POT1 proteins in green algae and land plants: DNA-binding properties and evidence of co-evolution with telomeric DNA

Telomeric DNA terminates with a single-stranded 3′ G-overhang that in vertebrates and fission yeast is bound by POT1 (Protection Of Telomeres). However, no in vitro telomeric DNA binding is associated with Arabidopsis POT1 paralogs. To further investigate POT1–DNA interaction in plants, we cloned POT1 genes from 11 plant species representing major branches of plant kingdom. Telomeric DNA binding was associated with POT1 proteins from the green alga Ostreococcus lucimarinus and two flowering plants, maize and Asparagus. Site-directed mutagenesis revealed that several residues critical for telomeric DNA recognition in vertebrates are functionally conserved in plant POT1 proteins. However, the plant proteins varied in their minimal DNA-binding sites and nucleotide recognition properties. Green alga POT1 exhibited a strong preference for the canonical plant telomere repeat sequence TTTAGGG with no detectable binding to hexanucleotide telomere repeat TTAGGG found in vertebrates and some plants, including Asparagus. In contrast, POT1 proteins from maize and Asparagus bound TTAGGG repeats with only slightly reduced affinity relative to the TTTAGGG sequence. We conclude that the nucleic acid binding site in plant POT1 proteins is evolving rapidly, and that the recent acquisition of TTAGGG telomere repeats in Asparagus appears to have co-evolved with changes in POT1 DNA sequence recognition

Heterologous Expression of Secreted Bacterial BPP and HAP Phytases in Plants Stimulates Arabidopsis thaliana Growth on Phytate

Phytases are specialized phosphatases capable of releasing inorganic phosphate from myo-inositol hexakisphosphate (phytate), which is highly abundant in many soils. As inorganic phosphorus reserves decrease over time in many agricultural soils, genetic manipulation of plants to enable secretion of potent phytases into the rhizosphere has been proposed as a promising approach to improve plant phosphorus nutrition. Several families of biotechnologically important phytases have been discovered and characterized, but little data are available on which phytase families can offer the most benefits toward improving plant phosphorus intake. We have developed transgenic Arabidopsis thaliana plants expressing bacterial phytases PaPhyC (HAP family of phytases) and 168phyA (BPP family) under the control of root-specific inducible promoter Pht1;2. The effects of each phytase expression on growth, morphology and inorganic phosphorus accumulation in plants grown on phytate hydroponically or in perlite as the only source of phosphorus were investigated. The most enzymatic activity for both phytases was detected in cell wall-bound fractions of roots, indicating that these enzymes were efficiently secreted. Expression of both bacterial phytases in roots improved plant growth on phytate and resulted in larger rosette leaf area and diameter, higher phosphorus content and increased shoot dry weight, implying that these plants were indeed capable of utilizing phytate as the source of phosphorus for growth and development. When grown on phytate the HAP-type phytase outperformed its BPP-type counterpart for plant biomass production, though this effect was only observed in hydroponic conditions and not in perlite. Furthermore, we found no evidence of adverse side effects of microbial phytase expression in A. thaliana on plant physiology and seed germination. Our data highlight important functional differences between these members of bacterial phytase families and indicate that future crop biotechnologies involving such enzymes will require a very careful evaluation of phytase source and activity. Overall, our data suggest feasibility of using bacterial phytases to improve plant growth in conditions of phosphorus deficiency and demonstrate that inducible expression of recombinant enzymes should be investigated further as a viable approach to plant biotechnology

\u3cem\u3eSelaginella moellendorffii\u3c/em\u3e telomeres: conserved and unique features in an ancient land plant lineage

Telomeres, the essential terminal regions of linear eukaryotic chromosomes, consist of G-rich DNA repeats bound by a plethora of associated proteins. While the general pathways of telomere maintenance are evolutionarily conserved, individual telomere complex components show remarkable variation between eukaryotic lineages and even within closely related species. The recent genome sequencing of the lycophyte Selaginella moellendorffii and the availability of an ever-increasing number of flowering plant genomes provides a unique opportunity to evaluate the molecular and functional evolution of telomere components from the early evolving non-seed plants to the more developmentally advanced angiosperms. Here we analyzed telomere sequence in S. moellendorffii and found it to consist of TTTAGGG repeats, typical of most plants. Telomere tracts in S. moellendorffii range from 1 to 5.5 kb, closely resembling Arabidopsis thaliana. We identified several S. moellendorffii genes encoding sequence homologs of proteins involved in telomere maintenance in other organisms, including CST complex components and the telomere-binding proteins, POT1 and the TRFL family. Notable sequence similarities and differences were uncovered among the telomere-related genes in some of the plant lineages. Taken together, the data indicate that comparative analysis of the telomere complex in early diverging land plants such as S. moellendorffii and green algae will yield important insights into the evolution of telomeres and their protein constituents

Non-Radioactive TRF Assay Modifications to Improve Telomeric DNA Detection Efficiency in Plants

The length of telomeric DNA is often considered a cellular biomarker of aging and general health status. Several telomere length measuring assays have been developed, of which the most common is the telomere restriction fragment (TRF) analysis, which typically involves the use of radioactively labeled oligonucleotide probes. While highly effective, this method potentially poses substantial health concerns and generates radioactive waste. Digoxigenin (DIG) alternatives to radioactive probes have been developed and used successfully in a number of assays. Here, we optimize the DIG protocol to measure telomere length in the model plant Arabidopsis thaliana and present evidence that this approach can be used successfully to efficiently and accurately measure telomere length in plants. Specifically, hybridization temperature of 42 °C instead of the typical 55 °C appears to generate stronger signals. In addition, DIG incorporation at 5′-end instead of 3′-end of the labeled oligonucleotide greatly enhances signal. We conclude that non-radioactive TRF assays can be as efficient as radioactive methods in detecting and measuring telomere length in plants, making this assay suitable for medical and research laboratories unable to utilize radioactivity due to hazardous waste disposal and safety concerns

The Arabidopsis Pot1 and Pot2 Proteins Function in Telomere Length Homeostasis and Chromosome End Protection

Pot1 (protection of telomeres 1) is a single-stranded telomere binding protein that is essential for chromosome end protection and telomere length homeostasis. Arabidopsis encodes two Pot1-like proteins, dubbed AtPot1 and AtPot2. Here we show that telomeres in transgenic plants expressing a truncated AtPot1 allele lacking the N-terminal oligonucleotide/oligosaccharide binding fold (P1ΔN) are 1 to 1.5 kb shorter than in the wild type, suggesting that AtPot1 contributes to the positive regulation of telomere length control. In contrast, telomere length is unperturbed in plants expressing the analogous region of AtPot2. A strikingly different phenotype is observed in plants overexpressing the AtPot2 N terminus (P2ΔC) but not the corresponding region in AtPot1. Although bulk telomeres in P2ΔC mutants are 1 to 2 kb shorter than in the wild type, these plants resemble late-generation telomerase-deficient mutants with severe growth defects, sterility, and massive genome instability, including bridged chromosomes and aneuploidy. The genome instability associated with P2ΔC mutants implies that AtPot2 contributes to chromosome end protection. Thus, Arabidopsis has evolved two Pot genes that function differently in telomere biology. These findings provide unanticipated information about the evolution of single-stranded telomere binding proteins

An Improved Gene Expression System to Generate Transgenic Arabidopsis Thaliana Plants Harboring a Bacillus Ginsengihumi Phytase Gene

We constructed a new vector system for heterologous gene expression in Arabidopsis thaliana. The construct contains a codon-optimized sequence encoding Bacillus ginsengihumi phytase behind an inducible plant-specific promoter for expression in root epithelial cells. The new vector introduced into the plant A. thaliana by Agrobacterium mediated transformation. We obtained several generations of transgenic A. thaliana plants with integrated Bacillus ginsengihumi phytase gene, as well as with an empty vector as a negative control. We tested several transgenic plants harboring the phyCg construct under the control of phosphate-starvation inducible Pht1;2 promoter and show that the phyCg gene is expressed at the mRNA level. Further characterization of these lines of plants will help us to design an improved transgenic strategy for the development of a root-specific heterologous system for the expression of bacterial phytases in plants

Analysis of \u3cem\u3eCarica papaya\u3c/em\u3e Telomeres and Telomere-Associated Proteins: Insights into the Evolution of Telomere Maintenance in Brassicales

Telomeres are terminal regions of linear eukaryotic chromosomes that are critical for genome stability and continued cell proliferation. The draft assembly of the papaya genome provides an opportunity to analyze and compare the evolution of telomeric DNA sequence composition and telomere maintenance machinery in this and other organisms of the Brassicales Order, which includes Arabidopsis. Here we investigate telomere size and sequence variation at papaya chromosome ends. As with most other plant species, papaya telomeres consist of TTTAGGG repeats. However, in contrast to members of the closely related Brassicaceae family, telomeres in papaya are ~10-fold longer. Sequence analysis reveals that many centromereproximal telomere repeats in papaya harbor nucleotide substitutions and insertions of Gs and Ts. In contrast, we found very few N-to-C substitutions, and even fewer instances of nucleotide deletion, suggesting that a six-nucleotide telomere repeat is not well tolerated. The papaya genome encodes single-copy sequence homologues of several genes involved in telomere maintenance and chromosome end protection, including the Telomerase Reverse Transcriptase (TERT) and Protection Of Telomeres (POT1). Notably, unlike Arabidopsis, which encodes six Telomere Repeat binding Factor-like (TRFL) proteins that bind double-stranded telomere DNA, papaya appears to encode only two such proteins. Thus, the more streamlined genome of papaya will provide an excellent resource for comparative and functional analysis of telomeres in plants

A C-terminal Myb extension domain defines a novel family of double-strand telomeric DNA-binding proteins in Arabidopsis

Little is known about the protein composition of plant telomeres. We queried the Arabidopsis thaliana genome data base in search of genes with similarity to the human telomere proteins hTRF1 and hTRF2. hTRF1/hTRF2 are distinguished by the presence of a single Myb-like domain in their C terminus that is required for telomeric DNA binding in vitro. Twelve Arabidopsis genes fitting this criterion, dubbed TRF-like (TRFL), fell into two distinct gene families. Notably, TRFL family 1 possessed a highly conserved region C-terminal to the Myb domain called Myb-extension (Myb-ext) that is absent in TRFL family 2 and hTRF1/hTRF2. Immunoprecipitation experiments revealed that recombinant proteins from TRFL family 1, but not those from family 2, formed homodimers and heterodimers in vitro. DNA binding studies with isolated C-terminal fragments from TRFL family 1 proteins, but not family 2, showed specific binding to double-stranded plant telomeric DNA in vitro. Removal of the Myb-ext domain from TRFL1, a family 1 member, abolished DNA binding. However, when the Myb-ext domain was introduced into the corresponding region in TRFL3, a family 2 member, telomeric DNA binding was observed. Thus, Myb-ext is required for binding plant telomeric DNA and defines a novel class of proteins in Arabidopsis

Selection of Efficient Taq DNA Polymerase to Optimize T-DNA Genotyping Method for Rapid Detection of Mutant Arabidopsis thaliana Plants

Plants harbor homologues of various animal genes involved in phosphorus metabolism, telomere biology and other cellular processes. Compared to experiments with many other multicellular organisms, research in the model plant Arabidopsis thaliana takes advantage of short generation time and an ever increasing arsenal of genetic and transgenic tools, including large collections of T-DNA knockout and activation lines. The availability of thousands of publicly available transgenic Arabidopsis lines provides a unique opportunity to address a number of important biological questions. However, identification of individual T-DNA mutant plants from a pool of seeds provided by a biological stock distribution center remains a laborious and time-consuming procedure. Here we compared a number of commercial Taq DNA polymerases commonly used for routine PCR genotyping to identify a single polymerase most suitable for genotyping T-DNA mutant plants. Our data indicate that Emerald Amp GT PCR Master Mix provides the most reliable, quick and simple DNA genotyping tool to determine the presence of a T-DNA insertion and to establish whether an individual A. thaliana plant is heterozygous or homozygous for the mutant allele