New insights on the role of HCN in root hair elongation through single cell proteomics

Root hairs are specialized structures involved in water and nutrient uptake by plants. They elongate from epidermal cells following a complex developmental program. β-cyanoalanine synthase (CAS), which is mainly involved in hydrogen cyanide (HCN) detoxification in Arabidopsis thaliana, plays a role in root hair elongation, as evidenced by the fact that cas-c1 mutants show a severe defect in root hair shape. In addition to root hairs, CAS C1 is expressed in the quiescent center and meristem. However, the cas-c1 mutation has no visible effect on either tissue, in both control and nutrient-deprivation conditions. To identify its role in root hair formation, we conducted single cell proteomics analysis by isolating root hair cells using Fluorescence-Activated Cell Sorting (FACS) from wild type and cas-c1 mutants. We also analyzed the presence of S-cyanylation, a protein post-translational modification (PTM) mediated by HCN and affecting cysteine residues and protein activity, in proteins of wild type and cas-c1 mutants. We found that several proteins involved in root hair development, related to the receptor kinase FERONIA signaling and to DNA methylation, are modified by this new post-translational modification.

EIN3 and RSL4 interfere with an MYB–bHLH–WD40 complex to mediate ethylene-induced ectopic root hair formation in Arabidopsis

The alternating cell specifications of root epidermis to form hair cells or nonhair cells in Arabidopsis are determined by the expression level of GL2, which is activated by an MYB–bHLH–WD40 (WER–GL3–TTG1) transcriptional complex. The phytohormone ethylene (ET) has a unique effect of inducing N-position epidermal cells to form root hairs. However, the molecular mechanisms underlying ET-induced ectopic root hair development remain enigmatic. Here, we show that ET promotes ectopic root hair formation through down-regulation of GL2 expression. ET-activated transcription factors EIN3 and its homolog EIL1 mediate this regulation. Molecular and biochemical analyses further revealed that EIN3 physically interacts with TTG1 and interferes with the interaction between TTG1 and GL3, resulting in reduced activation of GL2 by the WER–GL3–TTG1 complex. Furthermore, we found through genetic analysis that the master regulator of root hair elongation, RSL4, which is directly activated by EIN3, also participates in ET-induced ectopic root hair development. RSL4 negatively regulates the expression of GL2, likely through a mechanism similar to that of EIN3. Therefore, our work reveals that EIN3 may inhibit gene expression by affecting the formation of transcription-activating protein complexes and suggests an unexpected mutual inhibition between the hair elongation factor, RSL4, and the hair specification factor, GL2. Overall, this study provides a molecular framework for the integration of ET signaling and intrinsic root hair development pathway in modulating root epidermal cell specification.

Ethylene signaling increases reactive oxygen species accumulation to drive root hair initiation in Arabidopsis thaliana

Root hair initiation is a highly regulated aspect of root development. The plant hormone, ethylene, and its precursor, 1-amino-cyclopropane-1-carboxylic acid (ACC), induce formation and elongation of root hairs. We asked whether elevated ethylene induced root hair formation by increasing reactive oxygen species (ROS) synthesis in hair cells. Using confocal microscopy paired with redox biosensors and dyes, we demonstrated that treatments that elevate ethylene levels led to increased ROS accumulation in hair cells prior to root hair formation. In two ethylene-insensitive mutants, etr1-3 and ein3/eil1, there was no increase in root hair number or ROS accumulation. Conversely, etr1-7, a constitutive ethylene signaling receptor mutant, has increased root hair formation and ROS accumulation similar to ethylene-treated wild type seedlings. The rhd2-6 mutant, with a defect in the gene encoding a ROS synthesizing Respiratory Burst Oxidase Homolog C (RBOHC), showed impaired ethylene-dependent ROS synthesis and root hair formation and decreased RBOH enzyme activity compared to Col-0. To identify additional proteins that drive ROS induced root hair formation, we examined a time course root transcriptomic dataset examining Col-0 grown in the presence of ACC and identified PRX44 and other positively regulated transcripts that encode class III peroxidases (PRXs). The prx44-2 mutant has decreased root hair initiation and ROS accumulation when treated with ACC compared to Col-0 and pPRX44::GFP fluorescence is increased in response to ACC treatment. Together, these results support a model in which ethylene increases ROS accumulation through RBOHC and PRX44 to drive root hair formation.

Genetic and Molecular Analysis of Root Hair Development in Arabis alpina

Root hair formation in Arabidopsis thaliana is a well-established model system for epidermal patterning and morphogenesis in plants. Over the last decades, many underlying regulatory genes and well-established networks have been identified by thorough genetic and molecular analysis. In this study, we used a forward genetic approach to identify genes involved in root hair development in Arabis alpina, a related crucifer species that diverged from A. thaliana approximately 26–40 million years ago. We found all root hair mutant classes known in A. thaliana and identified orthologous regulatory genes by whole-genome or candidate gene sequencing. Our findings indicate that the gene-phenotype relationships regulating root hair development are largely conserved between A. thaliana and A. alpina. Concordantly, a detailed analysis of one mutant with multiple hairs originating from one cell suggested that a mutation in the SUPERCENTIPEDE1 (SCN1) gene is causal for the phenotype and that AaSCN1 is fully functional in A. thaliana. Interestingly, we also found differences in the regulation of root hair differentiation and morphogenesis between the species, and a subset of root hair mutants could not be explained by mutations in orthologs of known genes from A. thaliana. This analysis provides insight into the conservation and divergence of root hair regulation in the Brassicaceae.

Azospirillum brasilense stimulate the growth in Arabidopsis thaliana through the target of rapamycin protein

Azospirillum spp., one of the best studied genus of plant growth promoting rhizobacteria. These rhizobacteria are able to colonize hundreds of plant species and improve their growth, development and productivity. The target of rapamycin (TOR) protein is a central component of the TOR signaling pathway, which regulates cell growth and metabolism in response to environment cues in eukaryotes. In this study, the TOR function was analyzed in Arabidopsis thaliana L. plants inoculated with the rhizobacteria Azospirillum brasilense. Arabidopsis seedlings tor-es, which express an interference RNA in presence of estradiol and decrease TOR expression, showed an inhibition in the growth and lateral root formation, with or without 1x102 CFU/mL of the inoculum. In addition, a morphological analysis of the root showed an inhibition in the root hair formation. The results suggest that A. brasilense controls A. thaliana growth through TOR signaling pathway.

GTL1 is required for a robust root hair growth response to avoid nutrient overloading

Root hair growth is tuned in response to the environment surrounding plants. While most of previous studies focused on the enhancement of root hair growth during nutrient starvation, few studies investigated the root hair response in the presence of excess nutrients. We report that the post-embryonic growth of wild-type Arabidopsis plants is strongly suppressed with increasing nutrient availability, particularly in the case of root hair growth. We further used gene expression profiling to analyze how excess nutrient availability affects root hair growth, and found that RHD6 subfamily genes, which are positive regulators of root hair growth, are down-regulated in this condition. On the other hand, defects in GTL1 and DF1, which are negative regulators of root hair growth, cause frail and swollen root hairs to form when excess nutrients are supplied. Additionally, we observed that the RHD6 subfamily genes are mis-expressed in gtl1-1 df1-1. Furthermore, overexpression of RSL4, an RHD6 subfamily gene, induces swollen root hairs in the face of a nutrient overload, while mutation of RSL4 in gtl1-1 df1-1 restore root hair swelling phenotype. In conclusion, our data suggest that GTL1 and DF1 prevent unnecessary root hair formation by repressing RSL4 under excess nutrient conditions.

Alternative Oxidase Inhibition Impairs Tobacco Root Development and Root Hair Formation

Alternative oxidase (AOX) is the terminal oxidase of the mitochondrial respiratory electron transport chain in plant cells and is critical for the balance of mitochondrial hemostasis. In this study, the effect of inhibition of AOX with different concentrations of salicylhydroxamic acid (SHAM) on the tobacco root development was investigated. We show here that AOX inhibition significantly impaired the development of the main root and root hair formation of tobacco. The length of the main root of SHAM-treated tobacco was significantly shorter than that of the control, and no root hairs were formed after treatment with a concentration of 1 mM SHAM or more. The transcriptome analysis showed that AOX inhibition by 1 mM SHAM involved in the regulation of gene expression related to root architecture. A total of 5,855 differentially expressed genes (DEGs) were obtained by comparing SHAM-treated roots with control. Of these, the gene expression related to auxin biosynthesis and perception were significantly downregulated by 1 mM SHAM. Similarly, genes related to cell wall loosening, cell cycle, and root meristem growth factor 1 (RGF1) also showed downregulation on SHAM treatment. Moreover, combined with the results of physiological measurements, the transcriptome analysis demonstrated that AOX inhibition resulted in excessive accumulation of reactive oxygen species in roots, which further induced oxidative damage and cell apoptosis. It is worth noting that when indoleacetic acid (20 nM) and dimethylthiourea (10 mM) were added to the medium containing SHAM, the defects of tobacco root development were alleviated, but to a limited extent. Together, these findings indicated that AOX-mediated respiratory pathway plays a crucial role in the tobacco root development, including root hair formation.

Conserved and non-conserved functions of the rice homologs of the Arabidopsis trichome initiation-regulating MBW complex proteins

Background Trichome initiation in Arabidopsis is regulated by a MYB-bHLH-WD40 (MBW) transcriptional activator complex formed by the R2R3 MYB transcription factor GLABRA1 (GL1), MYB23 or MYB82, the bHLH transcription factor GLABRA3 (GL3), ENHANCER OF GLABRA3 (EGL3) or TRANSPARENT TESTA8 (TT8), and the WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1). However, the functions of the rice homologs of the MBW complex proteins remained uncharacterized. Results Based on amino acid sequence identity and similarity, and protein interaction prediction, we identified OsGL1s, OsGL3s and OsTTG1s as rice homologs of the MBW complex proteins. By using protoplast transfection, we show that OsGL1D, OsGL1E, OsGL3B and OsTTG1A were predominantly localized in the nucleus, OsGL3B functions as a transcriptional activator and is able to interact with GL1 and TTG1. By using yeast two-hybrid and protoplast transfection assays, we show that OsGL3B is able to interact with OsGL1E and OsTTG1A, and OsGL1E and OsTTG1A are also able to interact with GL3. On the other hand, we found that OsGL1D functions as a transcription activator, and it can interact with GL3 but not OsGL3B. Furthermore, our results show that expression of OsTTG1A in the ttg1 mutant restored the phenotypes including alternations in trichome and root hair formation, seed color, mucilage production and anthocyanin biosynthesis, indicating that OsTTG1A and TTG1 may have similar functions. Conclusion These results suggest that the rice homologs of the Arabidopsis MBW complex proteins are able to form MBW complexes, but may have conserved and non-conserved functions.

Transcriptome sequencing analysis of maize roots reveals the effects of substrate and root hair formation in a spatial context

Background Plant roots sense and respond to changes in their soil environment, but conversely contribute to rhizosphere organization through chemical, mechanical and biotic interactions. Transcriptomic profiling of plant roots can be used to assess how the plant adjusts its gene expression in relation to environment, genotype and rhizosphere processes; thus enabling us to achieve a better understanding of root-soil interactions. Methods We used a standardized soil column experimental platform to investigate the impact of soil texture (loam, sand) and root hair formation (wildtype, root hair defective rth3 mutant) in a spatial context (three sampling depths) and assessed maize root transcriptomic profiles using next-generation RNA sequencing. Results Substrate induced the largest changes in root gene expression patterns, affecting gene functions related to immunity, stress, growth and water uptake. Genes with column depth-related expression levels were associated with growth and plant defense. The influence of root hairs mainly manifested in differential expression of epidermal cell differentiation and cell wall organization, and defense response-related genes. Substrate type strongly modified the transcriptomic patterns related to column depth and root hair elongation, highlighting the strong impact of soil texture. Conclusions Our results demonstrate that substrate, sampling depth and plant genotype interactively affect maize gene expression, and suggest feedback processes between the plant, the soil and the microbiome. The obtained results form a foundational basis for the integration and interpretation of future experiments utilizing the same experimental platform.

Integrated analysis of plant gene expression and bacterial and protozoan community composition reveals changes related to root zonation, root cap and root hair formation

<p>This study was conducted within the framework of the DFG project SPP2089 “Rhizosphere Spatiotemporal Organization – a Key to Rhizosphere Functions” (project number 403641192).</p><p>As plant roots grow into the soil, the formation of biological gradients occurs at different spatial scales. It has been shown that plants recruit specific subsets of the soil bacterial community at their roots through excretion of mucilage at root tips and exudates at the sites of root hair formation. The promotion of or defense against certain bacterial taxa is also reflected in the composition of the protist communities that feed on bacteria.</p><p>Using high-throughput sequencing methods, we investigated emerging patterns in root gene expression in relation to bacterial and protozoan community structures. We found highly distinct root region specific patterns relating to differential root gene expression relating to growth, defense and transporter activity, as well as bacterial and protist (cercozoan) diversity. Root cap removal led to differently composed microbial communities, as well as a regulation of root genes relating to stress and defense. The lack of root hairs was only reflected in the amount of microbial carbon in soil and a small number of differentially expressed genes involved in cell wall processes.</p><p>We could show that the rhizosphere microbiome, is as dynamic as its environment. Root regions differentially affect microbial communities, which is also reflected in the expression of plant genes of categories relating to defense, immunity and stress. Our findings will further enhance our understanding of microbial root interactions at single root scale.</p>