Enhanced low oxygen survival in Arabidopsis through increased metabolic flux in the fermentative pathway

Abstract

We manipulated the enzyme activity levels of the alcohol fermentation pathway, pyruvate decarboxylase (PDC), and alcohol dehydrogenase (ADH) in Arabidopsis using sense and antisense overexpression of the corresponding genes (PDC1, PDC2, and ADH1). Transgenic plants were analyzed for levels of fermentation and evaluated for changes in hypoxic survival. Overexpression of either Arabidopsis PDC1 or PDC2 resulted in improved plant survival. In contrast, overexpression of Arabidopsis ADH1 had no effect on flooding survival. These results support the role of PDC as the control step in ethanol fermentation. Although ADH1 null mutants had decreased hypoxic survival, attempts to reduce the level of PDC activity enough to see an effect on plant survival met with limited success. The combination of flooding survival data and metabolite analysis allows identification of critical metabolic flux points. This information can be used to design transgenic strategies to improve hypoxic tolerance in plants.

Figure 1.

Under low oxygen stress conditions, plants activate three main fermentation pathways that use pyruvate as starting substrate.

Figure 2.

Activities of PDC, ADH, and LDH in roots and shoots of wild-type, transgenic, and null mutant Arabidopsis plants. Plants were grown for 4 weeks and then treated under aerobic (white column) or hypoxic (hatched column) conditions for 24 h as described (see “Materials and Methods”). Results shown are the mean of three replicates with ses from one representative experiment. A, PDC activity in roots. B, PDC activity in shoots. C, ADH activity in roots. D, ADH activity in shoots. E, LDH activity in roots. F, LDH activity in shoots.

Figure 3.

Effect of PDC and ADH manipulation on metabolic products of ethanol fermentation under hypoxic conditions. Plants were grown for 4 weeks and then hypoxically induced for 24 h. The levels of pyruvate, acetaldehyde, and ethanol were then determined (see “Materials and Methods”). Results shown are the mean of three replicates with ses from one representative experiment. Metabolite levels are presented for pyruvate in roots (A) and shoots (B), acetaldehyde content in the Murashige and Skoog growth medium (C), and ethanol in the medium (D), roots (E), and shoots (F). Values have been normalized against C24 wild type.

Figure 4.

Effect of PDC and ADH manipulation on other fermentative products under hypoxic conditions. Plants were grown for 4 weeks and hypoxically induced for 24 h, and then the levels of lactate and Ala were determined (see “Materials and Methods”). Results shown are the mean of three replicates with ses from one representative experiment. Metabolite levels are presented for lactate in roots (A), shoots (B), and Murashige and Skoog growth medium (C); and Ala in the medium (D), roots (E), and shoots (F). Values have been normalized against the C24 wild type.

Figure 5.

Survival assay results of Arabidopsis seedlings subjected to a low oxygen treatment (0.1% [v/v] O₂) with or without hypoxic (5% [v/v] O₂) pretreatment. Seedlings were exposed to two treatments: low oxygen (0.1% [v/v] O₂) conditions for 24 h without HPT (24H NHPT) or low oxygen for 48 h with a 24-h 5% (v/v) O₂ HPT (48H HPT). After these treatments, seedlings were transferred to vertical plates for a 2-week recovery period under normal growth chamber conditions. A representative plate for each environment was selected. Column 1, wild type (C24); column 2, PDC overexpressor (Ox-PDC1); and column 3, adh1^– mutant. The treatments are in row 1, 24-h NHPT; in row 2, 48-h NHPT; and in row 3, 48-h HPT.