Effects of Hypoxia on 13NH4+ Fluxes in Rice Roots1 : Kinetics and Compartmental Analysis

Techniques of compartmental (efflux) and kinetic influx analyses with the radiotracer 13NH4+ were used to examine the adaptation to hypoxia (15, 35, and 50% O2 saturation) of root N uptake and metabolism in 3-week-old hydroponically grown rice (Oryza sativa L., cv IR72) seedlings. A time-dependence study of NH4+ influx into rice roots after onset of hypoxia (15% O2) revealed an initial increase in the first 1 to 2.5 h after treatment imposition, followed by a decline to less than 50% of influx in control plants by 4 d. Efflux analyses conducted 0, 1, 3, and 5 d after the treatment confirmed this adaptation pattern of NH4+ uptake. Half-lives for NH4+ exchange with subcellular compartments, cytoplasmic NH4+ concentrations, and efflux (as percentage of influx) were unaffected by hypoxia. However, significant differences were observed in the relative amounts of N allocated to NH4+ assimilation and the vacuole versus translocation to the shoot. Kinetic experiments conducted at 100, 50, 35, and 15% O2 saturation showed no significant change in the Km value for NH4+ uptake with varying O2 supply. However, Vmax was 42% higher than controls at 50% O2 saturation, unchanged at 35%, and 10% lower than controls at 15% O2. The significance of these flux adaptations is discussed.

Selective Perturbation of Apical Membrane Traffic by Expression of Influenza M2, an Acid-activated Ion Channel, in Polarized Madin–Darby Canine Kidney Cells

The function of acidification along the endocytic pathway is not well understood, in part because the perturbants used to modify compartmental pH have global effects and in some cases alter cytoplasmic pH. We have used a new approach to study the effect of pH perturbation on postendocytic traffic in polarized Madin–Darby canine kidney (MDCK) cells. Influenza M2 is a small membrane protein that functions as an acid-activated ion channel and can elevate the pH of the trans-Golgi network and endosomes. We used recombinant adenoviruses to express the M2 protein of influenza virus in polarized MDCK cells stably transfected with the polymeric immunoglobulin (Ig) receptor. Using indirect immunofluorescence and immunoelectron microscopy, M2 was found to be concentrated at the apical plasma membrane and in subapical vesicles; intracellular M2 colocalized partly with internalized IgA in apical recycling endosomes as well as with the trans-Golgi network marker TGN-38. Expression of M2 slowed the rate of IgA transcytosis across polarized MDCK monolayers. The delay in transport occurred after IgA reached the apical recycling endosome, consistent with the localization of intracellular M2. Apical recycling of IgA was also slowed in the presence of M2, whereas basolateral recycling of transferrin and degradation of IgA were unaffected. By contrast, ammonium chloride affected both apical IgA and basolateral transferrin release. Together, our data suggest that M2 expression selectively perturbs acidification in compartments involved in apical delivery without disrupting other postendocytic transport steps.

Nitrate-Ammonium Synergism in Rice. A Subcellular Flux Analysis1

Many reports have shown that plant growth and yield is superior on mixtures of NO3− and NH4+ compared with provision of either N source alone. Despite its clear practical importance, the nature of this N-source synergism at the cellular level is poorly understood. In the present study we have used the technique of compartmental analysis by efflux and the radiotracer 13N to measure cellular turnover kinetics, patterns of flux partitioning, and cytosolic pool sizes of both NO3− and NH4+ in seedling roots of rice (Oryza sativa L. cv IR72), supplied simultaneously with the two N sources. We show that plasma membrane fluxes for NH4+, cytosolic NH4+ accumulation, and NH4+ metabolism are enhanced by the presence of NO3−, whereas NO3− fluxes, accumulation, and metabolism are strongly repressed by NH4+. However, net N acquisition and N translocation to the shoot with dual N-source provision are substantially larger than when NO3− or NH4+ is provided alone at identical N concentrations.

Altered Zn Compartmentation in the Root Symplasm and Stimulated Zn Absorption into the Leaf as Mechanisms Involved in Zn Hyperaccumulation in Thlaspi caerulescens

We investigated Zn compartmentation in the root, Zn transport into the xylem, and Zn absorption into leaf cells in Thlaspi caerulescens, a Zn-hyperaccumulator species, and compared them with those of a related nonaccumulator species, Thlaspi arvense. 65Zn-compartmental analysis conducted with roots of the two species indicated that a significant fraction of symplasmic Zn was stored in the root vacuole of T. arvense, and presumably became unavailable for loading into the xylem and subsequent translocation to the shoot. In T. caerulescens, however, a smaller fraction of the absorbed Zn was stored in the root vacuole and was readily transported back into the cytoplasm. We conclude that in T. caerulescens, Zn absorbed by roots is readily available for loading into the xylem. This is supported by analysis of xylem exudate collected from detopped Thlaspi species seedlings. When seedlings of the two species were grown on either low (1 μm) or high (50 μm) Zn, xylem sap of T. caerulescens contained approximately 5-fold more Zn than that of T. arvense. This increase was not correlated with a stimulated production of any particular organic or amino acid. The capacity of Thlaspi species cells to absorb 65Zn was studied in leaf sections and leaf protoplasts. At low external Zn levels (10 and 100 μm), there was no difference in leaf Zn uptake between the two Thlaspi species. However, at 1 mm Zn2+, 2.2-fold more Zn accumulated in leaf sections of T. caerulescens. These findings indicate that altered tonoplast Zn transport in root cells and stimulated Zn uptake in leaf cells play a role in the dramatic Zn hyperaccumulation expressed in T. caerulescens.