Biology subjects

Micol, J. L.

Publications and source records attributed to Micol, J. L..

8 recordsLinked to original sources

bioRxiv2024

Functional conservation and divergence of Arabidopsis VENOSA4 and human SAMHD1 in DNA repair

The human deoxyribonucleoside triphosphatase (dNTPase) Sterile alpha motif and histidine-aspartate domain containing protein 1 (SAMHD1) has a dNTPase-independent role in repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). Here, we show that VENOSA4 (VEN4), the probable Arabidopsis thaliana ortholog of SAMHD1, also functions in DSB repair by HR. The ven4 loss-of-function mutants showed increased DNA ploidy and deregulated DNA repair genes, suggesting DNA damage accumulation. Hydroxyurea, which blocks DNA replication and generates DSBs, induced VEN4 expression. The ven4 mutants were hypersensitive to hydroxyurea, with decreased DSB repair by HR. Metabolomic analysis of the strong ven4-0 mutant revealed depletion of metabolites associated with DNA damage responses. In contrast to SAMHD1, VEN4 showed no evident involvement in preventing R-loop accumulation. Our study thus reveals functional conservation in DNA repair by VEN4 and SAMHD1. One sentence summaryHuman SAMHD1 is involved in dNTP metabolism and DNA repair; the latter function is conserved in VEN4, its likely Arabidopsis ortholog.

plant biology↗

bioRxiv2024

Postembryonic developmental roles of the Arabidopsis KEULE gene

Cytokinesis in plant cells begins with the fusion of vesicles that transport cell wall materials at the center of the cell division plane, where the cell plate forms and expands radially until it fuses with the parental cell wall at the preprophase band. Vesicle fusion is facilitated by trans-SNARE complexes, with assistance from Sec1/Munc18 (SM) proteins. The SNARE protein KNOLLE and the SM protein KEULE are required for membrane fusion at the cell plate. Due to the crucial function of KEULE, all Arabidopsis (Arabidopsis thaliana) keule mutants identified to date are seedling lethal. Here, we identified the Arabidopsis serrata4-1 (sea4-1) and sea4-2 mutants, which carry recessive, hypomorphic alleles of KEULE. Homozygous sea4-1 and sea4-2 plants are viable and fertile but exhibit smaller rosettes and fewer leaves at bolting than the wild type. Their leaves are serrated, small, and undulated, with a complex venation pattern, develop necrotic patches, and undergo premature senescence. We established a likely relationship between these phenotypes and their defects in cytokinesis through reduced cell wall integrity and increased unfolded protein response. These findings shed light on the roles of KEULE in postembryonic development, particularly in the patterning of rosette leaves and leaf margins.

plant biology↗

bioRxiv2024

Overlapping roles of Arabidopsis INCURVATA11 and CUPULIFORMIS2 as Polycomb Repressive Complex 2 accessory proteins

Polycomb Repressive Complex 2 (PRC2) catalyzes the trimethylation of lysine 27 of histone H3 (H3K27me3) and plays a key role in epigenetic repression of gene expression in plants and animals. PRC2 core components have all been identified in Arabidopsis thaliana, with an expanding list of accessory proteins, some of which facilitate the recruitment of PRC2 to specific targets. INCURVATA11 (ICU11) is a 2-oxoglutarate and Fe2+-dependent dioxygenase that was previously shown to be a likely PRC2 accessory protein. In Tandem Affinity Purification (TAP)-based screens for interacting partners of ICU11 and its redundant paralog CUPULIFORMIS2 (CP2), we discovered that ICU11 interacts with four PRC2 core components, including EMBRYONIC FLOWER 2 (EMF2), and with the accessory proteins EMF1, TELOMERE REPEAT BINDING 1 (TRB1), TRB2, and TRB3. CP2 did not interact with PRC2 core components, nor with TRB1, TRB2, or TRB3, but did interact with TRB4 and TRB5. Both ICU11 and CP2 interacted with the nuclear proteins NAC DOMAIN CONTAINING PROTEIN 50 (NAC050), NAC052 and COP9 SIGNALOSOME SUBUNIT 1 (CSN1). Bimolecular Fluorescence Complementation (BiFC) assays revealed that ICU11 and CP2 both interact with the PRC2 core components CURLY LEAF and SWINGER, and the accessory proteins LIKE HETEROCHROMATIN PROTEIN 1, TRB1, and TRB3. ICU11 and CP2 did not interact with each other. Beyond their phenotypes, transcriptomic profiles revealed strong similarities between emf2-3 and the double mutant icu11-5 cp2-1, as well as with mutants in PRC2 core components. A significant proportion of the genes mis-regulated in icu11-5 cp2-1 are known to harbor H3K27me3 repressive marks in the wild type. Our results provide further evidence that ICU11 acts as a PRC2 accessory protein, and strongly suggest that CP2 plays a similar role.

plant biology↗

bioRxiv2023

The unequal functional redundancy of Arabidopsis INCURVATA11 and CUPULIFORMIS2 is not dependent on genetic background

The paralogous genes INCURVATA11 (ICU11) and CUPULIFORMIS2 (CP2) encode components of the epigenetic machinery in Arabidopsis and belong to the 2-oxoglutarate and Fe (II)-dependent dioxygenase superfamily. We previously inferred unequal functional redundancy between ICU11 and CP2 from a study of the synergistic phenotypes of the double mutant and sesquimutant combinations of icu11 and cp2 mutations, although they represented mixed genetic backgrounds. To avoid potential confounding effects arising from different genetic backgrounds, we generated the icu11-5 and icu11-6 mutants via CRISPR/Cas genome editing in the Col-0 background and crossed them to cp2 mutants in Col-0. The resulting mutants exhibited a postembryonic-lethal phenotype reminiscent of strong embryonic flower (emf) mutants. Double mutants involving icu11-5 and mutations affecting epigenetic machinery components displayed synergistic phenotypes, whereas cp2-3 did not besides icu11-5. Our results confirmed the unequal functional redundancy between ICU11 and CP2 and demonstrated that it is not allele or genetic background specific. An increase in sucrose content in the culture medium partially rescued the post-germinative lethality of icu11 cp2 double mutants and sesquimutants, facilitating the study of their morphological phenotypes throughout their life cycle, which include floral organ homeotic transformations. We thus established that the ICU11-CP2 module is required for proper flower organ identity.

genetics↗

bioRxiv2023

Analysis of Arabidopsis venosa4-0 supports the role of VENOSA4 in dNTP homeostasis

An imbalance in the deoxyribonucleoside triphosphate (dNTP) pool caused by an increase or decrease in the levels of any of the four dNTPs leads to increased DNA mutations, overloading DNA repair mechanisms. The human protein SAMHD1 (Sterile alpha motif and histidine-aspartate domain containing protein 1) functions as a dNTPase to maintain the balance of the dNTP pool, as well as in DNA repair. In eukaryotes, the limiting step in de novo dNTP synthesis is catalyzed by RIBONUCLEOTIDE REDUCTASE (RNR), which consists of two R1 and two R2 subunits. In Arabidopsis, RNR1 is encoded by CRINKLED LEAVES 8 (CLS8) and RNR2 by three paralogous genes, including TSO2 (TSO MEANING UGLY IN CHINESE 2). In plants, the de novo biosynthesis of purines occurs within the chloroplast, and DOV1 (DIFFERENTIAL DEVELOPMENT OF VASCULAR ASSOCIATED CELLS 1) catalyzes the first step of this pathway. Here, to explore the role of VENOSA4 (VEN4), the most likely Arabidopsis ortholog of human SAMHD1, we studied the ven4-0 mutant. The mutant leaf phenotype caused by the ven4-0 point mutation was stronger than those of T-DNA insertional ven4 mutations. Structural predictions suggested that the E249L amino acid substitution in the mutated VEN4-0 protein rigidifies its 3D structure compared to wild-type VEN4. The morphological phenotypes of the ven4, cls8, and dov1 single mutants were similar, and those of the ven4 tso2 and ven4 dov1 double mutants were synergistic. The ven4-0 mutant had reduced levels of four amino acids related to dNTP biosynthesis, including glutamine and glycine, which are precursors in the de novo purine biosynthesis pathway. Finally, despite its annotation in some databases, At5g40290, a paralog of VEN4, is likely a pseudogene. These observations support the previously proposed role of VEN4 in dNTP metabolism. Our results reveal a high degree of cross-kingdom functional conservation between VEN4 and SAMHD1 in dNTP homeostasis.

plant biology↗

bioRxiv2022

Versatile mapping-by-sequencing with Easymap v.2

MotivationMapping-by-sequencing combines Next Generation Sequencing (NGS) with classical genetic mapping by linkage analysis to establish gene-to-phenotype relationships. Although numerous tools have been developed to analyze NGS datasets, only a few are available for mapping-by-sequencing. One such tool is Easymap, a versatile, easy-to-use package that performs automated mapping of point mutations and small insertion/deletions (InDels), as well as large DNA insertions. ResultsHere, we describe Easymap v.2, which includes additional workflows to perform QTL-seq and variant density mapping analyses. Each mapping workflow can accommodate different experimental designs, including outcrossing and backcrossing, F2, M2, and M3 mapping populations, chemically induced mutation and natural variant mapping, input files containing single-end or paired-end reads of genomic or complementary DNA sequences, and alternative control sample files in FASTQ and VCF formats. Easymap v.2 can also be used as a variant analyzer in the absence of a mapping algorithm and includes a multi-threading option. Availability and implementationCode is available at http://genetics.edu.umh.es/resources/easymap/ Contactjlmicol@umh.es

plant biology↗

bioRxiv2022

The Arabidopsis ATP-Binding Cassette E protein ABCE2 is a conserved component of the translation machinery

O_LIATP-Binding Cassette E (ABCE) proteins dissociate cytoplasmic ribosomes after translation terminates, and contribute to ribosome recycling, thus linking translation termination to initiation. This function has been demonstrated to be essential in animals, fungi, and archaea, but remains unexplored in plants. C_LIO_LIIn most species, ABCE is encoded by a single-copy gene; by contrast, Arabidopsis thaliana has two ABCE paralogs, of which ABCE2 seems to conserve the ancestral function. We isolated apiculata7-1 (api7-1), a viable, hypomorphic allele of ABCE2,which has a pleiotropic morphological phenotype reminiscent of mutations affecting ribosome biogenesis factors and ribosomal proteins. We also studied api7-2, a null, recessive lethal allele of ABCE2. C_LIO_LICo-immunoprecipitation experiments showed that ABCE2 physically interacts with components of the translation machinery. An RNA-seq study of the api7-1 mutant showed increased responses to iron and sulfur starvation. We also found increased transcript levels of genes related to auxin signaling and metabolism. C_LIO_LIOur results support a conserved role for ABCE proteins in ribosome recycling in plants, as previously shown for the animal, fungal, and archaeal lineages. In plants, the ABCE2 protein seems important for general growth and vascular development, likely due to an indirect effect through auxin metabolism. C_LI

plant biology↗

bioRxiv2021

Easymap: a user-friendly software package for rapid mapping by sequencing of point mutations and large insertions

Mapping-by-sequencing strategies combine next-generation sequencing (NGS) with classical linkage analysis, allowing rapid identification of the causal mutations of the phenotypes exhibited by mutants isolated in a genetic screen. Computer programs that analyze NGS data obtained from a mapping population of individuals derived from a mutant of interest in order to identify a causal mutation are available; however, the installation and usage of such programs requires bioinformatic skills, modifying or combining pieces of existing software, or purchasing licenses. To ease this process, we developed Easymap, an open-source program that simplifies the data analysis workflows from raw NGS reads to candidate mutations. Easymap can perform bulked segregant mapping of point mutations induced by ethyl methanesulfonate (EMS) with DNA-seq or RNA-seq datasets, as well as tagged-sequence mapping for large insertions, such as transposons or T-DNAs. The mapping analyses implemented in Easymap have been validated with experimental and simulated datasets from different plant and animal model species. Easymap was designed to be accessible to all users regardless of their bioinformatics skills by implementing a user-friendly graphical interface, a simple universal installation script, and detailed mapping reports, including informative images and complementary data for assessment of the mapping results. One sentence summaryEasymap is a versatile user-friendly software tool that facilitates mapping-by-sequencing of large insertions and point mutations in plant and animal genomes

plant biology↗