CTP-mediated chloroplastic localization, and more importantly, RC2 can be widely applied in future chloroplastic metabolic engineering, particularly for crop plants.

BackgroundAlthough decreased protein expressions have been observed in NOA1 (Nitric Oxide Associated protein 1) deficient plants, the molecular mechanisms of how NOA1 regulates protein metabolism remain poorly understood. In this study, we have used a global comparative proteomic approach for both OsNOA1 suppression and overexpression transgenic lines under two different temperatures, in combination with physiological and biochemical analyses to explore the regulatory mechanisms of OsNOA1 in rice.ResultsIn OsNOA1-silenced or highly overexpressed rice, considerably different expression patterns of both chlorophyll and Rubisco as well as distinct phenotypes were observed between the growth temperatures at 22 °C and 30 °C. These observations led us to hypothesize there appears a narrow abundance threshold for OsNOA1 to function properly at lower temperatures, while higher temperatures seem to partially compensate for the changes of OsNOA1 abundance. Quantitative proteomic analyses revealed higher temperatures could restore 90% of the suppressed proteins to normal levels, whereas almost all of the remaining suppressed proteins were chloroplast ribosomal proteins. Additionally, our data showed 90% of the suppressed proteins in both types of transgenic plants at lower temperatures were located in the chloroplast, suggesting a primary effect of OsNOA1 on chloroplast proteins. Transcript analyses, along with in vitro pull-down experiments further demonstrated OsNOA1 is associated with the function of chloroplast ribosomes.ConclusionsOur results suggest OsNOA1 functions in a threshold-dependent manner for regulation of chloroplast proteins at lower temperatures, which may be mediated by interactions between OsNOA1 and chloroplast ribosomes.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1258-9) contains supplementary material, which is available to authorized users.

Location of the proteases would affect on protease stability and photorespiratory bypass pathway, while it is unsolved. Potato rbcS signal peptide was analyzed and constructed into the protease for study of their localization site. The tartronate semialdehyde reductase (EcTSR) proteins could be accurately and efficiently located in chloroplast only when this signal peptide was extended to 80 amino acids. The signal peptide would help malate synthase (CmMS) locate to the surface of chloroplast, to form granules on the outer membrane of chloroplast. The whole spectrum scanning showed that these proteins could enter chloroplast. A signal peptide named PCS1 (Peptide of self-cleavage site 1) carrying a self-cleavage site was designed, and sixteen amino acids from the blue pigment precursor protein of chloroplast positioning signal of Silene pratensis were added to the C-terminal of PCS1. Transient expression, Western blot analysis and full-spectrum scanning showed that PCS1 could locate the EcTSR to the chloroplast, after the removal of the signal peptide.