Effects of oxygen on nodule physiology and expression of nodulins in alfalfa

Abstract

Early nodulin 2 (ENOD2) transcripts and protein are specifically found in the inner cortex of legume nodules, a location that coincides with the site of a barrier to O2 diffusion. The extracellular glycoprotein that binds the monoclonal antibody MAC236 has also been localized to this site. Thus, it has been proposed that these proteins function in the regulation of nodule permeability to O2 diffusion. It would then be expected that the levels of ENOD2 mRNA/protein and MAC236 antigen would differ in nodules with different permeabilities to O2. We examined the expression of ENOD2 and other nodule-expressed genes in Rhizobium meliloti-induced alfalfa nodules grown under 8, 20, or 50% O2. Although there was a change in the amount of MAC236 glycoprotein, the levels of ENOD2 mRNA and protein did not differ significantly among nodules grown at the different [O2], suggesting that neither ENOD2 transcription nor synthesis is involved in the long-term regulation of nodule permeability. Moreover, although nodules from all treatments reduced their permeability to O2 as the partial pressure of O2 (pO2) was increased to 100%, the levels of extractable ENOD2 and MAC236 proteins did not differ from those measured at the growth pO2, further suggesting that if these proteins are involved in a short-term regulation of the diffusion barrier, they must be involved in a way that does not require increased transcription or protein synthesis.

Figure 1

Nitrogenase activities (A) and respiration rates (B) of nodulated roots of alfalfa exposed to gradual increases in pO₂ in a balance of Ar. Increases in pO₂ were from initial growth pO₂ of 8% (•), 20% (▪), and 50% (▴). Each point represents the mean of four replicates ± representative ses. Absolute initial values for TNA and respiration rates are shown in Table I.

Figure 2

RNA blot of total RNA from alfalfa nodules grown under 8, 20, or 50% O₂. A, Blot probed for MsENOD2 and Msc27 mRNAs. B, Blots probed for MsENOD40, MsLb3, and R. meliloti nifHDK mRNAs. Five micrograms of total nodule RNA was loaded per lane.

Figure 3

Immunological detection of different proteins on western blots. A, Leghemoglobin. Blot probed with anti-leghemoglobin antiserum that was previously exposed to alfalfa root proteins to remove cross-reacting antibodies. The leghemoglobin band is indicated by the arrow at 15 kD. Twenty micrograms of total protein was loaded per lane. B, ENOD2. Extracts from nodules grown at 8, 20, and 50% O₂ were subjected to electrophoresis on a 7.5% acrylamide gel and blotted onto nitrocellulose. The blot was probed with anti-ENOD2 antibody at 1 μg/mL. The major ENOD2 protein band is indicated by the arrow. C, MAC236 glycoprotein. Blot probed with MAC236 at 1:100 and monoclonal anti-ribosomal protein P0 at 1:200 dilution. The MAC236 glycoprotein is indicated by the black arrow, and the P0 protein (which served as a loading control) is indicated by the white arrow.

Figure 4

Quantification of MAC236 glycoprotein and ENOD2. A, Relative quantity of ENOD2 in nodules. B, Relative quantity of MAC236 glycoprotein in nodules. Bars represent the mean of three to four assays on each of six individual nodules. Error bars are sd for the six nodules.

Figure 5

Immunolabeling of ENOD2 protein in alfalfa nodules grown under different pO₂s. Following silver enhancement, immunogold labeling appeared as an opaque black deposit on intercellular spaces in the nodule parenchyma (arrowheads). The regions of the nodules depicted were comparable and are all in upper zone III. C, Outer cortex; E, nodule endodermis; Inf, infected cells in zone III. All views are magnified ×315. Bar, 50 μm. A, Labeled section of a nodule grown in 8% O₂. B, Labeled section of a nodule grown in 20% O₂. C, Labeled section of a nodule grown in 50% O₂.

Figure 6

Periodic acid Schiff's staining of paraffin-embedded alfalfa nodules that had been grown under different [O₂]. Zones I, II, and the periphery of zone III are shown. A, Longitudinal section of a nodule grown in 8% O₂. The arrow points to small starch grains in the nodule cortex. B, Longitudinal section of a nodule grown in 20% O₂. The arrow points to starch grains in the boundary layer. C, Longitudinal section of a nodule grown in 50% O₂.