The root form of ferredoxin-NADP oxidoreductase is expressed in rice coleoptiles for the assimilation of nitrate

Two ferredoxin-dependent proteins, nitrite reductase and glutamate synthase, play a role in nitrate assimilation during the anaerobic germination of rice (Oryza sativa L.). This paper reports the expression of the root form of ferredoxin-NADP⁺ oxidoreductase (FNR), the protein responsible for providing reduced ferredoxin in rice coleoptiles. Using an antibody against FNR, a protein with the expected molecular mass for root FNR (35 kDa) was recognized by Western blot analysis in extracts from aerobic and anaerobic coleoptiles. The enzyme is synthesized de novo, as shown by immunoprecipitation of the radiolabeled 35-kDa protein from anaerobic seedlings grown in the presence of [³⁵S]methionine. Northern blot analysis with specific probes for root and leaf FNR showed the presence of mRNA for the root form but not for the leaf form, in both aerobic and anaerobic rice coleoptiles. The inductive effect of exogenous nitrate on the expression of FNR is further evidence for the presence of the root type of FNR in rice coleoptiles. The importance of the expression of root FNR during the anaerobic development of rice seedlings is discussed. Received: 7 October 1996 / Accepted: 22 January 1997     

Induction of ferredoxin-NADP+ oxidoreductase and ferredoxin synthesis in pea root plastids during nitrate assimilation

A 14.5 kDa protein with antigenic components in common with pea leaf ferredoxin was detected on transblots of the soluble proteins of pea root plastids. The amount of this protein was found to increase during the induction of nitrate assimilation in pea roots, reaching a maximal level at 8–12 h. Concurrent with this, a fourfold increase in NADPH-dependent ferredoxin-NADP⁺ oxidoreductase (FNR) activity was observed corresponding to an increase in the amount of this protein detected immunologically on transblots using a leaf FNR antibody. These changes were not observed in plastids from roots of plants grown on ammonia or depleted of nitrogen. It is suggested that in addition to the already well reported induction by nitrate of nitrate reductase and nitrite reductase, there is a co-induction of a plastid located ferredoxin and FNR. Both these proteins are necessary for the transfer of reductant generated by the oxidative pentose phosphate pathway to nitrite reductase.     

Identification of microRNAs in rice root in response to nitrate and ammonium

Nitrate and ammonium are two major nitrogen(N) sources for higher plants,but they differ in utilization and signaling.Micro RNAs(mi RNAs) play an essential role in N signal transduction;however,knowledge remains limited about the regulatory role of mi RNAs responsive to different N sources,especially in crop plants.To get global overview on mi RNAs involved in N response in rice,we performed high-throughput small RNA-sequencing under different nitrate and ammonium treatments.The results demonstrated that only 16 and 11 mi RNAs were significantly induced by nitrate and ammonium under short-term treatment,respectively.However,60 differentially expressed mi RNAs were found between nitrate and ammonium under long-term cultivation.These results suggested that mi RNA response greatly differentiates between nitrate and ammonium treatments.Furthermore,44 mi RNAs were found to be differentially expressed between high-and low-N conditions.Our study reveals comprehensive expression profiling of mi RNAs responsive to different N sources and different N treatments,which advances our understanding on the regulation of different N signaling and homeostasis mediated by mi RNAs.     

Interaction of ferredoxin-NADP+ oxidoreductase with triazine dyes a rapid purification method by affinity chromatography

The triazine dyes, Cibacron blue F3GA and Procion red HE3B inhibited diaphorase activity of ferredoxin-NADP⁺ reductase, in a competitive manner with respect to NADPH. The K_i values were 1.5 and 0.2 μM, respectively. Binding of the dyes to the flavoprotein, as measured by difference spectroscopy, indicated an apparent stoichiometry of 1 mol dye/mol reductase and was prevented by NADP⁺ or high ionic strength. Chemical modification of a lysine residue and a carboxyl group at the NADP(H) binding site of the enzyme prevented complex formation with Procion red. Procion red showed a higher affinity for ferredoxin-NADP⁺ reductase than Cibacron blue. The K_d values were 1.9 and 5 μM, respectively. Once covalently linked to a Sepharose matrix, the triazine compounds specifically bind the flavoprotein. The interaction is partially electrostatic and partially hydrophobic. The enzyme can be eluted by high concentrations of salt or low concentrations of the corresponding coenzyme. The use of this affinity column allows the rapid purification of ferredoxin-NADP⁺ oxidoreductase from spinach leaves with good yields.     

Involvement of brassinosteroids in the gravitropic response of primary root of maize

Exogenously applied brassinolide (BL, 10(-9)-10(-5) M) increased gravitropic curvature in maize (Zea mays) primary roots. The BL-enhanced gravitropic curvature was clearly promoted in the presence of indole-3-acetic acid (IAA, 10(-10)-10(-8) M), indicating that BL is interactive with IAA during the gravitropic response. The interactive effect between BL and IAA was completely diminished by treatment of p-chlorophenoxy isobutric acid, an auxin action antagonist. The activation of the gravitropic response by BL in the absence and in the presence of IAA was nullified by application of 2, 3,5-triiodobenzoic acid, a polar auxin transport inhibitor. The data indicate that brassinosteroids (BRs) might be involved in auxin-mediated processes for the gravitropic response. Gas chromotography-selected ion-monitoring analysis revealed that maize primary roots contained approximately 0.3 ng g(-1) fresh weight castasterone as an endogenous BR. Exogenously applied castasterone also increased the gravitropic response of maize roots in an IAA-dependent manner. This study provides the first evidence, to our knowledge, for occurrence and gravitropic activity of BRs in plant roots.     

Biosynthesis of ferredoxin-NADP+ oxidoreductase. Evidence for the formation of a functional preholoenzyme in the cytoplasmic compartment

Biosynthesis of ferredoxin-NADP+ reductase in higher plants was investigated in relation with the mechanism of formation of the holoenzyme. The putative precursor of the flavoprotein, obtained after cell-free translation on a wheat germ extract primed with poly(A)-rich mRNA, was able to spontaneously bind free FAD, rendering a functional prereductase. The newly synthesized preholoenzyme showed diaphorase and cytochrome c reductase activities, an apparent molecular mass of 45 kDa, and contained FAD as the only flavin cofactor. It gave a positive reaction towards antisera against mature ferredoxin-NADP+ reductase. On the other hand, intracellular distribution of flavin-synthesizing enzymes indicates that FAD formation occurs in the cytoplasm; that is, in the same compartment as the site of reductase synthesis. On the basis of the preceding data a model is presented for the biosynthesis of the enzyme in vivo, involving conjugation of the apoprotein with FAD in the cytoplasm, followed by transport of the preholoreductase across the chloroplast envelope to reach its final destiny in the thylakoid membrane.     

Rapid procedure for the preparation of ferredoxin-NADP+ oxidoreductase in molecularly pure form at 36 kDa

Ferredoxin-NADP+ oxidoreductase (FNR, EC 1.18.1.2) was purified to molecular homogeneous form as judged by regular and sodium dodecyl sulfate (SDS)-electrophoresis using EDTA extraction of spinach thylakoids, followed by anion exchange on DEAE-cellulose, Procion Red HE 3B dye-ligand chromatography, and hydroxyapatite chromatography. By this procedure, within 1 week approx 7.5 mg of pure FNR, starting from 1 kg of spinach leaves, could be routinely obtained. By comparison with commercially available FNR and with aged preparations two different molecular forms of the enzyme were observed in SDS-electrophoresis. FNR prepared according to the described procedure revealed an apparent molecular mass of 36,000 Da, whereas all other tested preparations showed molecular masses of 3000 Da smaller. Migration in regular gel electrophoresis was the same for all preparations and zymogram stain indicated similar diaphorase activity of both the smaller and the larger forms.     

Isolation and characterization of a plant cDNA showing homology to animal glutathione peroxidases

A cDNA library from freshly isolated protoplasts was differentially screened using cDNAs from mesophyll cells, stressed leaf strips and cell suspension cultures. One of the selected clones, 6P229, turned out to encode a putative polypeptide showing homology to the btuE periplasmic protein of Escherichia coli and to animal selenium-dependent glutathione peroxidases. A major difference was that the putative selenocysteine in the active site was not encoded by the termination codon TGA. The 6P229 gene was found to be expressed in germinating seeds, in apex and in flowers, as well as in stressed tissues. This pattern of expression would be consistent with a key role in cellular metabolism such as defense against oxidative stresses.     

Free nucleotides in the primary root of maize

Free nucleotides of the primary root of maize were extracted with 5% HClO₄ and separated on a column of Dowex 1×8 ion exchanger in HCOO^? cycle. A two-step elution gradient (HCOOH, HCOOHNH₄) was used for the elution of the nucleotides. The incorporation of³²P into the nucleotides was followed at different time intervals and also in young and more mature root tissues. The nucleotides AMP, GMP, ADP, GDP, and ATP were identified. Labelled phosphorus was found in ATP after 30 s, in ADP after 3 min, and in AMP after 5 min incubation of the roots. More mature roots (18 days) contained higher amounts of AMP than the young ones (3 days).     

The function of chloroplast ferredoxin-NADP+ oxidoreductase positively regulates the accumulation of bamboo mosaic virus in Nicotiana benthamiana

A gene down-regulated in Nicotiana benthamiana after bamboo mosaic virus (BaMV) infection had high identity to the nuclear-encoded chloroplast ferredoxin NADP⁺ oxidoreductase gene (NbFNR). NbFNR is a flavoenzyme involved in the photosynthesis electron transport chain, catalysing the conversion of NADP⁺ into NADPH. To investigate whether NbFNR is involved in BaMV infection, we used virus-induced gene silencing to reduce the expression of NbFNR in leaves and protoplasts. After BaMV inoculation, the accumulation of BaMV coat protein and RNA was significantly reduced. The transient expression of NbFNR fused with orange fluorescent protein (OFP) localized in the chloroplasts and elevated the level of BaMV coat protein. These results suggest that NbFNR could play a positive role in regulating BaMV accumulation. Expressing a mutant that failed to translocate to the chloroplast did not assist in BaMV accumulation. Another mutant with a catalytic site mutation could support BaMV accumulation to some extent, but accumulation was significantly lower than that of the wild type. In an in vitro replication assay, the replicase complex with FNR inhibitor, heparin, the RdRp activity was reduced. Furthermore, BaMV replicase was revealed to interact with NbFNR in yeast two-hybrid and co-immunoprecipitation experiments. Overall, these results suggest that NbFNR localized in the chloroplast with functional activity could efficiently assist BaMV accumulation.