Catherine Bellini

AGO1 defines a novel locus of Arabidopsis controlling leaf development

An allelic series of the novel argonaute mutant (ago1‐1 to ago1‐6) of the herbaceous plant Arabidopsis thaliana has been isolated. The ago1 mutation pleotropically affects general plant architecture. The apical shoot meristem generates rosette leaves and a single stem, but axillary meristems rarely develop. Rosette leaves lack a leaf blade but still show adaxial/abaxial differentiation. Instead of cauline leaves, filamentous structures without adaxial/abaxial differentiation develop along the stem and an abnormal inflorescence bearing infertile flowers with filamentous organs is produced. Two independent T‐DNA insertions into the AGO1 locus led to the isolation of two corresponding genomic sequences as well as a complete cDNA. The AGO1 locus was mapped close to the marker mi291a on chromosome 1. Antisense expression of the cDNA resulted in a partial mutant phenotype. Sense expression caused some transgenic lines to develop goblet‐like leaves and petals. The cDNA encodes a putative 115 kDa protein with sequence similarity to translation products of a novel gene family present in nematodes as well as humans. No specific function has been assigned to these genes. Similar proteins are not encoded by the genomes of yeast or bacteria, suggesting that AGO1 belongs to a novel class of genes with a function specific to multicellular organisms.

The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants.

Reverse transcription-polymerase chain reaction (RT-PCR) approaches have been used in a large proportion of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably expressed genes, known as references. Statistical algorithms have been developed recently to help validate reference genes, and most studies of gene expression in mammals, yeast and bacteria now include such validation. Surprisingly, this important approach is under-utilized in plant studies, where putative housekeeping genes tend to be used as references without any appropriate validation. Using quantitative RT-PCR, the expression stability of several genes commonly used as references was tested in various tissues of Arabidopsis thaliana and hybrid aspen (Populus tremula x Populus tremuloides). It was found that the expression of most of these genes was unstable, indicating that their use as references is inappropriate. The major impact of the use of such inappropriate references on the results obtained by RT-PCR is demonstrated in this study. Using aspen as a model, evidence is presented indicating that no gene can act as a universal reference, implying the need for a systematic validation of reference genes. For the first time, the extent to which the lack of a systematic validation of reference genes is a stumbling block to the reliability of results obtained by RT-PCR in plants is clearly shown.

Phenotypic Plasticity of Adventitious Rooting in Arabidopsis Is Controlled by Complex Regulation of AUXIN RESPONSE FACTOR Transcripts and MicroRNA Abundance

The development of shoot-borne roots, or adventitious roots, is indispensable for mass propagation of elite genotypes. It is a complex genetic trait with a high phenotypic plasticity due to multiple endogenous and environmental regulatory factors. We demonstrate here that a subtle balance of activator and repressor AUXIN RESPONSE FACTOR (ARF) transcripts controls adventitious root initiation. Moreover, microRNA activity appears to be required for fine-tuning of this process. Thus, ARF17, a target of miR160, is a negative regulator, and ARF6 and ARF8, targets of miR167, are positive regulators of adventitious rooting. The three ARFs display overlapping expression domains, interact genetically, and regulate each others expression at both transcriptional and posttranscriptional levels by modulating miR160 and miR167 availability. This complex regulatory network includes an unexpected feedback regulation of microRNA homeostasis by direct and nondirect target transcription factors. These results provide evidence of microRNA control of phenotypic variability and are a significant step forward in understanding the molecular mechanisms regulating adventitious rooting.

Auxin and Light Control of Adventitious Rooting in Arabidopsis Require ARGONAUTE1

Adventitious rooting is a quantitative genetic trait regulated by both environmental and endogenous factors. To better understand the physiological and molecular basis of adventitious rooting, we took advantage of two classes of Arabidopsis thaliana mutants altered in adventitious root formation: the superroot mutants, which spontaneously make adventitious roots, and the argonaute1 (ago1) mutants, which unlike superroot are barely able to form adventitious roots. The defect in adventitious rooting observed in ago1 correlated with light hypersensitivity and the deregulation of auxin homeostasis specifically in the apical part of the seedlings. In particular, a clear reduction in endogenous levels of free indoleacetic acid (IAA) and IAA conjugates was shown. This was correlated with a downregulation of the expression of several auxin-inducible GH3 genes in the hypocotyl of the ago1-3 mutant. We also found that the Auxin Response Factor17 (ARF17) gene, a potential repressor of auxin-inducible genes, was overexpressed in ago1-3 hypocotyls. The characterization of an ARF17-overexpressing line showed that it produced fewer adventitious roots than the wild type and retained a lower expression of GH3 genes. Thus, we suggest that ARF17 negatively regulates adventitious root formation in ago1 mutants by repressing GH3 genes and therefore perturbing auxin homeostasis in a light-dependent manner. These results suggest that ARF17 could be a major regulator of adventitious rooting in Arabidopsis.

The Arabidopsis TONNEAU2 Gene Encodes a Putative Novel Protein Phosphatase 2A Regulatory Subunit Essential for the Control of the Cortical Cytoskeleton

In Arabidopsis ton2 mutants, abnormalities of the cortical microtubular cytoskeleton, such as disorganization of the interphase microtubule array and lack of the preprophase band before mitosis, markedly affect cell shape and arrangement as well as overall plant morphology. We present the molecular isolation of the TON2 gene, which is highly conserved in higher plants and has a vertebrate homolog of unknown function. It encodes a protein similar in its C-terminal part to B″ regulatory subunits of type 2A protein phosphatases (PP2As). We show that the TON2 protein interacts with an Arabidopsis type A subunit of PP2A in the yeast two-hybrid system and thus likely defines a novel subclass of PP2A subunits that are possibly involved in the control of cytoskeletal structures in plants.

Auxin Controls Arabidopsis Adventitious Root Initiation by Regulating Jasmonic Acid Homeostasis

Vegetative propagation of plants from shoots requires the development of adventitious roots that regenerate from stem cuttings. Multiple endogenous and environmental factors control this complex process; this study in the model plant Arabidopsis thaliana shows that adventitious rooting is an adaptive developmental response involving crosstalk between auxin and jasmonate signaling pathways. Vegetative shoot-based propagation of plants, including mass propagation of elite genotypes, is dependent on the development of shoot-borne roots, which are also called adventitious roots. Multiple endogenous and environmental factors control the complex process of adventitious rooting. In the past few years, we have shown that the auxin response factors ARF6 and ARF8, targets of the microRNA miR167, are positive regulators of adventitious rooting, whereas ARF17, a target of miR160, is a negative regulator. We showed that these genes have overlapping expression profiles during adventitious rooting and that they regulate each other’s expression at the transcriptional and posttranscriptional levels by modulating the homeostasis of miR160 and miR167. We demonstrate here that this complex network of transcription factors regulates the expression of three auxin-inducible Gretchen Hagen3 (GH3) genes, GH3.3, GH3.5, and GH3.6, encoding acyl-acid-amido synthetases. We show that these three GH3 genes are required for fine-tuning adventitious root initiation in the Arabidopsis thaliana hypocotyl, and we demonstrate that they act by modulating jasmonic acid homeostasis. We propose a model in which adventitious rooting is an adaptive developmental response involving crosstalk between the auxin and jasmonate regulatory pathways.

Towards a Systematic Validation of References in Real-Time RT-PCR

Real-time RT-PCR (also known as quantitative RT-PCR [qRT-PCR]) is a powerful tool for quantifying gene expression, combining both high sensitivity and specificity with efficient signal detection. It has relatively recently begun to be used to monitor gene expression in plants (reviewed in [Gachon et

Comprehensive Screening of Arabidopsis Mutants Suggests the Lysine Histidine Transporter 1 to Be Involved in Plant Uptake of Amino Acids

Plant nitrogen (N) uptake is a key process in the global N cycle and is usually considered a “bottleneck” for biomass production in land ecosystems. Earlier, mineral N was considered the only form available to plants. Recent studies have questioned this dogma and shown that plants may access organic N sources such as amino acids. The actual mechanism enabling plants to access amino acid N is still unknown. However, a recent study suggested the Lysine Histidine Transporter 1 (LHT1) to be involved in root amino acid uptake. In this study, we isolated mutants defective in root amino acid uptake by screening Arabidopsis (Arabidopsis thaliana) seeds from ethyl methanesulfonate-treated plants and seeds from amino acid transporter T-DNA knockout mutants for resistance against the toxic d-enantiomer of alanine (Ala). Both ethyl methanesulfonate and T-DNA knockout plants identified as d-Ala resistant were found to be mutated in the LHT1 gene. LHT1 mutants displayed impaired capacity for uptake of a range of amino acids from solutions, displayed impaired growth when N was supplied in organic forms, and acquired substantially lower amounts of amino acids than wild-type plants from solid growth media. LHT1 mutants grown on mineral N did not display a phenotype until at the stage of flowering, when premature senescence of old leaf pairs occurred, suggesting that LHT1 may fulfill an important function at this developmental stage. Based on the broad and unbiased screening of mutants resistant to d-Ala, we suggest that LHT1 is an important mediator of root uptake of amino acids. This provides a molecular background for plant acquisition of organic N from the soil.

Mutation in the Arabidopsis PASTICCINO1 gene, which encodes a new FK506-binding protein-like protein, has a dramatic effect on plant development

ABSTRACT The pasticcino (pas) mutants ofArabidopsis thaliana are a new class of plant developmental mutants; members of this class show ectopic cell proliferation in cotyledons, extra layers of cells in the hypocotyl, and an abnormal apical meristem. This phenotype is correlated with both cell division and cell elongation defects. There are three complementation groups ofpas mutants (pas1, pas2, andpas3, with, respectively 2, 1, and 4 alleles). Here we describe in more detail the pas1-1 allele, which was obtained by insertional mutagenesis. The PAS1 gene has been cloned and characterized; it encodes an immunophilin-like protein similar to the p59 FK506-binding protein (FKBP52). PAS1 is characterized by an FKBP-like domain and three tetratricopeptide repeat units. Although the presence of immunophilins in plants has already been demonstrated, the pas1-1 mutant represents the first inactivation of an FKBP-like gene in plants. PAS1expression is altered in pas1 mutants and in thepas2 and pas3 mutants. The expression of thePAS1 gene is increased in the presence of cytokinins, a class of phytohormones originally discovered because of their ability to stimulate cell division. These results are of particular relevance as they show for the first time that an FKBP-like protein plays an important role in the control of plant development.

Adventitious Roots and Lateral Roots: Similarities and Differences

In addition to its role in water and nutrient uptake, the root system is fundamentally important because it anchors a plant to its substrate. Although a wide variety of root systems exist across different species, all plants have a primary root (derived from an embryonic radicle) and different types of lateral roots. Adventitious roots, by comparison, display the same functions as lateral roots but develop from aerial tissues. In addition, they not only develop as an adaptive response to various stresses, such as wounding or flooding, but also are a key limiting component of vegetative propagation. Lateral and adventitious roots share key elements of the genetic and hormonal regulatory networks but are subject to different regulatory mechanisms. In this review, we discuss the developmental processes that give rise to lateral and adventitious roots and highlight knowledge acquired over the past few years about the mechanisms that regulate adventitious root formation.