Complementary DNA cloning and characterization of ferredoxin localized in bundle-sheath cells of maize leaves

Abstract

In maize (Zea mays L.) two leaf-specific ferredoxin (Fd) isoproteins, Fd I and Fd II, are distributed differentially in mesophyll and bundle-sheath cells. A novel cDNA encoding the precursor of Fd II (pFD2) was isolated by heterologous hybridization using a cDNA for Fd I (pFD1) as a probe. The assignment of the cDNAs to the Fds was verified by capillary liquid-chromatography/electrospray ionization-mass spectrometry. RNA-blot analysis demonstrated that transcripts for Fd I and Fd II accumulated specifically in mesophyll and bundle-sheath cells, respectively. The mature regions of pFD1 and pFD2 were expressed in Escherichia coli as functional Fds. Fd I and Fd II had similar redox potentials of -423 and -406 mV, respectively, but the Km value of Fd-NADP+ reductase for Fd II was about 3-fold larger than that for Fd I. Asparagine at position 65 of Fd II is a unique residue compared with Fd I and other Fds from various plants, which have aspartic acid or glutamic acid at the corresponding position as an electrostatic interaction site with Fd-NADP+ reductase. Substitution of asparagine-65 with aspartic acid increased the affinity of Fd II with Fd-NADP+ reductase to a level comparable to that of Fd I. These structural and functional differences of Fd I and Fd II may be related to their cell-specific expression in the leaves of a C4 plant.

Figure 1

Restriction map of maize Fd cDNAs (A), nucleotide sequence of the cDNA designated pFD2 (B), and comparison of the deduced amino acid sequences of maize Fd I and Fd II (C). A, The coding region is represented by an open box. Positions of the gene-specific probes that were used for RNA analysis are indicated by hatched bars. B, The amino acid sequence encoded by the open reading frame is shown below the nucleotide sequence. The determined N-terminal amino acid sequence and C-terminal residue of the mature form of Fd II (Hase et al., 1991a) are underlined. C, The amino acid sequence of maize Fd II deduced from pFD2 is compared with that of maize Fd I (Hase et al., 1991a). Gaps, denoted by dashes, have been inserted to achieve maximum homology. Identical amino acid residues between Fd I and Fd II are indicated by white letters on a black background.

Figure 2

Peptide mapping of Fd I and Fd II prepared from maize leaves. A, Purified Fd I and Fd II (2 μg each) and their mixture were subjected to nondenaturing PAGE with a linear gradient of 15% to 25% acrylamide (Kimata and Hase, 1989) and stained with Coomassie brilliant blue. B, The purified Fds were carboxymethylated and digested with lysylendopeptidase. The resulting peptides were subjected to capillary LC/ESI-MS. Three major peaks were obtained from the samples of both Fd I and Fd II, and their M_rs were measured (Table I).

Figure 3

RNA analysis of Fd I and Fd II transcripts in MC and BSC. Total RNAs (10 μg each) prepared from maize whole leaves (lane 1), MC protoplasts (lane 2), and BSC strands (lane 3) were subjected to electrophoresis on an agarose gel containing formamide and transferred to nylon membranes. The blots were probed with ³²P-labeled probes for genes for Fd I, Fd II, PEPC, and the Rubisco small subunit (see Methods).

Figure 4

Construction of plasmids for the expression of maize Fds (A) and the sequence of synthetic oligonucleotides for overexpression (B). pTrc99A-1, with a deletion of the PstI site in the polylinker region of the expression vector pTrc99A, was used for the construction. The region of pFD1 corresponding to the mature Fd I was amplified by PCR and inserted under the control of the trc promoter of pTrc99A-1 to yield pTMmFD1, as described in Methods. Codon usage of amino acids near the translation site of pTMmFD1 was altered by replacing the NcoI/PstI fragment with synthetic oligonucleotides shown in B to give pTMmFD1-1. Nucleotides altered from the original ones are denoted by lowercase letters. The expression plasmids for Fd II, pTMmFD2 and pTMmFD2-1, were constructed by replacing a PstI/XbaI fragment of pTMmFD1 and pTMmFD1-1 with a PstI/NheI fragment of pFD2, respectively.

Figure 5

Expression of maize Fds in E. coli JM105. Cells were transformed with the Fd-expression plasmids pTMmFD1, pTMmFD1-1, pTMmFD2, and pTMmFD2-1 and grown in the presence (+) or absence (−) of isopropyl-β-d(−)-thiogalactopyranoside (IPTG), as described in Methods. Whole cells from 10 μL of each bacterial culture were subjected to SDS-PAGE, and proteins were visualized by staining with Coomassie brilliant blue (A) or by immunolabeling with antimaize Fd I antibodies (Kimata and Hase, 1989; B and C).

Figure 6

Electron-transfer activity of wild-type and mutant Fds. Electron-transfer activity of Fd was assayed by measuring the rate of Cyt c (cyt.c) reduction as described in Methods. The values represent the amount (in micromoles) of Cyt c reduced in 800 μL of a reaction per minute. A typical example of three independent experiments is shown.