Substrate specificity of plastid phosphate transporters in a non-photosynthetic diatom and its implication in evolution of red alga-derived complex plastids.

The triose phosphate transporter (TPT) is one of the prerequisites to exchange metabolites between the cytosol and plastids. In this study, we demonstrated that the four plastid TPT homologues in the non-photosynthetic diatom Nitzschia sp. NIES-3581 were highly likely integrated into plastid envelope membranes similar to counterparts in the model photosynthetic diatom Phaeodactylum tricornutum, in terms of target membranes and C-terminal orientations. Three of the four Nitzschia TPT homologues are capable of transporting various metabolites into proteo-liposomes including triose phosphates (TPs) and phosphoenolpyruvate (PEP), the transport substrates sufficient to support the metabolic pathways retained in the non-photosynthetic diatom plastid. Phylogenetic analysis of TPTs and closely related transporter proteins indicated that diatoms and other algae with red alga-derived complex plastids possess only TPT homologues but lack homologues of the glucose 6-phosphate transporter (GPT), xylulose 5-phosphate transporter (XPT), and phosphoenolpyruvate transporter (PPT). Comparative sequence analysis suggests that many TPT homologues of red alga-derived complex plastids potentially have the ability to transport mainly TPs and PEP. TPTs transporting both TPs and PEP highly likely mediate a metabolic crosstalk between a red alga-derived complex plastid and the cytosol in photosynthetic and non-photosynthetic species, which explains the lack of PPTs in all the lineages with red alga-derived complex plastids. The PEP-transporting TPTs might have emerged in an early phase of endosymbiosis between a red alga and a eukaryote host, given the broad distribution of that type of transporters in all branches of red alga-derived complex plastid-bearing lineages, and have probably played a key role in the establishment and retention of a controllable, intracellular metabolic connection in those organisms.

Molecular identification of the phosphate transporter family 1 (PHT1) genes and their expression profiles in response to phosphorus deprivation and other abiotic stresses in Brassica napus.

Phosphate (Pi) transporters play critical roles in Pi acquisition and homeostasis. However, little is known about these transporters in oilseed rape. Therefore, the aim of the present study was to characterize the members of the PHT1 gene family in allotetraploid Brassica napus and to analyze their expression profiles in response to environmental stresses. In total, 49 PHT1 family members were identified in B. napus, including 27 genes in the A subgenome and 22 in the C subgenome. Most of the PHT1 proteins were predicted to localize to the plasma membrane. Phylogenetic analysis suggested that the members of the PHT1 gene family can be divided into seven clades, with the introns/exons and protein motifs conserved in each clade. Collinearity analysis revealed that most of the BnaPHT1 genes shared syntenic relationships with PHT1 members in Arabidopsis thaliana, B. rapa, and B. oleracea, and that whole-genome duplication (polyploidy) played a major driving force for BnaPHT1 evolution in addition to segmental duplication. Transcript abundance analysis showed that a broad range of expression patterns of individual BnaPHT1 genes occurred in response to phosphorus (P) deficiency. In addition, the expression levels of BnaPHT1 genes can be regulated by different nutrient stresses, including nitrogen (N), potassium (K), sulfur (S) and iron (Fe) stresses. Moveover, salt and drought stresses can regulate the transcript abundances of BnaPHT1s, as well as phytohormones including auxin and cytokinin. Gene coexpression analysis based on the RNA-seq data implied that BnaPHT1s might cooperate with each other as well as with other genes to regulate nutrient homeostasis in B. napus. Further analysis of the promoters revealed that GT-1, DRE and P1BS elements are widely distributed within the promoter regions of BnaPHT1 genes. Our results indicate that BnaPHT1s might be involved in cross-talk for sensing the external status of P, N, K, S and Fe, as well as salt and drought stresses. Moreover, these processes might be mediated by phytohormones. Our findings provide the first step in the complex genetic dissection of the Pi transport system in plants and implicate multiple transcriptional regulation, which probably refers to new roles of PHT1 genes in B. napus.

Isolation and expression analysis of the soybean GmPic gene.

The differential screening method was used to isolate the soy photoperiodic response-related genes and to further elucidate the molecular mechanisms of the soybean photoperiodic response. The light-sensitive species Zhong Dou 24 was used to receive long-time sunshine, short-time sunshine, and natural sunshine treatment. The cDNA-amplified fragment length polymorphism technique was used to screen the differentially expressed cDNA fragments. The rapid amplification of cDNA end technique was used to isolate the gene. Semi-quantitative reverse transcription polymerase chain reaction analysis was used to analyze the gene expression patterns in different light cycles. The gene had a total length of 983 bp, contained a complete open reading frame that encoded 248 amino acids, and shared homology with the mitochondrial phosphate transporter protein. The expression pattern analysis results showed that this gene was expressed in the early stages of soybean growth and development. The short-time sunshine inhibited its expression, whereas the long-time sunshine enhanced its expression. The differential screening method was used to isolate the soybean mitochondrial phosphate transporter gene. The gene may be used as a negative regulatory factor that is involved in the photoperiodic response of soybean.

Localization of a filarial phosphate permease that is up-regulated in response to depletion of essential Wolbachia endobacteria.

Wolbachia of filarial nematodes are essential, obligate endobacteria. When depleted by doxycycline worm embryogenesis, larval development and worm survival are inhibited. The molecular basis governing the endosymbiosis between Wolbachia and their filarial host is still being deciphered. In rodent filarial nematode Litomosoides sigmodontis, a nematode encoded phosphate permease gene (Ls-ppe-1) was up-regulated at the mRNA level in response to Wolbachia depletion and this gene promises to have an important role in Wolbachia-nematode endosymbiosis. To further characterize this gene, the regulation of phosphate permease during Wolbachia depletion was studied at the protein level in L. sigmodontis and in the human filaria Onchocerca volvulus. And the localization of phosphate permease (PPE) and Wolbachia in L. sigmodontis and O. volvulus was investigated in untreated and antibiotic treated worms. Depletion of Wolbachia by tetracycline (Tet) resulted in up-regulation of Ls-ppe-1 in L. sigmodontis. On day 36 of Tet treatment, compared to controls (Con), >98% of Wolbachia were depleted with a 3-fold increase in mRNA levels of Ls-ppe-1. Anti-Ls-PPE serum used in Western blots showed up-regulation of Ls-PPE at the protein level in Tet worms on day 15 and 36 of treatment. Immunohistology revealed the localization of Wolbachia and Ls-PPE in the embryos, microfilariae and hypodermis of L. sigmodontis female worms and up-regulation of Ls-PPE in response to Wolbachia depletion. Expression of O. volvulus phosphate permease (Ov-PPE) studied using anti-Ov-PPE serum, showed up-regulation of Ov-PPE at the protein level in doxycycline treated Wolbachia depleted O. volvulus worms and immunohistology revealed localization of Ov-PPE and Wolbachia and up-regulation of Ov-PPE in the hypodermis and embryos of doxycycline treated worms. Ls-PPE and Ov-PPE are upregulated upon Wolbachia depletion in same tissues and regions where Wolbachia are located in untreated worms, reinforcing a link between Wolbachia and this nematode encoded protein. The function of nematode phosphate permease in the endosymbiosis is unknown but could involve transportation of phosphate to Wolbachia, which encode all the genes necessary for de novo nucleotide biosynthesis. Electron microscopic localization of PPE and Wolbachia and RNAi mediated knock-down of PPE in filarial nematodes will bring further insights to the functions of PPE in the Wolbachia-nematode symbiosis.

[Phosphate permease gene as a marker for the specific identification of toxigenic fungus Fusarium cerealis].

We have developed phosphate permease gene sequence-based PCR detection system of Fusarium cerealis phytopathogenic fungus. Sequencing and analysis revealed that the gene displayed unique polymorphism and could serve to establish phylogenetic relations as well as a marker to design specific primers. The specificity assay has confirmed the absence of cross reactions with DNAs of closely related Fusarium species. The qPCR assay demonstrated the 10 pg detection limit of specific DNA per reaction.

Proteomic analysis of fructophilic properties of osmotolerant Candida magnoliae.

Candida magnoliae, an osmotolerant and erythritol producing yeast, prefers D-fructose to D-glucose as carbon sources. For the investigation of the fructophilic characteristics with respect to sugar transportation, a sequential extraction method using various detergents and ultracentrifugation was developed to isolate cellular membrane proteins in C. magnoliae. Immunoblot analysis with the Pma1 antibody and twodimensional electrophoresis analysis coupled with MS showed that the fraction II was enriched with membrane proteins. Eighteen proteins out of 36 spots were identified as membrane or membrane-associated proteins involved in sugar uptake, stress response, carbon metabolism, and so on. Among them, three proteins were significantly upregulated under the fructose supplying conditions. The hexose transporter was highly homologous to Ght6p in Schizosaccharomyces pombe, which was known as a predominant transporter for the fructose uptake of S. pombe because it exhibited higher affinity to D-fructose than D-glucose. The physicochemical properties of the ATP-binding cassette transporter and inorganic transporter explained their direct or indirect associations with the fructophilic behavior of C. magnoliae. The identification and characterization of membrane proteins involved in sugar uptake might contribute to the elucidation of the selective utilization of fructose to glucose by C. magnoliae at a molecular level.

Crystallization and preliminary X-ray diffraction analysis of human phosphate-binding protein.

Human phosphate-binding protein (HPBP) was serendipitously discovered by crystallization and X-ray crystallography. HPBP belongs to a eukaryotic protein family named DING that is systematically absent from the genomic database. This apoprotein of 38 kDa copurifies with the HDL-associated apoprotein paraoxonase (PON1) and binds inorganic phosphate. HPBP is the first identified transporter capable of binding phosphate ions in human plasma. Thus, it may be regarded as a predictor of phosphate-related diseases such as atherosclerosis. In addition, HPBP may be a potential therapeutic protein for the treatment of such diseases. Here, the purification, detergent-exchange protocol and crystallization conditions that led to the discovery of HPBP are reported.

Redundancy in the function of mitochondrial phosphate transport in Saccharomyces cerevisiae and Arabidopsis thaliana.

Most cellular ATP is produced within the mitochondria from ADP and Pi which are delivered across the inner-membrane by specific nuclearly encoded polytopic carriers. In Saccharomyces cerevisiae, some of these carriers and in particular the ADP/ATP carrier, are represented by several related isoforms that are distinct in their pattern of expression. Until now, only one mitochondrial Pi carrier (mPic) form, encoded by the MIR1 gene in S. cerevisiae, has been described. Here we show that the gene product encoded by the YER053C ORF also participates in the delivery of phosphate to the mitochondria. We have called this gene PIC2 for Pi carrier isoform 2. Overexpression of PIC2 compensates for the mitochondrial defect of the double mutant Deltamir1 Deltapic2 and restores phosphate transport activity in mitochondria swelling experiments. The existence of two isoforms of mPic does not seem to be restricted to S. cerevisiae as two Arabidopsis thaliana cDNAs encoding two different mPic-like proteins are also able to complement the double mutant Deltamir1 Deltapic2. Finally, we demonstrate that Pic2p is a mitochondrial protein and that its steady state level increases at high temperature. We propose that Pic2p is a minor form of mPic which plays a role under specific stress conditions.