Computer-calculated difference spectra were used to demonstrate the changes in Lb spectra under different conditions. Some conditions that increased absorbance in the 626 nm region (indicating Lb3+ accumulation) were root-fed ascorbate and dehydroascorbate, plant exposure to darkness, and nodule water immersion.

Reduction of ferric leghemoglobin to ferrous leghemoglobin in soybean nodules (Glycine max IL.] Meff. cv Woodworth) was studied using a spectrophotometer equipped with an in-cell space diffuse reflectance accessory. Nodule slices prepared and scanned under nitrogen gas showed a ferrous leghemoglobin absorption spectrum. Nodule slices equilibrated with 100% 02 or air exhibited two absorption bands characteristic of oxygenated leghemoglobin. The addition of CO shifted those bands to CO leghemoglobin absorption bands. Potassium ferricyanide was not effective in oxidizing ferrous to feffic leghemoglobin in nodule slices. However, ferric leghemoglobin was formed by treating the nodule slices with hydroxylamine, and this was confirmed by complexing the ferric leghemoglobin to acetate, fluoride, or nicotinic acid. The diminution of feffic leghemoglobin was monitored as a function of time, and in the presence of nicotinic acid, the conversion of ferric to ferrous leghemoglobin was monitored by the appearance of ferrous leghemoglobin nicotinate complex as a function of time. Ferric leghemoglobin reduction was also confirmed by direct transmission spectrophotometry. The evidence presented here suggests that ferrileghemoglobin reduction occurs in nodule slices.

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Response of 'Park' Kentucky bluegrass to inoculation with Klebsiella pneumoniae strain W-6 was tested under field conditions. Field inoculation did not increase nitrogenase activity measured in situ, but did increase the nitrogenase activity as measured using an excised root assay which included a 10-h incubation before the addition of acetylene. Fifteen lines composing 'Park' were grown in fritted-clay medium, inoculated with soil, and compared for nitrogenase activities using the excised roots assay. Significant differences were observed between two lines. Six lines were selected from the 15 lines, grown hydroponically, inoculated with soil, and assayed for nitrogenase activity using intact 105-day-old plants. Nitrogenase activities were immediately detectable and increased curvilinearly. Differences in nitrogenase activities among the six lines were detected within 1 h and significant differences were evident in 4 h. The six selected lines were also tested for levels of anthrone-reactive sugars in roots and root exudates of hydroponically grown plants. Significant differences were detected in levels of sugars in roots but not in root exudates. Possible correlations were sought among and within the three different experiments involving the six selected lines. With hydroponically grown plants, nitrogenase activities during the first 4 h were highly correlated with those after 24 h incubation. Correlations were found between nitrogenase activities in excised roots and soluble sugar concentrations in root tissue and root exudates.