Héctor Candela

Big impacts by small RNAs in plant development

The identification and study of small RNAs, including microRNAs and trans-acting small interfering RNAs, have added a layer of complexity to the many pathways that regulate plant development. These molecules, which function as negative regulators of gene expression, are now known to have greatly expanded roles in a variety of developmental processes affecting all major plant structures, including meristems, leaves, roots, and inflorescences. Mutants with specific developmental phenotypes have also advanced our knowledge of the biogenesis and mode of action of these diverse small RNAs. In addition, previous models on the cell autonomy of microRNAs may have to be revised as more data accumulate supporting their long distance transport. As many of these small RNAs appear to be conserved across different species, knowledge gained from one species is expected to have general application. However, a few surprising differences in small RNA function seem to exist between monocots and dicots regarding meristem initiation and sex determination. Integrating these unique functions into the overall scheme for plant growth will give a more complete picture of how they have evolved as unique developmental systems.

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.

Understanding synergy in genetic interactions

Synergy occurs when the contribution of two mutations to the phenotype of a double mutant exceeds the expectations from the additive effects of the individual mutations. The molecular characterization of genes involved in synergistic interactions has revealed that synergy mainly results from mutations in functionally related genes. Recent research in Arabidopsis thaliana has advanced our understanding of the scenarios resulting in synergistic phenotypes. Those involving homologous loci usually result from various levels of functional redundancy. Some of these loci fail to complement each other or become dose-dependent in certain multiple mutant combinations, suggesting that the effects of haploinsufficiency and redundancy can combine. Synergy involving non-homologous genes probably reflects the topology of the regulatory or metabolic networks and can arise when pathways that converge at a node are disrupted. The Hub genes provide a remarkable example, these genes have an extraordinary number of connections and mutations that interact with many unrelated pathways.

Ribosomes and translation in plant developmental control

Ribosomes play a basic housekeeping role in global translation. However, a number of ribosomal-protein-defective mutants show common and rare developmental phenotypes including growth defects, changes in leaf development, and auxin-related phenotypes. This suggests that translational regulation may be occurring during development. In addition, proteomic and bioinformatic analyses have demonstrated a high heterogeneity in ribosome composition. Although this might be a sign of unequal roles of individual ribosomal proteins, it does not explain every ribosomal-protein-defective phenotype. Moreover, comprehensive interpretations concerning the relationship between ribosomal-protein-defective phenotypes and molecular changes in ribosome status are lacking. In this review, we address these phenotypes based on three models, ribosome insufficiency, heterogeneity, and aberrancy, to consider how ribosomes play developmental roles. We propose that the three models are not mutually exclusive, and ribosomal-protein-defective phenotypes can be explained with one or more of these models. The three models with reference to genetic, biochemical, and bioinformatic knowledge will serve as a foundation for future studies of translational regulation.

The HVE/CAND1 gene is required for the early patterning of leaf venation in Arabidopsis.

The hemivenata-1 (hve-1) recessive allele was isolated in a search for natural variations in the leaf venation pattern of Arabidopsis thaliana, where it was seen to cause extremely simple venation in vegetative leaves and cotyledons, increased shoot branching, and reduced root waving and fertility, traits that are reminiscent of some mutants deficient in auxin signaling. Reduced sensitivity to exogenous auxin was found in the hve-1 mutant, which otherwise displayed a wild-type response to auxin transport inhibitors. The HVE gene was positionally cloned and found to encode a CAND1 protein. The hve-1 mutation caused mis-splicing of the transcripts of the HVE/CAND1 gene and a vein phenotype indistinguishable from that of hve-2 and hve-3, two putatively null T-DNA alleles. Inflorescence size and fertility were more affected by hve-2 and hve-3, suggesting that hve-1 is hypomorphic. The simple venation pattern of hve plants seems to arise from an early patterning defect. We found that HVE/CAND1 binds to CULLIN1, and that the venation patterns of axr1 and hve mutants are similar, which suggest that ubiquitin-mediated auxin signaling is required for venation patterning in laminar organs, the only exception being cauline leaves. Our analyses of double mutant and transgenic plants indicated that auxin transport and perception act independently to pattern leaf veins, and that the HVE/CAND1 gene acts upstream of ATHB-8 at least in higher order veins, in a pathway that involves AXR1, but not LOP1, PIN1, CVP1 or CVP2.

The art and design of genetic screens: maize

Maize (Zea mays) is an excellent model for basic research. Genetic screens have informed our understanding of developmental processes, meiosis, epigenetics and biochemical pathways — not only in maize but also in other cereal crops. We discuss the forward and reverse genetic screens that are possible in this organism, and emphasize the available tools. Screens exploit the well-studied behaviour of transposon systems, and the distinctive chromosomes allow an integration of cytogenetics into mutagenesis screens and analyses. The imminent completion of the maize genome sequence provides the essential resource to move seamlessly from gene to phenotype and back.

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.

The milkweed pod1 Gene Encodes a KANADI Protein That Is Required for Abaxial/Adaxial Patterning in Maize Leaves

Leaf primordia initiate from the shoot apical meristem with inherent polarity; the adaxial side faces the meristem, while the abaxial side faces away from the meristem. Adaxial/abaxial polarity is thought to be necessary for laminar growth of leaves, as mutants lacking either adaxial or abaxial cell types often develop radially symmetric lateral organs. The milkweed pod1 (mwp1) mutant of maize (Zea mays) has adaxialized sectors in the sheath, the proximal part of the leaf. Ectopic leaf flaps develop where adaxial and abaxial cell types juxtapose. Ectopic expression of the HD-ZIPIII gene rolled leaf1 (rld1) correlates with the adaxialized regions. Cloning of mwp1 showed that it encodes a KANADI transcription factor. Double mutants of mwp1-R with a microRNA-resistant allele of rld1, Rld1-N1990, show a synergistic phenotype with polarity defects in sheath and blade and a failure to differentiate vascular and photosynthetic cell types in the adaxialized sectors. The sectored phenotype and timing of the defect suggest that mwp1 is required late in leaf development to maintain abaxial cell fate. The phenotype of mwp1; Rld1 double mutants shows that both genes are also required early in leaf development to delineate leaf margins as well as to initiate vascular and photosynthetic tissues.

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.

Competency for shoot regeneration from Arabidopsis root explants is regulated by DNA methylation.

Plants exhibit high capacity to regenerate in three alternative pathways: tissue repair, somatic embryogenesis and de novo organogenesis. For most plants, de novo organ initiation can be easily achieved in tissue culture by exposing explants to auxin and/or cytokinin, yet the competence to regenerate varies among species and within tissues from the same plant. In Arabidopsis, root explants incubated directly on cytokinin-rich shoot inducing medium (SIM-direct), are incapable of regenerating shoots, and a pre-incubation step on auxin-rich callus inducing medium (CIM) is required to acquire competency to regenerate on the SIM. However the mechanism underlying competency acquisition still remains elusive. Here we show that the chromomethylase 3 (cmt3) mutant which exhibits significant reduction in CHG methylation, shows high capacity to regenerate on SIM-direct and that regeneration occurs via direct organogenesis. In WT, WUSCHEL (WUS) promoter, an essential gene for shoot formation, is highly methylated, and its expression on SIM requires pre-incubation on CIM. However, in cmt3, WUS expression induced by SIM-direct. We propose that pre-incubation on CIM is required for the re-activation of cell division. Following the transfer of roots to SIM, the intensive cell division activity continues, and in the presence of cytokinin leads to a dilution in DNA methylation that allows certain genes required for shoot regeneration to respond to SIM, thereby advancing shoot formation.