Simultaneous silencing of isoamylases ISA1, ISA2 and ISA3 by multi-target RNAi in potato tubers leads to decreased starch content and an early sprouting phenotype.

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.

Shuttling of (deoxy-) purine nucleotides between compartments of the diatom Phaeodactylum tricornutum.

Diatom 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.

The endosymbiont Amoebophilus asiaticus encodes an S-adenosylmethionine carrier that compensates for its missing methylation cycle.

All organisms require S-adenosylmethionine (SAM) as a methyl group donor and cofactor for various biologically important processes. However, certain obligate intracellular parasitic bacteria and also the amoeba symbiont Amoebophilus asiaticus have lost the capacity to synthesize this cofactor and hence rely on its uptake from host cells. Genome analyses revealed that A. asiaticus encodes a putative SAM transporter. The corresponding protein was functionally characterized in Escherichia coli: import studies demonstrated that it is specific for SAM and S-adenosylhomocysteine (SAH), the end product of methylation. SAM transport activity was shown to be highly dependent on the presence of a membrane potential, and by targeted analyses, we obtained direct evidence for a proton-driven SAM/SAH antiport mechanism. Sequence analyses suggest that SAM carriers from Rickettsiales might operate in a similar way, in contrast to chlamydial SAM transporters. SAM/SAH antiport is of high physiological importance, as it allows for compensation for the missing methylation cycle. The identification of a SAM transporter in A. asiaticus belonging to the Bacteroidetes phylum demonstrates that SAM transport is more widely spread than previously assumed and occurs in bacteria belonging to three different phyla (Proteobacteria, Chlamydiae, and Bacteroidetes).

Diatom plastids depend on nucleotide import from the cytosol.

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.

Lawsonia intracellularis contains a gene encoding a functional rickettsia-like ATP/ADP translocase for host exploitation.

ATP/ADP translocases are a hallmark of obligate intracellular pathogens related to chlamydiae and rickettsiae. These proteins catalyze the highly specific exchange of bacterial ADP against host ATP and thus allow bacteria to exploit their hosts' energy pool, a process also referred to as energy parasitism. The genome sequence of the obligate intracellular pathogen Lawsonia intracellularis (Deltaproteobacteria), responsible for one of the most economically important diseases in the swine industry worldwide, revealed the presence of a putative ATP/ADP translocase most similar to known ATP/ADP translocases of chlamydiae and rickettsiae (around 47% amino acid sequence identity). The gene coding for the putative ATP/ADP translocase of L. intracellularis (L. intracellularis nucleotide transporter 1 [NTT1(Li)]) was cloned and expressed in the heterologous host Escherichia coli. The transport properties of NTT1(Li) were determined by measuring the uptake of radioactively labeled substrates by E. coli. NTT1(Li) transported ATP in a counterexchange mode with ADP in a highly specific manner; the substrate affinities determined were 236.3 (+/- 36.5) microM for ATP and 275.2 (+/- 28.1) microM for ADP, identifying this protein as a functional ATP/ADP translocase. NTT1(Li) is the first ATP/ADP translocase from a bacterium not related to Chlamydiae or Rickettsiales, showing that energy parasitism by ATP/ADP translocases is more widespread than previously recognized. The occurrence of an ATP/ADP translocase in L. intracellularis is explained by a relatively recent horizontal gene transfer event with rickettsiae as donors.

Molecular and biochemical analysis of periplastidial starch metabolism in the cryptophyte Guillardia theta.

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 granule-bound 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 alpha-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.