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MATERIALS AND METHODS Four ferredoxin isoproteins were identifed in the C4 plant Zea mays L. by analysis of extracts from leaves, mesocotyls, and roots of the young seedlings. The relative amounts of the isoproteins isolated from the photosynthetic and nonphotosynthetic organs were different. All the isoproteins were present in the leaves of green and etfolated plants, whereas two out of the four isoproteins were not detected in the roots or in the mesocotyls. During the greening of effolated seedlings, the level of the two isoproteins unique to the leaf increased markedly. Analysis of the cellular and subcellular distribution of the two major leaf isoproteins showed that one isoprotein was present in the chloroplasts of both mesophyll and bundle sheath cells, whereas the other was only found in the chloroplasts of bundle sheath cells. This is the first report of the cell-specific expression of ferredoxin isoproteins in the leaves of a C4 plant.The presence of different molecular species of plant-type Fd in one organism has been reported in a variety of higher plants and algae (4,7,11,15,17), and variations in the Fd isoproteins with stages and conditions of growth have been observed (4,17,20). Although several attempts have been made to examine whether the Fd isoproteins have different functions in different aspects of Fd-linked metabolism, no conclusive evidence has been obtained for the biological significance ofthe isoproteins (4,7,19). Recently, the heterocyst of a blue-green alga, Anabaena variabilis, was shown to contain a specific Fd which is distinct from the Fd found in vegetative cells. The heterocyst Fd seemed to be involved in a specific interaction with nitrogenase (15).In the leaves of C4 plants, the metabolic process of photosynthesis is partitioned between two types of cell, BSC' and MC, and the intercellular compartmentalization ofthe assimilation of carbon and nitrogen is well documented (5). Furthermore, in the leaves of NADP-malic enzyme types of species, such as maize, almost all of capacity for non-cyclic electron flow is localized in the MC, and the PSII activity of the chloroplasts in the BSC is weak (5). In the course of a survey of Fd isoproteins in C4 plants, we Extraction and Partial Purification of FdDark-grown seedlings were divided into three parts, leaves including coleoptiles, mesocotyls and primary roots. Mesocotyl is the first internodal part formed above the seed storage tissues, which elongates considerably during growth in darkness. Ten g each of these tissues were ground with a mortar and pestle with 1 g of polyclar AT and a small amount of quartz sand in 40 ml of ice-cold extraction buffer (20 mM Tris-HCl (pH 7.5), 100 mm NaCl, 1 mM MgCl2, 1 mM EDTA, 1 mM PMSF, and 1%[v/v] 2-mercaptoethanol). The homogenate was filtered through two layers of cheese cloth and centrifuged at 12,000g for 10 min. The resulting supernatant was mixed with an excess amount of DEAE-cellulose (about 20 g wet weight) and the suspension of resin was packed in a column. After the column wa...

In maize (Zea mays L.), four ferredoxin (Fd) Recently, it was reported that nonphotosynthetic organs, such as maize roots and mesocotyls (12), and radish white roots (25) also contained Fd. These Fds are most probably involved in electron transfers from pyridine dinucleotides to

We have identified and characterized a nitrate-inducible ferredoxin (Fd) i n maize (Zea mays L.) roots by structural analysis of the purified protein and by cloning of i t s cDNA and gene. In maize Fd isoproteins are encoded by a small multigene family, and the nitrate-inducible Fd was identified as a novel isoprotein, designated Fd VI, which differed from any Fd I to Fd V identified to date. In the roots of seedlings cultured without nitrate, Fd VI was undetectable.However, during the induction of the capacity for nitrate assimilation, the amount of Fd VI increased markedly within 24 h. Concurrentiy, the levei of transcript for Fd VI increased, but more quickly, reaching a maximal leve1 within 2 h with kinetics similar to those of nitrite reductase and Fd-NADP+ reductase. Fd 111 was constitutively expressed in roots, and no such changes at the protein and mRNA levels were observed during the nitrate induction. I n the 5' flanking region of the gene for Fd VI only, we identified NIT-2 motifs, which are widely found in genes for enzymes related to nitrogen metabolism. These data indicate that Fd V I i s co-induced with the previously characterized enzymes involved i n nitrate assimilation, and they suggest that the novel Fd isoprotein, distinct from the constitutively expressed Fd, might play an important role as an electron carrier from NADPH to nitrite reductase and other Fd-dependent enzymes in root plastids.