Rapid repression of maize invertases by low oxygen. Invertase/sucrose synthase balance, sugar signaling potential, and seedling survival

Abstract

We show here that invertase gene expression and the invertase-sucrose (Suc) synthase ratio decrease abruptly in response to low oxygen in maize root tips. In addition to aiding in the conservation of carbon and possibly ATP, this response has the potential to directly affect sugar signaling relative to carbon flux. Experiments were motivated by the potential for a reduced invertase/Suc synthase balance to alter the impact of respiratory and/or membrane carbon flux on sugar signaling. Maize (Zea mays L.) seedlings with 5-cm primary roots were exposed to anoxic (0% [v/v] O2), hypoxic (3% [v/v] O2), and aerobic conditions. Rapid repression of the Ivr1 and Ivr2 maize invertases by low oxygen was evident in root tips within 3 h at both the transcript and activity levels. The speed and extent of this response increased with the degree of oxygen deprivation and differed with genotypes. This decrease in expression also contrasted markedly to that of other genes for respiratory Suc metabolism, such as Suc synthases, which typically increased or remained constant. Although previous work showed that the contrasting effects of sugars on Suc synthase genes were reflected in their regulation by hypoxia and anoxia, the same was not observed for the differentially sugar-responsive invertases. Theoretically advantageous reductions in the invertase/Suc synthase balance thus resulted. However, where this response was extreme (an Oh43 inbred), total sucrolytic capacity dropped below an apparent minimum and root tip viability was reduced. Paradoxically, only Oh43 seedlings showed survival levels >80% (versus <50%) after extreme, long-term stress, suggesting a possible advantage for multiple means of reducing sink activity. Overall, our results demonstrate a rapid change in the regulation and balance of invertases and Suc synthases that could have an immediate impact on limiting the extent of Suc cleavage and reducing the extent of concomitant, hexose-based sugar signaling under low oxygen.

Figure 1

Time course of the effects of low oxygen on invertase activity from root tips of intact maize seedlings under 0% (v/v) O₂ (anoxic), 3% (v/v) O₂ (hypoxic), or 21% (v/v) O₂ (aerobic) conditions for a hybrid (NK508) and two inbreds (W22 and Oh43). Soluble invertase activities are presented and insoluble activity was approximately 10% of these values (not shown). Treatments were initiated after 5 to 7 d of germination, when roots had reached approximately 5 cm. One-centimeter tips of primary roots (approximately 90 total, for approximately 0.6 g) were excised at each time point. Data are means ± se of three separate experiments, and values are plotted as percentage of maximum activity (determined as μm Glc g⁻¹ fresh weight h⁻¹). At 21% (v/v) O₂, soluble invertase activities for NK508, W22, and Oh43 were 282 (±8), 351 (±7), and 213 (±4) g⁻¹ fresh weight h⁻¹, respectively. Results were similar if expressed per unit of protein.

Figure 2

Time course of changes in Ivr1 mRNA levels from root tips of intact maize seedlings under 0% (v/v) O₂ (anoxic), 3% (v/v) O₂ (hypoxic), or 21% (v/v) O₂ (aerobic) conditions for a hybrid (NK508) and two inbreds (W22 and Oh43). Treatments and samples were as noted for Figure 1, and were from the same experiments. RNA gel blots were visualized by autoradiography, and the abundance of ³²P-mRNA was quantified with a phosphor imager (Molecular Dynamics). Ten micrograms of total RNA was loaded in each lane and uniformity was verified by visualization of rRNA bands (not shown) and by the constancy of Ivr1 mRNA levels under 21% (v/v) O₂ (bottom panels in each figure). For each experiment, data from the three oxygen treatments were compared in adjacent lanes on the same blot. Data represent the means ± se of three separate experiments. Identical blots were probed with Ivr2 in Figure 3.

Figure 3

Time course of changes in Ivr2 mRNA levels from root tips of intact maize seedlings under 0% (v/v) O₂ (anoxic), 3% (v/v) O₂ (hypoxic), or 21% (v/v) O₂ (aerobic) conditions for a hybrid (NK508) and two inbreds (W22, and Oh43). Blots were identical to those probed with Ivr1 in Figure 2, except that mRNA was hybridized with a cDNA for Ivr2. Visualization and quantification were also as in Figure 2. Data represent the means ± se of three separate experiments.

Figure 4

Time course of the effects of low oxygen on Suc synthase activity from root tips of intact maize seedlings under 0% (v/v) O₂ (anoxic), 3% (v/v) O₂ (hypoxic), or 21% (v/v) O₂ (aerobic) conditions for the Oh43 inbred, with data from the W22 inbred and an isogenic W22-sh1 mutant (null for the Sh1 gene) shown for comparison (the latter from Zeng et al., 1998). Treatments and samples were as in Figure 1.

Figure 5

Estimated capacity for Suc flux through Suc synthase versus invertase during the progression of low-oxygen events under anoxia. The relative invertase (IVR)/Suc synthase (SuSyn) balance and the estimated capacity for Suc cleavage (invertase + Suc synthase activities) were calculated from activity data not shown plus those presented in Figure 1 and those in Zeng et al. (1998). Data represent ranges.

Figure 6

Percentage seedling survival after 7 d (black bars) and root-tip survival after 12 and 24 h (black and white bars, respectively) of whole-seedling exposure to 0% (v/v) O₂ (anoxia) imposed as N₂ gas flow in darkness for hybrid NK508, and inbreds W22 and Oh43. Treatment was initiated 5 to 7 d after germination, when primary roots had reached approximately 5 cm. Capacity for regrowth was tested by replacing the N₂ flow with ambient air (21% [v/v] O₂) for an additional 6 d. Viability was appraised by the formation of new adventitious roots in addition to the maintenance of a turgid, growing shoot. Data represent means ± se of three separate experiments. Twenty-four hours after excision (not shown), less than 50% of NK508 root tips survived and all primary root tips from Oh43 had died.