The tonoplast monosaccharide transporter (TMT) family comprises three isoforms in Arabidopsis thaliana, and TMT-green fluorescent protein fusion proteins are targeted to the vacuolar membrane. TMT promoter-b-glucuronidase plants revealed that the TONOPLAST MONOSACCHARIDE TRANSPORTER1 (TMT1) and TMT2 genes exhibit a tissue-and cell type-specific expression pattern, whereas TMT3 is only weakly expressed. TMT1 and TMT2 expression is induced by drought, salt, and cold treatments and by sugar. During cold adaptation, tmt knockout lines accumulated less glucose and fructose compared with wild-type plants, whereas no differences were observed for sucrose. Cold adaptation of wild-type plants substantially promoted glucose uptake into isolated leaf mesophyll vacuoles. Glucose uptake into isolated vacuoles was inhibited by NH 4 þ , fructose, and phlorizin, indicating that transport is energy-dependent and that both glucose and fructose were taken up by the same carrier. Glucose import into vacuoles from two cold-induced tmt1 knockout lines or from triple knockout plants was substantially lower than into corresponding wild-type vacuoles. Monosaccharide feeding into leaf discs revealed the strongest response to sugar in tmt1 knockout lines compared with wild-type plants, suggesting that TMT1 is required for cytosolic glucose homeostasis. Our results indicate that TMT1 is involved in vacuolar monosaccharide transport and plays a major role during stress responses.
SummaryThe metabolic function of the plastidic ATP/ADP transporter (AATP) in heterotrophic plastids was examined in transgenic potato plants that exhibited increased or decreased amounts of the protein. Altered mRNA levels correlated with activities of the plastidic ATP/ADP transporter. Potato tubers with decreased plastidic ATP/ADP transporter activities exhibited reduced starch contents whereas sense lines accumulated increased amounts of tuber starch. Starch from wild-type tubers had an amylose content of 18.8%, starch from antisense plants contained 11.5-18.0% amylose, whereas starch from sense plants had levels of 22.7-27.0%. The differences in physiological parameters were accompanied with altered tuber morphology. These changes are discussed with respect to the stromal ATP supply during starch biosynthesis.
The Arabidopsis thaliana L. genome contains 58 membrane proteins belonging to the mitochondrial carrier family. Two mitochondrial carrier family members, here named AtNDT1 and AtNDT2, exhibit high structural similarities to the mitochondrial nicotinamide adenine dinucleotide (NAD ؉ ) carrierScNDT1 from bakers' yeast. Expression of AtNDT1 or AtNDT2 restores mitochondrial NAD ؉ transport activity in a yeast mutant lacking ScNDT. Localization studies with green fluorescent protein fusion proteins provided evidence that AtNDT1 resides in chloroplasts, whereas only AtNDT2 locates to mitochondria. Heterologous expression in Escherichia coli followed by purification, reconstitution in proteoliposomes, and uptake experiments revealed that both carriers exhibit a submillimolar affinity for NAD ؉ and transport this compound in a counterexchange mode. Among various substrates ADP and AMP are the most efficient counter-exchange substrates for NAD ؉ .Atndt1-and Atndt2-promoter-GUS plants demonstrate that both genes are strongly expressed in developing tissues and in particular in highly metabolically active cells. The presence of both carriers is discussed with respect to the subcellular localization of de novo NAD ؉ biosynthesis in plants and with respect to both the NAD ؉ -dependent metabolic pathways and the redox balance of chloroplasts and mitochondria.Nucleotides are metabolites of enormous importance for all living cells. They are the essential building blocks for DNA and RNA synthesis, energize most anabolic and many catabolic reactions, and fulfill critical functions in intracellular signal transduction (1, 2). Moreover, nucleotides serve as cofactors for a wide number of enzymes and are, with water, the most highly connected compounds within the metabolic network (3). Among these co-factors nicotinamide adenine dinucleotides are widely used for reductive/oxidative processes, playing important roles in the operation and control of a wide range of dehydrogenase activities. Accordingly, nucleotides are essential in nearly all cell organelles, and transport of these solutes into mitochondria, plastids, the endoplasmic reticulum, the Golgi apparatus, and peroxisomes has been observed (4 -7).Two types of nucleotide transport proteins have been identified to date at the molecular level: nucleotide transporter (NTT) 2 type carriers and members of the mitochondrial carrier family. The former transporters occur in plastids from all plants (8) and in a limited number of intracellular pathogenic bacteria (9). Most NTT-type carrier proteins catalyze an ATP/ADPϩP i counter-exchange mode of transport (10 -13), but several bacterial NTT proteins mediate either H ϩ /nucleotide transport or NAD ϩ /ADP counter-exchange (12,14,15). With the exception of the bacterial NAD ϩ /ADP carrier (14), all NTT proteins exhibit 12 predicted trans-membrane domains, whereas none of the NTT proteins share structural or domain similarities to members of the mitochondrial carrier family (11).Carriers belonging to the mitochondrial carrier family (MC...
Homologs of BT1 (the Brittle1 protein) are found to be phylogenetically related to the mitochondrial carrier family and appear to occur in both mono-and dicotyledonous plants. Whereas BT1 from cereals is probably involved in the transport of ADP-glucose, which is essential for starch metabolism in endosperm plastids, BT1 from a noncereal plant, Solanum tuberosum (StBT1), catalyzes an adenine nucleotide uniport when functionally integrated into the bacterial cytoplasmic membrane. Import studies into intact Escherichia coli cells harboring StBT1 revealed a narrow substrate spectrum with similar affinities for AMP, ADP, and ATP of about 300 -400 M. Transiently expressed StBT1-green fluorescent protein fusion protein in tobacco leaf protoplasts showed a plastidic localization of the StBT1. In vitro synthesized radioactively labeled StBT1 was targeted to the envelope membranes of isolated spinach chloroplasts. Furthermore, we showed by real time reverse transcription-PCR a ubiquitous expression pattern of the StBT1 in autotrophic and heterotrophic potato tissues. We therefore propose that StBT1 is a plastidic adenine nucleotide uniporter used to provide the cytosol and other compartments with adenine nucleotides exclusively synthesized inside plastids.
. 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...
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