Appearance of nitrite-reductase mRNA in mustard seedling cotyledons is regulated by phytochrome

Abstract

Nitrate reductase (NR, EC 1.6.6.1) and nitrite reductase (NIR, EC 1.7.7.1) are the key enzymes of nitrate reduction. It is well established that the appearance of these enzymes is "induced" by nitrate, and it is generally believed that NR is cytosolic while NIR is plastidic. In mustard (Sinapis alba L.) cotyledons we observed two isoforms of NIR (NIR1 and NIR2) using a chromato-focusing technique. Only one of them (NIR2) disappeared when the plastids were damaged by photooxidation in the presence of Norflurazon. It is concluded that NIR2 is plastidic while NIR1 is extraplastidic and not affected by photooxidation of the plastids. Both isoforms appear to have the same molecular weight (60 kilodaltons, kDa). Two distinct translation products which could be immunoprecipitated with NIR antiserum produced against total NIR from mustard were observed which differed slightly in molecular weight (60 versus 63 kDa). The 63-kDa polypeptide was considered to be the precursor of NIR2. While synthesis of NIR protein depended largely on nitrate, the levels of in-vitro-translatable NIR mRNAs were found to be either independent of nitrate and light (NIR1) or controlled by phytochrome only (NIR2). It appears that phytochrome strongly stimulates the level of mRNA while significant enzyme synthesis (NIR2) takes place only in the presence of relatively large amounts of nitrate. Since an increased enzyme level was strictly correlated with an increase of immunoresponsive NIR protein it is improbable that activation of a precursor plays a role. Rather, it is concluded that, in situ, nitrate controls translation.