The 33 kd protein of the photosynthetic oxygen‐evolving complex is synthesized in the cytoplasm as a larger precursor and transported into the thylakoid lumen via a stromal intermediate form. In this report we describe a reconstituted system in which the later stages of this import pathway can be studied in isolation. We demonstrate import of the 33 kd protein, probably as the intermediate form, into isolated pea thylakoids by a mechanism which is stimulated by the addition of ATP. The imported protein is processed to the mature size and is resistant to digestion by proteases. The thylakoidal protein transport system is specific in that non‐chloroplast proteins and precursors of stromal proteins are not imported.

The sulfonamide resistance gene from plasmid R46 encodes for a mutated dihydropteroate synthase insensitive to inhibition by sulfonamides. Its coding sequence was fused to the pea ribulose bisphosphate carboxylase/oxygenase transit peptide sequence. Incubation of isolated chloroplasts with the fusion protein synthesised in vitro, showed that the bacterial enzyme was transported to the chloroplast stroma and processed into a mature form. Expression of the gene fusion in transgenic plants resulted in a high level of resistance to sulfonamides. Direct selection of transformed shoots on leaf explants was efficient using sulfonamides as sole selective agents. Transformed shoots rooted normally on sulfonamides at concentrations toxic for untransformed ones. Sulfonamide resistance was transmitted to the progeny of transformed plants as a single Mendelian dominant character. These results demonstrate that this chimeric gene can be used as an efficient and versatile selectable marker for plant transformation.

The light-driven oxidation of water in the thylakoid membrane is carried out by photosystem II, a multisubunit complex containing both integral and extrinsic components. The wateroxidation reaction takes place on the lumenal side of the membrane and is catalysed by a group of proteins, often termed the oxygen-evolving complex, of which three proteins of 33, 23 and 16 kDa are particularly prominent [2,5]. The 33 kDa protein is believed to stabilise an essential manganese centre, whereas the 23 kDa and 16kDa proteins probably assume regulatory functions [ 1,6]. All three proteins are synthesised in the cytosol as larger precursors containing aminoterminal pre-sequences [11] and import into the thylakoid lumen is believed to take place by a two-step mechanism. Precursor proteins are initially transported into the stroma and cleaved to intermediate forms by a stromal processing peptidase, after which these intermediates are transferred across the thylakoid membrane and cleaved to the mature sizes by a second, thylakoid proces sing peptidase [ 3 ].We have determined the N-terminal amino acid sequence of the wheat 33 kDa protein, and have used specific antisera to isolate a full-length cDNA encoding the precursor protein from a wheat leaf library in 2gtll. The nucleotide and deduced amino acid sequences of this cDNA, p33K-2, are shown in Fig. 1. Amino acid residues 80-102 precisely match the sequence determined by N-terminal Edman degradation of the purified protein; the precursor protein therefore consists of a mature protein (246 residues) preceded by a pre-sequence of 79 amino acids. The mature wheat protein shows a high degree of conservation with the corresponding pea [9, 10] and spinach [7] proteins, with 82~o and 81~o of the residues being identical, respectively.The pre-sequence of the wheat 33 kDa protein is shorter than those of the corresponding spinach and pea proteins, by 5 and 2 residues, respectively. The intermediate cleavage site has yet to be determined, but it is believed that the envelope transfer and thylakoid transfer domains are of approximately equal length [3]. A comparison of the wheat and spinach pre-sequences is shown in Fig. 2; overall, these sequences exhibit considerably less homology (54 ~o residue identity) than do the mature sequences, but it is notable that the second, thylakoid transfer domains are more highly conserved than the envelope transfer domains. Only the amino-terminal sections of the envelope transfer domains are highly conserved, although it may be significant that the positions of basic residues are also conserved. The thylakoidThe nucleotide sequence data reported will appear in the EMBL GenBank and DDBJ Nucleotide Sequence Databases under the accession number X57408.