Nucleotide sequence of a cDNA encoding rice chloroplastic carbonic anhydrase

Plant Physiol. (1995) 107: 299-300

Plant Gene Regisfer

Nucleotide Sequence of a cDNA Encoding Rice Chloroplastic Carbonic Anhydrase Shoichi Suzuki and James N. Burnell*

Centre for Molecular Biotechnology, Queensland University of Technology, GPO Box 2434, Brisbane, Q, 4001, Queensland, Austral ia CA (EC 4.2.1.1) catalyzes the reversible hydration of CO, HCO, + H+ and according to the reaction CO, + H,O represents 1 to 2% of total leaf soluble protein (Okabe et al., 1984).Considerable differences have been reported to exist between the molecular mass of the holoenzyme in monocotyledonous and dicotyledonous species (4245 kD in monocotyledonous plants and between 140 and 250 kD in dicotyledonous plants), and smaller differences exist between the molecular masses of the subunits, 24 to 36 kD (see Sultemeyer et al., 1993, for review). Previous studies using antibodies have also shown that CA from monocotyledonous and dicotyledonous plant species differ in their antigenic cross-reactivity (Okabe et al., 1984; Burnell, 1990). Recently, the primary structure of spinach (Fawcett et al., 19901, pea (Roeske and Ogren, 1990; Majeau and Coleman, 1991), tobacco (Majeau and Coleman, 1992), and, most recently, Arabidopsis thaliana (Raines et al., 1992) have been reported, and the control of expression of maize (Burnell et al., 1990) and A. thaliana (Raines et al., 1992) CA have also been reported. To date only the CA from dicotyledonous plants has been characterized in any detail, and, except for brief reports on the CA of barley and wandering Jew (Tradescantia albiflora Kunth) (Atkins et al., 1972), monocotyledonous CA has largely been ignored. The characterization of rice (Oryza sativa) (a C3 monocotyledonous plant) CA has been conducted to allow comparison of plant CAs at the molecular level. A rice leaf cDNA library was constructed in AZAP and screened using a maize CA cDNA probe (data not shown) and 10 positively hybridizing phage plaques were isolated (Table I). The longest cDNA contained 1148 bp and an open reading frame encoding a preprotein of 273 amino acids. The processing site for the remova1 of the transit peptide was identified by N-terminal sequencing of the purified rice chloroplastic CA and indicated a transit peptide and a mature protein of 63 and 210 amino acid residues, respectively. The amino acid sequence of the mature rice protein exhibits between 59 and 63% identity with pea, tobacco, spinach, and Arabidopsis CA. The transit peptide of rice CA is considerably shorter than the transit peptide of dicotyledonous CAs. It may be significant that the amino acids deleted in the rice CA transit peptide compared with the dicotyledonous CA transit peptides are between amino acid residues 60 and 100 of spinach CA

Table 1. Characteristics of CA cDNA from rice Organism: Oryza sativa. Gene Product: Chloroplastic CA (EC 4.2.1.1). Techn iques: Purification of rice chloroplastic CA by (NH,),SO, fractionation, ion-exchange chromatography, and gel filtration; N-terminal sequencing; screening of a AZAP cDNA library constructed from green leaves using maize CA cDNA; in vivo excision; double-stranded plasmid sequencing in pBluescript and dideoxy sequencing of both strands. Method of Identification: Comparison of the deduced amino acid sequence with the Nterminal amino acid sequence of purified rice chloroplastic CA. Comparison of the deduced amino acid sequence with previously described CA sequences (spinach, tobacco, pea, and Arabidopsis). Features of the cDNA: This clone is 1148 nucleotides in length, consisting of a 35nucleotide 5' untranslated region, an open reading frame of 81 9 bp, and a 297-nucleotide 3' untranslated region. Regulation: Light-stimulated accumulation in leaf tissue of a 1.2-kb transcript as determined by northern analysis. Features of the Protein: The cDNA contains an open reading frame of 273 amino acids that encodes a 63-amino acid transit peptide and a 210-amino acid mature protein. Subcellular Location: Chloroplastic.

(see Fawcett et al., 1990). These amino acids contain a large number of acidic residues and have been suggested as unlikely components of a transit peptide of other plant CAs (Fawcett et al., 1990). In addition, rice CA has a 10amino acid deletion at the C-terminal end of the mature CA compared with dicotyledonous CAs. Those amino acid residues of pea CA shown by site-directed mutagenesis studies (Provart et al., 1993) to be necessary for enzyme activity ( C ~ S " ~GluZo4, , HisZz0,CysZz3,G~U''~;numbering of amino acids according to pea CA) are a11 conserved in rice CA, whereas those residues that were not essential for enzyme activity ( H ~ s and ' ~ ~HisZo9)are not conserved in rice.

* Corresponding author; fax 61-7-864-1534.

Abbreviation: CA, carbonic anhydrase. 299

300

Suzuki and Burnell

Received July 5, 1994; accepted August 22, 1994. Copyright Clearance Center: 0032-0889/95/107/0299/02. 'The GenBank accession number for the sequence reported in this article is U08404.

LITERATURE ClTED

Atkins CA, Patterson BD, Graham D (1972) Plant carbonic anhydrases. 11. Preparation and some properties of monocotyledon and dicotyledon enzyme types. Plant Physiol 5 0 218-223 Burnell JN (1990) Immunological study of carbonic anhydrase in C, and C, plants using antibodies to maize cytosolic and spinach chloroplastic carbonic anhydrase. Plant Cell Physiol 31: 423-427 Bumell JN, Suzuki I, Sugiyama T (1990) Light induction and the effect of nitrogen status upon the activity of carbonic anhydrase in maize leaves. Plant Physiol 94: 384-387 Fawcett TW, Browse JA, Volokita M, Bartlett SG (1990) Spinach carbonic anhydrase primary structure deduced from the sequence of a cDNA clone. J Biol Chem 265 5414-5427

Plant Physiol. Vol. 107, 1995

Majeau N, Coleman JR (1991) Isolation and charactcrization of a cDNA coding for pea chloroplastic carbonic anhj,drase. Plant Physiol 95: 264-268 Majeau N, Coileman JR (1992) Nucleotide sequence of a complementary DNA encoding tobacco chloroplastic carbonic anhydrase. Plant Physiol 100: 1077-1078 Okabe K, Yang S, Tsuzuki M, Miyachi S (1984) Carbonic anhydrase: its content in spinach leaves and its taxonoinic diversity studied with anti-spinach leaf carbonic anhydrase antibody. Plant Sci Lett 33: 145-153 Provart NJ, Majeau N, Coleman JR (1993) Characterization of pea chloroplastic carbonic anhydrase. Expression in E ;chevichiu coli and site-directed mutagenesis. Plant Mo1 Biol 2 2 937-943 Raines CA, Horsnell PR, Holder C, Lloyd JC (199:!) Arubidopsis thaliann carbonic anhydrase: cDNA sequence and 'zffect of CO, on mRNA levels. Plant Mo1 Biol 2 0 1143-1148 Roeske CA, Ogren WL (1990) Nucleotide sequence of pea cDNA encoding chloroplast carbonic anhydrase. Nucleic Acid Res 1 8 3413 Sultemeyer D, Schmidt C, Fock HP (1993)Carbonic anhydrases in higher plants and aquatic microorganisms. Physiol Plant 88: 179-190