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Respiratory nitrate reductase (NR) from Bradyrhizobium sp. (Lupinus) USDA 3045 has biochemical properties of the membrane-bound NR type. However, in the completely sequenced rhizobium genomes only genes for the periplasmic type of dissimilatory NR were found. Therefore purification and identification of the enzyme by tandem mass spectrometry (MS/MS) was under taken. MS/MS spectra representing 149 unique tryptic peptides derived from purified 137-kDa subunit matched the NCBInr-deposited NarG sequences. MS/MS sequencing of two other SDS/PAGE bands (65- and 59-kDa) identified them as derivatives of the NarH-type protein. Applying additional validation criteria, 73% of the sequence of the NarG subunit (902 aa) and 52% of NarH sequence (266 aa) was assembled (UniProt KB acc. no. P85097 and P85098). This is the first unambiguous identification of an active NarGH-like NR in rhizobia. Moreover, arguments are provided here for the existence of a functional enzyme of this type also among other rhizobial species, basing on immunoblot screening and the presence of membrane-associated NR-active electrophoretic forms.
Induction of nitrate reductase (NR) activity in coralloid roots of Cycas revoluta was observed after 8 h incubation in 0.02 M KNO3. Other plants growing near Cycas showed a higher level of NR immediately when incubated in KNO3. In contrast to NR, intact coralloid roots showed very high nitrogenase activity (~1.2 to 1.6 µmol C2H4 g fresh wt-1 h-1) under both light and dark conditions as compared to transverse sections of roots. Localization of NR and nitrogenase was tested in coralloid roots using different sets of roots and also in the endophyte. Our results showed that NR activity was mainly due to the endophyte (Anabaena cycadeae); coralloid roots lacked it, as no NR activity was observed in chloramphenicol-treated intact root samples.
In this study, investigated were the effects of NaCl (60 mmol/dm-3) and NaCl supplemented with different salts (5 mmol/dm-3 CaCl2, CaSO4, CaCO3, KCl), on growth of two maize varieties (Cyrkon and Limko). After 7 days of cultivation in nutrient solution the growth response to salinity of both maize varieties was similar. NaCl led to a dramatic decrease in growth of plants (approx. 50% reduction in fresh and dry weight of root, and 70% reduction in fresh weight of shoot). Addition of extra Ca2+ or K+ to nutrient solution containing NaCl did not definitely improve the growth parameters of maize. However, among the tested salts, CaCl2 had a beneficial visual effect on maize seedlings. In other cases the plants showed noticeable symptoms of salt damage. In long term exposure to salinity (two weeks) growth of Cyrkon was more inhibited than Limko. Comparison of growth responses in short-term exposure to salinity (7 days) with long-term (14 days) showed that in Cyrkon variety the negative effects of NaCl were intensified and addition of CaCl2 to salinized solution had not positive effects on growth. On the contrary, in Limko variety, there was a significant improvement in growth (especially in root dry weight). This fact indicates that during longer exposure to salinity Limko was able to adapt to those conditions. Salinity caused a significant decrease in leaf nitrate reductase activity (60% and 30% reduction respectively in Limko and Cyrkon). Addition of CaCl2 to salinized nutrient solution resulted in greater enzyme inhibition in Cyrkon (50% decline in relation to plants grown under sole NaCl), and 30% increase in Limko. Inhibition of nitrate reductase activity did not cause a decrease in concentration of soluble protein in maize leaves.
Nitrate reductase activity in gibberellic acid and kinetin treated mustard (Brassica juncea Coss. cv. T-59 ‘Varuna’) seedlings, grown in the presence or absence of light and/or NO₃ was investigated. While both light and NO₃, alone could induce NR activity, their combination showed additive effects. Kinetin treatment significantly promoted both light- and NO₃- induced NR activities, assayed by either in vivo or in vitro techniques, whereas, gibberellic acid was almost ineffective. In the absence of both light and NO₃, however, phytohormones alone could not induce NR activity. Both light-induced and NO₃ induced NR fractions had a pH optima of 7.5, preferred NADH as an electron donor (NADH: NADPH ratio 2.5) and Km values for NO₃ was 0.2 mM. Actinomycin D, cycloheximide and tungstate were equally effective in suppressing the development of NR activity after exposure to light or NO₃. These results indicate that two independent NR fractions operate, with apparently identical properties but separate control mechanisms.
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