Evidence for the critical role of sucrose synthase for anoxic tolerance of maize roots using a double mutant

Abstract

The induction of the sucrose synthase (SuSy) gene (SuSy) by low O2, low temperature, and limiting carbohydrate supply suggested a role in carbohydrate metabolism under stress conditions. The isolation of a maize (Zea mays L.) line mutant for the two known SuSy genes but functionally normal showed that SuSy activity might not be required for aerobic growth and allowed the possibility of investigating its importance during anaerobic stress. As assessed by root elongation after return to air, hypoxic pretreatment improved anoxic tolerance, in correlation with the number of SuSy genes and the level of SuSy expression. Furthermore, root death in double-mutant seedlings during anoxic incubation could be attributed to the impaired utilization of sucrose (Suc). Collectively, these data provide unequivocal evidence that Suc is the principal C source and that SuSy is the main enzyme active in Suc breakdown in roots of maize seedlings deprived of O2. In this situation, SuSy plays a critical role in anoxic tolerance.

Figure 1

Effect of HPT on roots from inbred W22 line of the Sh1Sus1 homozygous wild-type (1) and the sh1sus1 double-mutant (2) genotype. Intact seedlings were placed overnight on floats over nutrient medium bubbled with 3% O₂ in N₂ (HPT) or with 50% O₂ in N₂ (NHPT). After a subsequent 24-h anoxic treatment, the seedlings were removed and placed between two layers of moist filter paper for 48 h.

Figure 2

SuSy protein levels in roots from the Sh1Sus1 homozygous wild type, the sh1Sus1 single mutant, and the sh1sus1 double mutant. Root-tip extracts containing 20 μg of protein were analyzed by SDS-PAGE. Detection of SuSy protein on immunoblots was carried out using a polyclonal antiserum that detected both SuSy subunits. The molecular mass (92 kD) of the SS1 subunit is indicated to the right of the digitized image of the original gels.

Figure 3

Proteins synthesized in line W22 roots of the Sh1Sus1 (A) and the sh1sus1 (B) genotype during HPT. Proteins synthesized during HPT were analyzed by two-dimensional IEF/SDS-PAGE. The numbers to the left indicate the position of the molecular mass markers (in kilodaltons). The arrows indicate the position of SuSy.

Figure 4

Native gel analysis of HK, FK, ADH, and LDH in root extracts from NHPT and HPT double mutants. Extracts containing 20 μg of protein were analyzed on nondenaturing polyacrylamide gels and stained for activity, as described in Methods. The figure is a digitized image of the original gels.

Figure 5

Time course of ethanol produced by excised root tips from the Sh1Sus1 homozygous wild type (A) and the sh1sus1 double mutant (B) during anoxic incubation in nutrient medium supplemented with 100 mm Glc. Open symbols indicate root tips from HPT seedlings; closed symbols indicate root tips from NHPT seedlings. Each point is the mean ± sd of three independent determinations.

Figure 6

Changes in Fru (A) and Suc (B) content in excised root tips from NHPT seedlings of the Sh1Sus1 homozygous wild-type (•) and the sh1sus1 double-mutant (▪) genotype during anoxic incubation in nutrient medium supplemented with 100 mm Glc. Each point is the mean ± sd of three independent determinations.