Biochem. J. (1999) 337, 523–530 (Printed in Great Britain) 523 Occurrence and expression of members of the ferritin gene family in cowpeas Alison J. WARDROP, Ronald E. WICKS and Barrie ENTSCH1 Division of Molecular and Cellular Biology, School of Biological Sciences, University of New England, Armidale, NSW 2351, Australia Ferritin gene expression has been demonstrated in a variety of also provide preliminary evidence for a family of ferritin genes in plants including maize, Arabidopsis, cowpeas, soybeans, beans soybeans (Glycine max) related to that in cowpeas. We conclude and peas. Most available evidence shows that the mature protein that a family of genes is probably present in all higher plants. We is located in plastids and its production is under gene trans- have used quantitative reverse transcriptase-mediated PCR to criptional control. In maize, two different ferritin genes have show that each of the four members of the cowpea ferritin gene been identified; they were found to express protein under different family expresses mRNA in leaves and roots under normal growth physiological conditions. Only single gene products have been with a complete nutrient supply. The results clearly show a found until now in the other plants, with the exception of marked differential pattern of mRNA levels formed during cowpeas (Vigna unguiculata). Our previous work with cowpeas development from the four genes. We conclude that the com- [Wicks and Entsch (1993) Biochem. Biophys. Res. Commun. position of plant ferritin molecules from plant leaf extracts is 192, 813–819] showed the existence of a family of at least three probably a complex mixture of subunits, which might be different ferritin genes, each coding for a protein subunit with a unique in roots and in leaves. amino acid sequence. Here we report the discovery of a fourth active gene in cowpeas and present the full cDNA sequences for Key words: gene family, gene expression, plant ferritins, reverse two of the four known members of the cowpea gene family. We transcriptase-PCR. INTRODUCTION for soybeans [6], beans [11], peas [8,12] and Arabidopsis thaliana Ferritins are multimeric proteins (24 subunits) that are present in [13]. In A. thaliana [13] the iron-mediated oxidative stress plants, animals and micro-organisms. In all cases the protein is transcriptional response described for maize [10] has been responsible for the storage and distribution of iron within cells observed, but there is no evidence for the existence of an abscisic [1]. Mammalian ferritins have been extensively characterized. acid-regulated ferritin gene. In previous studies we have es- Two active genes code for the functionally different subunit tablished that at least three cowpea (Vigna unguiculata) ferritin types, H and L, which assemble in various ratios in the final genes are expressed in developing leaves. Two of these genes code protein depending on the tissue in which the protein is found [2]. for protein products with substantial sequence divergence from Until recently, only one functional type of plant ferritin subunit the third product, which is closely related to the other legume has been described; this subunit displays characteristics of both subunits [14]. The possibility exists that members of this gene H-type and L-type animal ferritin subunits [2]. On the basis of family in cowpeas might be differentially expressed in a manner sequence comparisons, plant and animal ferritins seem to be analogous to that for maize or animal ferritins, and that all derived from a common evolutionary ancestor [3]. Although plants have a complex family of ferritin genes. ferritin probably performs the same function in plants and Here we present evidence for four distinct transcribed members animals, most experimental evidence supports the regulation of of the cowpea ferritin gene family, and also provide preliminary ferritin synthesis in plants at the level of transcription, as opposed evidence from PCR-amplifcation of soybean DNA that this gene to translation, in animals, and plant ferritins are located in family is not unique to cowpeas. For each of four known genes plastids as opposed to the cytoplasm in animals [4–6]. In animals, in cowpeas we have used quantitative reverse transcriptase- iron regulates ferritin expression through a non-coding region of mediated PCR (RT–PCR) techniques to estimate the levels of mRNA (the iron response element) that has not been detected in mRNA during the growth and expansion of cowpea leaves as all reported plant ferritin sequences [7]. well as in roots. Results show clear patterns of differential Coding regions for plant ferritin subunits are longer than accumulation of mRNA species. those for animal ferritins; an extra sequence at the N-terminus of the plant protein subunit encodes a transit peptide responsible MATERIALS AND METHODS for plastid targeting, and also an extension peptide of approx. 24 Materials amino acid residues that is specific to plant ferritins and found in the mature peptide [3,8]. In maize, two different ferritin genes Agarose for gel electrophoresis was obtained from Pharmacia have been identified that seem to be differentially expressed in Biotech (Uppsala, Sweden). Restriction enzymes, reverse trans- response to abscisic acid or iron-mediated oxidative stress [9,10]. criptase (MMLV RT, RNase H Minus) and DNase (RQ1 These genes are almost identical (95–96%) in the protein-coding RNase-free DNase) were all obtained from Promega Corporation region. In dicotyledons, single gene products have been reported (Madison, WI, U.S.A.). Radioactive [α-$#P]dATP was supplied Abbreviations used: MMLV, Moloney murine leukaemia virus; RT–PCR, reverse transcriptase-mediated PCR. 1 To whom correspondence should be addressed (e-mail bentsch!metz.une.edu.au). The nucleotide sequence data reported will appear in DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession numbers AF052057, AF052058, AF052512, AF052513 and AF052511. # 1999 Biochemical Society 524 A. J. Wardrop, R. E. Wicks and B. Entsch by Bresatec (Adelaide, SA, Australia). All other reagents were of were taken from leaves at lamina lengths specified in Table 3, or A.R. grade. Escherichia coli strains JM105 and DH5α [15] were from root tips (2 cm). All total RNA preparations were treated used for routine cloning; E. coli LE392 [15] was used for the with RNase-free DNase. RNA quality was checked by gel construction of cDNA libraries in phage λgt10 from Pharmacia electrophoresis and then quantified by spectrophotometry. (Uppsala, Sweden). Cowpea (V. unguiculata var N41) and soybean (G. max var Valder) seeds were obtained from the NSW cDNA synthesis Department of Agriculture (Tamworth, NSW, Australia). First-strand cDNA (forming a DNA\RNA duplex) was syn- thesized from 1 µg of total RNA by the method of Dallman and Plant growth Porter [18] with MMLV reverse transcriptase and oligo(dT) in Cowpea seedlings were grown in moist vermiculite in a green- the presence of a ribonuclease inhibitor. Reaction conditions house under conditions of natural light and at ambient tem- were set to give a maximum yield of cDNA. After annealing at perature. Growth was supported by a complete mineral mixture 37 mC, 200 units of MMLV reverse transcriptase was added and supplemented with extra soluble iron as FeEDTA. Leaf samples the reaction was left for 60 min at 37 mC. A second 200 units of were taken at specified lamina lengths (see Table 3) and placed enzyme was added and the reaction was left for a further 60 min on ice before being transferred to liquid nitrogen for storage. at 37 mC, followed by inactivation of the enzyme at 70 mC for Soybean seedlings were grown under the same conditions, with 10 min. The final solution was made to 100 µl; 4 µl samples were leaf samples taken at 5–7 cm lamina length for the preparation of used in each PCR reaction. DNA or RNA. Competitive templates Cloning and sequencing of cowpea ferritin cDNA Templates were fragments of genomic cowpea DNA that had Total RNA was prepared from expanding cowpea leaves (5–7 cm been subcloned in pUC18. The genomic fragments for CP1 and in lamina length) by the method of Chomczynski and Sacchi [16]. CP2 contained small introns and were used unchanged (see Table Poly(A)+ RNA was isolated from total RNA by using a poly(dT) 2). The genomic fragments for CP3 and CP5 contained large magnetic bead capture system (Promega PolyATract2 mRNA introns and were engineered to remove most of the intron (by Isolation System; Promega Corporation). Clones for the two using the Clontech Mutagenesis Kit and specific oligonucleotides reported cowpea ferritin cDNA species (CP2 and CP3) were to loop out a large segment of DNA) so that a suitable fragment isolated from two independent lambda cDNA libraries. One size was obtained (see Table 2). The templates were used as library was constructed from 4 µg of poly(A)+ RNA with the linearized pUC18, and stock solutions from 8 to 8i10( ag\µl cDNA Synthesis System Plus kit (Amersham International, UK) (expressed as concentration of unique template DNA) were – first-strand synthesis primed with oligo-dT, and the cDNA was prepared by 10-fold serial dilutions. packaged into lambda gt10 phage particles using the Promega Packagene2 Lambda DNA Packaging System. For the second PCR library, cDNA was synthesized from 3 µg of poly(A)+ RNA (oligo-dT priming) using Moloney murine leukaemia virus Components of the PCR reactions were tested for contaminants (MMLV) reverse transcriptase, and EcoRI\NotI adaptors were by performing 40-cycle reactions without added DNA template. Each reaction mixture contained, dNTPs (200 µM final con- added (Pharmacia Biotech cDNA Synthesis Kit; Pharmacia, µ centration of each), 0.5 M each primer, 1.5 mM MgCl#, PCR Sweden). The cDNA was cut with EcoRI, inserted into lambda \ \ \ gt10 and packaged using the Promega Packagene2 Lambda buffer [67 mM Tris HCl (pH 8.8) 16.6 mM (NH%)#SO% 0.2 mg\ml gelatin\0.45% (v\v) Triton X-100], 1 unit of Taq DNA Packaging System.