Transcriptional regulation of plant phosphate transporters.

Phosphorus is acquired by plant roots primarily via the high-affinity inorganic phosphate (Pi) transporters. The transcripts for Pi transporters are highly inducible upon Pi starvation, which also results in enhanced Pi uptake when Pi is resupplied. Using antibodies specific to one of the tomato Pi transporters (encoded by LePT1), we show that an increase in the LePT1 transcript under Pi starvation leads to a concurrent increase in the transporter protein, suggesting a transcriptional regulation for Pi acquisition. LePT1 protein accumulates rapidly in tomato roots in response to Pi starvation. The level of transporter protein accumulation depends on the Pi concentration in the medium, and it is reversible upon resupply of Pi. LePT1 protein accumulates all along the roots under Pi starvation and is localized primarily in the plasma membranes. These results clearly demonstrate that plants increase their capacity for Pi uptake during Pi starvation by synthesis of additional transporter molecules.

Tomato phosphate transporter genes are differentially regulated in plant tissues by phosphorus.

Phosphorus is a major nutrient acquired by roots via high-affinity inorganic phosphate (Pi) transporters. In this paper, we describe the tissue-specific regulation of tomato (Lycopersicon esculentum L.) Pi-transporter genes by Pi. The encoded peptides of the LePT1 and LePT2 genes belong to a family of 12 membrane-spanning domain proteins and show a high degree of sequence identity to known high-affinity Pi transporters. Both genes are highly expressed in roots, although there is some expression of LePT1 in leaves. Their expression is markedly induced by Pi starvation but not by starvation of nitrogen, potassium, or iron. The transcripts are primarily localized in root epidermis under Pi starvation. Accumulation of LePT1 message was also observed in palisade parenchyma cells of Pi-starved leaves. Our data suggest that the epidermally localized Pi transporters may play a significant role in acquiring the nutrient under natural conditions. Divided root-system studies support the hypothesis that signal(s) for the Pi-starvation response may arise internally because of the changes in cellular concentration of phosphorus.

Differential expression of TPS11, a phosphate starvation-induced gene in tomato.

Plants respond to phosphate (Pi) deficiency through adaptive mechanisms involving several morphological, biochemical and molecular changes. In this study, we have characterized the structure and expression of a tomato (Lycopersicon esculentum L.) phosphate starvation-induced gene (TPSI1). A 3.5 kb genomic fragment containing the 474 bp TPSI1 transcript was isolated. The TPSI1 transcript contains an open reading frame of 174 nucleotides encoding a 58 amino acid polypeptide. TPSI1 is an intron-less gene and only one copy could be detected in the tomato genome. The promoter region of TPSI1 contains several conserved sequences found in phosphate starvation induced genes of yeast. The TPSI1 transcripts are rapidly induced in roots and leaves during Pi starvation. A significant increase in the TPSI1 mRNA was observed in cell cultures and roots after 3 and 12 h of Pi starvation, respectively. Induction of the TPSI1 gene appears to be a response specific to Pi starvation since it is not affected by starvation of other nutrients (nitrogen, potassium and iron). The amount of TPSI1 transcript decreased rapidly when Pi-starved tomato plants were resupplied with Pi. These results suggest that TPSI1 gene expression may be a part of the early Pi starvation response mechanism in plants.

Phosphate transporters from the higher plant Arabidopsis thaliana.

Two cDNAs (AtPT1 and AtPT2) encoding plant phosphate transporters have been isolated from a library prepared with mRNA extracted from phosphate-starved Arabidopsis thaliana roots, The encoded polypeptides are 78% identical to each other and show high degree of amino acid sequence similarity with high-affinity phosphate transporters of Saccharomyces cerevisiae, Neurospora crassa, and the mycorrhizal fungus Glomus versiforme. The AtPT1 and AtPT2 polypeptides are integral membrane proteins predicted to contain 12 membrane-spanning domains separated into two groups of six by a large charged hydrophilic region. Upon expression, both AtPT1 and AtPT2 were able to complement the pho84 mutant phenotype of yeast strain NS219 lacking the high-affinity phosphate transport activity. AtPT1 and AtPT2 are representatives of two distinct, small gene families in A. thaliana. The transcripts of both genes are expressed in roots and are not detectable in leaves. The steady-state level of their mRNAs increases in response to phosphate starvation.

Characterization of the unique intron-exon junctions of Euglena gene(s) encoding the polyprotein precursor to the light-harvesting chlorophyll a/b binding protein of photosystem II.

The precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II (LHCPII) is a polyprotein containing multiple copies of LHCPII covalently joined by a decapeptide linker. cDNA and genomic clones encoding the 5' and 3' end of a 6.6 kb LHCPII mRNA were sequenced. A 3.1 kb genomic region encoding 1.05 kb of the 5' end of LHCPII mRNA contains 4 introns. A 7.6 kb genomic region encoding 3.3 kb of the 3' end of LHCPII mRNA contains 10 introns. The 5' and 3' ends of the 14 identified Euglena introns lacked the conserved dinucleotides (5'-GT and AG-3') found at the termini of virtually every characterized nuclear pre-mRNA intron. A common consensus splice site selection sequence could not be identified. The Euglena introns do not have the structural characteristics of group I and group II introns. The only structural feature common to all Euglena introns was the ability of short stretches of nucleotides at the 5' and 3' ends of the introns to base pair, forming a stable stem-loop with the 5' and 3' splice site juxtaposed for splicing but displaced by 2 nucleotides. The 26 nucleotide sequence at the 5' end of LHCPII mRNA is absent from the genomic sequence and identical to the 5' end of one of the small Euglena SL-RNAs indicating that it is post-transcriptionally added by trans-splicing.

The presequence of Euglena LHCPII, a cytoplasmically synthesized chloroplast protein, contains a functional endoplasmic reticulum-targeting domain.

The precursor to the Euglena light-harvesting chlorophyll a/b-binding protein of photosystem II (pLHCPII) is unique; it is a polyprotein, synthesized on membrane-bound ribosomes and transported to the Golgi apparatus prior to chloroplast localization. A cDNA corresponding to the 5' end of LHCPII mRNA has been isolated and sequenced. The deduced amino acid sequence of this cDNA indicates that Euglena pLHCPII contains a 141-amino acid N-terminal extension. The N-terminal extension contains three hydrophobic domains and a potential signal peptidase cleavage site at amino acid 35. Cotranslational processing by canine microsomes removed approximately 35 amino acids from an in vitro synthesized 33-kDa pLHCPII composed of a 141-amino acid N-terminal extension and a 180-amino acid partial LHCPII unit truncated at the beginning of the third membrane-spanning hydrophobic domain. Processed pLHCPII was degraded by exogenous protease, indicating that it had not been translocated to the microsomal lumen. Extraction with 0.1 M Na2CO3, pH 11.5, did not remove the processed pLHCPII from the microsomal membrane. A stop-transfer membrane anchor sequence appears to anchor the nascent protein within the membrane, preventing translocation into the lumen. Taken together, these results provide biochemical evidence for a functional cleaved signal sequence within the N-terminal extension of a Euglena cytoplasmically synthesized chloroplast-localized protein.

Characterization of a Euglena gene encoding a polyprotein precursor to the light-harvesting chlorophyll a/b-binding protein of photosystem II.

A 7.4 kb segment of a Euglena nuclear gene encoding a portion of the polyprotein precursor to the light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCPII) has been isolated and sequenced. Nine exons can be assembled into a continuous open reading frame encoding 113 amino acids of the C-terminus of an LHCPII, followed by 4 complete LHCPIIs. The intron-exon junctions required to maintain maximum amino acid sequence homology between the LHCPIIs encoded by the Euglena genomic clone and Arabidopsis LHCPII do not conform to the consensus splice site sequences present in most eukaryotic organisms. Three types of LHCPIIs are contained within the polyprotein precursor. There is greater than 90% sequence identity at both the protein and nucleic acid level within a type while between types, there is less than 65% sequence identity. All Euglena LHCPIIs contain three hydrophobic membrane-spanning domains in the same positions as found in other LHCPIIs. Hybridization of genomic Southern blots with a probe encoding LHCPII suggests that the Euglena genome encodes a large number (30-50) of LHCPIIs. A probe derived from the 3' end of the sequenced genomic clone hybridizes with equal intensity to 2-3 genomic fragments on Southern blots. The probe encoding LHCPII hybridizes to RNAs of 9.5 and 6.6 kb on northern blots of total RNA while the 3'-end probe hybridizes only to the 6.6 kb RNA. The 6.6 kb LHCPII band detected on northern blots appears to be composed of transcription products derived from 2-3 LHCPII genes. Euglena appears to be unique in that a large number of LHCPIIs are encoded by a small number (3-5) of polyprotein genes.