Investigating the Electron Transfer Systems of the Endoplasmic Reticulum Membrane in Plants

Laura Wayne

Within the endoplasmic reticulum membrane there are two electron transfer systems (ETS): NADPH:cytochrome P450 reductase (ATR) transfers electrons to cytochrome P450 monooxygenases and NADH:cytochrome b5 reductase (CBR1) transfers electrons to cytochrome b5 (CYB5), which then transfers reductant to fatty acid desaturases and hydroxylases. The CYB5 ETS is required by the castor bean (Ricinus communis) fatty acid hydroxylase (RcFAH12) in order to synthesize ricinoleic acid. Ricinoleic acid is a valuable industrial feedstock but extraction of ricinoleic acid from the toxic castor beans is unattractive. Expressing the RcFAH12 in Arabidopsis has resulted in low yields of ricinoleic acid. Previously, it was shown that a mutation in CBR1 resulted in a differential decrease of ricinoleic acid and polyunsaturated fatty acids, implying that the fatty acid desaturases were outcompeting RcFAH12 for the limited reductant available. To test if reductant supply was limiting ricinoleic acid accumulation, castor and Arabidopsis CBR1 were each expressed in RcFAH12 Arabidopsis. The castor CYB5 proteins were also heterologously expressed, as well as the complete ETS. In all experiments, the endogenous Arabidopsis ETS was sufficient in supplying reductant to RcFAH12. Previous studies have shown redundancy between the CBR1 and the ATR ETS. However, the previously reported cbr1-1 mutant had lower levels of !-linolenic acid in the seeds, suggesting that this redundancy is incomplete. A complete knockout of CBR1 demonstrated that CBR1 is not redundant with ATR in pollen tube growth, seed filling, and germination. However, mutations in ATR1 and ATR2 exacerbated the male gametophyte defect of cbr1-2, suggesting that there is minimal redundancy. The atr1 and atr2 single mutants had no visual phenotype, but the double homozygotes were lethal, indicating that at least one of these two proteins is needed for essential functions. Segregation of ATR2 in the homozygous atr1 background led to a male gametophyte defect while segregation of ATR1 in the homozygous atr2 background resulted in embryo lethality. These results indicate that ATR1 and ATR2 have acquired distinct roles in male gametophyte function and embryo development. Distinguishing the roles of ATR and CBR1 will lead to a better understanding of the ETS of the ER membrane in Arabidopsis.

Investigating the Electron Transfer Systems of the Endoplasmic Reticulum Membrane in Plants

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