Stephan Eberhard
email: stephan.eberhard@ibpc.fr

Sona Valuchova

Julie Ravat

Jaroslav Fulneček

Pascale Jolivet

Sandrine Bujaldon

Stéphane Lemaire

Francis-André Wollman

Maria Teresa Teixeira

Karel Riha

Jaroslav Fulne Ček

St

D Lemaire

Teresa Teixeira

Xu

Molecular characterization of Chlamydomonas reinhardtii telomeres and telomerase mutants

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Photosynthetic algae are in the highlight of basic and applied research, not only because of their core role for Earth's biosphere in oxygen evolution and carbon fixation but also because of their increased use in biotechnology for the production of proteins, bulk chemicals, and high-value molecules [START_REF] Scaife | Establishing Chlamydomonas reinhardtii as an industrial biotechnology host[END_REF][START_REF] Scranton | Chlamydomonas as a model for biofuels and bio-products production[END_REF]. Thus, a detailed understanding of algal physiology, including their cell cycle, cell growth, and genome integrity, is of critical importance. Chlamydomonas reinhardtii, also referred to as the "photosynthetic yeast" [START_REF] Rochaix | Chlamydomonas reinhardtii as the photosynthetic yeast[END_REF], is the most prominent model organism in the green algae lineage. It is widely used for biotechnological applications as well as to study fundamental processes, such as photosynthesis and cilia structure and function [START_REF] Harris | Chlamydomonas as a model organism[END_REF][START_REF] Sasso | From molecular manipulation of domesticated Chlamydomonas reinhardtii to survival in nature[END_REF]. It has a fully sequenced nuclear genome of 111 Mb distributed over 17 chromosomes [START_REF] Merchant | The Chlamydomonas genome reveals the evolution of key animal and plant functions[END_REF] and is amenable to powerful genetic approaches [START_REF] Harris | The Chlamydomonas Sourcebook (Second Edition)[END_REF]. Although genetic transformation is available in this organism, targeted nuclear genome modification is still not straightforward, but the emerging CRISPR/Cas9 technology might help solve this limitation [START_REF] Findinier | The dynamin-like protein Fzl promotes thylakoid fusion and resistance to light stress in Chlamydomonas reinhardtii[END_REF].

In eukaryotes, telomeres are repeated sequences found at the extremities of linear chromosomes. They are important for chromosome integrity and may limit cell proliferation capacity in some organisms. By progressively shortening with each cell cycle because of the end replication problem, telomeres eventually become too short and trigger a cell cycle arrest termed replicative senescence [START_REF] Lundblad | A mutant with a defect in telomere elongation leads to senescence in yeast[END_REF][START_REF] Harley | Telomeres shorten during ageing of human fibroblasts[END_REF]. Most unicellular eukaryotes and germ, stem, and cancer cells in multicellular organisms counteract telomere shortening by expressing telomerase, an enzyme that adds de novo telomere sequences and allows for an unlimited proliferation potential [START_REF] Pfeiffer | Replication of telomeres and the regulation of telomerase[END_REF][START_REF] Wu | Telomerase mechanism of telomere synthesis[END_REF]. Despite the crucial functions of telomeres and telomerase in maintaining genome stability and controlling cell proliferation in many model organisms, including plants, ciliates, fungi, and mammals [START_REF] Fulcher | If the cap fits, wear it: An overview of telomeric structures over evolution[END_REF], telomere biology in algae remains to be investigated in depth.

To our knowledge, only a handful of studies on C. reinhardtii telomeres have been published. Early studies published in the 90s showed that (i) C. reinhardtii telomeres are composed of TTTTAGGG repeats, which are different from the Arabidopsis-type TTTAGGG sequence [START_REF] Petracek | Chlamydomonas telomere sequences are A+T-rich but contain three consecutive G-C basepairs[END_REF]; (ii) the size of cloned telomeric repeats ranges from 300 to 600 bp [START_REF] Petracek | Chlamydomonas telomere sequences are A+T-rich but contain three consecutive G-C basepairs[END_REF][START_REF] Hails | Isolation of a Chlamydomonas reinhardtii telomere by functional complementation in yeast[END_REF]; (iii) they form G-quadruplex structures in vitro [START_REF] Petracek | Chlamydomonas reinhardtii telomere repeats form unstable structures involving guanine-guanine base pairs[END_REF]; and (iv) the Gbp1 protein binds in vitro to singlestranded telomere sequences through two RNA recognition motifs, with a preference for RNA when Gbp1 is monomeric and for DNA when it is dimeric [START_REF] Petracek | A Chlamydomonas protein that binds single-stranded G-strand telomere DNA[END_REF]Johnston et al, 1999). More recently, bioinformatics studies focused on the evolutionary relationships of telomere sequences in green algae [START_REF] Fulnečkov Á | Transitions between the Arabidopsis-type and the human-type telomere sequence in green algae (clade Caudivolvoxa, Chlamydomonadales)[END_REF][START_REF] Fulnečkov Á | Dynamic evolution of telomeric sequences in the green algal order Chlamydomonadales[END_REF]. Finally, a broad study of telomerase activity in green algae revealed that telomerase activity in C. reinhardtii extracts is low or not detectable (Fulnečkov á et al, 2013).

To gain a better understanding of C. reinhardtii telomere structure and maintenance, we investigated telomere sequence and end structure, analyzed telomere length distribution across different reference strains, identified CrTERT, the gene encoding the catalytic subunit of telomerase, and provided a genetic analysis of telomerase function, thus opening new avenues of research on telomere dynamics, proliferation potential, and genome integrity in C. reinhardtii.

Results

C. reinhardtii telomeric repeats are mostly nondegenerate with few low-frequency variants

In their seminal article, [START_REF] Petracek | Chlamydomonas telomere sequences are A+T-rich but contain three consecutive G-C basepairs[END_REF] cloned and sequenced a limited number of C. reinhardtii telomeric repeats, revealing their canonical TTTTAGGG sequence [START_REF] Petracek | Chlamydomonas telomere sequences are A+T-rich but contain three consecutive G-C basepairs[END_REF]. Telomeric repeats are also identifiable in 18 of 34 chromosome ends on the available v5.5 genome sequence of C. reinhardtii (https:// phytozome.jgi.doe.gov; Fig S1A). As the sequenced genome shows some telomeric repeat variations, we analyzed telomeric repeat sequences on a larger scale and looked for putative variants of the canonical telomere sequence. We amplified telomeres by a PCRbased method [START_REF] Forstemann | Telomerase-dependent repeat divergence at the 3' ends of yeast telomeres[END_REF] using a forward primer specific to a conserved subtelomere-telomere junction common to 10 telomeres from eight different chromosomes (Fig S1A and B). The reverse primer was universal and annealed to a sequence of cytosines artificially added at the 39 end of the telomeres by terminal transferase reaction. After cloning into a plasmid and sequencing, we analyzed 32 telomere sequences, encompassing 709 repeats. We found that ~90% (n = 636) of the repeats corresponded to the canonical sequence TTTTAGGG. We also detected variants such as TTTAGGG (corresponding to the canonical Arabidopsis thaliana sequence, n = 37; either at the subtelomere-telomere junction, n = 24; or elsewhere, n = 13) or TTTTTAGGG (n = 13) and TTTTGGG (n = 8) (Table 1 andFig S1B). These three variants were found in at least two independent clones at the same position in the telomere sequence, thus likely representing true low-frequency variants and not sequencing errors. We also detected sequence variants that occurred only in single clones (n = 15) and for which PCR and/or sequencing errors can, therefore, not be ruled out. We conclude that C. reinhardtii telomeric repeats are mostly nondegenerate with few lowfrequency variants.

C. reinhardtii telomeres form a protective structure and a subset ends with a 39 overhang, whereas another subset bears blunt ends

The protective structure formed by telomeric DNA bound by specific proteins is critical for telomere functions [START_REF] Palm | How shelterin protects mammalian telomeres[END_REF]. To test the presence of such a structure at C. reinhardtii telomeres, we performed a micrococcal nuclease (MNase) digestion of nuclei and asked whether telomere DNA would be protected from its activity. When nuclei were subjected to increasing amounts of MNase, nucleosomal DNA was protected from digestion and migrated at ~150 bp based on ethidium bromide staining (Fig 1A,left), as expected [START_REF] Clark | Nucleosome positioning, nucleosome spacing and the nucleosome code[END_REF]. Intermediate digestion products migrated in a typical ladder pattern corresponding to di-nucleosomes, trinucleosomes, and higher order structures (Fig 1A, left and middle, asterisks in the lane with one unit of MNase). Strikingly, Southern blot hybridization with a radioactive telomeric probe revealed a diffuse pattern, suggesting that telomeric DNA was protected from MNase digestion in a noncanonical manner (Fig 1A,right). As a control, the same membrane was stripped and probed for 18S rDNA, which showed the canonical nucleosome structure (Fig 1A,middle,asterisks). The size of the protected telomeric DNA was in the range of 200-700 bp, which could correspond to the full telomere length. This result suggests that telomeric DNA might be fully associated with and protected by a noncanonical nucleosomal structure or by other protein complexes, similar to telosomes as observed in yeasts, for example [START_REF] Wright | Saccharomyces telomeres assume a non-nucleosomal chromatin structure[END_REF][START_REF] Greenwood | Fission yeast telosomes: Noncanonical histone-containing chromatin structures dependent on shelterin and RNA[END_REF].

The chromosome end structure determines the protection strategies used to cap the telomere. In many species, telomeres end with a 59 to 39 single-stranded overhang, important for the t-loop structure in human telomeres, telomerase recruitment, and binding of specific capping proteins, such as the CST and Ku complexes and POT1 [START_REF] Palm | How shelterin protects mammalian telomeres[END_REF][START_REF] Teixeira | CST meets shelterin to keep telomeres in check[END_REF][START_REF] Wellinger | Everything you ever wanted to know about Saccharomyces cerevisiae telomeres: Beginning to end[END_REF]. As it was reported that the Gbp1 protein preferentially binds single-stranded C. reinhardtii telomeric DNA (Johnston et al, 1999), the presence of a 39 overhang would be consistent with a role of Gbp1 at telomeres, possibly protecting them from degradation and fusions similarly to telomere capping proteins in other species. To experimentally test the presence of a 39 overhang at C. reinhardtii telomeres, we performed primer extension telomere repeat amplification (PETRA) [START_REF] Heacock | Molecular analysis of telomere fusions in Arabidopsis: Multiple pathways for chromosome end-joining[END_REF]. PETRA requires the annealing of an adaptor primer (PETRA-T) to the overhang. After primer extension, the telomere was PCR-amplified using a unique subtelomeric forward primer and a reverse primer (PETRA-A) complementary to a tag sequence present in PETRA-T (Figs 1B and S1C). Successful amplification by PETRA is indicative of the presence of a 39 overhang. Using primers specific for three different telomeres (1R, 9R, and 10R), we found robust amplification of PETRA products in three C. reinhardtii strains (CC4350+, T222+, and CC125+), strongly suggesting that these telomeres have a 39 overhang of at least 12 nucleotides, corresponding to the size of the annealed part of PETRA-T to the overhang (Figs 1B and S1C). Control with prior treatment of the samples with exonuclease I (ExoI) showed a decreased PETRA efficiency, indicating that the PETRA-T primer indeed requires 39 overhang for annealing. The presence of a 39 overhang at telomeres was further confirmed by native in-gel hybridization assay, which detected exonuclease T (ExoT)-dependent singlestranded DNA on the G-rich strand of the telomeres of strain T222+, but not on the C-rich strand (Figs 1C and S1D).

As it was shown that a subset of A. thaliana telomeres displays blunt ends instead of 39 overhangs [START_REF] Kazda | Chromosome end protection by blunt-ended telomeres[END_REF], we asked whether blunt-ended telomeres also exist in C. reinhardtii because the PETRA and in-gel experiments do not exclude this possibility. To test this, we applied the hairpin assay, which was successfully used in A. thaliana to detect blunt-ended telomeres [START_REF] Kazda | Chromosome end protection by blunt-ended telomeres[END_REF]. Briefly, a synthetic hairpin DNA can be ligated to both strands of the telomeres, only if they are blunt ended. After digestion with AluI at a site in the subtelomeres, the ligated products migrate as a doublesized fragment compared with the unligated control in denaturing conditions. Cleavage of the ligated product by BamHI, using a restriction site designed in the hairpin, can then show that the slow migrating product was indeed generated by ligation to the hairpin (Fig 1D,left). Ligation products of a higher molecular weight were clearly detected using this hairpin assay in C. reinhardtii strain CC4350+ as in A. thaliana (Fig 1D,right,arrows) and they were abolished by cleaving the hairpin by BamHI or pretreatment with T7 exonuclease that generates 39 single-stranded DNA protrusions at DNA ends. This demonstrates that a fraction of telomeres in C. reinhardtii is blunt ended.

Taken together, our structural analysis of telomeres indicates that as in A. thaliana, chromosome ends in C. reinhardtii are composed of two subsets, one ending with a 39 overhang and the other with a blunt end.

Terminal restriction fragment (TRF) analysis of C. reinhardtii telomeres

To study telomere length distributions and their possible regulations, we optimized a TRF analysis for C. reinhardtii to accurately measure telomere length from populations of cells.

We first measured telomere length in three independent biological replicates of strains T222+ and S24-, two isogenic reference strains differing only in their mating type [START_REF] Gallaher | Chlamydomonas genome resource for laboratory strains reveals a mosaic of sequence variation, identifies true strain histories, and enables strain-specific studies[END_REF]. We found that telomere fragments spread as a smear over a large range of lengths, from ~200 to ~1,200 bp (Fig 1E). The two strains displayed a significant difference in their average telomere length (mean ± SD: T222+ = 539 ± 54 bp; N = 18 and S24-= 710 ± 12 bp; N = 5). To demonstrate that the detected smeary signal corresponded to terminal fragments, we digested the genomic DNA (gDNA) with exonuclease Bal31 before restriction digestion [START_REF] Fajkus | Techniques in plant telomere biology[END_REF] and indeed observed that with increasing incubation times with Bal31, the signal progressively decreased in size until it nearly disappeared after 10 min (Figs 1F and S1E). The migration of a band at ~200 bp was not altered even with the longest Bal31 treatment, indicating that it stemmed from interstitial telomere repeats located within the genome. Because this sharp band did not cross-react with a probe targeting TG microsatellite sequences (Fig S1F), it most probably corresponded to bona fide telomere sequence-containing region(s) of the genome and not to nonspecific cross hybridizations.

Telomere length distribution is stable in different standard growth conditions C. reinhardtii has been widely used as a model organism to study photosynthetic processes because of its ability to grow in different metabolic regimes [START_REF] Harris | The Chlamydomonas Sourcebook (Second Edition)[END_REF]. Under strictly phototrophic conditions (minimum medium in the light), photosynthesis is the only metabolic process providing ATP and reducing power to growing cells. In strictly heterotrophic conditions in the dark, C. reinhardtii can survive by respiring the acetate contained in Trisacetate-phosphate (TAP) medium. In mixotrophic conditions, that is, TAP medium in the light, cells use a combination of photosynthesis and respiration to grow. Because in other organisms, environmental conditions can regulate telomere length [START_REF] Walmsley | Genetic control of chromosome length in yeast[END_REF][START_REF] Von Zglinicki | Role of oxidative stress in telomere length regulation and replicative senescence[END_REF][START_REF] Epel | Accelerated telomere shortening in response to life stress[END_REF][START_REF] Romano | Environmental stresses disrupt telomere length homeostasis[END_REF], we asked whether telomeres vary in length and/or size distributions in response to different standard growth conditions.

We first tested whether cells displayed different telomere lengths during a standard growth kinetic in TAP medium, from inoculation to exponential and then stationary phase, sampled at different time points over a period of 8 d. We observed no significant difference in telomere length between the samples (Fig 2A). Prolonged incubation in stationary phase for up to 15 d also did not affect telomere length (Fig 2B). Thus, telomere length was not altered either in exponential growth in replete medium or in the absence of growth, during nutrient depletion, and with any other property of saturated cultures, even over a prolonged period.

We also asked whether stimulating cell growth could affect telomere length. Because of the multiple fission mode of cell division of C. reinhardtii [START_REF] Cross | The Chlamydomonas cell cycle[END_REF], actively growing cells might spend less time in each phase of the cell cycle, and we reasoned that on average, telomerase might thus be less active. To test this hypothesis, a TAP culture was constantly maintained in exponential growth phase by serial dilutions over a period of 10 d. Telomere length did not significantly change (Fig 2C), and therefore, high division rate did not affect telomere length or distribution.

Finally, we checked telomere length distributions in cultures grown in either strictly phototrophic, strictly heterotrophic, or in-gel hybridization assay of telomeres from strain T222+ using a G-probe (oT0958, left) or a C-probe (oT0959, right). Most of the native signal, when hybridized with the C-probe, was absent when the gDNA was pretreated with ExoT. Lower panel: The native gel was then denatured and transferred to a membrane, which was then hybridized with the same probes. The uncropped gel and membrane are shown in Fig S1D . (D) Hairpin ligation assay on C. reinhardtii CC4350+ strain and A. thaliana. A scheme of the assay is shown (left). Digestion with BamHI, which removes the ligated hairpin and pretreatment with T7 exonuclease ("T7 exo."), which resects the 59 end of a duplex DNA, are used as controls. (E) T222+ and S24-strains were subcloned and three subclones were independently grown in liquid culture until stationary phase and subsequently analyzed by TRF Southern blot hybridization. (F) gDNA was subjected to Bal31 digestion for 0 to 10 min. Digested products were column-purified and then processed for TRF analysis. 0: no Bal31 digestion, but gDNA was column-purified before digestion by the restriction enzymes. NP: gDNA was directly analyzed by TRF, with No column Purification. Dashed line: smear corresponding to telomeres. Star: Bal31-insensitive band, corresponding to interstitial telomeric repeat (see also Fig S1E). mixotrophic conditions for 7 d in liquid medium (~20 population doublings) but found no significant difference between the conditions (Fig 2D). As telomeres might reach a new steadystate level with a slower kinetic, we repeated the experiment over a period of 60 d (~200 population doublings) but again did not detect changes in telomere length regardless of the growth conditions (Fig S2).

These experiments demonstrated that C. reinhardtii has an active telomere maintenance mechanism and that telomere length distribution is robust with regards to perturbation in metabolic regimes under a variety of standard laboratory growth conditions.

C. reinhardtii reference strains show important differences in telomere length and size distributions Even though telomere length distribution was very stable under different growth conditions for a given strain (Fig 2), we did observe a reproducible and significant difference in mean telomere length between the three laboratory reference strains CC4350+, T222+, and CC125+ by PETRA (Figs 1B and S1C) and between T222+ and S24-by TRF Southern blot (Fig 1E). We thus wondered if related C. reinhardtii strains displayed inter-strain differences in telomere length distribution. To test this, we took advantage of the recent sequencing of many closely related reference strains widely used in different laboratories across the world and which display up to 2% genetic divergence [START_REF] Gallaher | Chlamydomonas genome resource for laboratory strains reveals a mosaic of sequence variation, identifies true strain histories, and enables strain-specific studies[END_REF]. We performed TRF analysis on 12 related C. reinhardtii strains to characterize their telomeres (Figs 3A and S3A). Strikingly, steadystate telomere lengths were highly variable from strain to strain, ranging from 378 ± 24 bp (mean ± SD, N = 4) in CC125+ to 3.2 ± 1.1 kb (N = 3) in cw15.J14+, encompassing nearly one order of magnitude (Fig 3B). Telomere length did not correlate with genome divergence (genetically close strains are depicted with the same color), and we did not find any obvious genomic region, as described by [START_REF] Gallaher | Chlamydomonas genome resource for laboratory strains reveals a mosaic of sequence variation, identifies true strain histories, and enables strain-specific studies[END_REF], that would cosegregate with longer or shorter telomeres. In particular, neither the mating type nor the presence or absence of a cell wall correlated with telomere length variations. The average telomere length in strain cw15.J14+ was particularly striking, and we asked whether the signal could stem from internal telomere repeats. A Bal31 exonuclease treatment time course showed the signal decreasing in size demonstrating that this signal indeed corresponded to terminal repeats (Fig S3B,right). In addition to length variations, some strains, such as CC503+ and CC1010+, displayed multimodal telomere length distributions (Figs 3A and B,and S3A), and the multiple peaks corresponded to terminal fragments and not internal ones (Fig S3B,left).

Interestingly, the interstitial band at ~200 bp, which was present in 11 tested C. reinhardtii reference strains was absent A liquid culture of T222+ cells was grown to exponential phase (2 × 10 6 cells/ml), a sample of cells was harvested and the remaining cells diluted with fresh media to 5 × 10 4 cells/ml. This serial dilution was repeated 10 times. Samples corresponding to dilutions 1, 3, 6, 8, and 10 were then analyzed by TRF Southern blot hybridization. Plate: Cells were directly scraped from 1-wk-old streaks on TAP Petri dishes, without liquid culture. (D) Telomere length distributions in different metabolic growth conditions. The cells were grown for 6 d to stationary phase either in heterotrophic conditions in TAP medium in the dark, in mixotrophic conditions in TAP medium in low (LL) or higher light (HL), or in pure photo-autotrophic conditions in minimum (MIN) medium under HL.

from the S1D2-(CC2290-) strain (Fig 3A,star). S1D2-is an interfertile but divergent C. reinhardtii strain, often used for genetic mapping purposes [START_REF] Gross | Extensive restriction fragment length polymorphisms in a new isolate of Chlamydomonas reinhardtii[END_REF][START_REF] Vysotskaia | Development and characterization of genome-wide single nucleotide polymorphism markers in the green alga Chlamydomonas reinhardtii[END_REF]. Thus, the interstitial telomere sequence might have emerged in a subset of C. reinhardtii species or conversely might have been lost in S1D2-.

Identification of the gene encoding the catalytic subunit of telomerase

Telomerase is a holoenzyme comprising at least a reversetranscriptase catalytic subunit and a template RNA, which are sufficient for in vitro telomerase activity (Lingner et al, 1997a). These core actors are associated with multiple other proteins, required for its recruitment, processivity, and regulation [START_REF] Lewis | Telomerase and telomere-associated proteins: Structural insights into mechanism and evolution[END_REF]. As the catalytic subunit of telomerase (e.g., hTERT in human, AtTERT in A. thaliana, and Est2 in Saccharomyces cerevisiae) is conserved, we sought to identify the gene encoding this subunit in C. reinhardtii and to characterize the contribution of telomerase to telomere length maintenance. Nucleotide BLAST searches in C. reinhardtii genome failed to find similarity to most of the shelterin or shelterin-like genes and telomerase-associated genes from human, A. thaliana, and S. cerevisiae, except for CBF5 from A. thaliana (also CBF5 in C. reinhardtii), corresponding to the dyskerin gene.

Gene model Cre04.g213652 of the C. reinhardtii nuclear genome (Phytozome v5.5; https://phytozome.jgi.doe.gov/pz/#) has a predicted N-terminal part of the corresponding protein showing partial sequence similarity with the RNA-binding domain of telomerase from a number of organisms (Fig 4A). However, the available gene model extends over 25 kb, contains 28 introns and is predicted to encode a 5,019-aa protein, much larger than telomerase from A. thaliana (1,123 aa), maize (1,188 aa), iris (1,295 aa), and rice (1,261 aa), overall suggesting that the current gene model is probably incorrect. In addition, two sequencing gaps and the presence of TG and CCAC satellites both in introns and exons cloud the structure of the putative gene. Although expressed sequence tags from cDNA libraries supported the validity of some parts of the conserved 59 and 39 regions, no expressed sequence tag was found for the central part of the gene model in the available C. reinhardtii expression libraries. As stated above, nucleotide sequence alignments failed to detect similarity with telomerase catalytic subunit genes of other organisms. We thus performed PSI-Blast alignments of the C-terminal protein domain of the putative C. reinhardtii telomerase with telomerase from plants using PRALINE (http:// www.ibi.vu.nl/programs/pralinewww). The alignments showed strong similarity to the C-terminal catalytic reverse-transcriptase domain of A. thaliana (e-value = 3 × 10 -36 ), maize (e-value = 4 × 10 -35 ), iris (e-value = 1 × 10 -36 ), and rice (e-value = 3 × 10 -24 ) (Fig 4B). The conserved C motif (mC) in organisms ranging from S. cerevisiae to A. thaliana and humans, including two critical aspartates for telomerase catalytic activity (Lingner et al, 1997b;[START_REF] Nakamura | Telomerase catalytic subunit homologs from fission yeast and human[END_REF][START_REF] Oguchi | Molecular cloning and characterization of AtTERT, a telomerase reverse transcriptase homolog in Arabidopsis thaliana[END_REF] showed strong sequence conservation with a corresponding motif in the putative C. reinhardtii protein (Fig 4B andC). Motif E (mE) was conserved to a lesser degree, whereas no clear conservation of motifs mA, mD, and motif 1 and 2 (Lingner et al, 1997b;[START_REF] Oguchi | Molecular cloning and characterization of AtTERT, a telomerase reverse transcriptase homolog in Arabidopsis thaliana[END_REF] (C) The mC motif of C. reinhardtii shows strong sequence similarity with the mC motif containing two catalytically essential aspartates in yeast and human telomerases (Lingner et al, 1997b;Ogushi et al, 1999) putative RNA-binding domain of the gene and was named tel-m1. LMJ.RY0402.209904 has an insertion in the putative CDS of the putative catalytic C-terminal domain and was named tel-m2. LMJ.RY0402.105594 has an insertion in an intron in a nonconserved region between these two domains and was named tel-m4. Although the insertions in these three mutants were already mapped by the work of [START_REF] Li | An indexed, mapped mutant library enables reverse genetics studies of biological processes in Chlamydomonas reinhardtii[END_REF] with a confidence of 95% for tel-m1 and tel-m4 and 73% for tel-m2, we verified that all three mutants indeed had the insertion at the predicted loci, using PCR with primers targeting the gene and/or the inserted paromomycin resistance marker (Fig S4A andB). For all three mutants, the obtained PCR products were gel-excised, sequenced, and shown to correspond to the expected genomic region. We also backcrossed mutants tel-m1 and tel-m2 with the paromomycin-sensitive T222+ strain and analyzed the segregation of the paromomycin resistance phenotype in tetrads after germination of the diploids (Fig S4C). Paromomycin resistance systematically segregated with a 2:2 ratio in the haploid offspring, suggesting that the functional marker was not inserted at multiple loci in the genome. Correct 2:2 segregation of the mating locus in the offspring of the tetrads was checked by PCR (Fig S4D ).

We then analyzed the telomere length of the three mutant strains. All three mutants showed significantly shorter telomeres when compared with the parental CC4533-strain used by [START_REF] Li | An indexed, mapped mutant library enables reverse genetics studies of biological processes in Chlamydomonas reinhardtii[END_REF] to construct the CliP library (Figs 5A and S5A; mean ± SD, tel-m1: 373 ± 25 bp, N = 4, tel-m2: 383 ± 30 bp, N = 4, and tel-m4: 387 ± 12 bp, N = 2, compared with CC4533-: 614 ± 41 bp, N = 3). We verified that the shorter telomere length in mutants tel-m1, tel-m2, and tel-m4 was not simply due to the transformation protocol used to generate the CliP library or to the insertion of the paromomycin marker itself. Telomere length was measured in another mutant from the CliP library, harboring an insertion elsewhere in the genome (on chromosome 1), and was comparable with the parental CC4533strain (Fig 5A,"control" and Fig S5D).

We conclude that although gene model Cre04.g213652 might be wrong in its predicted structure and will require further study to be corrected, this genomic region indeed harbors the gene encoding for the catalytic subunit of telomerase in C. reinhardtii, and we propose to rename it CrTERT.

Telomere rearrangement and maintenance in long-term cultures of telomerase mutants

Because telomeres shortened in telomerase-negative cells, we wondered whether the cells would experience replicative senescence after an extended period of growth, when telomeres reach a critically short length. We thus grew the telomerase mutants tel-m1 and tel-m2 as well as the reference strain for 2 mo (~200 population doublings), with dilutions into fresh TAP medium every 5 d. We did not observe CrTERT mutant cultures dying out or any obvious growth defect at any time point. However, TRF analysis of these long-term cultures of tel-m1 and tel-m2 showed alterations of their telomere length distribution (Fig 5B,compare with Fig 5A): additional discrete bands appeared at sizes above 1 kb (red dots), and a signal that extended up to the wells was detected (vertical red line). Interestingly, the three independent cultures of the tel-m2 mutant gave similar but distinct patterns with respect to the discrete bands and the high molecular weight signal. The tel-m1 mutant also displayed some additional bands, albeit not to the extent of tel-m2. Overall, these altered TRF patterns observed in prolonged cultures of telomerase mutants are reminiscent of TRF patterns observed for cells with telomerase-independent maintenance pathways (e.g., type II survivors of telomerase-negative yeast cells or alternative lengthening of telomeres (ALT)-like telomerase-negative cancer cells. See the Discussion section.)

Telomeres shorten progressively in telomerase mutants

The initial CliP telomerase mutants might have accumulated additional, potentially suppressor, mutations, which could interfere with the proper assessment of the mutant phenotype. Importantly, the presence of suppressor mutations could explain why these mutants did not show any discernible growth defects in standard growth conditions or any sign of senescence after prolonged culture.

To outcross potential suppressor mutations and gain a kinetic perspective on telomere shortening in the telomerase mutants, we backcrossed mutants tel-m1 and tel-m2 with a wild-type strain of opposite mating type (T222+) and, after germination of the diploids, studied the telomere length distribution of the obtained tetrads. Backcrossing a mutant cell with a telomerase-positive strain should allow telomerase to elongate the shortest telomeres brought in by the mutant strain. The subsequent meiosis would then shuffle the chromosomes and the telomeres in the four haploid cells, independently of the mutant or wild-type status of the telomerase gene. We thus expect that immediately after meiosis in the diploid, the four haploid cells would have similar and nearly wild-type average telomere length. After culture, the telomere length in the four progenies should vary according to the status of the CrTERT gene.

Strikingly, measurement of telomere length in the four haploid progenies of the tel-m1-x T222+ cross after 21 d, the earliest time point we could obtain, showed that two of them displayed on average longer telomeres and the other two shorter telomeres, which corresponded to the telomerase mutants as assessed by paromomycin resistance (Fig 5C,"21 d"). After 21 more days, the telomeres of the telomerase-positive cultures maintained or increased their average length, whereas the telomerase-negative cultures displayed further shortening of their telomeres (Fig 5C,"42 d," and Fig S5B). A similar result was observed for the progenies of the cross tel-m2-x T222+ (Figs 5D andS5C). Telomere length could not be assessed in the dormant diploid state, as diploid cells could not be grown. These results strongly argued against the possibility that the shorter telomeres observed in tel-m1 and tel-m2 were due to additional mutations in the genome because they would not necessarily have co-segregated with the paromomycin marker. We also noted the presence of other bands and peaks in the smear, which were likely the result of segregating parental telomeres of very different lengths during meiosis (black dots in Figs 5C andD, andS5B).

Although no growth defect was observed for the initial tel-m1 and tel-m2 mutants, analysis of the haploid progeny from backcrosses between tel-m1 and tel-m2 with the wild-type T222+ strain (n = 4 independent tetrads, with eight telomerase-negative haploids) showed that four of the eight telomerase-negative haploid progenies experienced growth defects and then massive cell death, typical of replicative senescence (highlighted in red in the table of Fig S5E). Strikingly, for each of these four telomerase-negative progenies that experienced massive cell death, some cells managed to form colonies again at very low frequency (Fig S5E,left) and thus corresponded to postsenescence survivors. The four other telomerase-negative haploid progenies did not display any growth defect (highlighted in green in the table of Fig S5E). Individual colonies of postsenescent survivors kept on solid media showed cycles of moderate growth and subsequent cell death. This complex and dynamic survivor phenotype will be investigated in future studies.

Discussion

In this study, we provide a detailed molecular characterization of C. reinhardtii telomeres by investigating their sequence, end structure, and length distribution. We also identify CrTERT, the gene encoding the catalytic subunit of telomerase, and find that mutants of this gene experience telomere shortening and can enter replicative senescence.

Telomere sequence and end structure

Because telomere-bound proteins specifically interact with telomere sequences [START_REF] Palm | How shelterin protects mammalian telomeres[END_REF][START_REF] Fulcher | If the cap fits, wear it: An overview of telomeric structures over evolution[END_REF], the variability of telomeric repeat motif can have functional consequences [START_REF] Arneric | Tel1 kinase and subtelomere-bound Tbf1 mediate preferential elongation of short telomeres by telomerase in yeast[END_REF][START_REF] Marzec | Nuclear-receptor-mediated telomere insertion leads to genome instability in ALT cancers[END_REF]. C. reinhardtii telomeric repeats are mostly nondegenerate, with few lowfrequency variants, notably repeats of the canonical A. thaliana type (TTTAGGG), possibly as a remnant of the ancestral motif in the green lineage [START_REF] Fulnečkov Á | Dynamic evolution of telomeric sequences in the green algal order Chlamydomonadales[END_REF]. The overall low occurrence of variants (Table 1) suggests that C. reinhardtii telomerase is a highfidelity reverse transcriptase, in contrast to telomerase from other unicellular eukaryotes such as S. pombe or S. cerevisiae [START_REF] Zakian | Telomeres: Beginning to understand the end[END_REF].

Analysis of the available genome sequences of C. reinhardtii strains and our TRF analysis suggests the presence of interstitial telomeric repeats, which might be due to chromosome end-to-end fusion over the course of evolution [START_REF] Meyne | Distribution of non-telomeric sites of the (TTAGGG)n telomeric sequence in vertebrate chromosomes[END_REF][START_REF] Azzalin | Human intrachromosomal telomeric-like repeats: Sequence organization and mechanisms of origin[END_REF][START_REF] Uchida | Interstitial telomerelike repeats in the Arabidopsis thaliana genome[END_REF][START_REF] Gaspin | Distribution of short interstitial telomere motifs in two plant genomes: Putative origin and function[END_REF][START_REF] Aksenova | Expansion of interstitial telomeric sequences in yeast[END_REF]. But whether they might have any functional role, for example, as binding site for transcription factors [START_REF] Platt | Rap1 relocalization contributes to the chromatin-mediated gene expression profile and pace of cell senescence[END_REF] remains to be investigated.

The protective function of telomeres is not solely provided by their sequence and associated proteins but also by the structure formed at the termini. Similar to A. thaliana, C. reinhardtii telomeres seem to comprise two subsets with different end structures: 39 overhangs and blunt ends. The latter might correspond to telomeres replicated by the leading strand synthesis, which naturally generates a blunt end. Although we cannot exclude that the structures we detect might represent transient blunt ends that then undergo further processing, the very short relative duration of the S phase in C. reinhardtii cell cycle rather suggests that the blunt ends are stable structures. Although 39 overhangs are common to many eukaryotic species, blunt ends have been observed in plants, mostly in angiosperms but not in the moss Physcomitrella patens [START_REF] Kazda | Chromosome end protection by blunt-ended telomeres[END_REF]. Our discovery that the blunt ends are present in green algae suggests that this structure is of much older evolutionary origins than previously thought.

Intra-strain stability and inter-strain variations in telomere length distribution

Telomere length is regulated by multiple pathways, as shown by exhaustive screens performed in S. cerevisiae [START_REF] Askree | A genome-wide screen for Saccharomyces cerevisiae deletion mutants that affect telomere length[END_REF][START_REF] Gatbonton | Telomere length as a quantitative trait: Genomewide survey and genetic mapping of telomere length-control genes in yeast[END_REF][START_REF] Ungar | A genome-wide screen for essential yeast genes that affect telomere length maintenance[END_REF][START_REF] Chang | Genome-wide analysis to identify pathways affecting telomere-initiated senescence in budding yeast[END_REF], including nucleic acid metabolism, DNA replication, chromatin modification, and protein degradation, among others. In addition, telomere length is also sensitive to both internal and environmental cues [START_REF] Walmsley | Genetic control of chromosome length in yeast[END_REF][START_REF] Von Zglinicki | Role of oxidative stress in telomere length regulation and replicative senescence[END_REF][START_REF] Epel | Accelerated telomere shortening in response to life stress[END_REF][START_REF] Cetin | How to survive aneuploidy[END_REF][START_REF] Romano | Environmental stresses disrupt telomere length homeostasis[END_REF][START_REF] Fulcher | If the cap fits, wear it: An overview of telomeric structures over evolution[END_REF][START_REF] Millet | Cell populations can use aneuploidy to survive telomerase insufficiency[END_REF][START_REF] Millet | Aneuploidy as a mechanism of adaptation to telomerase insufficiency[END_REF]. We found no change in telomere length distribution when C. reinhardtii cells were grown in a wide variety of physiologically relevant laboratory conditions, including growth phases, carbon sources, and light conditions. Although we cannot exclude that other harsher growth conditions or internal signaling (e.g., DNA damage or replication stress) might induce an alteration in telomere length or structure, this result suggests that the mechanisms maintaining telomere length homeostasis are highly robust and efficient.

In stark contrast, closely related strains of C. reinhardtii displayed very different telomere length profiles, similar to variations observed in different strains, isolates, or ecotypes of other species [START_REF] Walmsley | Genetic control of chromosome length in yeast[END_REF][START_REF] Burr | Pinning down loose ends: Mapping telomeres and factors affecting their length[END_REF][START_REF] Zhu | Telomere length regulation in mice is linked to a novel chromosome locus[END_REF][START_REF] Shakirov | Length regulation and dynamics of individual telomere tracts in wild-type Arabidopsis[END_REF][START_REF] Raices | Uncoupling of longevity and telomere length in C. elegans[END_REF][START_REF] Liti | Segregating YKU80 and TLC1 alleles underlying natural variation in telomere properties in wild yeast[END_REF][START_REF] Fulcher | Genetic architecture of natural variation of telomere length in Arabidopsis thaliana[END_REF] and suggesting that a complex network of genetic regulation controls telomere length. A detailed functional genetic approach to map the regions of the genome responsible for telomere length variation could identify pathways regulating telomere length.

Overall, the diversity of telomere length distributions observed in these reference strains highlights the plasticity of telomere length regulation and the phenotypic heterogeneity of C. reinhardtii reference strains.

Identification of CrTERT encoding the catalytic subunit of telomerase

Sequence similarity and functional analyses of three independent mutant alleles suggest that the gene model Cre04.g213652 corresponds to, or at least encompasses, the gene encoding the catalytic subunit of telomerase, required to maintain telomere length in C. reinhardtii. We propose to rename it CrTERT.

Multiple lines of evidence support this conclusion. First, the predicted protein shares significant sequence similarity with the RNA-binding domain of telomerase from other organisms in its N terminus. Second, we find a very strong conservation of the C-terminal domain of the proposed CrTERT protein, including two essential aspartates, with the catalytic domain of telomerase not only from plants (maize, A. thaliana, soya, and iris) but also from yeast and human. Third, three independent mutants (tel-m1, tel-m2, and tel-m4) with different insertions of the paromomycin resistance marker in CrTERT, including within its RNA-binding domain (tel-m1) and its catalytic domain (tel-m2) display significantly shorter telomeres than the parental CC4533-strain, which is not the case for other independent mutants from the CliP library located in loci unrelated to telomerase. Finally, telomeres shortened progressively in paromomycin-resistant progenies. However, as we are as of yet unable to detect the mRNA corresponding to CrTERT by either Northern blotting or RT-qPCR (Reverse Transcription Quantitative PCR), possibly because of its low expression, we could not assess CrTERT expression in our study. The identification of additional components of the telomerase holoenzyme and telomere-associated proteins will be the focus of future work.

Telomere shortening, replicative senescence, and alternative maintenance pathways After prolonged liquid cultures of multiple independent tel-m1 and tel-m2 mutants, we observed a drastically altered TRF pattern: discrete bands above 1 kb and a continuous smear of high molecular weight fragments up to the wells. These new TRF signals could correspond to extremely long telomeres, as seen for strain cw15.J14+ and also to DNA molecules with abnormal structures, such as G-quartets, other secondary structures, or single-stranded DNA. These rearrangements might be produced by alternative mechanisms of telomere maintenance or elongation and are reminiscent of telomere profiles observed in type II postsenescent yeast cells [START_REF] Lundblad | An alternative pathway for yeast telomere maintenance rescues est1-senescence[END_REF], ALT cancer cells [START_REF] Cesare | Alternative lengthening of telomeres: Models, mechanisms and implications[END_REF][START_REF] Shay | Cancer. Cancer and telomeres: An ALTernative to telomerase[END_REF], or ALT A. thaliana cell lines [START_REF] Zellinger | Ku suppresses formation of telomeric circles and alternative telomere lengthening in Arabidopsis[END_REF][START_REF] Akimcheva | Genome stability in Arabidopsis cells exhibiting alternative lengthening of telomeres[END_REF], in which telomerase-independent recombination mechanisms can lead to very long and heterogeneous telomeres, thus sustaining long-term cell divisions. In these described cases, telomerase is not expressed, telomeres undergo sister-chromatid, and interchromosome homologous recombination using gene conversion, break-induced replication, rolling circle amplification, or yet unknown mechanisms.

Another line of evidence suggesting the occurrence of postsenescence survivors of telomerase-negative cells in C. reinhardtii came from the analysis of the offspring of backcrosses of the tel-m1 and tel-m2 mutants with T222+ reference strain. The CrTERT-mutant progenies experience telomere shortening, and 50% of them eventually stopped growing after about 6 mo on solid media, a phenotype consistent with replicative senescence. The other 50% of telomerase-negative progenies have not yet entered senescence at the date of publication of this work (>21 mo). The cells that experienced senescence and generated first generation survivors then showed a complex pattern of moderate growth, followed by cell death and emergence of a new generation of clonal survivors. We do not yet understand the dynamics and variability of the senescence phenotype in these backcrossed haploid progenies. We speculate that for the initial CliP mutants, additional mutations could have been generated that might have acted as suppressors of the senescence phenotype. This would also explain why no growth defect was observed for the initial CliP mutants even after more than 2 yr of maintenance on solid media, whereas senescence, cell death, and postsenescent survivors could be observed after backcrossing these mutants and selecting telomerase-negative haploid progenies. Alternatively, the initial CliP mutants might have already been postsenescence survivors from the beginning. In a future work, it will be interesting to characterize postsenescence survivors by assessing hallmarks of human ALT cancers, including circular extrachromosomal telomeric DNA and up-regulation of telomeric repeat-containing RNA (TERRA) [START_REF] Cesare | Alternative lengthening of telomeres: Models, mechanisms and implications[END_REF][START_REF] Arora | Telomere elongation chooses TERRA ALTernatives[END_REF].

Although some fundamental aspects of its telomeres share similarities to other eukaryotes, C. reinhardtii shows a unique combination of telomeric properties that distinguishes it from any other model organism. The characterization of its telomeres at the level of sequence, end structure, length distribution, and maintenance by telomerase or alternative mechanisms provided by this study is an essential step to propose C. reinhardtii as a valuable model organism for telomere biology research.

Materials and Methods

Strains and growth conditions

Strains T222+, S24-, CC124-, CC125+, CC503+, CC1009-, CC1010+, and CC4425+ (D66) are described in [START_REF] Gallaher | Chlamydomonas genome resource for laboratory strains reveals a mosaic of sequence variation, identifies true strain histories, and enables strain-specific studies[END_REF]. Strains cw15.J3and cw.J14-are cell wall-less strains obtained by crossing. Strains CC620+, CC521+, and CC4350+ are described in the Chlamydomonas Resource Center (https://www.chlamycollection.org/). Strain CC4533is described in the work by [START_REF] Li | An indexed, mapped mutant library enables reverse genetics studies of biological processes in Chlamydomonas reinhardtii[END_REF]. Strain S1D2 is described in the work by [START_REF] Gross | Extensive restriction fragment length polymorphisms in a new isolate of Chlamydomonas reinhardtii[END_REF], [START_REF] Harris | Chlamydomonas as a model organism[END_REF][START_REF] Vysotskaia | Development and characterization of genome-wide single nucleotide polymorphism markers in the green alga Chlamydomonas reinhardtii[END_REF]. Unless stated otherwise, the cells were grown under continuous illumination either on plates or in agitated 200-ml liquid cultures in TAP medium [START_REF] Harris | The Chlamydomonas Sourcebook (Second Edition)[END_REF] under low-light, that is, 8 μE⋅m -2 ⋅s -1 or higher light (HL), that is, 80 μE⋅m -2 ⋅s -1 . The A. thaliana ecotype Columbia (Col-0) plant was used for the hairpin assay.

gDNA extraction

Unless stated otherwise, the cells were grown in liquid cultures to early stationary phase (~2 × 10 7 cells⋅mL -1 ) and 150 ml was collected by centrifugation (5,000g, 5 min). The pellet was frozen at -80°C. The cells were then thawed at room temperature and 5 ml of preheated buffer AP1 with RNase (QIAGEN DNA Plant Maxi Kit) was added and cells lysed at 65°C for 2 h. After lysis, gDNA was extracted according to the manufacturer's protocol (QIAGEN DNA Plant Maxi Kit). For PETRA and hairpin assays, gDNA was extracted using the CTAB method as described in [START_REF] Borevitz | Large-scale identification of single-feature polymorphisms in complex genomes[END_REF].

Telomere PCR and sequencing

Bulk gDNA was denatured at 95°C during 5 min. End labeling reactions (total volume 6 μl) contained 100 ng of bulk gDNA, 1× New England Biolabs restriction buffer 4, dCTP 100 μM, and 1 unit of terminal transferase (New England Biolabs, NEB) and was carried out at 37°C during 30 min, then 65°C during 10 min, and 94°C during 5 min. The end-labeled telomeres were then amplified with the primers 169M (poly-G-containing primer) and oT1090 targeting the subtelomere/telomere junction common to 10 telomeres of eight chromosomes (Table S1). PCR reactions (40 μl) contained the endlabeled DNA, 200 μM of dNTPs, primers at 0.5 μM for oT1090 and 0.75 μM for 169M, 1× Taq Mg-free buffer (NEB), and 2.5 U of standard Taq polymerase (NEB). The PCR conditions were as follows: 94°C 3 min; 32 cycles of 94°C 20 s, 60°C 40 s, and 68°C 20 s; and 68°C 5 min.

For sequencing, PCR products were ligated for 1 h at 16°C in a pDrive plasmid. 2 μl of the ligation product was transformed into competent bacteria (PCR cloning kit; QIAGEN). Bacteria were plated on LB + ampicillin (100 μg/ml) + IPTG (50 μM) + X-gal (80 μg/ml) medium overnight at 37°C. Plasmids were extracted and purified (Millipore Plasmid Miniprep 96 Kit and Manifold) after 24 h of culture of white colonies in 1 ml of LB 2X + ampicillin (100 μg/ml) in a 96-well microplate. DNA insertion in plasmids was verified by EcoRI (NEB) digestion. Plasmids were Sanger-sequenced with the M13-PU primer (Eurofins Genomics).

Isolation of nuclei

Nuclear fraction was prepared from cell wall-mutant CC4350+. Exponentially growing cells (2 d in liquid culture) were gently spun and thoroughly resuspended in 90 ml buffer A per liter of culture (25 mM Hepes-NaOH, pH 7.5, 20 mM KCl, 20 mM MgCl 2 , 600 mM sucrose, 10% glycerol, and 5 mM DTT). Triton X-100 was first diluted in 10 ml of buffer A per liter of culture and subsequently added drop wise to the cells while swirling them gently, to a final concentration of 0.5%. Nuclei were pelleted at 800 g for 2-4 min. Using a paintbrush, the pellet was gently resuspended in fresh buffer A without Triton X-100. After centrifugation, the integrity of nuclei (1-5 μl) was checked by fluorescent microscopy using DAPI/vectashield (5 μl) staining. Nuclei were resuspended in buffer B (2.5% Ficoll 400, 0.5 M sorbitol, 0.008% spermidine, 50% glycerol, and 1 mM DTT), using 1-2 ml per 200 ml of original culture volume. For storage, nuclei were frozen in liquid nitrogen and stored at -80°C.

MNase hypersensitivity assay

MNase hypersensitivity assay was based on [START_REF] Lodha | Analysis of chromatin structure in the control regions of the chlamydomonas HSP70A and RBCS2 genes[END_REF]. 1 ml of nuclei isolated from C. reinhardtii strain CC4350+ cw15 mt+ in buffer B was thawed on ice. To collect nuclei, the sample was spun at maximal speed for 15 s and resuspended in 500 μl of 1× MN buffer (50 mM Tris-HCl, pH 8.0, and 5 mM CaCl 2 ). Reactions of total volume of 110 μl were carried out in 1× MN buffer using 60 μl of sample and different amounts of MNase units (Fermentas). gDNA from C. reinhardtii CC4350+ (~750 ng) was used as a control for enzyme activity and digested with 15 U of nuclease. The samples were incubated 3 min at room temperature, and reactions were stopped by adding 110 μl of STOP buffer (1% SDS and 50 mM EDTA). Proteins were then denatured for 45 min at 65°C and DNA was extracted using 500 μl of phenol:chloroform:isoamyl alcohol (25:24:1) using Phase Trap A (Peqlab). Aqueous phase was precipitated by adding 42 μl of 3 M NaOAc and 840 μl of 96% ethanol. DNA was pelleted by centrifugation for 10 min at maximal speed. The pellet was washed with 70% ethanol, dried, and resuspended in 25 μl H 2 O. 6× loading dye (6 μl) was added prior loading onto 1.5% agarose gel. DNA was stained with ethidium bromide (1% solution; AppliChem), blotted onto uncharged membrane (Amersham), and hybridized with a (T 4 AG 3 ) 3 probe. After scanning, the membrane was stripped and reprobed using 18S-derived probe.

PETRA and hairpin assay

Telomere length of individual chromosomes was determined by Primer Extension Telomere Repeat Amplification (PETRA) as previously described [START_REF] Watson | Germline replications and somatic mutation accumulation are independent of vegetative life span in Arabidopsis[END_REF]. For primer extension by the Φ29 polymerase, we used the C. reinhardtii-specific PETRA-T oligonucleotide 59-CTCTAGACTGTGAGACTTGGACTACCCTAAAACCCT-39 (Table S1). For specific chromosome arms, we used subtelomeric oligonucleotides 1R: 59-TACTTGTGTGTGCTGTGCGT-39, 9R: 59-ACAG-CACAATACAGTATATA-39, and 10R: 59-AACGTCCTCGTGAGACCACC-39 (Table S1). The hairpin assay for detecting blunt-ended telomeres was performed as previously described [START_REF] Kazda | Chromosome end protection by blunt-ended telomeres[END_REF]. Southern hybridization was performed with a [ 32 P]ATP-labeled (TTTTAGGG) 3 probe. PETRA membrane was also hybridized with [ 32 P]ATP-labeled 1-kb ladder (Thermo Fisher Scientific).

TRF and in-gel hybridization analyses 2 μg of gDNA was digested in 300 μl with a cocktail of six restriction enzymes (PstI, BamHI, MnlI, FokI, TaqI, and MspI; 20 units each). Digestion products were isopropanol precipitated, resuspended in loading buffer (gel loading dye, Purple 6X, New England Biolabs) and resolved on a 1.5% agarose gel for 4 h at 150 V. The gel was then soaked in a denaturation bath (0.4 M NaOH and 1 M NaCl) for 20 min and transferred overnight by capillarity to a charged nylon membrane (Hybond XL; GE Healthcare). The CHSB Chlamydomonas telomere-specific oligonucleotide probe (Fulneckova et al, 2013) (59-GTTTTAGGGTTTTAGGGTTTTAGGGTTTTAG-39, Table S1) was 32 Plabeled at the 59 terminus with ATP (γ-32 P) by the T4 polynucleotide kinase (New England Biolabs). The membrane was hybridized using the Rapid-hyb Buffer protocol (GE Healthcare). In brief, the membrane was prehybridized at 42°C in Rapid-hyb buffer for 1 h, then the radioactive probe (20 pmol) was added, and the incubation was continued for 1 h. The membrane was washed consecutively with 5× SSC, 0.5% SDS (42°C for 10 min); 5× SSC, 0.1% SDS (42°C for 20 min); and 1× SSC, 0.1% SDS (25°C for 30 min). A phosphor screen was exposed to the membrane and imaged with a Typhoon FLA 9500 scanner (GE Healthcare). Average telomere length was assessed using ImageJ 1.49v (NIH) by measuring the peak of the telomere length distribution signal. For multimodal telomere length profiles, the multiple peaks were measured. We used TeloTool, a software for TRF analysis with a built-in probe intensity correction algorithm [START_REF] Gohring | TeloTool: A new tool for telomere length measurement from terminal restriction fragment analysis with improved probe intensity correction[END_REF], to verify that unequal telomeric probe binding was negligible in our conditions. For in-gel hybridization analysis, gDNA was digested by the cocktail of enzymes following the same procedure, with some samples being pretreated with ExoT (50 units for 2 h at 25°C; New England Biolabs), which degrades single-stranded 39 DNA extension and generates blunt ends, similarly to ExoI. Samples were then run in a 1× Trisborate EDTA (TBE) 0.75% agarose gel in 1× TBE buffer for 18 h at 20 V. The gel was then dried and hybridized overnight at 37°C with radioactively labeled probes (oT0958, G-probe and oT0959, C-probe, Table S1) in hybridization buffer (5× SSC, 5 μM inorganic pyrophosphate, 1 mM Na 2 HPO 4 , 5× Denhardt's solution, 40 nM ATP, and 20 μg/ml salmon sperm DNA). The gel was then washed three times for 30 min at room temperature with 0.25× SSC and imaged as for Southern blots. For loading controls, the gel was then transferred in denaturing conditions on a charged nylon membrane and hybridized again with the same probes.

Figure 1 .

 1 Figure 1. Structural characterization of C. reinhardtii telomeres. (A) Characterization of C. reinhardtii telosomes by MNase digestion of chromatin (left panel; "EtBr": ethidium bromide staining of the migration gel) and Southern analysis with a telomeric specific probe (right panel; (TTTTAGGG) 3 : radiolabeled probe). The membrane was then stripped and rehybridized with an 18S rDNA probe (middle panel). Lanes correspond to increasing amounts of MNase units; "Pr": control where nuclei were digested with 60 MNase units after protein removal. (B) PETRA was used to amplify three different telomeres (1R, 9R, and 10R) from strain CC4350+ and analyzed by Southern blot hybridization using the telomere-specific probe (TTTTAGGG) 3 (see also Fig S1C). Negative controls include omission of the primer extension step by Φ29 polymerase and pretreatment of the samples with ExoI. (C) Upper panel: Native

Figure 2 .

 2 Figure2. Telomere length distribution is stable under various growth conditions. (A) Telomere length distributions of T222+ strain at different growth stages of liquid cultures. T222+ cells were harvested at early exponential (1), mid-exponential (2), late exponential (3), and early (4, 5) and late (6) stationary phases and analyzed by TRF Southern blot hybridization. (B) Telomere length distributions of prolonged cultures in stationary phase. The cells were harvested after 1, 5, 8, and 15 d after reaching stationary phase and compared with late exponential cultures. (C) Telomere length distributions of serial dilutions of rapidly growing cells. A liquid culture of T222+ cells was grown to exponential phase (2 × 10 6 cells/ml), a sample of cells was harvested and the remaining cells diluted with fresh media to 5 × 10 4 cells/ml. This serial dilution was repeated 10 times. Samples corresponding to dilutions 1, 3, 6, 8, and 10 were then analyzed by TRF Southern blot hybridization. Plate: Cells were directly scraped from 1-wk-old streaks on TAP Petri dishes, without liquid culture. (D) Telomere length distributions in different metabolic growth conditions. The cells were grown for 6 d to stationary phase either in heterotrophic conditions in TAP medium in the dark, in mixotrophic conditions in TAP medium in low (LL) or higher light (HL), or in pure photo-autotrophic conditions in minimum (MIN) medium under HL.

  was found in the predicted C. reinhardtii protein. Other well-conserved regions in the C-terminal part with no assigned motif are also depicted in Fig 4B.To demonstrate that the genomic region Cre04.g213652 indeed contains the gene encoding the catalytic subunit of telomerase of C. reinhardtii, we selected three strains harboring insertions of the paromomycin resistance cassette within the putative gene from the recently created CliP library of mapped insertional mutants[START_REF] Li | An indexed, mapped mutant library enables reverse genetics studies of biological processes in Chlamydomonas reinhardtii[END_REF] (https://www.chlamylibrary.org) (Fig 4A). LMJ.RY0402.077111 has an insertion in a putative intron near the region encoding the

Figure 3 .

 3 Figure 3. Vast differences in telomere length distributions in C. reinhardtii reference strains. (A) Telomeres of recently sequenced C. reinhardtii reference strains (Gallaher et al, 2015) were analyzed by TRF analysis. Strains sharing the same name color are closely related genetically, whereas strains with different colors are more divergent. Dashed vertical lines indicate independent gels. Star: S1D2-strain does not display the band at ~200 bp. cw15 and cw92 indicate mutations that led to cell wall-less strains. (B) Mean and SD of telomere length for each strain as calculated by analysis of Southern blots from the indicated number of independent biological replicates (N).

Figure 4 .

 4 Figure 4. Identification of the CrTERT gene encoding the catalytic subunit of telomerase in C. reinhardtii.(A) The protein corresponding to the predicted gene model Cre04.g213652.t1.1 of the available C. reinhardtii nuclear genome harbors an annotated Nterminal domain with significant similarities to the RNA template-binding domain of telomerases from other organisms. The C-terminal domain shows strong similarities with the catalytic domain of this enzyme in other organisms. Mutants tel-m1 (LMJ.RY0402.077111) and tel-m2 (LMJ.RY0402.209904) from the CliP library have reported insertions in either the RNA-binding or the catalytic domain, respectively. Mutant tel-m4 (LMJ.RY0402.105594) has an insertion in between these two domains. (B) PSIblast alignments show strong amino-acid sequence similarity of the catalytic domain of telomerases from many organisms with the putative C. reinhardtii protein. Similarity score ranges from 0 (light blue) to 9 and * (red) indicates identity. The motifs B9, C, and E (mB9, mC, and mE) described byLingner et al (1997b), Ogushi et al (1999) show strong conservation in C. reinhardtii, including two catalytic aspartates, essential for telomerase function in other organisms (red asterisks). Conservation can also be observed downstream of mE between CrTERT and the other telomerases. (C) The mC motif of C. reinhardtii shows strong sequence similarity with the mC motif containing two catalytically essential aspartates in yeast and human telomerases(Lingner et al, 1997b; Ogushi et al, 1999). Cr, C. reinhardtii; At, A. thaliana; Os, O. sativa; Zm, Z. mays; lt, I. tectorum; Sc, S. cerevisiae; Hs, H. sapiens.

Figure 5 .

 5 Figure 5. Insertional mutants of the CrTERT gene have shorter telomeres. (A) Mutants tel-m1 and tel-m2 have shorter telomeres in TRF analyses (three independent subclones are shown). Control: mutant LMJ.RY0402.239308 from the CliP library, which has an insertion in a gene unrelated to CrTERT. Paromomycin resistance phenotype is indicated ("[Paro S/R ]"; "S": sensitive, and "R": resistant). (B) Prolonged liquid cultures of telomerase mutants lead to rearranged TRF patterns. Cells were cultured in liquid medium for 2 mo before TRF analysis. Additional bands and slow-migrating DNA molecules (red dots and dotted vertical line, respectively) are indicated for tel-m1 and tel-m2 and are not present in the CC4533reference strain TRF pattern. (C) Tetrad analysis of the cross between tel-m1 and T222+ shows a 2:2 cosegregation of paromomycin resistance and shortened telomeres after 21 and 42 d after the cross (see also Fig S5B). Mating types "mt+" and "mt-" are indicated. (D) Tetrad analysis of the cross between tel-m2 and T222+ shows a 2:2 cosegregation of paromomycin resistance and shortened telomeres after ~80 d after the cross (see also Fig S5C). Mating types "mt+" and "mt-" are indicated.

Table 1 .

 1 Frequency of telomeric repeat motifs determined by telomere PCR and sequencing of 32 independent clones.

	Sequence	n	Frequency
	TTTTAGGG	636	89.7%
	TTTAGGG	37	5.2%
	TTTTTAGGG	13	1.8%
	TTTTGGG	8	1.1%
	Others	15	2.1%

  . Cr, C. reinhardtii; At, A. thaliana; Os, O. sativa; Zm, Z. mays; lt, I. tectorum; Sc, S. cerevisiae; Hs, H. sapiens.

Chlamydomonas telomeres and telomerase Eberhard et al. https://doi.org/10.26508/lsa.201900315 vol 2 | no 3 | e201900315

Acknowledgements

We thank Erin Henninger for her help in setting up the in-gel experiment. We thank the Chlamydomonas Mutant Library Group at Princeton University, the Carnegie Institution for Science, and the Chlamydomonas Resource Center at the University of Minnesota for providing the indexed Chlamydomonas insertional mutants. This work was supported by the Agence Nationale pour la Recherche (ANR) grant "AlgaTelo" (ANR-17-CE20-0002-01) to Z Xu, la Fondation de la Recherche M édicale (MTT " équipe labellis ée") and the ANR grant "InTelo" (ANR-16-CE12-0026) to MT Teixeira, the "Initiative d'Excellence" program from the French State (Grant "DYNAMO," ANR-11-LABX-0011), and by the Ministry of Education, Youth and Sports of the Czech Republic, European Regional Development Fund-Project "REMAP" (No. CZ.02.1.01/0.0/0.0/15_003/0000479) to K Riha.

Supplementary Information

Supplementary Information is available at https://doi.org/10.26508/lsa. 201900315.

Author Contributions

S Eberhard: conceptualization, formal analysis, supervision, investigation, methodology, and writing-original draft, review, and editing. S Valuchova: conceptualization, formal analysis, investigation, methodology, and writing-review and editing. J Ravat: formal analysis, investigation, and writing-review and editing. J Fulneček: formal analysis, investigation, methodology, and writing-review and editing. P Jolivet: formal analysis, investigation, methodology, and writing-review and editing. S Bujaldon: formal analysis, investigation, and writing-review and editing. SD Lemaire: formal analysis and writing-original draft, review, and editing. F-A Wollman: conceptualization, formal analysis, and writing-review and editing. MT Teixeira: conceptualization, formal analysis, and writing-review and editing. K Riha: conceptualization, formal analysis, supervision, and writing-review and editing. Z Xu: conceptualization, formal analysis, supervision, investigation, methodology, and writing-original draft, review, and editing.

Conflict of Interest Statement

The authors declare that they have no conflict of interest.


Christian D Hoyos
email: christian@gmail.com

Juan C Duque
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Andrés F Barco

Élise Vareilles
email: elise.vareilles@mines-albi.fr

A Search Engine Optimization Recommender System

Search Engine Optimization reefers to the process of improving the position of a given website in a web search engine results. This is typically done by adding a set of parameters and metadata to the hypertext files of the website. As nowadays the majority of the web-content creators are non-experts, automation of the search engine optimization process becomes a necessity. On this regard, this paper presents a recommender system to improve search engine optimization based on the site's content and creator's preferences. It exploits text analysis for labels and tags, artificial intelligence for deducing content intention and topics, and case-based reasoning for generating recommendations of parameters and metadata. Recommendations are given in natural language using a predefined set of sentences.

Introduction

Normally, web content creators require their websites to be easily found by content consumers through search engines [START_REF] Killoran | How to use search engine optimization techniques to increase website visibility[END_REF]. They do so by setting parameters and adding metadata to the hypertext source files of the websites. These parameters and metadata allow the algorithms of the search engines to index and retrieve data of millions of websites in an efficient way [START_REF] Kiryakov | Semantic annotation, indexing, and retrieval[END_REF]. For instance, parameters about the intention of the website allow to classify content and metadata stating the location is useful to customize content or restrict access. Further, this information makes possible for the search engine to rank the results of a query by priority. As reported by Chitika [START_REF]The value of google result positioning[END_REF], configuring websites for correct indexing is a key element of their success. This configuration of values is called Search Engine Optimization (SEO). Now, although every website is implemented following a standard, namely HTML, there is no standard for web page ranking as each search engine (Google, Yahoo, Bing, etc) implements its own ranking system. This implies that improving the indexing position of a website requires an expert on both the content as well as on the search engine ranking system.

On this regard, this paper proposes an expert recommendation system in charge of performing SEO for a given web page 4 targeting the Google search engine. It uses artificial intelligence to deduce the intention and content topic of the web page, it uses text analysis over labels and tags in order to classify and comparison, and it uses casebased reasoning to provide recommendations for improving SEO on the web page.

The documents is structured as follows. The overall behavior of the system, and its architecture, are presented in Section 2. Each of the modules of the system are described in Section 3. An experimental test and its results are shown in Section 4. Conclusions are presented in Section 5.

Overview

To provide recommendations for indexation of a web page, aspects such as content topic, keywords, intention of the (authors') web page, metadata, related web pages and the specific raking system of the search engine, should be taken into account. These aspects allow the expert system to understand the website communication goals and to create recommendations that respect the search engine implementation. The expert system proposed here tries to unveil the previous aspects using three modules in charge of analysis and one module in charge of recommendation generation (see Figure 1).

The systems receives three inputs, two of which are optional. The first input of the system is either an HTML source file or an hyperlink (URL to an HTML). If the HTML contains scripts or CSS definitions, they are ignore are they not provide useful information for the indexation. Hyperlinks should be accessible from the web. The second input is the topic of the web page, which is an optional value. The last input value is the intention of the web page and it is as well optional. It is worth noticing that having explicitly defined topic and intention will help the system's accuracy and performance (no topic and intention processing). Having the inputs, the system executes the following steps and throws as output a web page score and its recommendations.

First, the web page is analyzed using text analysis over the HTML source code. The analysis throws an score depending on the presence or absence of 22 of the more important factors for indexation according to Google [START_REF] Fernández | Google's pagerank and beyond: The science of search engine rankings[END_REF][START_REF]The value of google result positioning[END_REF]. These factors add positive values to the score when present and negative values when not. This is the first source of knowledge to build a recommendation of a web page.

Once the text analysis is done, a topic and intention analysis is performed using the IBM Watson system (a state-of-the-art artificial intelligent API) [START_REF] Vohra | The new era of watson computing this article introduces cognitive computing using ibm watson and how to leverage cognitive computing with ecm centric solutions[END_REF]. The topic and intention are useful in two ways. At the one hand, they allows to classify the content of the web page. And, on the other hand, they are basis a case-based reasoning recommendation executed in the last step.

Next, using the obtained topic and intention as keywords, the system performs a search query in the Google search engine and retrieves the first 10 pages from the result. It then proceeds by analyzing each web page in the aim of extracting key values, such as keywords and metadata, that made those pages the 10 first ranked pages of Google. This is an implementation of case-based reasoning [START_REF] Kolodner | Case-Based Reasoning[END_REF] and are the second source of knowledge to build a recommendation of a web page. Finally, the system builds a recommendation using HTML code and natural language [4] using predefined sentences. They are based on the hypertext files of the website. As nowadays the majority of the web-content creators are non-experts, automation of the search engine optimization process becomes a necessity. On this regard, this paper presents a recommender system to improve search engine optimization based on the site's content and creator's preferences. It exploits text analysis for labels and tags, artificial intelligence for deducing content intention and topics, and case-based reasoning for generating recommendations of parameters and metadata. Recommendations are given in natural language using a predefined set of sentences.

Introduction

Normally, web content creators require their websites to be easily found by content consumers through search engines [START_REF] Killoran | How to use search engine optimization techniques to increase website visibility[END_REF]. They do so by setting parameters and adding metadata to the hypertext source files of the websites. These parameters and metadata allow the algorithms of the search engines to index and retrieve data of millions of websites in an efficient way [START_REF] Kiryakov | Semantic annotation, indexing, and retrieval[END_REF]. For instance, parameters about the intention of the website allow to classify content and metadata stating the location is useful to customize content or restrict access. Further, this information makes possible for the search engine to rank the results of a query by priority. As reported by Chitika [START_REF]The value of google result positioning[END_REF], configuring websites for correct indexing is a key element of their success. This configuration of values is called Search Engine Optimization (SEO). Now, although every website is implemented following a standard, namely HTML, there is no standard for web page ranking as each search engine (Google, Yahoo, Bing, etc) implements its own ranking system. This implies that improving the indexing position of a website requires an expert on both the content as well as on the search engine ranking system.

On this regard, this paper proposes an expert recommendation system in charge of performing SEO for a given web page 4 targeting the Google search engine. It uses artificial intelligence to deduce the intention and content topic of the web page, it uses text analysis over labels and tags in order to classify and comparison, and it uses casebased reasoning to provide recommendations for improving SEO on the web page.

The documents is structured as follows. The overall behavior of the system, and its architecture, are presented in Section 2. Each of 1 Universidad de San Buenaventura Cali. Santiago de Cali, Colombia. email: {christian, juan.duque}@gmail.com

Overview

To provide recommendations for indexation of a web page, aspects such as content topic, keywords, intention of the (authors') web page, metadata, related web pages and the specific raking system of the search engine, should be taken into account. These aspects allow the expert system to understand the website communication goals and to create recommendations that respect the search engine implementation. The expert system proposed here tries to unveil the previous aspects using three modules in charge of analysis and one module in charge of recommendation generation (see Figure 1). The systems receives three inputs, two of which are optional. The first input of the system is either an HTML source file or an hyperlink (URL to an HTML). If the HTML contains scripts or CSS definitions, they are ignore are they not provide useful information for the indexation. Hyperlinks should be accessible from the web. The second input is the topic of the web page, which is an optional value. The last input value is the intention of the web page and it is as well optional. It is worth noticing that having explicitly defined topic and intention will help the system's accuracy and performance (no topic and intention processing). Having the inputs, the system executes the following steps and throws as output a web page score and its recommendations.

First, the web page is analyzed using text analysis over the HTML source code. The analysis throws an score depending on the presence or absence of 22 of the more important factors for indexation according to Google [START_REF] Fernández | Google's pagerank and beyond: The science of search engine rankings[END_REF][START_REF]The value of google result positioning[END_REF]. These factors add positive values to the score when present and negative values when not. This is the first source of knowledge to build a recommendation of a web page.

Once the text analysis is done, a topic and intention analysis is performed using the IBM Watson system (a state-of-the-art artificial intelligent API) [START_REF] Vohra | The new era of watson computing this article introduces cognitive computing using ibm watson and how to leverage cognitive computing with ecm centric solutions[END_REF]. The topic and intention are useful in two ways. At the one hand, they allows to classify the content of the web page. And, on the other hand, they are basis a case-based reasoning recommendation executed in the last step.

Next, using the obtained topic and intention as keywords, the system performs a search query in the Google search engine and retrieves the first 10 pages from the result. It then proceeds by analyzing each web page in the aim of extracting key values, such as keywords and metadata, that made those pages the 10 first ranked pages of Google This is an implementation of case-based reasoning [START_REF] Kolodner | Case-Based Reasoning[END_REF] Figure 1. Recommendation System Architecture.

the identified negative evaluated factors (e.g., missing tags) and the extracted data from the first 10 pages (e.g., new keywords).

System's Core

The recommendation system is divided in four modules.

Module 1: HTML analysis

This module focuses in labels and metadata of the web page HTML source files. In particular, it looks for specific information that is related with the Google ranking system and 22 key aspects in specific labels as <meta name=...>. These aspects include keywords definition, char-set codification, description of web page, copyright, content duplication and broken links, among others. Each factor has associated a positive value if included in the source file and a negative value if not. The Table 1 present some of the key aspects and its respective values. Note: It is important to know that one of the most important factor in the Google search engine is the value determined by the PageRank algorithm [START_REF] Bianchini | Inside pagerank[END_REF]. This algorithm takes into account the number and quality of other web pages pointing to the web page in reference.

Simply put, the more pages on the web point at the referenced page the better. More points are given if the other web page is high ranked. This works as a kind of endorsement. The PageRank is not included in the recommendation system analysis as it is not based in HTML tags and metadata.

Module 2: Intention and topic deduction

The intention and topic is deduced from the content, meaning that only the text within the labels <body> ... </body> are analyzed. Both intention and topic are deduced using the IBM Watson system through its public API only if no user input is given. Watson is, en essence, an on-line system that exploits several techniques from artificial intelligence to provide services as speech to text, natural language understanding and query answer system, emotion and sentiment analysis, translator and visual recognition [START_REF] Ferrucci | Introduction to "This is Watson[END_REF][START_REF] Vohra | The new era of watson computing this article introduces cognitive computing using ibm watson and how to leverage cognitive computing with ecm centric solutions[END_REF].

The topic and intention are deduced by Watson using Natural Language Understanding/Classification for the analysis of text. In the case of the topic, classification is done through a set of categories, concepts and keywords. In case of the intention the system classifies according to how positive or negative is the web page. Then the analysis assigns one of the following labels to the page: Very Positive, Positive, Neutral, Negative and Very Negative. Each of these labels are connected to numerical values thrown by Watson, as presented in Table 2

Module 3: Case-based reasoning

The set of categories, concepts, keywords and the intention are used for constructing a search query in the aim of obtaining similar web pages. The main idea is to extract the parameters used by top ranked web pages, the first 10 pages in Google's search engine, that address the same topic and has the same intention. Potentially, those 10 pages include data in their HTML files that made them the first ranked by the search engine. Arguably, using the same or similar parameters (as new keywords or tags), will help to improve indexation of other pages. For instance, adding keywords that were not previously included in the web page but that are a common for most of the 10 retrieved pages.

Note:

The system only retrieves the first 10 pages for two reasons. At the one hand, according to the literature, the probability of user access to a web page ranked after 10th position is around 1% [START_REF]The value of google result positioning[END_REF]. Thus, the system obtains only those web pages that are likely to have high user access rate. And, at the other hand, the analysis of more pages may reduce its efficiency. Consider that each of the 10 pages is analyzed using the same techniques. Ergo, the process, plus comparison, must be executed 11 times, which is time consuming.

Module 4: Natural language recommendation

Recommendation are build with structured predefined sentences of the form: target factor + recommendation over factor + explanation of recommendation + example in HTML. Each recommendation is classified into four categories in according with its importance:

• Black: Critical recommendation to be applied for basic indexation in Google search engine. • Red: Not following the recommendation may significantly affect the position of the web page in the results. • Yellow: Not following the recommendation may moderately affect the position of the web page in the results. • Blue: Not following the recommendation may minimally affect the position of the web page in the results.

Test

Two type of tests have been made; tests using public web pages and tests using an authors' web page.

Public websites tests

In these tests, five topics have been chosen and the following five queries have been designed. The first three results of each query have been feed to the system with automatic execution. Table 3 shows the number of recommendations of each found page. From the results we conclude the following.

• There is no direct relation between the number of recommendations and the score of the web page. Indeed, it depends on what factor is being recommended and its impact on the score. For instance, a web page may have few recommendations on factors, but one of the factor is being repeated within the page ergo reducing the score significantly. • Of the five queries, three of them show tendency of score decreasing, which is expected. The first pages of the other two queries do not have high score but may be affected by other factor not taken into account, mainly traffic and results of PageRank algorithm. • The best ranked pages are part of sites like Wikipedia. In fact, two of the found web pages are from Wikipedia and are the top ranked pages. This is mostly due to the many external and self-reference hyperlinks of the site.

Authors' designed web page

For these tests, a web page created by the authors is feed to the system in three different round. Recommendations (from rounds one and two) are implemented before the next round (rounds two and three). The designed page is a basic HTML file, without styles or scripts, used to show the improvement of a given web page through the system's recommendations. The title of the web page is the "The fall of JQuery", and addresses the descend of developers using JQuery.

Figure 2 show the recommendations of the first round (in Spanish5 ) with different colors for their importance. As an example of the result, first line of recommendation states "You should use labels h1, h2, h3...h6 more often, as they help defining the importance of conent within the page". The values thrown by the system in the three rounds show an evolution of the web page through the recommendations. As expected, for a web page with no external links referencing at it, the system assigns low score and several recommendations for the first run, augmenting score and decreasing recommendations. Bear in mind that the number of recommendations is lower than the tests in the previous sections given that the content of the designed web page is not as big and does not have as many links as the other pages.

Conclusions

Although the internals of web search engines are very similar, each of them implements different ranking system for indexing web pages. In consequence, the identification of factors that are included in the ranking systems, and its tuning by means of hypertext (metadata), is critical for the success of a given web page. In this context, tags, topic and intention are relevant for recommending changes in the aim of improving results position.

This paper proposed a recommender system for improving the search optimization of a web page in the Google's search engine. The system evaluates 22 main factors used by Google search engine to classify the web pages (ranking them). The system represents a positive contribution because:

• Basic and fundamental factors are handled so that the search engine can identify the content and structure of the web page. • Each recommendation explains with details and examples, and in natural language, how the improvement of a factor in the website can be made. • An user without much experience in SEO can make use of the recommendation system as it is intuitive. • Recommendations are different for each factor and each web page (customized recommendations). • The analysis and recommendations are made based on the top 10 bests indexed sites in Google, that deal with the same topic and intention (instance of case-based reasoning).

Figure 2 .

 2 Figure 2. Recommendations for first round to "The fall of JQuery" web page..

Table 1 .

 1 Evaluated factors and scoring.

	Label	Description	Benefit Penalty
	F1	User of keywords in tag title.	13,5	-16,8
	F2	Connection among keywords (in-	13,5	-16,8
		terrelated)
	F3	Low density on keywords (not too	10,5	-16,8
		many)
	F4	Description in tag meta with a	10,5	-16,8
		maximum of 200 words.
	F5	Excessive use of meta and alt	10,5	-16,8
		tags.
	F6	Definition codification in tag	13,5	-12,6
		char-set.
	F7	Avoid the use of tag refresh	7,5	-16,8
	F8	Use of tag alt in <img> and	12	-12,6
		<input>
	F9	No broken URLs in source file	13,5	-10,5
	F10	Use of tag H (h1, h2, h3)	10,5	-12,6
	F11	Exceeding maximum number of	6	-14,7
		characters in tag title
	F12	Use of tag keyword with maxi-	12	-8,4
		mum of 200 characters.
	F13	Percentage (between 5 and 20) of	10,5	-8,4
		keywords in text
	F14	Hyperlinks to pages of the same	13,5	-4,2
		website
	F15	Content strongly connected to the	10,6	-6,3
		web page topic and keywords
	F16	Duplicated content.	10,5	-6,3
	F17	Use of strong, bold and	12	-4,2
		italic for fonts.
	F18	Use of cache-control tag.	9	0
	F19	Keywords in URL.	6	-6,3
	F20	Use of keywords in numbered lists.	7,5	-4,2
	F21	Use of tag author.	3	-2,1
	F22	Definition of tag copyright	3	-2,1

Table 2 .

 2 . Tablewith this

	Label	Min Max
	Very Positive	0.6	1
	Positive	02	0.6
	Neutral	-0.2	0.2
	Negative	-0.6	-0.2
	Very Negative	-1	-0.6

Table 3 .

 3 Test results with five queries.

	Query	Index	# Reco	Score
		1	43	191
	Football critic	2	18	215
		3	63	292
		1	32	494
	Mediterranean food	2	14	111
		3	33	114
		1	25	306
	Vaccinations for cats	2	16	439
		3	23	171
		1	36	222
	Contamination of the Oceans	2	17	67
		3	48	50
		1	66	600
	Renewable energies	2	65	223
		3	10	90

Table 4 .

 4 Table 4 presents the results of the three rounds of execution. Follow-up of authors' web page.

	Round	# Recommendations	Score
	1	13	-116
	2	5	100
	3	0	167

The system interface is in Spanish as it is being used in a multimedia engineering program in Colombia.