Pedro Robles

The Arabidopsis thaliana Homolog of Yeast BRE1 Has a Function in Cell Cycle Regulation during Early Leaf and Root Growth

Chromatin modification and transcriptional activation are novel roles for E3 ubiquitin ligase proteins that have been mainly associated with ubiquitin-dependent proteolysis. We identified HISTONE MONOUBIQUITINATION1 (HUB1) (and its homolog HUB2) in Arabidopsis thaliana as RING E3 ligase proteins with a function in organ growth. We show that HUB1 is a functional homolog of the human and yeast BRE1 proteins because it monoubiquitinated histone H2B in an in vitro assay. Hub knockdown mutants had pale leaf coloration, modified leaf shape, reduced rosette biomass, and inhibited primary root growth. One of the alleles had been designated previously as ang4-1. Kinematic analysis of leaf and root growth together with flow cytometry revealed defects in cell cycle activities. The hub1-1 (ang4-1) mutation increased cell cycle duration in young leaves and caused an early entry into the endocycles. Transcript profiling of shoot apical tissues of hub1-1 (ang4-1) indicated that key regulators of the G2-to-M transition were misexpressed. Based on the mutant characterization, we postulate that HUB1 mediates gene activation and cell cycle regulation probably through chromatin modifications.

Genetic analysis of leaf form mutants from the Arabidopsis Information Service collection

Abstract Although a vast inventory of morphological mutants of Arabidopsis thaliana is available, only some have been used for genetic studies of leaf development. Such is the case with the Arabidopsis Information Service (AIS) Form Mutants collection, assembled by A. R. Kranz and currently stored at the Nottingham Arabidopsis Stock Centre, which includes a large number of mutant lines, most of which have been little studied. With the aim of contributing to the genetic dissection of leaf ontogeny, we have subjected 57 mutant lines isolated by others to genetic analysis; 47 of which were from the AIS collection. These are characterized by vegetative leaves of abnormal shape or size, and were chosen as candidates for mutations in genes required for leaf morphogenesis. The mutant phenotypes studied were shown to be inherited as single recessive Mendelian traits and were classified into 10 phenotypic classes. These mutant strains were found to fall into 37 complementation groups, 7 of which corresponded to known genes. Results of the phenotypic analysis and data on the genetic interactions of these mutants are presented, and their possible developmental defects discussed.

Differential contributions of ribosomal protein genes to Arabidopsis thaliana leaf development

In Arabidopsis thaliana, mutations in genes encoding ribosomal proteins (r-proteins) perturb various developmental processes. Whether these perturbations are caused by overall ribosome insufficiency or partial dysfunction of the ribosome caused by deficiency of a particular ribosomal protein is not known. To distinguish these possibilities, a comparative study using several r-protein mutants was required. Here, we identified mutations in 11 r-protein genes from previously isolated denticulata and pointed-leaves mutants. Most of these mutations were associated with pointed leaves, with reduced growth due to a decrease in the number or size of palisade mesophyll and pavement cells. In addition, leaf abaxialization was usually observed when these r-protein mutations were combined with asymmetric leaves1 (as1) and as2 mutations. These results suggest that the establishment of leaf polarity is highly sensitive to ribosome functionality in general. However, several r-protein mutants showed a preference towards a specific developmental defect. For example, rpl4d mutations did not affect cell proliferation but caused strong abaxialization of leaves in the as1 and as2 backgrounds. On the other hand, rps28b enhanced leaf abaxialization of as2 to a weaker extent than expected on the basis of its negative effect on cell proliferation. In addition, hypomorphic rps6a alleles had the strongest effects on most of the phenotypes examined. These findings suggest that deficiencies in these three r-protein genes lead to production of dysfunctional ribosomes. Depending on their structural abnormalities, dysfunctional ribosomes may affect translation of specific transcripts involved in the regulation of some leaf developmental processes.

HIGH-THROUGHPUT GENETIC MAPPING IN ARABIDOPSIS THALIANA

Abstract To facilitate rapid determination of the chromosomal location of novel mutations, we have improved current approaches to gene mapping using microsatellite length polymorphisms. The high-throughput linkage analysis method described here allows a novel gene to be tested for linkage against the whole genome of a multicellular eukaryote, Arabidopsis thaliana, in a single polyacrylamide gel. The procedure is based on the simultaneous co-amplification of 21 microsatellites in a single tube, using a multiplex PCR mix containing 21 primer pairs, each including one oligonucleotide labeled with one of three fluorescent dyes that have different emission wavelengths. The amplification products, which range in number from 21 to 42, depending on the genotype of the individual being tested, are electrophoresed in a single lane on a polyacrylamide gel. The use of an automated fragment analyzer makes it possible to perform linkage analysis on a one gel-one gene basis using DNA samples from 19 F2 individuals obtained from an outcross involving a mutant and a wild-type that is genetically polymorphic with respect to the ecotype in which the mutant was generated. Discrimination of the amplification products is facilitated not only by labeling with different fluorochromes, but also by prior testing of different sequences for the ability to prime the amplification of each microsatellite, in order to ensure that multiplex PCR yields compatible amplification products of non-overlapping size. The method is particularly useful in large-scale mutagenesis projects, as well as for routine mapping of single mutants, since it reveals the map position of a gene less than 24 h after the F2 individuals to be analyzed have become available. The concepts employed here can easily be extended to other biological systems.

The RON1/FRY1/SAL1 gene is required for leaf morphogenesis and venation patterning in Arabidopsis

To identify genes involved in vascular patterning in Arabidopsis (Arabidopsis thaliana), we screened for abnormal venation patterns in a large collection of leaf shape mutants isolated in our laboratory. The rotunda1-1 (ron1-1) mutant, initially isolated because of its rounded leaves, exhibited an open venation pattern, which resulted from an increased number of free-ending veins. We positionally cloned the RON1 gene and found it to be identical to FRY1/SAL1, which encodes an enzyme with inositol polyphosphate 1-phosphatase and 3′ (2′),5′-bisphosphate nucleotidase activities and has not, to our knowledge, previously been related to venation patterning. The ron1-1 mutant and mutants affected in auxin homeostasis share perturbations in venation patterning, lateral root formation, root hair length, shoot branching, and apical dominance. These similarities prompted us to monitor the auxin response using a DR5-GUS auxin-responsive reporter transgene, the expression levels of which were increased in roots and reduced in leaves in the ron1-1 background. To gain insight into the function of RON1/FRY1/SAL1 during vascular development, we generated double mutants for genes involved in vein patterning and found that ron1 synergistically interacts with auxin resistant1 and hemivenata-1 but not with cotyledon vascular pattern1 (cvp1) and cvp2. These results suggest a role for inositol metabolism in the regulation of auxin responses. Microarray analysis of gene expression revealed that several hundred genes are misexpressed in ron1-1, which may explain the pleiotropic phenotype of this mutant. Metabolomic profiling of the ron1-1 mutant revealed changes in the levels of 38 metabolites, including myoinositol and indole-3-acetonitrile, a precursor of auxin.

A Role for AUXIN RESISTANT3 in the Coordination of Leaf Growth

The characteristically flat structure of Arabidopsis thaliana vegetative leaves requires coordinating the growth of the epidermal, palisade mesophyll, spongy mesophyll and vascular tissues. Mutations disrupting such coordination or the specific growth properties of any of these tissues can cause hyponasty, epinasty, waviness or other deviations from flatness. Here, we show that the incurvata6 (icu6) semi-dominant allele of the AUXIN RESISTANT3 (AXR3) gene causes leaf hyponasty. Cotyledons and leaves of icu6/AXR3 plants exhibited reduced size of adaxial pavement cells, and abnormal expansion of palisade mesophyll cells. Enhanced auxin responses in the adaxial domain of icu6/AXR3 developing cotyledons and leaves correlated with increased cell divisions in the adaxial epidermis. Leaf incurvature in icu6/AXR3 leaves was alleviated by loss-of-function alleles of the ASYMMETRIC LEAVES1 (AS1) and AS2 genes, which restrict the expression of class I KNOX genes to the shoot apical meristem and regulate cell proliferation in leaf primordia. Taken together, our results suggest that an interaction between auxin responses and the AS1-AS2 pathway coordinates tissue growth during Arabidopsis thaliana leaf expansion.

Low-Resolution Mapping of Untagged Mutations

The mapping method detailed here is based on the multiplex polymerase chain reaction (PCR) coamplification of 32 molecular markers, using fluorescently labeled oligonucleotides as primers. For the genotyping of a single plant from a mapping population, only two simultaneous amplifications are required, the products of which are finally electrophoresed in an automated DNA sequencer controlled by fragment analysis software. An analysis of the genotypes of 50 plants allows mapping of the mutation of interest within a candidate genomic interval of about 15 cM (3 Mb, corresponding to about 40 BAC clones).

Arabidopsis MDA1, a nuclear-encoded protein, functions in chloroplast development and abiotic stress responses.

Most chloroplast and mitochondrial proteins are encoded by nuclear genes, whose functions remain largely unknown because mutant alleles are lacking. A reverse genetics screen for mutations affecting the mitochondrial transcription termination factor (mTERF) family in Arabidopsis thaliana allowed us to identify 75 lines carrying T-DNA insertions. Two of them were homozygous for insertions in the At4g14605 gene, which we dubbed MDA1 (MTERF DEFECTIVE IN Arabidopsis1). The mda1 mutants exhibited altered chloroplast morphology and plant growth, and reduced pigmentation of cotyledons, leaves, stems and sepals. The mda1 mutations enhanced salt and osmotic stress tolerance and altered sugar responses during seedling establishment, possibly as a result of reduced ABA sensitivity. Loss of MDA1 function caused up-regulation of the RpoTp/SCA3 nuclear gene encoding a plastid RNA polymerase and modified the steady-state levels of chloroplast gene transcripts. Double mutant analyses indicated that MDA1 and the previously described mTERF genes SOLDAT10 and RUG2 act in different pathways. Our findings reveal a new role for mTERF proteins in the response to abiotic stress, probably through perturbed ABA retrograde signalling resulting from a disruption in chloroplast homeostasis.

Analysis of ven3 and ven6 reticulate mutants reveals the importance of arginine biosynthesis in Arabidopsis leaf development

Arabidopsis thaliana reticulate mutants exhibit differential pigmentation of the veinal and interveinal leaf regions, a visible phenotype that often indicates impaired mesophyll development. We performed a metabolomic analysis of one ven6 (venosa6) and three ven3 reticulate mutants that revealed altered levels of arginine precursors, namely increased ornithine and reduced citrulline levels. In addition, the mutants were more sensitive than the wild-type to exogenous ornithine, and leaf reticulation and mesophyll defects of these mutants were completely rescued by exogenous citrulline. Taken together, these results indicate that ven3 and ven6 mutants experience a blockage of the conversion of ornithine into citrulline in the arginine pathway. Consistent with the participation of VEN3 and VEN6 in the same pathway, the morphological phenotype of ven3 ven6 double mutants was synergistic. Map-based cloning showed that the VEN3 and VEN6 genes encode subunits of Arabidopsis carbamoyl phosphate synthetase (CPS), which is assumed to be required for the conversion of ornithine into citrulline in arginine biosynthesis. Heterologous expression of the Arabidopsis VEN3 and VEN6 genes in a CPS-deficient Escherichia coli strain fully restored bacterial growth in minimal medium, demonstrating the enzymatic activity of the VEN3 and VEN6 proteins, and indicating a conserved role for CPS in these distinct and distant species. Detailed study of the reticulate leaf phenotype in the ven3 and ven6 mutants revealed that mesophyll development is highly sensitive to impaired arginine biosynthesis.

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.