miR396-targeted AtGRF transcription factors are required for coordination of cell division and differentiation during leaf development in Arabidopsis

In plants, cell proliferation and polarized cell differentiation along the adaxial–abaxial axis in the primordium is critical for leaf morphogenesis, while the temporal–spatial relationships between these two processes remain largely unexplored. Here, it is reported that microRNA396 (miR396)-targeted Arabidopsis growth-regulating factors (AtGRFs) are required for leaf adaxial–abaxial polarity in Arabidopsis. Reduction of the expression of AtGRF genes by transgenic miR396 overexpression in leaf polarity mutants asymmetric leaves1 (as1) and as2 resulted in plants with enhanced leaf adaxial–abaxial defects, as a consequence of reduced cell proliferation. Moreover, transgenic miR396 overexpression markedly decreased the cell division activity and the expression of cell cycle-related genes, but resulted in an increased percentage of leaf cells with a higher ploidy level, indicating that miR396 negatively regulates cell proliferation by controlling entry into the mitotic cell cycle. miR396 is mainly expressed in the leaf cells arrested for cell division, coinciding with its roles in cell cycle regulation. These results together suggest that cell division activity mediated by miR396-targeted AtGRFs is important for polarized cell differentiation along the adaxial–abaxial axis during leaf morphogenesis in Arabidopsis

Genetic variability in Quercus suber L. somatic embryogenesis

Mestrado em Microbiologia MolecularAs culturas de tecidos celulares ou tecidos de plantas comportam-se, em muitos aspectos, de modo semelhante aos microorganismos (por exemplo, em ambos os casos são requeridas condições de meios de cultura e assépsia adequados, apresentam curvas de crescimento sigmóide, podendo também ocorrer variações genéticas). A caracterização e análise da variabilidade genética destas culturas são, actualmente, efectuadas com base em marcadores moleculares, muitos dos quais foram desenvolvidos inicialmente para estudos em microrganismos ou células animais (como por exemplo a “Polymerase Chain Reaction”) e mais tarde transferidas para a análise da variabilidade genética em culturas celulares de plantas (por exemplo, como forma de assegurar a fidelidade clonal). A fidelidade clonal é o factor de maior importância na comercialização de material micropropagado obtido por métodos de cultura de tecidos “in vitro”. Este facto é da maior importância nos programas de melhoramento florestal, dado que a micropropagação de plantas superiores é um meio rápido de produção de stocks de plantas clonais para programas de reflorestação e conservação de germoplasma elite ou de elevado interesse ecológico. Contudo, devido ao longo ciclo de vida das espécies lenhosas, uma análise alargada de parâmetros genéticos e fenotípicos é essencial, especialmente quando as plantas derivam de embriogénese somática, processo no qual se consideram as células como estando sobre condições de stress (por exemplo, exposição a auxinas), bem como estados de ciclo celular repetitivos. Atendendo a estas considerações, utilizámos embriões somáticos de Quercus suber L. (genótipo QsG3) obtidos a partir de explantes de folha de uma árvore adulta, os quais foram mantidos no nosso laboratório por um ano. A variabilidade genética dos embriões somáticos e das plantas resultantes foi avaliada por dois marcadores moleculares: “histone H3 promoter type I element” e “RAPD”. Não foi encontrada variabilidade genética de acordo com estes dois marcadores durante todo o processo de embriogénese, assegurando assim a reprodutibilidade deste processo “in vitro”. Concluindo, os marcadores moleculares usados neste trabalho podem representar uma ajuda adicional como técnicas para assegurar variabilidade genética em complemento com outras técnicas. ABSTRACT: Plant cell or tissue cultures behave, in many aspects, in a similar way to microorganisms (e.g. all require a suitable culture medium and asseptic conditions, they present a sigmoid growth curve and genetic variation may occur in those cultures). The characterization and analysis of genetic variability of these cultures is presently performed by molecular markers, many of which were first developed for microorganisms or animal cell studies (e.g. Polymerase Chain Reaction) and later transferred to analysis of genetic variability in plant cell cultures (e.g. to assess clonal fidelity). In fact, clonal fidelity is a major concern in commercial micropropagation using in vitro tissue cultures. This is particularly important in forest breeding programs as micropropagation of tree species since it offers a rapid means of producing clonal planting stock for forestation programmes and conservation of elite and rare germplasm. But due to the long period of woody species life-cycle, a screening for genetic and phenotypical parameters of micropropagated plants is essential, in particular when plants derived from somatic embryogenesis, where cells may be considered to be under stressing conditions (e.g. auxins), as also under repetitive cell cycles. Within this scope, we used Quercus suber L. somatic embryos (Gs3 genotype) achieved from leaf explants of a mature adult plant, maintained in our laboratory for one year. Genetic variability of somatic embryos and emblings was evaluated by using two molecular markers: histone H3 promoter type I element and RAPD. We found no genetic variability according with these two markers during the whole process of embryogenesis, assessing the reliability of this in vitro regeneration process. In conclusion, the molecular markers used in this work may represent an added value as tools of genetic variability assessment in complement with other techniques

Caractérisation de la famille des protéines Kinases de type NIMA chez les plantes et analyse fonctionnelle de PNek1, une NEK du peuplier (Populus tremula X P. Alba clone 717 I-B4)

Les protéines kinases de type NIMA (NIMA related kinases - Neks) forment une famille relativement bien conservée chez les eucaryotes. Plusieurs d’entre elles, comme la protéine kinase NIMA d’Aspergillus nidulans et la protein Nek2 de mammifères ont fait l’objet d’études suffisamment approfondies pour les impliquer dans la régulation du cycle cellulaire. L’objectif du présent travail était de caractériser la famille des Neks chez les plantes, et plus particulièrement de déterminer le rôle de PNek1, une Nek de peuplier. J’ai identifié neuf PtNeks chez Populus trichocarpa, sept AtNeks chez Arabidopsis thaliana et six OsNeks chez Oryza sativa. L’analyse phylogénétique et leur distribution chromosomique suggèrent une descendance unique chez les plantes, probablement à partir de Nek1. L’analyse du profil d’expression transcriptionnelle indique que la régulation de l’expression des Neks est liée aux patrons de développement basipétal de la feuille et vasculaire de la plante. Plus particulièrement, l’analyse de l’expression du gène PNek1 révèle une concordance exacte avec les sites de production de l’auxine, du développement basipétal de la feuille et de l’initiation du système vasculaire. PNek1 n’est toutefois pas induite par la signalisation de l’auxine. La surexpression de PNek1 chez Arabidopsis induit des anomalies importantes au niveau de l’inflorescence, empêchant même la fertilisation de la fleur. Au plan cellulaire, PNek1 est localisé dans le nucléole et s’accumule lors de la phase G2 précédant la mitose. L’accumulation de PNek1 est aussi induite lors d’un stress génotoxique au point de contrôle en G1/S. Des résultats récents de double hybride indiquent que PNek1 pourrait être impliquée dans la maturation de l’ARNm puisqu’elle interagit avec DBR1, une protéine directement impliquée dans l’épissage. Le présent travail offre une perspective inédite de la littérature des Neks comme régulateurs du cycle cellulaire. Le contexte biologique particulier du peuplier m’a aussi conduit à associer les Neks au développement d’organes complexes. Cette approche et ces observations représentent donc en soit une contribution originale, se distinguant des nombreuses études antérieures faites chez les mammifères, où seule leur relation au cycle cellulaire a été étudiée.The NIMA-related kinases family (Neks) is well conserved among eukaryotes. Several studies, especially on Aspergillus nidulans NIMA and mammalian Nek2, have tagged them as cell cycle regulators. The objective pursued in this work was to characterise the plant Nek family and, more specifically, to identify a possible role for PNek1, a Nek from poplar tree. Here, I describe nine PtNeks in Populus trichocarpa, seven AtNeks in Arabidopsis thaliana and six OsNeks in Oryza sativa. Phylogenetic analysis in addition to their chromosomal distribution suggest a unique origin for all plant Neks. Exhaustive transcript expression analysis indicates that plant Neks regulation is related to the basipetal and vascular plant development patterns. Moreover, PNek1 promoter expression analysis reveals a striking similarity with sites of auxin production, basipetal leaf development and vascular initiation. However, PNek1 is not induced by auxin signalling. PNek1 overexpression in Arabidopsis induces severe inflorescence anomalies, which can lead to flower sterility. At the cell level, PNek1 is localised in the nucleoli and accumulates during the G2 phase before the onset for mitosis. PNek1 transcript accumulation could also be induced by a genotoxic stress at the G1/S checkpoint. Yeast two-hybrid experiments indicate that PNek1 could be involved in mRNA maturation since it interacts with DBR1, a protein directly involved in RNA splicing. This work offers a unique perspective to the actual Neks literature as cell cycle regulators. The particular biological context of poplar trees also brought me to associate Neks with complex organ development. This approach and these observations represent an original contribution, distinguishing itself from the numerous mammalians studies which only looked at their relation to the cell cycle regulation

Systematic Screen of Histone H4 in Arabidopsis thaliana

Histones regulate diverse processes in eukaryotes and consequently, can have widespread effects on organismal fitness and development. Histones are a dynamic target for a variety of post-translational modifications (PTMs) and the assessment of histone function has typically been accomplished by mutating enzymes that catalyze and/or recognize these PTMs (i.e., writers and readers, respectively). Although considerable information has been gained in the past several decades by using this strategy, multiple issues such as writer/reader redundancy, unidentified writers/readers of histone PTMs, and writers/readers with additional non-histone targets can preclude the identification of new roles for histones and complicate the assessment of mutant phenotypes. To bypass these issues and provide a complementary strategy to study histones, large-scale histone replacement systems have been developed and optimized in yeast and fly model systems. However, such systems have never been implemented in plants in part due to the difficulty in eliminating endogenous histone genes that are typically present in many copies and different locations in plant genomes. Here, we present the development of a genetic strategy for the plant model organism Arabidopsis thaliana in which the expression of endogenous histone H4 can be completely replaced with modified H4 transgenes. We use histone H4, which is a single variant histone in plants that is encoded by the largest number of genes (8) among all functionally-distinct histone proteins, as a proof-of-concept for an experimental system allowing the direct assessment of histone function in plants. Our CRISPR/Cas9-based strategy allows for the simultaneous targeting of many histone genes for the generation of a background depleted of endogenous histone expression. We validated our platform by showing that a single transformation with our modified H4 transgenes can restore a wild-type phenotype, demonstrating that our system can be used for the rapid establishment of histone replacement in plants. Using this strategy, we established a collection of plants expressing different H4 point mutants targeting residues that may be post-translationally modified in vivo. To demonstrate the utility of this new H4 mutant collection, we screened it to uncover substitutions in H4 that alter flowering time, rosette morphology, DNA replication, chromatin structure, and gene silencing. We identified different mutations in the tail (H4R17A) and the globular domain (H4R36A, H4R39K, H4R39A, and H4K44A) of H4 that strongly accelerate the floral transition. Additionally, we used machine learning to identify H4 mutations that alter different morphometric traits in vegetative tissue. Finally, we identified several novel roles for H4 tail and globular domain residues in the regulation of endoreduplication, chromatin condensation, and transposon silencing. After these broad screens for histone function, we then performed targeted analyses of H4R17A mutants to determine a molecular mechanism responsible for the early flowering displayed by these mutants. We found that a conserved regulatory relationship between H4R17 and the ISWI chromatin remodeling complex in plants is responsible for the phenotypes observed in H4R17A mutants. Similar to other biological systems, H4R17 regulates nucleosome spacing via ISWI, and mutation of H4R17 results in large-scale changes to global nucleosome positioning and gene expression, leading to altered development. Overall, this work provides a large set of H4 mutants to the plant epigenetics community that can be used to systematically assess histone H4 function in A. thaliana and a blueprint to replicate this strategy for studying other histone proteins in plants. As this resource represents the largest collection of H4 point mutants in a multicellular organism, our work will enable new insights into the regulation of chromatin by histone H4 in multicellular eukaryotes