Distinct early transcriptional regulations by turgor and osmotic potential in the roots of Arabidopsis.

In a context of climate change, deciphering signaling pathways driving plant adaptation to drought, changes in water availability, and salt is key. A crossing point of these plant stresses is their impact on plant water potential (Ψ), a composite physico-chemical variable reflecting the availability of water for biological processes such as plant growth and stomatal aperture. The Ψ of plant cells is mainly driven by their turgor and osmotic pressures. Here we investigated the effect of a variety of osmotic treatments on the roots of Arabidopsis plants grown in hydroponics. We used, among others, a permeating solute as a way to differentiate variations on turgor from variations in osmotic pressure. Measurement of cortical cell turgor pressure with a cell pressure probe allowed us to monitor the intensity of the treatments and thereby preserve the cortex from plasmolysis. Transcriptome analyses at an early time point (15 min) showed specific and quantitative transcriptomic responses to both osmotic and turgor pressure variations. Our results highlight how water-related biophysical parameters can shape the transcriptome of roots under stress and provide putative candidates to explore further the early perception of water stress in plants.

pin2 mutant agravitropic root phenotype is conditional and nutrient-sensitive.

Plants have the capacity to sense and adapt to environmental factors using the phytohormone auxin as a major regulator of tropism and development. Among these responses, gravitropism is essential for plant roots to grow downward in the search for nutrients and water. We discovered a new mutant allele of the auxin efflux transporter PIN2 that revealed that pin2 agravitropic root mutants are conditional and nutrient-sensitive. We describe that nutrient composition of the medium, rather than osmolarity, can revert the agravitropic root phenotype of pin2. Indeed, on phosphorus- and nitrogen-deprived media, the agravitropic root defect was restored independently of primary root growth levels. Slow and fast auxin responses were evaluated using DR5 and R2D2 probes, respectively, and revealed a strong modulation by nutrient composition of the culture medium. We evaluated the role of PIN and AUX auxin transporters and demonstrated that neither PIN3 nor AUX1 are involved in this process. However, we observed the ectopic expression of PIN1 in the epidermis in the pin2 mutant background associated with permissive, but not restrictive, conditions. This ectopic expression was associated with a restoration of the asymmetric accumulation of auxin necessary for the reorientation of the root according to gravity. These observations suggest a strong regulation of auxin distribution by nutrients availability, directly impacting root's ability to drive their gravitropic response.

High-throughput and automatic structural and developmental root phenotyping on Arabidopsis seedlings.

BACKGROUND: High-throughput phenotyping is crucial for the genetic and molecular understanding of adaptive root system development. In recent years, imaging automata have been developed to acquire the root system architecture of many genotypes grown in Petri dishes to explore the Genetic x Environment (GxE) interaction. There is now an increasing interest in understanding the dynamics of the adaptive responses, such as the organ apparition or the growth rate. However, due to the increasing complexity of root architectures in development, the accurate description of the topology, geometry, and dynamics of a growing root system remains a challenge. RESULTS: We designed a high-throughput phenotyping method, combining an imaging device and an automatic analysis pipeline based on registration and topological tracking, capable of accurately describing the topology and geometry of observed root systems in 2D + t. The method was tested on a challenging Arabidopsis seedling dataset, including numerous root occlusions and crossovers. Static phenes are estimated with high accuracy ([Formula: see text] and [Formula: see text] for primary and second-order roots length, respectively). These performances are similar to state-of-the-art results obtained on root systems of equal or lower complexity. In addition, our pipeline estimates dynamic phenes accurately between two successive observations ([Formula: see text] for lateral root growth). CONCLUSIONS: We designed a novel method of root tracking that accurately and automatically measures both static and dynamic parameters of the root system architecture from a novel high-throughput root phenotyping platform. It has been used to characterise developing patterns of root systems grown under various environmental conditions. It provides a solid basis to explore the GxE interaction controlling the dynamics of root system architecture adaptive responses. In future work, our approach will be adapted to a wider range of imaging configurations and species.

Root Membrane Ubiquitinome under Short-Term Osmotic Stress.

Osmotic stress can be detrimental to plants, whose survival relies heavily on proteomic plasticity. Protein ubiquitination is a central post-translational modification in osmotic-mediated stress. In this study, we used the K-Ɛ-GG antibody enrichment method integrated with high-resolution mass spectrometry to compile a list of 719 ubiquitinated lysine (K-Ub) residues from 450 Arabidopsis root membrane proteins (58% of which are transmembrane proteins), thereby adding to the database of ubiquitinated substrates in plants. Although no ubiquitin (Ub) motifs could be identified, the presence of acidic residues close to K-Ub was revealed. Our ubiquitinome analysis pointed to a broad role of ubiquitination in the internalization and sorting of cargo proteins. Moreover, the simultaneous proteome and ubiquitinome quantification showed that ubiquitination is mostly not involved in membrane protein degradation in response to short osmotic treatment but that it is putatively involved in protein internalization, as described for the aquaporin PIP2;1. Our in silico analysis of ubiquitinated proteins shows that two E2 Ub-conjugating enzymes, UBC32 and UBC34, putatively target membrane proteins under osmotic stress. Finally, we revealed a positive role for UBC32 and UBC34 in primary root growth under osmotic stress.

Responses to Systemic Nitrogen Signaling in Arabidopsis Roots Involve trans-Zeatin in Shoots.

Plants face temporal and spatial variation in nitrogen (N) availability. This includes heterogeneity in soil nitrate (NO3-) content. To overcome these constraints, plants modify their gene expression and physiological processes to optimize N acquisition. This plasticity relies on a complex long-distance root-shoot-root signaling network that remains poorly understood. We previously showed that cytokinin (CK) biosynthesis is required to trigger systemic N signaling. Here, we performed split-root experiments and used a combination of CK-related mutant analyses, hormone profiling, transcriptomic analysis, NO3- uptake assays, and root growth measurements to gain insight into systemic N signaling in Arabidopsis thaliana By comparing wild-type plants and mutants affected in CK biosynthesis and ABCG14-dependent root-to-shoot translocation of CK, we revealed an important role for active trans-zeatin (tZ) in systemic N signaling. Both rapid sentinel gene regulation and long-term functional acclimation to heterogeneous NO3- supply, including NO3- transport and root growth regulation, are likely mediated by the integration of tZ content in shoots. Furthermore, shoot transcriptome profiling revealed that glutamate/glutamine metabolism is likely a target of tZ root-to-shoot translocation, prompting an interesting hypothesis regarding shoot-to-root communication. Finally, this study highlights tZ-independent pathways regulating gene expression in shoots as well as NO3- uptake activity in response to total N deprivation.

The Casuarina NIN gene is transcriptionally activated throughout Frankia root infection as well as in response to bacterial diffusible signals.

Root nodule symbioses (RNS) allow plants to acquire atmospheric nitrogen by establishing an intimate relationship with either rhizobia, the symbionts of legumes or Frankia in the case of actinorhizal plants. In legumes, NIN (Nodule INception) genes encode key transcription factors involved in nodulation. Here we report the characterization of CgNIN, a NIN gene from the actinorhizal tree Casuarina glauca using both phylogenetic analysis and transgenic plants expressing either ProCgNIN::reporter gene fusions or CgNIN RNAi constructs. We have found that CgNIN belongs to the same phylogenetic group as other symbiotic NIN genes and CgNIN is able to complement a legume nin mutant for the early steps of nodule development. CgNIN expression is correlated with infection by Frankia, including preinfection stages in developing root hairs, and is induced by culture supernatants. Knockdown mutants were impaired for nodulation and early root hair deformation responses were severely affected. However, no mycorrhizal phenotype was observed and no induction of CgNIN expression was detected in mycorrhizas. Our results indicate that elements specifically required for nodulation include NIN and possibly related gene networks derived from the nitrate signalling pathways.

Inhibition of auxin signaling in Frankia species-infected cells in Casuarina glauca nodules leads to increased nodulation.

Actinorhizal symbioses are mutualistic interactions between plants and the soil bacteria Frankia spp. that lead to the formation of nitrogen-fixing root nodules. The plant hormone auxin has been suggested to play a role in the mechanisms that control the establishment of this symbiosis in the actinorhizal tree Casuarina glauca. Here, we analyzed the role of auxin signaling in Frankia spp.-infected cells. Using a dominant-negative version of an endogenous auxin-signaling regulator, INDOLE-3-ACETIC ACID7, we established that inhibition of auxin signaling in these cells led to increased nodulation and, as a consequence, to higher nitrogen fixation per plant even if nitrogen fixation per nodule mass was similar to that in the wild type. Our results suggest that auxin signaling in Frankia spp.-infected cells is involved in the long-distance regulation of nodulation in actinorhizal symbioses.

Identification of potential transcriptional regulators of actinorhizal symbioses in Casuarina glauca and Alnus glutinosa.

BACKGROUND: Trees belonging to the Casuarinaceae and Betulaceae families play an important ecological role and are useful tools in forestry for degraded land rehabilitation and reforestation. These functions are linked to their capacity to establish symbiotic relationships with a nitrogen-fixing soil bacterium of the genus Frankia. However, the molecular mechanisms controlling the establishment of these symbioses are poorly understood. The aim of this work was to identify potential transcription factors involved in the establishment and functioning of actinorhizal symbioses. RESULTS: We identified 202 putative transcription factors by in silico analysis in 40 families in Casuarina glauca (Casuarinaceae) and 195 in 35 families in Alnus glutinosa (Betulaceae) EST databases. Based on published transcriptome datasets and quantitative PCR analysis, we found that 39% and 26% of these transcription factors were regulated during C. glauca and A. glutinosa-Frankia interactions, respectively. Phylogenetic studies confirmed the presence of common key transcription factors such as NSP, NF-YA and ERN-related proteins involved in nodule formation in legumes, which confirm the existence of a common symbiosis signaling pathway in nitrogen-fixing root nodule symbioses. We also identified an actinorhizal-specific transcription factor belonging to the zinc finger C1-2i subfamily we named CgZF1 in C. glauca and AgZF1 in A. glutinosa. CONCLUSIONS: We identified putative nodulation-associated transcription factors with particular emphasis on members of the GRAS, NF-YA, ERF and C2H2 families. Interestingly, comparison of the non-legume and legume TF with signaling elements from actinorhizal species revealed a new subgroup of nodule-specific C2H2 TF that could be specifically involved in actinorhizal symbioses. In silico identification, transcript analysis, and phylogeny reconstruction of transcription factor families paves the way for the study of specific molecular regulation of symbiosis in response to Frankia infection.

Heart of endosymbioses: transcriptomics reveals a conserved genetic program among arbuscular mycorrhizal, actinorhizal and legume-rhizobial symbioses.

To improve their nutrition, most plants associate with soil microorganisms, particularly fungi, to form mycorrhizae. A few lineages, including actinorhizal plants and legumes are also able to interact with nitrogen-fixing bacteria hosted intracellularly inside root nodules. Fossil and molecular data suggest that the molecular mechanisms involved in these root nodule symbioses (RNS) have been partially recycled from more ancient and widespread arbuscular mycorrhizal (AM) symbiosis. We used a comparative transcriptomics approach to identify genes involved in establishing these 3 endosymbioses and their functioning. We analysed global changes in gene expression in AM in the actinorhizal tree C. glauca. A comparison with genes induced in AM in Medicago truncatula and Oryza sativa revealed a common set of genes induced in AM. A comparison with genes induced in nitrogen-fixing nodules of C. glauca and M. truncatula also made it possible to define a common set of genes induced in these three endosymbioses. The existence of this core set of genes is in accordance with the proposed recycling of ancient AM genes for new functions related to nodulation in legumes and actinorhizal plants.

Auxin carriers localization drives auxin accumulation in plant cells infected by Frankia in Casuarina glauca actinorhizal nodules.

Actinorhizal symbioses are mutualistic interactions between plants and the soil bacteria Frankia that lead to the formation of nitrogen-fixing root nodules. Little is known about the signaling mechanisms controlling the different steps of the establishment of the symbiosis. The plant hormone auxin has been suggested to play a role. Here we report that auxin accumulates within Frankia-infected cells in actinorhizal nodules of Casuarina glauca. Using a combination of computational modeling and experimental approaches, we establish that this localized auxin accumulation is driven by the cell-specific expression of auxin transporters and by Frankia auxin biosynthesis in planta. Our results indicate that the plant actively restricts auxin accumulation to Frankia-infected cells during the symbiotic interaction.