In Vitro Analysis of Metabolite Transport Proteins

Roell, Marc-Sven; Kuhnert, Franziska; Zamani-Nour, Shirin; Weber, Andreas P.M.

doi:10.1007/978-1-4939-7225-8_6

Cited by 2 publications

(2 citation statements)

References 12 publications

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“…Ultimately, the isolated membrane was re-suspended in 50 mM HEPES (KOH, pH 271 7.5), 5 mM EDTA, 2 mM DTT together with protease inhibitors (for detailed 272 procedure see Furbank et al, 2001 andRoell et al, 2017). Finally, the protein 273 concentration was measured utilizing the Pierce BCA Protein Assay Kit 274 (ThermoFisher Scientific, Germany) following the manufacturer's instructions.…”

Section: Total Leaf Membrane Protein Isolation 263mentioning

confidence: 99%

“…Finally, the uptake of radio-labeled substrate was 297 measured as counts per minute (CPM) by scintillation counting. To correct for 298 background and false positives, the entire experiment was repeated using 299 proteoliposomes without pre-loading of the substrate of interest (for detailed 300 procedure see Roell et al, 2017). The uptake data were further assessed relative to 301 both internal standards and total protein content (mg) in each sample.…”

Section: Radioactive Labeled [ 14 C]-malate Uptake Measurementmentioning

confidence: 99%

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Overexpression of the chloroplastic 2-oxoglutarate/malate transporter in rice disturbs carbon and nitrogen homeostasis

Zamani-Nour

Walker

et al. 2020

Preprint

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36The chloroplastic oxaloacetate/malate transporter (OMT1 or DiT1) takes part in the 37 malate valve that protects chloroplasts from excessive redox poise through export of 38 malate and import of oxaloacetate (OAA). Together with the glutamate/malate 39 transporter (DCT1 or DiT2), it connects carbon with nitrogen assimilation, by 40 providing α-ketoglutarate for the GS/GOGAT reaction and exporting glutamate to the 41 cytoplasm. OMT1 further plays a prominent role in C 4 photosynthesis. OAA resulting 42 from PEP-carboxylation is imported into the chloroplast, reduced to malate by 43 plastidic NADP-MDH, and then exported for transport to bundle sheath cells. Both 44 transport steps are catalyzed by OMT1, at the rate of net carbon assimilation. 45 Therefore, to engineer C 4 photosynthesis into C 3 crops, OMT1 must be expressed in 46 high amounts on top of core C 4 metabolic enzymes. We report here high-level 47 expression of ZmOMT1 from maize in rice (Oryza sativa ssp. indica IR64). Increased 48 activity of the transporter in transgenic rice was confirmed by reconstitution of 49 transporter activity into proteoliposomes. Unexpectedly, over-expression of ZmOMT1 50 in rice negatively affected growth, CO 2 assimilation rate, total free amino acid 51 contents, TCA cycle metabolites, as well as sucrose and starch contents. 52Accumulation of high amounts of aspartate and the impaired growth phenotype of 53 OMT1 rice lines could be suppressed by simultaneous over-expression of ZmDiT2. 54Implications for engineering C 4 -rice are discussed. 55 56 Keywords: C 4 -rice, oxaloacetate/malate transporter, glutamate/malate transporter, 57 photosynthesis, carbon and nitrogen assimilation, gas exchange 58 59 60 61 62 63 64 65 Population growth, climate change, and lack of arable land are placing greater 66 dependence on crop yield improvement. However, crop demand is already outpacing 67 the yield gains achieved by conventional breeding and hence, step-wise changes in 68 crop yield are needed (Kromdijk and Long, 2016). Rice (Oryza sativa L.) is a C 3 plant 69 that belongs to the Gramineae family and is one of the most important staple crops in 70 the world. Its highest consumption is in Asia (Muthayya et al., 2014) where 60% of 71 the world population exists (Bai et al., 2018), with the highest and lowest rates of 72 poverty and income, respectively (FAO, 2017). Therefore, boosting rice yield and 73 performance is an important goal for improving the quality of life for a large share of 74 the global population. Engineering the C 3 crop rice to perform C 4 photosynthesis 75 would greatly improve rice productivity by up to 50% per year (Wang et al., 2016), 76 through maximizing the conversion of the captured solar energy into chemical energy 77 and biomass (Hibberd et al., 2008). 78 C 3 photosynthesis performs both initial carbon fixation and Calvin-Benson cycle 79 reactions in the mesophyll (M). In C 4 photosynthesis, initial carbon fixation and the 80 Calvin-Benson cycle are carried out separately in M and one or more layers of 81 ...

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Section: Total Leaf Membrane Protein Isolation 263mentioning

confidence: 99%

Section: Radioactive Labeled [ 14 C]-malate Uptake Measurementmentioning

confidence: 99%

Overexpression of the chloroplastic 2-oxoglutarate/malate transporter in rice disturbs carbon and nitrogen homeostasis

Zamani-Nour

Walker

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

Overexpression of the chloroplastic 2-oxoglutarate/malate transporter disturbs carbon and nitrogen homeostasis in rice

Zamani-Nour

Lin

Walker

et al. 2020

Journal of Experimental Botany

Self Cite

View full text Add to dashboard Cite

The chloroplastic 2-oxaloacetate/malate transporter (OMT1 or DiT1) takes part in the malate valve that protects chloroplasts from excessive redox poise through export of malate and import of oxaloacetate (OAA). Together with the glutamate/malate transporter (DCT1 or DiT2), it connects carbon with nitrogen assimilation, by providing 2-oxoglutarate for the GS/GOGAT reaction and exporting glutamate to the cytoplasm. OMT1 further plays a prominent role in C4 photosynthesis: OAA resulting from PEP-carboxylation is imported into the chloroplast, reduced to malate by plastidic NADP-MDH, and then exported for transport to bundle sheath cells. Both transport steps are catalyzed by OMT1, at the rate of net carbon assimilation. Therefore, to engineer C4 photosynthesis into C3 crops, OMT1 must be expressed in high amounts on top of core C4 metabolic enzymes. We report here high-level expression of ZmOMT1 from maize in rice (Oryza sativa ssp. indica IR64). Increased activity of the transporter in transgenic rice was confirmed by reconstitution of transporter activity into proteoliposomes. Unexpectedly, over-expression of ZmOMT1 in rice negatively affected growth, CO2 assimilation rate, total free amino acid contents, TCA cycle metabolites, as well as sucrose and starch contents. Accumulation of high amounts of aspartate and the impaired growth phenotype of OMT1 rice lines could be suppressed by simultaneous over-expression of ZmDiT2. Implications for engineering C4-rice are discussed.

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In Vitro Analysis of Metabolite Transport Proteins

Cited by 2 publications

References 12 publications

Overexpression of the chloroplastic 2-oxoglutarate/malate transporter in rice disturbs carbon and nitrogen homeostasis

Overexpression of the chloroplastic 2-oxoglutarate/malate transporter in rice disturbs carbon and nitrogen homeostasis

Overexpression of the chloroplastic 2-oxoglutarate/malate transporter disturbs carbon and nitrogen homeostasis in rice

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