Diatoms are ecologically important algae that acquired their plastids by secondary endosymbiosis, resulting in a more complex cell structure and an altered distribution of metabolic pathways when compared with organisms with primary plastids. Diatom plastids are surrounded by 4 membranes; the outermost membrane is continuous with the endoplasmic reticulum. Genome analyses suggest that nucleotide biosynthesis is, in contrast to higher plants, not located in the plastid, but in the cytosol. As a consequence, nucleotides have to be imported into the organelle. However, the mechanism of nucleotide entry into the complex plastid is unknown. We identified a high number of putative nucleotide transporters (NTTs) in the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum and characterized the first 2 isoforms (NTT1 and NTT2). GFP-based localization studies revealed that both investigated NTTs are targeted to the plastid membranes, and that NTT1 most likely enters the innermost plastid envelope via the stroma. Heterologously expressed NTT1 acts as a proton-dependent adenine nucleotide importer, whereas NTT2 facilitates the counter exchange of (deoxy-)nucleoside triphosphates. Therefore, these transporters functionally resemble NTTs from obligate intracellular bacteria with an impaired nucleotide metabolism rather than ATP/ADP exchanging NTTs from primary plastids. We suggest that diatoms harbor a specifically-adapted nucleotide transport system and that NTTs are the key players in nucleotide supply to the complex plastid.chloroplast ͉ complex plastids ͉ nucleotide synthesis ͉ nucleotide transport

Some of the most important bacterial pathogens of humans can only replicate within eukaryotic cells. These obligate intracellular bacteria have developed sophisticated mechanisms to interact with and exploit their hosts. Prime examples of obligate intracellular bacterial pathogens are members of the orders Chlamydiales and Rickettsiales (hereafter referred to as chlamydiae and rickettsiae, respectively). These phylogenetically largely unrelated groups of microorganisms employ nucleotide transport (NTT) proteins, which import nucleotides or allow parasitization of their hosts' energy pool by exchanging bacterial ADP for host ATP (6,10,16,17,26,30,45,51). Among bacteria, NTT proteins are unique to chlamydiae and rickettsiae and were, in addition, only found in plastids of plants and algae (30,39,50,57).NTT proteins have been classified into the ATP/ADP antiporter family AAA by Saier and coworkers (TC number 2.A.12 in the Transport Classification Database [44]). Yet, recent studies showed that NTT proteins comprise transporters with highly dissimilar transport modes and substrate affinities. An alternative classification of NTT proteins according to transport mode was therefore proposed, subdividing the NTT protein family into three classes; class I contains nucleotide antiporters, class II contains proton-driven nucleotide symporters, and class III contains NAD ϩ /ADP antiporters (17). Bacterial and plastidic NTT proteins are fundamentally different from the analogous ADP/ATP carriers of the mitochondrial carrier family with respect to structure and transport characteristics (25,(41)(42)(43)57). In contrast to ATP/ADP translocases of NTT protein family class I, which enable bacterial energy parasitism, mitochondrial ADP/ ATP carriers function in the reverse direction, exporting newly synthesized ATP from the mitochondrial matrix to the host cytosol in exchange for ADP.Using BlastP (2) against the nonredundant protein sequences at GenBank/EMBL/DDBJ in order to find as-yetunrecognized NTT proteins, we recently identified a gene coding for an NTT protein most similar to known chlamydial and rickettsial ATP/ADP translocases in the genome sequence of Lawsonia intracellularis PHE/MN1-00. L. intracellularis is a gram-negative, microaerophilic, obligate intracellular bacterium belonging to the Deltaproteobacteria. L. intracellularis enters the host cell via induced phagocytosis; the phagosome is quickly degraded, and Lawsonia resides directly in the cytoplasm (27). L. intracellularis is an important veterinary pathogen causing proliferative enteropathy (ileitis) in many mammals but mostly in pigs (27,37,46). Proliferative enteropathy is characterized by a progressive proliferation of immature intestinal epithelial cells (enterocytes) following infection with L. intracellularis. The disease, which can persist for several weeks, leads to anorexia, diarrhea, reduced growth of infected animals, and decreased reproductive performance (27,34,46).

Starch in synchronously grown Guillardia theta cells accumulates throughout the light phase, followed by a linear degradation during the night. In contrast to the case for other unicellular algae such as Chlamydomonas reinhardtii, no starch turnover occurred in this organism under continuous light. The gene encoding granulebound starch synthase (GBSS1), the enzyme responsible for amylose synthesis, displays a diurnal expression cycle. The pattern consisted of a maximal transcript abundance around the middle of the light phase and a very low level during the night. This diurnal regulation of GBSS1 transcript abundance was demonstrated to be independent of the circadian clock but tightly light regulated. A similar yet opposite type of regulation pattern was found for two ␣-amylase isoforms and for one of the two plastidic triose phosphate transporter genes investigated. In these cases, however, the transcript abundance peaked in the night phase. The second plastidic triose phosphate transporter gene had the GBSS1 mRNA abundance pattern. Quantification of the GBSS1 activity revealed that not only gene expression but also total enzyme activity exhibited a maximum in the middle of the light phase. To gain a first insight into the transport processes involved in starch biosynthesis in cryptophytes, we demonstrated the presence of both plastidic triose phosphate transporter and plastidic ATP/ADP transporter activities in proteoliposomes harboring either total membranes or plastid envelope membranes from G. theta. These molecular and biochemical data are discussed with respect to the environmental conditions experienced by G. theta and with respect to the unique subcellular location of starch in cryptophytes.

SummaryDiatom plastids show several peculiarities when compared with primary plastids of higher plants or algae. They are surrounded by four membranes and depend on nucleotide uptake because, unlike in plants, nucleotide de novo synthesis exclusively occurs in the cytosol. Previous analyses suggest that two specifically adapted nucleotide transporters (NTTs) facilitate the required passage of nucleotides across the innermost plastid membrane. However, nucleotide transport across the additional plastid membranes remains to be clarified.Phylogenetic studies, transport assays with the recombinant protein as well as GFP-based targeting analyses allowed detailed characterization of a novel isoform (PtNTT5) of the six NTTs of Phaeodactylum tricornutum.PtNTT5 exhibits low amino acid similarities and is only distantly related to all previously characterized NTTs. However, in a heterologous expression system, it acts as a nucleotide antiporter and prefers various (deoxy-) purine nucleotides as substrates. Interestingly, PtNTT5 is probably located in the endoplasmic reticulum, which in diatoms also represents the outermost plastid membrane.PtNTT5, with its unusual transport properties, phylogeny and localization, can be taken as further evidence for the establishment of a sophisticated and specifically adapted nucleotide transport system in diatom plastids.

Isoamylases hydrolyse (1–6)-alpha-D-glucosidic linkages in starch and are involved in both starch granule formation and starch degradation. In plants, three isoamylase isoforms with distinct functions in starch synthesis (ISA1 and ISA2) and degradation (ISA3) have been described. Here, we created transgenic potato plants with simultaneously decreased expression of all three isoamylases using a chimeric RNAi construct targeting all three isoforms. Constitutive expression of the hairpin RNA using the 35S CaMV promoter resulted in efficient silencing of all three isoforms in leaves, growing tubers, and sprouting tubers. Neither plant growth nor tuber yield was effected in isoamylase-deficient potato lines. Interestingly, starch metabolism was found to be impaired in a tissue-specific manner. While leaf starch content was unaffected, tuber starch was significantly reduced. The reduction in tuber starch content in the transgenic plants was accompanied by a decrease in starch granules size, an increased sucrose content and decreased hexose levels. Despite the effects on granule size, only little changes in chain length composition of soluble and insoluble glucose polymers were detected. The transgenic tubers displayed an early sprouting phenotype that was accompanied by an increased level of sucrose in parenchyma cells below the outgrowing bud. Since high sucrose levels promote sprouting, we propose that the increased number of small starch granules may cause an accelerated turnover of glucan chains and hence a more rapid synthesis of sucrose. This observation links alterations in starch structure/degradation with developmental processes like meristem activation and sprout outgrowth in potato tubers.