New Insights into the Enhancement of Adventitious Root Formation Using N,N'-Bis(2,3-methylenedioxyphenyl)urea.

Adventitious rooting is a process of postembryonic organogenesis strongly affected by endogenous and exogenous factors. Although adventitious rooting has been exploited in vegetative propagation programs for many plant species, it is a bottleneck for vegetative multiplication of difficult-to-root species, such as many woody species. The purpose of this research was to understand how N,N'-bis-(2,3-methylenedioxyphenyl)urea could exert its already reported adventitious rooting adjuvant activity, starting from the widely accepted knowledge that adventitious rooting is a hormonally tuned progressive process. Here, by using specific in vitro bioassays, histological analyses, molecular docking simulations and in vitro enzymatic bioassays, we have demonstrated that this urea derivative does not interfere with polar auxin transport; it inhibits cytokinin oxidase/dehydrogenase (CKX); and, possibly, it interacts with the apoplastic portion of the auxin receptor ABP1. As a consequence of this dual binding capacity, the lifespan of endogenous cytokinins could be locally increased and, at the same time, auxin signaling could be favored. This combination of effects could lead to a cell fate transition, which, in turn, could result in increased adventitious rooting.

Adventitious Root Formation in Tree Species.

Adventitious root formation is a postembryonic organogenesis process induced by differentiated cells other than those specified to develop roots [...].

Comprehensive analysis of the isomiRome in the vegetative organs of the conifer Pinus pinaster under contrasting water availability.

An increasing number of microRNAs (miRNAs) and miRNA-related sequences produced during miRNA biogenesis, comprising the isomiRome, have been recently highlighted in different species as critical mediators of environmental stress responses. Conifers have some of the largest known genomes but an extensive characterization of the isomiRome from any conifer species has been lacking. We provide here a comprehensive overview of the Pinus pinaster isomiRome expressed in roots, stem and needles under well-watered and drought conditions. From the 13,441 unique small RNA sequences identified, 2,980 were annotated as canonical miRNAs or miRNA* and the remaining were classified as isomiRNA or miRNA-like sequences. A survey of their expression patterns highlighted roots as the most responsive organ under drought, where specific sequences of which a 24-nt novel miRNA stood out, were strongly down-regulated. Given the putative roles of the miRNA-targeted transcripts validated specifically in root tissues, some of the miRNAs, conserved and novel, are shortlisted as potential regulators of drought response. These results provide a valuable resource for comparative studies between gymnosperms and angiosperms. Furthermore, it evidences high transferability of the isomiRome between pine species being a useful basis for further molecular regulation and physiological studies, and especially those focused on adaptation to drought conditions.

Expression Levels of Genes Encoding Proteins Involved in the Cell Wall-Plasma Membrane-Cytoskeleton Continuum Are Associated With the Maturation-Related Adventitious Rooting Competence of Pine Stem Cuttings.

Stem cutting recalcitrance to adventitious root formation is a major limitation for the clonal propagation or micropropagation of elite genotypes of many forest tree species, especially at the adult stage of development. The interaction between the cell wall-plasma membrane and cytoskeleton may be involved in the maturation-related decline of adventitious root formation. Here, pine homologs of several genes encoding proteins involved in the cell wall-plasma membrane-cytoskeleton continuum were identified, and the expression levels of 70 selected genes belonging to the aforementioned group and four genes encoding auxin carrier proteins were analyzed during adventitious root formation in rooting-competent and non-competent cuttings of Pinus radiata. Variations in the expression levels of specific genes encoding cell wall components and cytoskeleton-related proteins were detected in rooting-competent and non-competent cuttings in response to wounding and auxin treatments. However, the major correlation of gene expression with competence for adventitious root formation was detected in a family of genes encoding proteins involved in sensing the cell wall and membrane disturbances, such as specific receptor-like kinases (RLKs) belonging to the lectin-type RLKs, wall-associated kinases, Catharanthus roseus RLK1-like kinases and leucine-rich repeat RLKs, as well as downstream regulators of the small guanosine triphosphate (GTP)-binding protein family. The expression of these genes was more affected by organ and age than by auxin and time of induction.

A Perspective on Adventitious Root Formation in Tree Species.

Adventitious root formation is an organogenic process, regulated at several levels, that is crucial for the successful vegetative propagation of numerous plants. In many tree species, recalcitrance to adventitious root formation is a major limitation in the clonal propagation of elite germplasms. Information on the mechanisms underlying the competence for adventitious root formation is still limited. Therefore, increasing our understanding of the mechanisms that enable differentiated somatic cells to switch their fates and develop into root meristematic cells, especially those involved in cell developmental aging and maturation, is a priority in adventitious root-related research. The dynamic cell wall-cytoskeleton, along with soluble factors, such as cellular signals or transcriptional regulators, may be involved in adult cell responses to intrinsic or extrinsic factors, resulting in maintenance, induction of root meristematic cell formation, or entrance into another differentiating pathway.

Cellular dynamics during maturation-related decline of adventitious root formation in forest tree species.

Adventitious root formation is a process in which roots are induced, from determined or differentiated cells that have not been specified to develop a root, at positions where they do not normally occur during development. In forest tree species, a decline in the capacity to form adventitious roots from similar cell types in stem cuttings is associated with tree age and maturity. This decline limits the success of vegetative propagation of selected adult trees. The joint action of local signals and a dynamic cascade of regulatory changes in gene expression, resulting in stereotypical cell division patterns, regulate cell fate changes that enable a somatic differentiated cell to reactivate meristem programs toward the induction of an adventitious root meristem.

Molecular Dissection of the Regenerative Capacity of Forest Tree Species: Special Focus on Conifers.

Somatic embryogenesis (SE) and organogenesis have become leading biotechnologies for forest tree improvement and the implementation of multi-varietal forestry. Despite major advances in clonal propagation using these technologies, many forest tree species, such as conifers, show a low regeneration capacity. Developmental factors such as genotype, the type and age of the explant or tissue, and the age and maturity of the mother tree are limiting factors for the success of propagation programs. This review summarizes recent research on the molecular pathways involved in the regulation of key steps in SE and organogenesis of forest tree species, mainly conifers. The interaction between auxin and stress conditions, the induction of cell identity regulators and the role of cell wall remodeling are reviewed. This information is essential to develop tools and strategies to improve clonal propagation programs for forest tree species.

The GRAS gene family in pine: transcript expression patterns associated with the maturation-related decline of competence to form adventitious roots.

BACKGROUND: Adventitious rooting is an organogenic process by which roots are induced from differentiated cells other than those specified to develop roots. In forest tree species, age and maturation are barriers to adventitious root formation by stem cuttings. The mechanisms behind the respecification of fully differentiated progenitor cells, which underlies adventitious root formation, are unknown. RESULTS: Here, the GRAS gene family in pine is characterized and the expression of a subset of these genes during adventitious rooting is reported. Comparative analyses of protein structures showed that pine GRAS members are conserved compared with their relatives in angiosperms. Relatively high GRAS mRNA levels were measured in non-differentiated proliferating embryogenic cultures and during embryo development. The mRNA levels of putative GRAS family transcription factors, including Pinus radiata's SCARECROW (SCR), PrSCR, and SCARECROW-LIKE (SCL) 6, PrSCL6, were significantly reduced or non-existent in adult tissues that no longer had the capacity to form adventitious roots, but were maintained or induced after the reprogramming of adult cells in rooting-competent tissues. A subset of genes, SHORT-ROOT (PrSHR), PrSCL1, PrSCL2, PrSCL10 and PrSCL12, was also expressed in an auxin-, age- or developmental-dependent manner during adventitious root formation. CONCLUSIONS: The GRAS family of pine has been characterized by analyzing protein structures, phylogenetic relationships, conserved motifs and gene expression patterns. Individual genes within each group have acquired different and specialized functions, some of which could be related to the competence and reprogramming of adult cells to form adventitious roots.

Direct reprogramming of adult somatic cells toward adventitious root formation in forest tree species: the effect of the juvenile-adult transition.

Cellular plasticity refers, among others, to the capability of differentiated cells to switch the differentiation process and acquire new fates. One way by which plant cell plasticity is manifested is through de novo regeneration of organs from somatic differentiated cells in an ectopic location. However, switching the developmental program of adult cells prior to organ regeneration is difficult in many plant species, especially in forest tree species. In these species, a decline in the capacity to regenerate shoots, roots, or embryos from somatic differentiated cells is associated with tree age and maturation. The decline in the ability to form adventitious roots from stem cuttings is one of the most dramatic effects of maturation, and has been the subject of investigations on the basic nature of the process. Cell fate switches, both in plants and animals, are characterized by remarkable changes in the pattern of gene expression, as cells switch from the characteristic expression pattern of a somatic cell to a new one directing a new developmental pathway. Therefore, determining the way by which cells reset their gene expression pattern is crucial to understand cellular plasticity. The presence of specific cellular signaling pathways or tissue-specific factors underlying the establishment, maintenance, and redirection of gene expression patterns in the tissues involved in adventitious root formation could be crucial for cell fate switch and for the control of age-dependent cellular plasticity.

Genetic control of functional traits related to photosynthesis and water use efficiency in Pinus pinaster Ait. drought response: integration of genome annotation, allele association and QTL detection for candidate gene identification.

BACKGROUND: Understanding molecular mechanisms that control photosynthesis and water use efficiency in response to drought is crucial for plant species from dry areas. This study aimed to identify QTL for these traits in a Mediterranean conifer and tested their stability under drought. RESULTS: High density linkage maps for Pinus pinaster were used in the detection of QTL for photosynthesis and water use efficiency at three water irrigation regimes. A total of 28 significant and 27 suggestive QTL were found. QTL detected for photochemical traits accounted for the higher percentage of phenotypic variance. Functional annotation of genes within the QTL suggested 58 candidate genes for the analyzed traits. Allele association analysis in selected candidate genes showed three SNPs located in a MYB transcription factor that were significantly associated with efficiency of energy capture by open PSII reaction centers and specific leaf area. CONCLUSIONS: The integration of QTL mapping of functional traits, genome annotation and allele association yielded several candidate genes involved with molecular control of photosynthesis and water use efficiency in response to drought in a conifer species. The results obtained highlight the importance of maintaining the integrity of the photochemical machinery in P. pinaster drought response.

Epigenetic regulation of adaptive responses of forest tree species to the environment.

Epigenetic variation is likely to contribute to the phenotypic plasticity and adaptative capacity of plant species, and may be especially important for long-lived organisms with complex life cycles, including forest trees. Diverse environmental stresses and hybridization/polyploidization events can create reversible heritable epigenetic marks that can be transmitted to subsequent generations as a form of molecular "memory". Epigenetic changes might also contribute to the ability of plants to colonize or persist in variable environments. In this review, we provide an overview of recent data on epigenetic mechanisms involved in developmental processes and responses to environmental cues in plant, with a focus on forest tree species. We consider the possible role of forest tree epigenetics as a new source of adaptive traits in plant breeding, biotechnology, and ecosystem conservation under rapid climate change.

Towards decoding the conifer giga-genome.

Several new initiatives have been launched recently to sequence conifer genomes including pines, spruces and Douglas-fir. Owing to the very large genome sizes ranging from 18 to 35 gigabases, sequencing even a single conifer genome had been considered unattainable until the recent throughput increases and cost reductions afforded by next generation sequencers. The purpose of this review is to describe the context for these new initiatives. A knowledge foundation has been acquired in several conifers of commercial and ecological interest through large-scale cDNA analyses, construction of genetic maps and gene mapping studies aiming to link phenotype and genotype. Exploratory sequencing in pines and spruces have pointed out some of the unique properties of these giga-genomes and suggested strategies that may be needed to extract value from their sequencing. The hope is that recent and pending developments in sequencing technology will contribute to rapidly filling the knowledge vacuum surrounding their structure, contents and evolution. Researchers are also making plans to use comparative analyses that will help to turn the data into a valuable resource for enhancing and protecting the world's conifer forests.

CsSCL1 is differentially regulated upon maturation in chestnut microshoots and is specifically expressed in rooting-competent cells.

The Castanea sativa SCL1 gene (CsSCL1) has previously been shown to be induced by auxin during adventitious root (AR) formation in rooting-competent microshoots. However, its expression has not previously been analyzed in rooting-incompetent shoots. This study focuses on the regulation of CsSCL1 during maturation and the role of the gene in the formation of AR. The expression of CsSCL1 in rooting-incompetent microshoots and other tissues was investigated by quantitative reverse transcriptase--polymerase chain reaction. The analysis was complemented by in situ hybridization of the basal segments of rooting-competent and --incompetent microshoots during AR induction, as well as in AR and lateral roots. It was found that CsSCL1 is upregulated by auxin in a cell-type- and phase-dependent manner during the induction of AR. In root-forming shoots, CsSCL1 mRNA was specifically located in the cambial zone and derivative cells, which are rooting-competent cells, whereas in rooting-incompetent shoots the hybridization signal was more diffuse and evenly distributed through the phloem and parenchyma. CsSCL1 expression was also detected in lateral roots and axillary buds. The different CsSCL1 expression patterns in rooting-competent and -incompetent microshoots, together with the specific location of transcripts in cell types involved in root meristem initiation and in the root primordia of AR and lateral roots, indicate an important role for the gene in determining whether certain cells will enter the root differentiation pathway and its involvement in meristem maintenance.

Carcinoma epidermoide infiltrante de clítoris en paciente de 30 años, no ligado a infección por virus del papiloma humano / Invasive squamous cell carcinoma of the vulva in a 30-year-old woman not associated with human papillomavirus infection

El carcinoma epidermoide invasor de vulva es excepcional antes de los 40 años. Presentamos un el caso de un carcinoma epidermoide diferenciado de clítoris en una paciente de 30 años, nulípara, que debutó con una clínica de vulvodinia de 2 años de evolución. A la exploración, se detectó una tumoración sospechosa en el clítoris, que se biopsió y correspondió a un carcinoma epidermoide invasor de vulva estadio Ia. Se practicó una cirugía conservadora (tumorectomía con márgenes de seguridad y ganglio centinela) con buena recuperación de la paciente y reinserción de ella a la vida laboral normal en un plazo inferior a los 30 días (AU)

Invasive squamous cell carcinoma of the vulva is exceptional before the age of 40 years. We present a differentiated squamous carcinoma of the clitoris in a 30-year-old nulliparous woman who presented with vulvodynia for the past 2 years. On examination, a suspicious tumor was detected in the clitoris. Biopsy revealed an invasive squamous cell carcinoma of the vulva, stage Ia. Conservative surgery (tumorectomy with safety margins and sentinel node) was performed with good patient recovery and return to normal working life before 30 days (AU)

Reprogramming adult cells during organ regeneration in forest species.

The possibility of regenerating whole plants from somatic differentiated cells emphasizes the plasticity of plant development. Cell-type respecification during regeneration can be induced in adult tissues as a consequence of injuries, changes in external or internal stimuli or changes in positional information. However, in many plant species, switching the developmental program of adult cells prior to organ regeneration is difficult, especially in forest species. Besides its impact on forest productivity, basic information on the flexibility of cell differentiation is necessary for a comprehensive understanding of the epigenetic control of cell differentiation and plant development. Studies of reprogramming adult cells in terms of regulative expression changes of selected genes will be of great interest to unveil basic mechanisms regulating cellular plasticity.

Characterization and expression of a Pinus radiata putative ortholog to the Arabidopsis SHORT-ROOT gene.

We characterized a Pinus radiata D. Don putative ortholog to the Arabidopsis thaliana (L.) Heynh. SHORT--ROOT gene (AtSHR) and analyzed its expression in different organs during vegetative development and in response to exogenous auxin during adventitious rooting. The predicted protein sequence contained domains characteristic of the GRAS protein family and showed a strong similarity to the SHORT--ROOT (SHR) proteins. Quantitative reverse transcriptase--polymerase chain reaction (qRT-PCR) and in situ hybridization showed that the gene is predominantly expressed in roots, root primordia and in the cambial region of hypocotyl cuttings. Increased mRNA levels were observed, independently of the presence or absence of exogenous auxin, in the cambial region and rooting competent cells of hypocotyl cuttings within the first 24 h of adventitious rooting, before the activation of cell divisions and the organization of the adventitious root meristem. The expression pattern in organs and during adventitious rooting was similar to that of a Pinus radiata SCARECROW-LIKE (PrSCL1) gene, except that PrSCL1 is induced in response to exogenous auxin. Results suggest that the Pinus radiata SHORT-ROOT (PrSHR) gene has a role in root meristem formation and maintenance and in the cambial region of hypocotyl cuttings.

Two SCARECROW-LIKE genes are induced in response to exogenous auxin in rooting-competent cuttings of distantly related forest species.

We characterized SCARECROW-LIKE genes induced by auxin in rooting-competent cuttings of two distantly related forest species (Pinus radiata D. Don and Castanea sativa Mill.) before the activation of cell division that results in adventitious root formation. The predicted protein sequences contain domains characteristic of the GRAS protein family and show a strong similarity to the SCARECROW-LIKE proteins, indicating conserved functions of these proteins. Quantitative RT-PCR analysis showed that these genes are expressed at relatively high levels in roots. Induction of increased mRNA levels in rooting-competent cuttings of both species in response to exogenous auxin was observed within the first 24 h of the root induction process, a time when cell reorganization takes place, but before the resumption of cell division and the appearance of adventitious root primordia. These results suggest that SCARECROW-LIKE genes play a role during the earliest stages of adventitious root formation.

Age-related loss of rooting capability in Arabidopsis thaliana and its reversal by peptides containing the Arg-Gly-Asp (RGD) motif.

We describe here an experimental system to study the age-related decline of adventitious root formation in Arabidopsis thaliana L. (Heynh), ecotype Landsberg erecta (Ler). The system is based on the different rooting capacity of hypocotyls from de-rooted juvenile (12-day-old) and adult (26-day-old) plants. Hypocotyls from de-rooted juvenile plants rooted readily within a week of culture, and the rooting process was not dependent on exogenous auxin. In contrast, hypocotyls from de-rooted adult plants rooted poorly and only after longer periods of time. Exogenously applied auxin had no effect on rooting of hypocotyls from de-rooted adult plants. Rooting capacity, although correlated with the transition to flowering, did not depend on this transition. Root induction declined in a similar manner when the transition to flowering was delayed, either genetically with the fve mutant or physiologically with short days. The results showed that rooting of hypocotyls from de-rooted adult plants depended on the effect of peptides containing the RGD motif. Both the percentage of rooting and the number of roots were largely increased when the hypocotyls were treated transiently with the RGD peptide. The effect of the RGD peptide was a necessary, but not sufficient, condition for rooting of hypocotyls from de-rooted adult plants.