P.M. Aparicio-Tejo

Nitrogen fixation and leghemoglobin content during vegetative growth of alfalfa

Summary Fluorometric assay for leghemoglobin (LHb) was superior in selectivity and sensitivity to conventional colorimetric procedures. Concentration of LHb in alfalfa ( Medicago sativa L. cv. Aragon) nodules decreased significantly during the vegetative growth period of plants (between 4 and 20-weeks-old). This decline was not selective for LHb since the ratio LHb/total soluble protein remained constant. LHb represented about 30 % of the nodule soluble protein. Nitrogenase activity, estimated as acetylene reduction activity (ARA), fell along with LHb and total nodule soluble protein. However, ARA per unit LHb consistently declined during the period of study, and the correlation between ARA and LHb was not linear but exponential. Results indicate that LHb is not causal in the development of nitrogenase activity, suggesting that other factors than quantitative changes of total nodule LHb (e.g. those concerning the functional state of LHb) might be implicated in the declining of nitrogenase activity with ageing of nodules.

Root nodule enzymes of ammonia metabolism from Medicago sativa L. as influenced by nitrate levels

Plants of alfalfa (Medicago sativa L.) were subjected to six levels of nitrate ranging from 0 to 200 mM. Then, nodule content of soluble protein and activities of the nodule enzymes, nitrogenase (C(2)H(2) reduction activity), glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.1.14), and NADH/NAD(+)-glutamate dehydrogenases (EC 1.4.1.2) were determined. Decline of glutamate synthase activity closely paralleled that of nitrogenase. In contrast, glutamine synthetase activity reached a minimum at 20mM nitrate but began to recover at higher nitrate concentrations. Patterns of NADH-(aminating) and NAD(+)-(deaminating) glutamate dehydrogenase activities were opposed with a great increase (2- to 5-fold) in the aminating direction at highest levels of nitrate. Results indicate a main role of glutamine synthetase/glutamate synthase pathway in the assimilation of fixed N. In addition, increase of the activities of glutamine synthetase and NADH-glutamate dehydrogenase concomitant with an almost fully suppressed nitrogenase and decreased NAD(+)-glutamate dehydrogenase activities at above 20mM nitrate reflect: (a) rapid induced senescence of nodules and (b) the production of ammonia by alternative means as nitrate reduction or oxidative deamination of amino acids.

Levels of ammonia, nitrite, and nitrate in alfalfa root nodules supplied with nitrate

Summary Protein content, protease activity, and levels of NH 4 + , NO 2 - and NO 3 - were determined in the plant and bacteroid fractions of alfalfa ( Medicago sativa L. cv. Aragon) nodules supplied with NO 3 - (0—200mM). Protein of the host cell cytosol represented approximately 70 % of the total nodule protein. The effect of NO 3 - on plant cytosolic protein closely paralleled that on bacteroid protein. Protein of both nodule fractions remained constant upon exposure to NO 3 - concentrations below 20 mM and was reduced by 30% above 20 mM NO 3 - . Patterns of proteolytic activity were inversely correlated to those of the corresponding protein for both bacteroids and cytosol. The nodule level of NH 4 + was basically unaffected by increasing added NO3- whereas NO 2 - accumulated in both bacteroids and plant cytosol, although to a greater extent in the latter. Likewise, NO 3 - accumulated in the cytosol with much higher relative values than those of NO 2 - . Only trace or null amounts of NO 3 - were found in the corresponding bacteroids. Linear correlations either between bacteroidal and cytosolic NO 2 - , or between NO 2 - in both fractions and cytosolic NO 3 - levels were highly significant. The overall results indicate that the whole nodule capacity of NO 2 - reduction is not sufficient to avoid accumulation of NO 2 - , and support the view that NO 2 - plays a more important role than NH 4 + or NO 3 - itself as inhibitor of nodule activity.

Dormancy Mechanism and the Effect of Scarification in the Germination of Halimium halimifolium Seeds

Germination of Halimium halimifolium seeds was very low during the experimental period. The scarification treatment enhanced the germination significantly at all the three temperatures tested, indicating that H. halimifolium seeds are endowed with complex dormancy mechanisms. The highest temperature (30°C) showed an adverse effect which can be correlated with a secondary dormancy at embryo level. n nThe lipidic nature of H. halimifolium exotesta is probably the first factor causing dormancy in this species, acting both as a water and oxygen barrier. We can conclude that H halimifolium shows a tegumentary dormancy, which is presumably located at exotesta level.