Growth-inhibiting effects of the unconventional plant APYRASE 7 of Arabidopsis thaliana influences the LRX/RALF/FER growth regulatory module.

Plant cell growth involves coordination of numerous processes and signaling cascades among the different cellular compartments to concomitantly enlarge the protoplast and the surrounding cell wall. The cell wall integrity-sensing process involves the extracellular LRX (LRR-Extensin) proteins that bind RALF (Rapid ALkalinization Factor) peptide hormones and, in vegetative tissues, interact with the transmembrane receptor kinase FERONIA (FER). This LRX/RALF/FER signaling module influences cell wall composition and regulates cell growth. The numerous proteins involved in or influenced by this module are beginning to be characterized. In a genetic screen, mutations in Apyrase 7 (APY7) were identified to suppress growth defects observed in lrx1 and fer mutants. APY7 encodes a Golgi-localized NTP-diphosphohydrolase, but opposed to other apyrases of Arabidopsis, APY7 revealed to be a negative regulator of cell growth. APY7 modulates the growth-inhibiting effect of RALF1, influences the cell wall architecture and -composition, and alters the pH of the extracellular matrix, all of which affect cell growth. Together, this study reveals a function of APY7 in cell wall formation and cell growth that is connected to growth processes influenced by the LRX/RALF/FER signaling module.

Gibberellins negatively regulate the development of Medicago truncatula root system.

The root system displays a remarkable plasticity that enables plants to adapt to changing environmental conditions. This plasticity is tightly linked to the activity of root apical meristems (RAMs) and to the formation of lateral roots, both controlled by related hormonal crosstalks. In Arabidopsis thaliana, gibberellins (GAs) were shown to positively control RAM growth and the formation of lateral roots. However, we showed in Medicago truncatula that GAs negatively regulate root growth and RAM size as well as the number of lateral roots depending at least on the MtDELLA1 protein. By using confocal microscopy and molecular analyses, we showed that GAs primarily regulate RAM size by affecting cortical cell expansion and additionally negatively regulate a subset of cytokinin-induced root expansin encoding genes. Moreover, GAs reduce the number of cortical cell layers, resulting in the formation of both shorter and thinner roots. These results suggest contrasting effects of GA regulations on the root system architecture depending on plant species.

Mutations in the Arabidopsis ROL17/isopropylmalate synthase 1 locus alter amino acid content, modify the TOR network, and suppress the root hair cell development mutant lrx1.

The growth and development of organisms must be tightly controlled and adjusted to nutrient availability and metabolic activities. The Target of Rapamycin (TOR) network is a major control mechanism in eukaryotes and influences processes such as translation, mitochondrial activity, production of reactive oxygen species, and the cytoskeleton. In Arabidopsis thaliana, inhibition of the TOR kinase causes changes in cell wall architecture and suppression of phenotypic defects of the cell wall formation mutant lrx1 (leucine-rich repeat extensin 1). The rol17 (repressor of lrx1 17) mutant was identified as a new suppressor of lrx1 that induces also a short root phenotype. The ROL17 locus encodes isopropylmalate synthase 1, a protein involved in leucine biosynthesis. Dependent on growth conditions, mutations in ROL17 do not necessarily alter the level of leucine, but always cause development of the rol17 mutant phenotypes, suggesting that the mutation does not only influence leucine biosynthesis. Changes in the metabolome of rol17 mutants are also found in plants with inhibited TOR kinase activity. Furthermore, rol17 mutants show reduced sensitivity to the TOR kinase inhibitor AZD-8055, indicating a modified TOR network. Together, these data suggest that suppression of lrx1 by rol17 is the result of an alteration of the TOR network.

Cytokinins and the CRE1 receptor influence endogenous gibberellin levels in Medicago truncatula.

Gibberellins (GAs) and cytokinins (CKs) are hormones that play antagonistic roles in several developmental processes in plants. However, there has been little exploration of their reciprocal interactions. Recent work in Medicago truncatula has revealed that GA signalling can regulate CK levels and response in roots. Here, we examine the reciprocal interaction, by assessing how CKs and the CRE1 (Cytokinin Response 1) CK receptor may influence endogenous GA levels. Real-Time RT-PCR analyses revealed that the expression of key GA biosynthesis genes is regulated in response to a short-term CK treatment and requires the CRE1 receptor. Similarly, GA quantifications indicated that a short-term CK treatment decreases the GA1 pool in wild-type plants and that GA levels are increased in the cre1 mutant compared to the wild-type. These data suggest that the M. truncatula CRE1-dependent CK signaling pathway negatively regulates bioactive GA levels.

DELLA1-Mediated Gibberellin Signaling Regulates Cytokinin-Dependent Symbiotic Nodulation.

In legume plants, low-nitrogen soils promote symbiotic interactions with rhizobial bacteria, leading to the formation of nitrogen-fixing root nodules. Among critical signals regulating this developmental process are bacterial Nod Factors (NFs) and several plant hormones, including cytokinins (CKs) and gibberellins (GAs). Here, we show in Medicago truncatula that GA signaling mediated by DELLA1 decreases the amount of bioactive CKs in roots and negatively impacts the Cytokinin Response1 (CRE1)-dependent NF activation of a subset of CK-signaling genes as well as of the CK-regulated Nodulation Signaling Pathway2 and Ethylene Response Factor Required for Nodulation1 early nodulation genes. Consistently, a dominant-active DELLA1 protein can partially rescue the reduced nodulation of the cre1 mutant and triggers the formation of nodule-like structures when expressed in the root cortex or in the root epidermis. This suggests a model where the DELLA1-mediated GA signaling interplays with the CRE1-dependent CK pathway to regulate early nodulation in response to both NF and CK signals critical for this symbiotic interaction.

Root Development and Endosymbioses: DELLAs Lead the Orchestra.

DELLA proteins, acting as integrators of gibberellin (GA) action, are emerging as key regulators of root system architecture. Recent studies have revealed how they dictate the dynamics of root growth and are required for the establishment of root endosymbioses with rhizobial bacteria and mycorrhizal fungi. Like conductors, DELLAs can thereby harmonize root development depending on soil environments.

DELLA-mediated gibberellin signalling regulates Nod factor signalling and rhizobial infection.

Legumes develop symbiotic interactions with rhizobial bacteria to form nitrogen-fixing nodules. Bacterial Nod factors (NFs) and plant regulatory pathways modulating NF signalling control rhizobial infections and nodulation efficiency. Here we show that gibberellin (GA) signalling mediated by DELLA proteins inhibits rhizobial infections and controls the NF induction of the infection marker ENOD11 in Medicago truncatula. Ectopic expression of a constitutively active DELLA protein in the epidermis is sufficient to promote ENOD11 expression in the absence of symbiotic signals. We show using heterologous systems that DELLA proteins can interact with the nodulation signalling pathway 2 (NSP2) and nuclear factor-YA1 (NF-YA1) transcription factors that are essential for the activation of NF responses. Furthermore, MtDELLA1 can bind the ERN1 (ERF required for nodulation 1) promoter and positively transactivate its expression. Overall, we propose that GA-dependent action of DELLA proteins may directly regulate the NSP1/NSP2 and NF-YA1 activation of ERN1 transcription to regulate rhizobial infections.

Comparative transcriptomic analysis of salt adaptation in roots of contrasting Medicago truncatula genotypes.

Evolutionary diversity can be driven by the interaction of plants with different environments. Molecular bases involved in ecological adaptations to abiotic constraints can be explored using genomic tools. Legumes are major crops worldwide and soil salinity is a main stress affecting yield in these plants. We analyzed in the Medicago truncatula legume the root transcriptome of two genotypes having contrasting responses to salt stress: TN1.11, sampled in a salty Tunisian soil, and the reference Jemalong A17 genotype. TN1.11 plants show increased root growth under salt stress as well as a differential accumulation of sodium ions when compared to A17. Transcriptomic analysis revealed specific gene clusters preferentially regulated by salt in root apices of TN1.11, notably those related to the auxin pathway and to changes in histone variant isoforms. Many genes encoding transcription factors (TFs) were also differentially regulated between the two genotypes in response to salt. Among those selected for functional studies, overexpression in roots of the A17 genotype of the bHLH-type TF most differentially regulated between genotypes improved significantly root growth under salt stress. Despite the global complexity of the differential transcriptional responses, we propose that an increase in this bHLH TF expression may be linked to the adaptation of M. truncatula to saline soil environments.

Dual involvement of a Medicago truncatula NAC transcription factor in root abiotic stress response and symbiotic nodule senescence.

Legume crops related to the model plant Medicago truncatula can adapt their root architecture to environmental conditions, both by branching and by establishing a symbiosis with rhizobial bacteria to form nitrogen-fixing nodules. Soil salinity is a major abiotic stress affecting plant yield and root growth. Previous transcriptomic analyses identified several transcription factors linked to the M. truncatula response to salt stress in roots, including NAC (NAM/ATAF/CUC)-encoding genes. Over-expression of one of these transcription factors, MtNAC969, induced formation of a shorter and less-branched root system, whereas RNAi-mediated MtNAC969 inactivation promoted lateral root formation. The altered root system of over-expressing plants was able to maintain its growth under high salinity, and roots in which MtNAC969 was down-regulated showed improved growth under salt stress. Accordingly, expression of salt stress markers was decreased or induced in MtNAC969 over-expressing or RNAi roots, respectively, suggesting a repressive function for this transcription factor in the salt-stress response. Expression of MtNAC969 in central symbiotic nodule tissues was induced by nitrate treatment, and antagonistically affected by salt in roots and nodules, similarly to senescence markers. MtNAC969 RNAi nodules accumulated amyloplasts in the nitrogen-fixing zone, and were prematurely senescent. Therefore, the MtNAC969 transcription factor, which is differentially affected by environmental cues in root and nodules, participates in several pathways controlling adaptation of the M. truncatula root system to the environment.

The LOB-like transcription factor Mt LBD1 controls Medicago truncatula root architecture under salt stress.

Lateral root (LR) formation and emergence are influenced by the environment and determines the architecture of the root system in the soil. Whereas auxins appear as the main hormone controlling LR initiation, patterning and emergence, abscisic acid (ABA) is the key hormone mediating the effect of the environment on root architecture. Hormone signaling act through transcription factors (TFs) and the Medicago truncatula LOB-like TF LBD1 was shown to be auxin-inducible but repressed by the HD-Zip I TF MtHB1 in response to salt stress and ABA during LR formation. Here, we demonstrate that the constitutive expression of Mt LBD1 in Medicago roots alters their global architecture when the plant is subjected to salt stress. Hence, LBD1 may control the final form of the root system in the soil environment.

Environmental regulation of lateral root emergence in Medicago truncatula requires the HD-Zip I transcription factor HB1.

The adaptation of root architecture to environmental constraints is a major agricultural trait, notably in legumes, the third main crop worldwide. This root developmental plasticity depends on the formation of lateral roots (LRs) emerging from primary roots. In the model legume Medicago truncatula, the HD-Zip I transcription factor HB1 is expressed in primary and lateral root meristems and induced by salt stress. Constitutive expression of HB1 in M. truncatula roots alters their architecture, whereas hb1 TILLING mutants showed increased lateral root emergence. Electrophoretic mobility shift assay, promoter mutagenesis, and chromatin immunoprecipitation-PCR assays revealed that HB1 directly recognizes a CAATAATTG cis-element present in the promoter of a LOB-like (for Lateral Organ Boundaries) gene, LBD1, transcriptionally regulated by auxin. Expression of these genes in response to abscisic acid and auxin and their behavior in hb1 mutants revealed an HB1-mediated repression of LBD1 acting during LR emergence. M. truncatula HB1 regulates an adaptive developmental response to minimize the root surface exposed to adverse environmental stresses.

The TOR pathway modulates the structure of cell walls in Arabidopsis.

Plant cell growth is limited by the extension of cell walls, which requires both the synthesis and rearrangement of cell wall components in a controlled fashion. The target of rapamycin (TOR) pathway is a major regulator of cell growth in eukaryotes, and inhibition of this pathway by rapamycin reduces cell growth. Here, we show that in plants, the TOR pathway affects cell wall structures. LRR-extensin1 (LRX1) of Arabidopsis thaliana is an extracellular protein involved in cell wall formation in root hairs, and lrx1 mutants develop aberrant root hairs. rol5 (for repressor of lrx1) was identified as a suppressor of lrx1. The functionally similar ROL5 homolog in yeast, Ncs6p (needs Cla4 to survive 6), was previously found to affect TOR signaling. Inhibition of TOR signaling by rapamycin led to suppression of the lrx1 mutant phenotype and caused specific changes to galactan/rhamnogalacturonan-I and arabinogalactan protein components of cell walls that were similar to those observed in the rol5 mutant. The ROL5 protein accumulates in mitochondria, a target of the TOR pathway and major source of reactive oxygen species (ROS), and rol5 mutants show an altered response to ROS. This suggests that ROL5 might function as a mitochondrial component of the TOR pathway that influences the plant's response to ROS.

Identification of transcription factors involved in root apex responses to salt stress in Medicago truncatula.

The root apex contains meristematic cells that determine root growth and architecture in the soil. Specific transcription factor (TF) genes in this region may integrate endogenous signals and external cues to achieve this. Early changes in transcriptional responses involving TF genes after a salt stress in Medicago truncatula (Mt) roots were analysed using two complementary transcriptomic approaches. Forty-six salt-regulated TF genes were identified using massive quantitative real-time RT-PCR TF profiling in whole roots. In parallel, Mt16K+ microarray analysis revealed 824 genes (including 84 TF sequences) showing significant changes (p < 0.001) in their expression in root apexes after a salt stress. Analysis of salt-stress regulation in root apexes versus whole roots showed that several TF genes have more than 30-fold expression differences including specific members of AP2/EREBP, HD-ZIP, and MYB TF families. Several salt-induced TF genes also respond to other abiotic stresses as osmotic stress, cold and heat, suggesting that they participate in a general stress response. Our work suggests that spatial differences of TF gene regulation by environmental stresses in various root regions may be crucial for the adaptation of their growth to specific soil environments.

The modified flavonol glycosylation profile in the Arabidopsis rol1 mutants results in alterations in plant growth and cell shape formation.

Flavonoids are secondary metabolites known to modulate plant growth and development. A primary function of flavonols, a subgroup of flavonoids, is thought to be the modification of auxin fluxes in the plant. Flavonols in the cell are glycosylated, and the repressor of lrx1 (rol1) mutants of Arabidopsis thaliana, affected in rhamnose biosynthesis, have a modified flavonol glycosylation profile. A detailed analysis of the rol1-2 allele revealed hyponastic growth, aberrant pavement cell and stomatal morphology in cotyledons, and defective trichome formation. Blocking flavonoid biosynthesis suppresses the rol1-2 shoot phenotype, suggesting that it is induced by the modified flavonol profile. The hyponastic cotyledons of rol1-2 are likely to be the result of a flavonol-induced increase in auxin concentration. By contrast, the pavement cell, stomata, and trichome formation phenotypes appear not to be induced by the modified auxin distribution. Together, these results suggest that changes in the composition of flavonols can have a tremendous impact on plant development through both auxin-induced and auxin-independent processes.

The Arabidopsis sweetie mutant is affected in carbohydrate metabolism and defective in the control of growth, development and senescence.

SUMMARY: Sugars modulate many vital metabolic and developmental processes in plants, from seed germination to flowering, senescence and protection against diverse abiotic and biotic stresses. However, the exact mechanisms involved in morphogenesis, developmental signalling and stress tolerance remain largely unknown. Here we report the characterization of a novel Arabidopsis thaliana mutant, sweetie, with drastically altered morphogenesis, and a strongly modified carbohydrate metabolism leading to elevated levels of trehalose, trehalose-6-phosphate and starch. We additionally show that the disruption of SWEETIE causes significant growth and developmental alterations, such as severe dwarfism, lancet-shaped leaves, early senescence and flower sterility. Genes implicated in sugar metabolism, senescence, ethylene biosynthesis and abiotic stress were found to be upregulated in sweetie. Our physiological, biochemical, genetic and molecular data indicate that the mutation in sweetie was nuclear, single and recessive. The effects of metabolizable sugars and osmolytes on sweetie morphogenesis were distinct; in light, sweetie was hypersensitive to sucrose and glucose during vegetative growth and a partial phenotypic reversion took place in the presence of high sorbitol concentrations. However, SWEETIE encodes a protein that is unrelated to any known enzyme involved in sugar metabolism. We suggest that SWEETIE plays an important regulatory function that influences multiple metabolic, hormonal and stress-related pathways, leading to altered gene expression and pronounced changes in the accumulation of sugar, starch and ethylene.

The Arabidopsis root hair cell wall formation mutant lrx1 is suppressed by mutations in the RHM1 gene encoding a UDP-L-rhamnose synthase.

Cell and cell wall growth are mutually dependent processes that must be tightly coordinated and controlled. LRR-extensin1 (LRX1) of Arabidopsis thaliana is a potential regulator of cell wall development, consisting of an N-terminal leucine-rich repeat domain and a C-terminal extensin-like domain typical for structural cell wall proteins. LRX1 is expressed in root hairs, and lrx1 mutant plants develop distorted root hairs that often swell, branch, or collapse. The aberrant cell wall structures found in lrx1 mutants point toward a function of LRX1 during the establishment of the extracellular matrix. To identify genes that are involved in an LRX1-dependent developmental pathway, a suppressor screen was performed on the lrx1 mutant, and two independent rol1 (for repressor of lrx1) alleles were isolated. ROL1 is allelic to Rhamnose Biosynthesis1, which codes for a protein involved in the biosynthesis of rhamnose, a major monosaccharide component of pectin. The rol1 mutations modify the pectic polysaccharide rhamnogalacturonan I and, for one allele, rhamnogalacturonan II. Furthermore, the rol1 mutations cause a change in the expression of a number of cell wall-related genes. Thus, the lrx1 mutant phenotype is likely to be suppressed by changes in pectic polysaccharides or other cell wall components.

The enl mutants enhance the lrx1 root hair mutant phenotype of Arabidopsis thaliana.

The development of root hairs serves as an excellent model to study cell growth using both cytological and genetic approaches. In the past, we have characterized LRX1, an extracellular protein of Arabidopsis consisting of an LRR-domain and a structural extensin domain. LRX1 is specifically expressed in root hairs and lrx1 mutants show severe deficiencies in root hair development. In this work, we describe the characterization of enl (enhancer of lrx1) mutants that were isolated in a visual screen of an ethylmethanesulfonate -mutagenized lrx1 line for plants exhibiting an enhanced lrx1 phenotype. Four recessive enl mutants were analyzed, three of which define new genetic loci involved in root hair development. The mutations at the enl loci and lrx1 result in additive phenotypes in enl/lrx1 double mutants. One enl mutant is affected in the ACTIN2 gene and encodes a protein with a 22 amino acid deletion at the C-terminus. The comparison of molecular and phenotypic data of different actin2 alleles suggests that the truncated ACTIN2 protein is still partially functional.

Whole-genome comparison of leucine-rich repeat extensins in Arabidopsis and rice. A conserved family of cell wall proteins form a vegetative and a reproductive clade.

We have searched the Arabidopsis and rice (Oryza sativa) genomes for homologs of LRX1, an Arabidopsis gene encoding a novel type of cell wall protein containing a leucine-rich repeat (LRR) and an extensin domain. Eleven and eight LRX (LRR/EXTENSIN) genes have been identified in these two plant species, respectively. The LRX gene family encodes proteins characterized by a short N-terminal domain, a domain with 10 LRRs, a cysteine-rich motif, and a variable C-terminal extensin-like domain. Phylogenetic analysis performed on the conserved domains indicates the existence of two major clades of LRX proteins that arose before the eudicot/monocot divergence and then diversified independently in each lineage. In Arabidopsis, gene expression studies by northern hybridization and promoter::uidA fusions showed that the two phylogenetic clades represent a specialization into "reproductive" and "vegetative" LRXs. The four Arabidopsis genes of the "reproductive" clade are specifically expressed in pollen, whereas the seven "vegetative" genes are predominantly expressed in various sporophytic tissues. This separation into two expression classes is also supported by previous studies on maize (Zea mays) and tomato (Lycopersicon esculentum) LRX homologs and by information on available rice ESTs. The strong conservation of the amino acids responsible for the putative recognition specificity of the LRR domain throughout the family suggests that the LRX proteins interact with similar ligands.

ACTIN2 is essential for bulge site selection and tip growth during root hair development of Arabidopsis.

Root hairs develop as long extensions from root epidermal cells. After the formation of an initial bulge at the distal end of the epidermal cell, the root hair structure elongates by tip growth. Because root hairs are not surrounded by other cells, root hair formation provides an excellent system for studying the highly complex process of plant cell growth. Pharmacological experiments with actin filament-interfering drugs have provided evidence that the actin cytoskeleton is an important factor in the establishment of cell polarity and in the maintenance of the tip growth machinery at the apex of the growing root hair. However, there has been no genetic evidence to directly support this assumption. We have isolated an Arabidopsis mutant, deformed root hairs 1 (der1), that is impaired in root hair development. The DER1 locus was cloned by map-based cloning and encodes ACTIN2 (ACT2), a major actin of the vegetative tissue. The three der1 alleles develop the mutant phenotype to different degrees and are all missense mutations, thus providing the means to study the effect of partially functional ACT2. The detailed characterization of the der1 phenotypes revealed that ACT2 is not only involved in root hair tip growth, but is also required for correct selection of the bulge site on the epidermal cell. Thus, the der1 mutants are useful tools to better understand the function of the actin cytoskeleton in the process of root hair formation.