Molecular and Biochemical Analysis of the Plastidic ADP-glucose Transporter (ZmBT1) from Zea mays

Abstract

. 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.Cereal crops accumulate starch in seed endosperm plastids as main energy reserve. The pathway of starch synthesis in cereal endosperms is unique and requires enzyme isoforms that are not present in other tissues or non-cereal plants. The ability of heterotrophic plastids to utilize cytosolic precursors to support their biosynthetic and catabolic pathways depends on the presence of specific transporters in the plastid envelope. In cereal endosperms, the ADP-glucose pyrophosphorylase (AGPase), 2 which catalyzes the first committed and rate-limiting step in starch biosynthesis, is mainly localized in the cytosol with a total AGPase activity of about 85-95% (2). Therefore, ADP-glucose (ADP-Glc) is synthesized in the cytosol of cereal endosperms as the main precursor for starch synthesis and has to be subsequently imported into the storage plastids.Several maize (Zea mays L.) endosperm mutants affected in starch quality or quantity were used to elucidate critical steps in amyloplast starch synthesis. The Waxy gene, which encodes a starch-granule-bound starch synthase involved in amylose synthesis (3) and the Shrunken-2 and Brittle-2 genes, which encode subunits of the AGPase (4 -6), were shown to be important for starch synthesis in maize. The Brittle-1 (BT1) maize mutant was identified in 1926 (7, 8) and corresponding endosperm is severely reduced in starch content, which results in kernels with a collapsed angular appearance at maturity. The BT1 protein from Z. mays (ZmBT1) belongs to the mitochondrial carrier family and is located in the amyloplast envelope membrane (9, 10). The absence of ZmBT1 correlates with a 12-fold higher level of ADP-Glc in the cytosol of BT1 mutant endosperm than in wild-type endosperm (11) and BT1 mutant kernels accumulate about 80% less starch than wild-type kernels (12). The incorporation of externally applied ADP-Glc into starch in amyloplasts isolated from BT1 mutant endosperm was reduced to about 25% compared with wild-type amyloplasts (13). These results indicate that ZmBT1 is involved in the transport of ADP-Glc into maize endosperm plastids (14), but up to now, no d...

The Import and Export Business in Plastids: Transport Processes across the Inner Envelope Membrane

“…In cereals, this metabolite is synthesized in the cytosol and therefore has to be imported into amyloplasts. This theory was finally proved to be correct by direct measurement of the transport activity of the maize BT1 protein after heterologous expression in Escherichia coli (Kirchberger et al, 2007; but notice the different function of BT1 in plants other than cereals [ Fig. 1; Table I]).…”

“…This enables the determination of the transport activity by measuring the import or export of radiolabeled substances into or out of intact E. coli cells. Several plastid transporters have been analyzed using this approach including the NTTs and BT1 from maize and Arabidopsis (Tjaden et al, 1998;Kirchberger et al, 2007). As the production of membrane proteins in E. coli is in some cases difficult to achieve, yeast cells have been widely used for this purpose as an alternative.…”

Delivery of Prolamins to the Protein Storage Vacuole in Maize Aleurone Cells

“…To estimate cross-contamination between the two tissues, we extracted RNA and amplified transcripts of Brittle1 (Bt1) and Viviparous1 (Vp1), which are genes preferentially expressed in the starchy endosperm and aleurone cells, respectively (Shannon et al, 1998;Cao et al, 2007;Kirchberger et al, 2007). Quantification via real-time RT-PCR showed a 12.8-fold enrichment of Bt1 transcripts in starchy endosperm samples and 29.7-fold enrichment of Vp1 transcripts in aleurone samples, indicating very low cross-contamination between the two cell types ( Figures 1A and 1B).…”

Recent progress toward understanding the role of starch biosynthetic enzymes in the cereal endosperm

“…Although UDP is the preferred nucleoside diphosphate substrate for SuSy, ADP could also be an acceptor molecule of this sucrolytic enzyme to produce ADPGlc [12][13][14]. Cytosolic ADPGlc is then thought to be transported into the amyloplast by means of Brittle-1, a membrane protein located in the envelope membranes of amyloplasts [15], whose absence results in reduced starch content [16,17]. For a summary of the transporters found from the plastid envelope membrane, see [18].…”

“…It starts with individual chains as the first level, which are formed by ADP-glucose through (1→4)-α-glycosidic linkages. Those chains in amylopectin have been further denoted as C chains having the reducing end, A chains (degree of polymerization, DP, [6][7][8][9][10][11][12] carrying no branches, B1 chains (DP [13][14][15][16][17][18][19][20][21][22][23][24] carrying A chains, B2 chains (DP DOI 10.1515/amylase-2017-0006 [25][26][27][28][29][30][31][32][33][34][35][36] carrying B1 chains, B3 chains (DP > 36) carrying B2 chains, and so on (for a recent review, see [2]). Although still occasionally one finds statements to the contrary, amylose is not solely linear, but contains a small but significant number of long-chain branches [3].…”