Sara Jover-Gil

Mutations in the MicroRNA Complementarity Site of the INCURVATA4 Gene Perturb Meristem Function and Adaxialize Lateral Organs in Arabidopsis

Here, we describe how the semidominant, gain-of-function icu4-1 and icu4-2 alleles of the INCURVATA4 (ICU4) gene alter leaf phyllotaxis and cell organization in the root apical meristem, reduce root length, and cause xylem overgrowth in the stem. The ICU4 gene was positionally cloned and found to encode the ATHB15 transcription factor, a class III homeodomain/leucine zipper family member, recently named CORONA. The icu4-1 and icu4-2 alleles bear the same point mutation that affects the microRNA complementarity site of ICU4 and is identical to those of several semidominant alleles of the class III homeodomain/leucine zipper family members PHABULOSA and PHAVOLUTA. The icu4-1 and icu4-2 mutations significantly increase leaf transcript levels of the ICU4 gene. The null hst-1 allele of the HASTY gene, which encodes a nucleocytoplasmic transporter, synergistically interacts with icu4-1, the double mutant displaying partial adaxialization of rosette leaves and carpels. Our results suggest that the ICU4 gene has an adaxializing function and that it is down-regulated by microRNAs that require the HASTY protein for their biogenesis.

The microRNA pathway genes AGO1, HEN1 and HYL1 participate in leaf proximal-distal, venation and stomatal patterning in Arabidopsis.

We isolated Arabidopsis thaliana mutants with incurved vegetative leaves. Positional cloning of incurvata8 (icu8), icu9 and icu15 has identified them as new loss-of-function alleles of the HYPONASTIC LEAVES1 (HYL1), ARGONAUTE1 (AGO1) and HUA ENHANCER1 (HEN1) genes, respectively, which encode known components of the microRNA pathway. The morphological and histological characterization of these mutants and of dicer-like1-9 indicates that small RNAs participate in the proximal-distal and adaxial-abaxial patterning of leaves, as well as in stomatal number establishment. The abnormal vasculature of ago1 and hyl1 leaves also suggests a role for AGO1 and HYL1 in venation patterning. Our mutants expand the allelic series of AGO1, HYL1 and HEN1, and might help to understand the developmental and cellular significance of miRNA-mediated posttranscriptional regulation.

Leaf phenomics: a systematic reverse genetic screen for Arabidopsis leaf mutants

The study and eventual manipulation of leaf development in plants requires a thorough understanding of the genetic basis of leaf organogenesis. Forward genetic screens have identified hundreds of Arabidopsis mutants with altered leaf development, but the genome has not yet been saturated. To identify genes required for leaf development we are screening the Arabidopsis Salk Unimutant collection. We have identified 608 lines that exhibit a leaf phenotype with full penetrance and almost constant expressivity and 98 additional lines with segregating mutant phenotypes. To allow indexing and integration with other mutants, the mutant phenotypes were described using a custom leaf phenotype ontology. We found that the indexed mutation is present in the annotated locus for 78% of the 553 mutants genotyped, and that in half of these the annotated T-DNA is responsible for the phenotype. To quickly map non-annotated T-DNA insertions, we developed a reliable, cost-effective and easy method based on whole-genome sequencing. To enable comprehensive access to our data, we implemented a public web application named PhenoLeaf (http://genetics.umh.es/phenoleaf) that allows researchers to query the results of our screen, including text and visual phenotype information. We demonstrated how this new resource can facilitate gene function discovery by identifying and characterizing At1g77600, which we found to be required for proximal-distal cell cycle-driven leaf growth, and At3g62870, which encodes a ribosomal protein needed for cell proliferation and chloroplast function. This collection provides a valuable tool for the study of leaf development, characterization of biomass feedstocks and examination of other traits in this fundamental photosynthetic organ.

Multi-gene silencing in Arabidopsis: a collection of artificial microRNAs targeting groups of paralogs encoding transcription factors

Functional redundancy often hampers the analysis of gene families. To overcome this difficulty, we constructed Arabidopsis thaliana lines that expressed artificial microRNAs designed to simultaneously target two to six paralogous genes encoding members of transcription factor families. Of the 576 genes that we chose as targets, only 122 had already been functionally studied at some level. As a simple indicator of the inhibitory effects of our amiRNAs on their targets, we examined the amiRNA-expressing transgenic lines for morphological phenotypes at the rosette stage. Of 338 transgenes tested, 21 caused a visible morphological phenotype in leaves, a proportion that is much higher than that expected as a result of insertional mutagenesis. Also, our collection probably represents many other mutant phenotypes, not just those in leaves. This robust, versatile method enables functional examination of redundant transcription factor paralogs, and is particularly useful for genes that occur in tandem.

Loss of function of Arabidopsis microRNA-machinery genes impairs fertility, and has effects on homologous recombination and meiotic chromatin dynamics

MicroRNAs (miRNAs) are ~22-nt single-stranded noncoding RNAs with regulatory roles in a wide range of cellular functions by repressing eukaryotic gene expression at a post-transcriptional level. Here, we analyzed the effects on meiosis and fertility of hypomorphic or null alleles of the HYL1, HEN1, DCL1, HST and AGO1 genes, which encode miRNA-machinery components in Arabidopsis. Reduced pollen and megaspore mother cell number and fertility were shown by the mutants analyzed. These mutants also exhibited a relaxed chromatin conformation in male meiocytes at the first meiotic division, and increased chiasma frequency, which is likely to be due to increased levels of mRNAs from key genes involved in homologous recombination. The hen1-13 mutant was found to be hypersensitive to gamma irradiation, which mainly causes double-strand breaks susceptible to be repaired by homologous recombination. Our findings uncover a role for miRNA-machinery components in Arabidopsis meiosis, as well as in the repression of key genes required for homologous recombination. These genes seem to be indirect miRNA targets.

Arabidopsis MAS2, an Essential Gene That Encodes a Homolog of Animal NF-κ B Activating Protein, Is Involved in 45S Ribosomal DNA Silencing

Arabidopsis MAS2, an essential gene, encodes an animal NKAP homolog that localizes to the nucleolar organizer region, interacts with ribosome biogenesis and splicing factors, and affects 45S rDNA expression. Ribosome biogenesis requires stoichiometric amounts of ribosomal proteins and rRNAs. Synthesis of rRNAs consumes most of the transcriptional activity of eukaryotic cells, but its regulation remains largely unclear in plants. We conducted a screen for ethyl methanesulfonate-induced suppressors of Arabidopsis thaliana ago1-52, a hypomorphic allele of AGO1 (ARGONAUTE1), a key gene in microRNA pathways. We identified nine extragenic suppressors as alleles of MAS2 (MORPHOLOGY OF AGO1-52 SUPPRESSED2). Positional cloning showed that MAS2 encodes the putative ortholog of NKAP (NF-κ B activating protein), a conserved eukaryotic protein involved in transcriptional repression and splicing in animals. The mas2 point mutations behave as informational suppressors of ago1 alleles that cause missplicing. MAS2 is a single-copy gene whose insertional alleles are embryonic lethal. In yeast two-hybrid assays, MAS2 interacted with splicing and ribosome biogenesis proteins, and fluorescence in situ hybridization showed that MAS2 colocalizes with the 45S rDNA at the nucleolar organizer regions (NORs). The artificial microRNA amiR-MAS2 partially repressed MAS2 and caused hypomethylation of 45S rDNA promoters as well as partial NOR decondensation, indicating that MAS2 negatively regulates 45S rDNA expression. Our results thus reveal a key player in the regulation of rRNA synthesis in plants.

Members of the DEAL subfamily of the DUF1218 gene family are required for bilateral symmetry but not for dorsoventrality in Arabidopsis leaves

Most plant leaves exhibit bilateral symmetry, which has been hypothesized as an inevitable consequence of the existence of the proximodistal and dorsoventral axes. No gene has been described that affects leaf bilateral symmetry but not dorsoventrality in Arabidopsis thaliana. We screened for viable insertional mutations that affect leaf morphology, and out of more than 700 mutants found only one, desigual1-1 (deal1-1), that exhibited bilateral symmetry breaking but no obvious defects in dorsoventrality. We found that deal1-1 is an allele of VASCULATURE COMPLEXITY AND CONNECTIVITY (VCC). Several overlapping regulatory pathways establish the interspersed lobes and indentations along the margin of Arabidopsis thaliana leaves. These pathways involve feedback loops of auxin, the PIN-FORMED1 (PIN1) auxin efflux carrier, and the CUP-SHAPED COTYLEDON2 (CUC2) transcriptional regulator. Early vcc (deal1) leaf primordia fail to acquire bilateral symmetry and instead form ectopic lobes and sinuses. The vcc leaves show aberrant recruitment of marginal cells expressing properly polarized PIN1, resulting in misplaced auxin maxima. Normal PIN1 polarization requires CUC2 expression and CUC2 genetically interacts with VCC; VCC also affects CUC2 expression. VCC has a domain of unknown function, DUF1218, and localizes to the endoplasmic reticulum membrane. VCC acts partially redundantly with its two closest paralogs, DEAL2 and DEAL3, in early leaf margin patterning and is required for bilateral symmetry, but its loss of function does not visibly affect dorsoventrality.

Complete genome sequence of a novel virus, classifiable within the Potyviridae family, which infects passion fruit (Passiflora edulis)

We report the complete nucleotide sequence of a new member of the Potyviridae family isolated from passion fruit plants grown in Israel, called Passiflora edulis symptomless virus (PeSV). The PeSV genome is 9,928 nucleotides long and encodes a 3,173 amino acids polyprotein that is predicted to be proteolytically cleaved into 10 mature peptides. Our phylogenetic analysis shows that PeSV represents a new species, and is most closely related to rose yellow mosaic virus (RoYMV). According to currently accepted criteria for genus demarcation, both viruses should be assigned as representative isolates of new species in the recently approved genus, Roymovirus, in the Potyviridae family.