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MicroRNAs are key regulators in the development processes or stress responses in plants. In the last decade, several conserved or non-conserved microRNAs have been identified in Medicago truncatula. Different strategies leading to the inactivation of microRNAs in plants have been described. Here, we propose a protocol for an effective inactivation of microRNAs using a STTM strategy in M. truncatula transgenic roots.

This decade introduced "omics" approaches, such as genomics, transcriptomics, proteomics, and metabolomics in association with reverse and forward genetic approaches, developed earlier, to try to identify molecular pathways involved in the development or in the response to environmental conditions as well as in animals and plants. This review summarizes studies that utilized "omics" strategies to unravel the root development in the model legume Medicago truncatula and how external factors such as soil mineral status or the presence of bacteria and fungi affect root system architecture in this species. We also compare these "omics" data to the knowledges concerning the Arabidopsis thaliana root development, nowadays considered as the model of allorhiz root systems. However, unlike legumes, this species is unable to interact with soil nitrogen-fixing rhizobia and arbuscular-mycorrhizal (AM) fungi to develop novel root-derived symbiotic structures. Differences in root organization, development, and regulatory pathways between these two model species have been highlighted.

Two inorganic phosphate (Pi) uptake mechanisms operate in streptophytes and chlorophytes, the two lineages of green plants. PHOSPHATE TRANSPORTER B (PTB) proteins are hypothesized to be the Na + /Pi symporters catalysing Pi uptake in chlorophytes, whereas PHOSPHATE TRANSPORTER 1 (PHT1) proteins are the H + /Pi symporters that carry out Pi uptake in angiosperms. PHT1 proteins are present in all streptophyte lineages. However, Pi uptake in streptophyte algae and marine angiosperms requires Na + influx, suggesting that Na + /Pi symporters also function in some streptophytes. We tested the hypothesis that Na + /Pi symporters exist in streptophytes. We identified PTB sequences in streptophyte genomes. Core PTB proteins are present at the plasma membrane of the liverwort Marchantia polymorpha. The expression of M. polymorpha core PTB proteins in the Saccharomyces cerevisiae pho2 mutant defective in high-affinity Pi transport rescues growth in low-Pi environments. Moreover, levels of core PTB mRNAs of M. polymorpha and the streptophyte alga Coleochaete nitellarum are higher in low-Pi than in Pi-replete conditions, consistent with a role in Pi uptake from the environment. We conclude that land plants inherited two Pi uptake mechanisms - mediated by the PTB and PHT1 proteins, respectively - from their streptophyte algal ancestor. Both systems operate in parallel in extant early diverging land plants., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

To discover mechanisms that controlled the growth of the rooting system in the earliest land plants, we identified genes that control the development of rhizoids in the liverwort Marchantia polymorpha. 336,000 T-DNA transformed lines were screened for mutants with defects in rhizoid growth, and a de novo genome assembly was generated to identify the mutant genes. We report the identification of 33 genes required for rhizoid growth, of which 6 had not previously been functionally characterized in green plants. We demonstrate that members of the same orthogroup are active in cell wall synthesis, cell wall integrity sensing, and vesicle trafficking during M. polymorpha rhizoid and Arabidopsis thaliana root hair growth. This indicates that the mechanism for constructing the cell surface of tip-growing rooting cells is conserved among land plants and was active in the earliest land plants that existed sometime more than 470 million years ago [1, 2]., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The colonization of the land by plants, sometime before 470 million years ago, was accompanied by the evolution tissue systems [1-3]. Specialized structures with diverse functions-from nutrient acquisition to reproduction-derived from single cells in the outermost layer (epidermis) were important sources of morphological innovation at this time [2, 4, 5]. In extant plants, these structures may be unicellular extensions, such as root hairs or rhizoids [6-9], or multicellular structures, such as asexual propagules or secretory hairs (papillae) [10-12]. Here, we show that a ROOTHAIR DEFECTIVE SIX-LIKE (RSL) class I basic helix-loop-helix transcription factor positively regulates the development of the unicellular and multicellular structures that develop from individual cells that expand out of the epidermal plane of the liverwort Marchantia polymorpha; mutants that lack MpRSL1 function do not develop rhizoids, slime papillae, mucilage papillae, or gemmae. Furthermore, we discovered that RSL class I genes are also required for the development of multicellular axillary hairs on the gametophyte of the moss Physcomitrella patens. Because class I RSL proteins also control the development of rhizoids in mosses and root hairs in angiosperms [13, 14], these data demonstrate that the function of RSL class I genes was to control the development of structures derived from single epidermal cells in the common ancestor of the land plants. Class I RSL genes therefore controlled the generation of adaptive morphological diversity as plants colonized the land from the water., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Real-time quantitative polymerase chain reaction (qPCR) has become widely used as a method to compare gene transcript levels across different conditions. However, selection of suitable reference genes to normalize qPCR data is required for accurate transcript level analysis. Recently, Marchantia polymorpha has been adopted as a model for the study of liverwort development and land plant evolution. Identification of appropriate reference genes has therefore become a necessity for gene expression studies. In this study, transcript levels of eleven candidate reference genes have been analyzed across a range of biological contexts that encompass abiotic stress, hormone treatment and different developmental stages. The consistency of transcript levels was assessed using both geNorm and NormFinder algorithms, and a consensus ranking of the different candidate genes was then obtained. MpAPT and MpACT showed relatively constant transcript levels across all conditions tested whereas the transcript levels of other candidate genes were clearly influenced by experimental conditions. By analyzing transcript levels of phosphate and nitrate starvation reporter genes, we confirmed that MpAPT and MpACT are suitable reference genes in M. polymorpha and also demonstrated that normalization with an inappropriate gene can lead to erroneous analysis of qPCR data.

In vascular plants, strigolactones (SLs) are known for their hormonal role and for their role as signal molecules in the rhizosphere. SLs are also produced by the moss Physcomitrella patens, in which they act as signaling factors for controlling filament extension and possibly interaction with neighboring individuals. To gain a better understanding of SL action at the cellular level, we investigated the effect of exogenously added molecules (SLs or analogs) in moss growth media. We used the previously characterized Ppccd8 mutant that is deficient in SL synthesis and showed that SLs affect moss protonema extension by reducing caulonema cell elongation and mainly cell division rate, both in light and dark conditions. Based on this effect, we set up bioassays to examine chemical structure requirements for SL activity in moss. The results suggest that compounds GR24, GR5, and 5-deoxystrigol are active in moss (as in pea), while other analogs that are highly active in the control of pea branching show little activity in moss. Interestingly, the karrikinolide KAR1, which shares molecular features with SLs, did not have any effect on filament growth, even though the moss genome contains several genes homologous to KAI2 (encoding the KAR1 receptor) and no canonical homologue to D14 (encoding the SL receptor). Further studies should investigate whether SL signaling pathways have been conserved during land plant evolution.

Purpose: Clinical evaluation of Alphacor keratoprosthesis in patients at high risk of corneal allograft rejection., Design: Retrospective case series., Patients and Methods: Alphacor implantation was performed via a two-step procedure with intrastromal insertion followed by secondary exposure of the optic after 6 months. Visual acuity and occurrence of postoperative complications were evaluated., Results: Fourteen eyes of 14 patients underwent Alphacor keratoprosthesis implantation. Mean follow-up was 15.6 ± 5.6 months (from 2 to 24 months). Postoperative mean visual acuity gain was 2.5 ± 3.1 lines (from 0 to +11 lines). Visual acuity was superior or equal to 20/200 in 21% of cases. Seven cases of stromal melt (50%) occurred, of which one case (7.1%) experienced spontaneous extrusion of the implant, three cases (21.4%) required tectonic penetrating keratoplasty, and three patients underwent lamellar keratoplasty on top of the implant. Three cases (21.4%) of retroprosthetic membrane were observed and successfully managed. One patient (7.1%) developed late endophthalmitis., Conclusion: Alphacor is an alternative to corneal allograft in cases of corneal blindness at high risk of allograft failure. Throughout the preoperative evaluation, it is imperative to take into account the limitations of this keratoprosthesis in terms of indications, and additional studies are necessary in order to determine and refine the optimal surgical technique for implantation as well as the efficacy of postoperative treatments., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Strigolactones are a novel class of plant hormones controlling shoot branching in seed plants. They also signal host root proximity during symbiotic and parasitic interactions. To gain a better understanding of the origin of strigolactone functions, we characterised a moss mutant strongly affected in strigolactone biosynthesis following deletion of the CAROTENOID CLEAVAGE DIOXYGENASE 8 (CCD8) gene. Here, we show that wild-type Physcomitrella patens produces and releases strigolactones into the medium where they control branching of protonemal filaments and colony extension. We further show that Ppccd8 mutant colonies fail to sense the proximity of neighbouring colonies, which in wild-type plants causes the arrest of colony extension. The mutant phenotype is rescued when grown in the proximity of wild-type colonies, by exogenous supply of synthetic strigolactones or by ectopic expression of seed plant CCD8. Thus, our data demonstrate for the first time that Bryophytes (P. patens) produce strigolactones that act as signalling factors controlling developmental and potentially ecophysiological processes. We propose that in P. patens, strigolactones are reminiscent of quorum-sensing molecules used by bacteria to communicate with one another.

Purpose: To assess corneal epithelial cell survival after keratoplasty., Methods: Corneal impression cytology (CIC) was performed on sex-mismatched corneal transplants. Fluorescent in situ hybridization (FISH) with sex chromosome-specific probes was performed to identify epithelial cell mosaicism and therefore allocate the donor or recipient origin of the cells. Twenty-four samples of corneal epithelial cells derived from 21 transplanted patients were analyzed. All patients received post-operative treatment using dexamethasone eye drops, with progressive tapering over 18 months, and nine patients also received 2% cyclosporine eye drops., Results: Out of the 24 samples reaching quality criteria, sex mosaicism was found in 13, demonstrating the presence of donor-derived cells at the center of the graft for at least 211 days post keratoplasty. Kaplan-Meier analysis established a median survival of donor corneal epithelial cells of 385 days. Although not statistically significant, the disappearance of donor cells seemed to be delayed and the average number of persistent cells appeared to be greater when 2% cyclosporine was used topically as an additional immunosuppressive therapy., Conclusions: The combination of corneal impressions and FISH analysis is a valuable tool with negligible side effects to investigate the presence of epithelial cell mosaicism in sex-mismatched donor transplants. Epithelial cells survived at the center of the graft with a median survival of more than one year, suggesting slower epithelial turnover than previously described.

Purpose: To assess the use of a new polygonal trephination pattern for penetrating keratoplasty (PK) assisted by femtosecond laser., Design: Prospective, nonrandomized clinical study., Methods: Sixteen eyes underwent decagonal PK. Nine had Fuchs dystrophy, 4 had pseudophakic bullous keratopathy, 1 had experienced trauma, 1 had corneal amyloidosis, and 1 had keratoconus. A Femtec (Tecnolas PerfectVision) laser was used to create decagonal penetrating cuts on both donor and recipient corneas. All patients were evaluated for uncorrected visual acuity, best spectacle-corrected visual acuity, pachymetry, topography, and endothelial cell density. Scanning electron microscopy was performed on corneal tissue after surgery., Results: All eyes were treated successfully without intraoperative complications. The mean follow-up ± standard deviation was 9.75 ± 3.5 months. Mean postoperative best spectacle-corrected visual acuity was 20/53, and there was a significant improvement in both uncorrected visual acuity (P = .0019) and best spectacle-corrected visual acuity (P = .001). At 6 months, mean ± standard deviation manifest astigmatism was 1.90 ± 1.20 diopters. Mean endothelial cell density was 1502 ± 458 cells/mm². Scanning electron microscopy displayed straight decagonal cut margins and minor remaining tissue bridges., Conclusions: Use of the decagonal trephination profile was effective and safe to perform PK. Short-term visual results and refractive results are encouraging compared with those of conventional PK studies. Longer-term follow-up and comparative studies are necessary to determine precisely advantages the and optimal surgical settings of this technique., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purpose: Anterior segment imaging using optical coherence tomography (OCT) time domain technology has been used for many years. When it appeared, it was a promising technique in the analysis of the anterior segment, making it possible to reach a definition of the ocular structures comparable with histology. Now with new-generation OCT, it is possible to perform high-definition and three-dimensional imaging., Material and Method: A 3D OCT-1000 (Topcon, Tokyo, Japan) parameterized to obtain high-definition and 3D imaging of the iridocorneal structures., Results: We present a collection of images obtained using this technique., Discussion: Based on a very sophisticated tool for retinal and glaucoma imagery, it is now possible to obtain very-high-quality imaging of the anterior segment, which, with its great versatility, increases the value of acquiring this type of device., Conclusions: The potential of 3D OCT-1000, already a very good exam for the posterior segment, and the only one to perform anterior segment three-dimensional imaging in a single acquisition, should not be neglected.

Objective: Evaluation of the effectiveness of arcuate keratotomy performed with femtosecond laser for correction of postkeratoplasty astigmatism., Methods: This retrospective clinical study included 11 eyes. All cases underwent arcuate keratotomy using femtosecond laser. Outcome measures included visual acuity and endothelial cell density as well as refractive, keratometric, and topographic astigmatism. The incision depth was also evaluated by OCT-3., Results: With a mean follow-up of 7.4+/-6.7 months, uncorrected visual acuity was not modified and the mean best corrected visual acuity significantly improved from 1.68+/-1.59 lines (p=0.007). The mean preoperative refractive cylinder was 5.18+/-1.15D, decreasing to 3.41+/-1.93D (p=0.045) after laser-arcuate keratotomy. The reduction of preoperative keratometric (7.79+/-3.69D) and topographic (7.98+/-2.41D) astigmatism was higher, with a decrease to 4.81+/-2.52D (p=0.021) and 4.36+/-2.59D (p=0.005) postoperatively. Endothelial cell density was not modified after surgery. The difference between achieved and planned incision depth was -10.7+/-63.5mum. All cases were uncomplicated., Discussion: Arcuate keratotomies performed with the femtosecond laser were effective in reducing postkeratoplasty astigmatism and has a number of advantages over conventional techniques. However, efficacy could be improved by a more accurate nomogram., Conclusion: Arcuate keratotomy performed with femtosecond laser is a relatively easy, safe, and effective means of treating postkeratoplasty astigmatism. Given our small sample, much larger series are needed to provide more confident estimates of astigmatism reduction proportions and to adjust correction parameters.

Purpose: To compare the effectiveness of arcuate keratotomy (AK) performed with a femtosecond laser (FSL) or Hanna keratome (Moria, Anthony, France) for correction of postkeratoplasty astigmatism., Design: Prospective, randomized study., Methods: This clinical study included 20 eyes. Two groups of 10 eyes underwent AK using an FLS or keratome. Refractive and keratometric astigmatism were evaluated before surgery and 6 months after surgery. The astigmatic changes in the 2 groups were measured through arithmetic and vector analysis (Alpins method)., Results: Six months after surgery, the mean uncorrected and corrected visual acuities did not change significantly. The mean preoperative refractive cylinder was 8.6 +/- 3.0 diopters (D) and 6.7 +/- 2.1 D, decreasing to 3.9 +/- 2.4 D and 4.7 +/- 2.4 D after laser AK and mechanized AK, respectively. The mean arithmetic change was significantly higher after laser AK, with a decrease of -55.4 +/- 20.7% (P = .011). Vector analysis showed a systematic undercorrection of astigmatism in both groups with a refractive correction index of 0.82 and 0.90 after laser AK and mechanized AK, respectively. Although no statistically significant differences were detected, a wider spread of angle of error and an almost significant difference of mean absolute angle of error (P = .052) suggest a larger misalignment of treatment during mechanized AK. All cases were uncomplicated after laser AK, 1 microperforation occurred and 1 case with off-center incisions occurred after mechanized AK., Conclusions: AK performed with the femtosecond laser was effective in reducing postkeratoplasty astigmatism and has some advantages over conventional techniques. However, efficacy could be improved by a more accurate nomogram and alignment of treatment.