Abstract
Physiological studies on the Brittle1 maize mutant have provided circumstantial evidence that ZmBT1 (Zea mays Brittle1 protein) is involved in the ADP-Glc transport into maize endosperm plastids, but up to now, no direct ADP-Glc transport mediated by ZmBT1 has ever been shown. The heterologous synthesis of ZmBT1 in Escherichia coli cells leads to the functional integration of ZmBT1 into the bacterial cytoplasmic membrane. ZmBT1 transports ADP-Glc in counterexchange with ADP with apparent affinities of about 850 and 465 mum, respectively. Recently, a complete ferredoxin/thioredoxin system has been identified in cereal amyloplasts and BT1 has been proposed as a potential Trx target protein (Balmer, Y., Vensel, W. H., Cai, N., Manieri, W., Schurmann, P., Hurkman, W. J., and Buchanan, B. B. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 2988-2993). Interestingly, we revealed that the transport activity of ZmBT1 is reversibly regulated by redox reagents such as diamide and dithiothreitol. The expression of ZmBT1 is restricted to endosperm tissues during starch synthesis, whereas a recently identified BT1 maize homologue, the ZmBT1-2, exhibits a ubiquitous expression pattern in hetero- and autotrophic tissues indicating different physiological roles for both maize BT1 isoforms. BT1 homologues are present in both mono- and dicotyledonous plants. Phylogenetic analyses classify the BT1 family into two phylogenetically and biochemically distinct groups. The first group comprises BT1 orthologues restricted to cereals where they mediate the ADP-Glc transport into cereal endosperm storage plastids during starch synthesis. The second group occurs in mono- and dicotyledonous plants and is most probably involved in the export of adenine nucleotides synthesized inside plastids.
Highlights
Heterologous Expression of ZmBT1 in E. coli Cells—We showed previously that the heterologous synthesis of the plastidic StBT1 homologue in E. coli leads to the functional integration of StBT1 into the bacterial cytoplasmic membrane [15]
The ADP-Glc required for starch synthesis in plastids of cereal endosperm is mainly synthesized in the cytosol and has to be transported across the plastid envelope [2, 51, 52]
We have investigated the biochemical characteristics of the maize BT1 homologue ZmBT1 (Fig. 1)
Summary
Cultivation of Plants—Maize plants (Z. mays L.) were grown in a greenhouse at 22–26 °C and watered once a day. The expression plasmid (pET 16b, Novagen, Heidelberg, Germany) encoding the recombinant ZmBT1 protein with an additional N-terminal tag of 10 histidine residues was constructed as follows: the cDNA coding the entire ZmBT1 was generated by PCR from first strand cDNA of maize endosperm tissue using Pfu DNA polymerase (Invitrogen). Transport Assays with E. coli—IPTG-induced E. coli cells (100 l) harboring the ZmBT1 expression plasmid (or the given controls) were added to 100 l of potassium phosphate buffer (50 mM, pH 7.0) containing radioactively labeled ADP or ADPGlc. Uptake of nucleotides was carried out at 30 °C in an Eppendorf reaction vessel incubator and terminated after the indicated time periods by transferring the cells to a 0.45-m membrane filter (mixed cellulose ester, 25 mm diameter; Schleicher & Schuell, Dassel, Germany) under vacuum [23]. E. coli cells were incubated with potassium phosphate buffer (50 mM, pH 7.0) containing 1 M [14C]ADP-Glc as a transport substrate. The phylogenetic tree was created with PhyML [26] after aligning the sequences with Muscle [27]
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