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Soybean plants form symbiotic nitrogen-fixing nodules with specific rhizobia bacteria. The root hair is the initial infection site for the symbiotic process before the nodules. Since roots and nodules grow in soil and are hard to perceive, little knowledge is available on the process of soybean root hair deformation and nodule development over time. In this study, adaptive microrhizotrons were used to observe root hairs and to investigate detailed root hair deformation and nodule formation subjected to different rhizobia densities. The result showed that the root hair curling angle increased with the increase of rhizobia density. The largest curling angle reached 268° on the 8th day after inoculation. Root hairs were not always straight, even in the uninfected group with a relatively small angle (<45°). The nodule is an organ developed after root hair curling. It was inoculated from curling root hairs and swelled in the root axis on the 15th day after inoculation, with the color changing from light (15th day) to a little dark brown (35th day). There was an error between observing the diameter and the real diameter; thus, a diameter over 1 mm was converted to the real diameter according to the relationship between the perceived diameter and the real diameter. The diameter of the nodule reached 5 mm on the 45th day. Nodule number and curling number were strongly related to rhizobia density with a correlation coefficient of determination of 0.92 and 0.93, respectively. Thus, root hair curling development could be quantified, and nodule number could be estimated through derived formulation.

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The tight regulation of protein composition within the plasma membranes of plant cells is crucial for the proper development of plants and for their ability to respond to a changing environment. Upon being endocytosed, integral membrane proteins can be secreted, sorted into multivesicular bodies/late endosomes, and degraded in the lytic vacuole, or recycled back to the plasma membrane to continue functioning. The evolutionarily conserved retromer complex has attracted the interest of plant cell biologists for over a decade as it has emerged as a key regulator of the trafficking of endocytosed integral plasma membrane proteins. Recently, a related recycling complex that shares a subunit with retromer was described in metazoan species. Named "retriever", homologs to the proteins that comprise this new recycling complex and its accessory proteins are found within plant lineages. Initial experiments indicate that there is conservation of function between metazoan and plant retriever proteins, suggesting that it is prudent to re-evaluate the available plant retromer data with the added potential of a plant retriever complex.

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Root resource acquisition strategies play a crucial role in understanding plant water uptake and drought adaptation. However, the interrelationships among mycorrhizal associations, root hair development, and fine root strategies, as well as the disparities between C3 and C4 grasses, remain largely unknown. A pot experiment was conducted to determine leaf gas exchange, root morphology, root hair, mycorrhizal fungi, and biomass allocation of three C4 grasses and four C3 grasses, common species of grasslands in Northeast China, under the control and drought conditions. Compared to the C3 grasses, the C4 grasses increased specific surface area by decreasing tissue density, yet exhibited root hair factor at only 21 % of the C3 grasses. Under the drought conditions, the C4 grasses exhibited more intense and extensive adjustments in root traits, characterized by shifts toward a more conservative morphology with increased root diameter and tissue density, as well as reduced mycorrhizal colonization rates. These adaptations led to a decrease in root absorptive function, which was compensated in the C4 grasses by greater root biomass partitioning and root hair factor. Variances in root strategies between plants functional groups were closely related to leaf photosynthetic rate, water and nitrogen use efficiency. We observed that the C4 grasses prefer direct acquisition of soil resources through the fine root pathway over the root hair or mycorrhizal pathway, suggesting a 'do-it-yourself' approach. These findings provide valuable insights into how plant communities of different photosynthetic types might respond to future climate change.

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Combinatorial interactions between different regulators diversify and enrich the chance of transcriptional regulation in eukaryotic cells. However, a dose-dependent functional switch of homologous transcriptional repressors has rarely been reported. Here, we show that SHY2, an auxin/indole-3-acetic acid (Aux/IAA) repressor, exhibits a dose-dependent bimodal role in auxin-sensitive root-hair growth and gene transcription in Arabidopsis, whereas other Aux/IAA homologs consistently repress the auxin responses. The co-repressor (TOPLESS [TPL])-binding affinity of a bimodal Aux/IAA was lower than that of a consistently repressing Aux/IAA. The switch of a single amino acid residue in the TPL-binding motif between the bimodal form and the consistently repressing form switched their TPL-binding affinity and transcriptional and biological roles in auxin responses. Based on these data, we propose a model whereby competition between homologous repressors with different co-repressor-binding affinities could generate a bimodal output at the transcriptional and developmental levels.

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Root hairs (RHs) are an innovation of vascular plants whose development is coordinated by endogenous and environmental cues, such as ethylene and light conditions. However, the potential crosstalk between ethylene and light conditions in RH development is unclear. We report that Arabidopsis constitutive photomorphogenic 1 (COP1) integrates ethylene and light signaling to mediate RH development. Darkness suppresses RH development largely through COP1. COP1 inhibits both cell fate determination of trichoblast and tip growth of RHs based on pharmacological, genetic, and physiological analyses. Indeed, COP1 interacts with and catalyzes the ubiquitination of ACS2 and ACS6. COP1- or darkness-promoted proteasome-dependent degradation of ACS2/6 leads to a low ethylene level in underground tissues. The negative role of COP1 in RH development by downregulating ethylene signaling may be coordinated with the positive role of COP1 in hypocotyl elongation by upregulating ethylene signaling, providing an evolutionary advantage for seedling fitness.

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Reactive oxygen species (ROS) are essential signaling molecules that enable cells to respond rapidly to a range of stimuli. The ability of plants to recognize various stressors, incorporate a variety of environmental inputs, and initiate stress-response networks depends on ROS. Plants develop resilience and defensive systems as a result of these processes. Root hairs are central components of root biology since they increase the surface area of the root, anchor it in the soil, increase its ability to absorb water and nutrients, and foster interactions between microorganisms. In this review, we specifically focused on root hair cells and we highlighted the identification of ROS receptors, important new regulatory hubs that connect ROS production, transport, and signaling in the context of two hormonal pathways (auxin and ethylene) and under low temperature environmental input related to nutrients. As ROS play a crucial role in regulating cell elongation rates, root hairs are rapidly gaining traction as a very valuable single plant cell model for investigating ROS homeostasis and signaling. These promising findings might soon facilitate the development of plants and roots that are more resilient to environmental stressors.

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Root hair (RH) cells are important for the growth and survival of seedlings. They favor plant-microbe interactions and nutrients uptake. When invading the soil, RH cells have to penetrate a dense medium exhibiting a variety of physical properties, such as mechanical resistance, that impact the growth and survival of plants. Here we investigate the effect of the mechanical resistance of the culture medium on RH-physical and phenotypical parameters such as length, time, and speed of growth. We also analyze the impact of the environment on nuclear dynamics. We show that the RH growth rate and the nucleus speed decrease similarly as mechanical resistance increases while the time of growth of RH cells is invariable. Moreover, during RH growth, the nucleus-to-tip distance was found to decrease when the stiffness of the environment was increased. Along this line, using Latrunculin B treatment in liquid growth media, we could internally slow down RH growth to reach speeds similar to those observed in stiff solid media while the nucleus-to-tip distance was only slightly affected, supporting thus the idea of a specific effect of mechanical resistance of the environment on nucleus dynamics.

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Polyamines are ubiquitous biomolecules with a number of established functions in eukaryotic cells. In plant cells, polyamines have previously been linked to abiotic and biotic stress tolerance, as well as to the modulation of programmed cell death (PCD), with contrasting reports on their pro-PCD and pro-survival effects. Here, we used two well-established platforms for the study of plant PCD, Arabidopsis thaliana suspension cultures cells and the root hair assay, to examine the roles of the polyamines spermine and spermidine in the regulation of PCD. Using these systems for precise quantification of cell death rates, we demonstrate that both polyamines can trigger PCD when applied exogenously at higher doses, whereas at lower concentrations they inhibit PCD induced by both biotic and abiotic stimuli. Furthermore, we show that concentrations of polyamines resulting in inhibition of PCD generated a transient ROS burst in our experimental system, and activated the expression of oxidative stress- and pathogen response-associated genes. Finally, we examined PCD responses in existing Arabidopsis polyamine synthesis mutants, and identified a subtle PCD phenotype in Arabidopsis seedlings deficient in thermo-spermine. The presented data show that polyamines can have a role in PCD regulation; however, that role is dose-dependent and consequently they may act as either inhibitors, or inducers, of PCD in Arabidopsis.

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ELONGATED HYPOCOTYL 5 (HY5) is a major light-associated transcription factor involved in plant growth and development. In Arabidopsis (Arabidopsis thaliana), the role of HY5 is very well-defined in regulating primary root growth and lateral root formation; however, information regarding its role in root hair development is still lacking, and little is known about the genetic pathways regulating this process. In this study, we investigated the role of HY5 and its associated components in root hair development. Detailed analysis of root hair phenotype in wild-type (WT) and light signaling mutants in light and dark conditions revealed the importance of light-dependent HY5-mediated root hair initiation. Altered auxin levels in the root apex of the hy5 mutant and interaction of HY5 with promoters of root hair developmental genes were responsible for differential expression of root hair developmental genes and phenotype in the hy5 mutant. The partial complementation of root hair in the hy5 mutant after external supplementation of auxin and regaining of root hair in PIN-FORMED 2 (pin2) and PIN-FORMED 2 (pin3) mutants after grafting suggested that the auxin-mediated root hair development pathway requires HY5. Furthermore, miR397b overexpression (miR397bOX) and CRISPR/Cas9-based mutants (miR397bCR) indicated miR397b targets genes encoding Reduced Residual Arabinose (RRA1/RRA2), which in turn regulate root hair growth. The regulation of the miR397b- (RRA1/RRA2) module by HY5 demonstrated its indirect role by targeting root hair cell wall genes. Together, this study demonstrated that HY5 controls root hair development by integrating auxin signalling and other miRNA-mediated pathways.

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Redox changes of pyridine nucleotides in cellular compartments are highly dynamic and their equilibria are under the influence of various reducing and oxidizing reactions. To obtain spatiotemporal data on pyridine nucleotides in living plant cells, typical biochemical approaches require cell destruction. To date, genetically encoded fluorescent biosensors are considered to be the best option to bridge the existing technology gap, as they provide a fast, accurate, and real-time readout. However, the existing pyridine nucleotides genetically encoded fluorescent biosensors are either sensitive to pH change or slow in dissociation rate. Herein, we employed the biosensors which generate readouts that are pH stable for in planta measurement of NADH/NAD+ ratio and NADPH level. We generated transgenic Arabidopsis lines that express these biosensors in plastid stroma and cytosol of whole plants and pollen tubes under the control of CaMV 35S and LAT52 promoters, respectively. These transgenic biosensor lines allow us to monitor real-time dynamic changes in NADH/NAD+ ratio and NADPH level in the plastids and cytosol of various plant tissues, including pollen tubes, root hairs, and mesophyll cells, using a variety of fluorescent instruments. We anticipate that these valuable transgenic lines may allow improvements in plant redox biology studies.

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Compacted soil layers adversely affect rooting depth and access to deeper nutrient and water resources, thereby impacting climate resilience of crop production and global food security. Root hair plays well-known roles in facilitating water and nutrient acquisition. Here, we report that root hair also contributes to root penetration into compacted layers. We demonstrate that longer root hair, induced by elevated auxin response during a root compaction response, improves the ability of rice roots to penetrate harder layers. This compaction-induced auxin response in the root hair zone is dependent on the root apex-expressed auxin synthesis gene OsYUCCA8 (OsYUC8), which is induced by compaction stress. This auxin source for root hair elongation relies on the auxin influx carrier AUXIN RESISTANT 1 (OsAUX1), mobilizing this signal from the root apex to the root hair zone. Mutants disrupting OsYUC8 and OsAUX1 genes exhibit shorter root hairs and weaker penetration ability into harder layers compared with wild type (WT). Root-hair-specific mutants phenocopy these auxin-signaling mutants, as they also exhibit an attenuated root penetration ability. We conclude that compaction stress upregulates OsYUC8-mediated auxin biosynthesis in the root apex, which is subsequently mobilized to the root hair zone by OsAUX1, where auxin promotes root hair elongation, improving anchorage of root tips to their surrounding soil environment and aiding their penetration ability into harder layers.

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The phytohormone auxin plays a pivotal role in governing plant growth and development. Although the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX (TIR1/AFB) receptors function in both the nucleus and cytoplasm, the mechanism governing the distribution of TIR1/AFBs between these cellular compartments remains unknown. In this study, we demonstrate that auxin-mediated oxidation of TIR1/AFB2 is essential for their targeting to the nucleus. We showed that small active molecules, reactive oxygen species (ROS) and nitric oxide (NO), are indispensable for the nucleo-cytoplasmic distribution of TIR1/AFB2 in trichoblasts and root hairs. Further studies revealed that this process is regulated by the FERONIA receptor kinase-NADPH oxidase signaling pathway. Interestingly, ROS and NO initiate oxidative modifications in TIR1C140/516 and AFB2C135/511, facilitating their subsequent nuclear import. The oxidized forms of TIR1C140/516 and AFB2C135/511 play a crucial role in enhancing the function of TIR1 and AFB2 in transcriptional auxin responses. Collectively, our study reveals a novel mechanism by which auxin stimulates the transport of TIR1/AFB2 from the cytoplasm to the nucleus, orchestrated by the FERONIA-ROS signaling pathway.

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Plant genetics plays a key role in determining root hair initiation and development. A complex network of genetic interactions therefore closely monitors and influences root hair phenotype and morphology. The significance of these genes can be studied by employing, for instance, loss-of-function mutants, overexpression plant lines, and fluorescently labeled constructs. Confocal laser scanning microscopy is a great tool to visually observe and document these morphological features. This chapter elaborates the techniques involved in handling of microscopic setup to acquire images displaying root hair distribution along the fully elongated zone of Arabidopsis thaliana roots. Additionally, we illustrate an approach to visualize early fate determination of epidermal cells in the root apical meristem, by describing a method for imaging YFP tagged transgenic plant lines.

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Plant Elicitor Peptides (Peps) induce plant immune responses and inhibit root growth through their receptors PEPR1 and PEPR2, two receptor-like kinases. In our study, we found a previously unknown function of Peps that enhance root hair growth in a PEPRs-independent manner. When we characterized the expression patterns of PROPEP genes, we found several gene promoters of PROPEP gene family were particularly active in root hairs. Furthermore, we observed that PROPEP2 is vital for root hair development, as disruption of PROPEP2 gene led to a significant reduction in root hair density and length. We also discovered that PROPEP2 regulates root hair formation via the modulation of CPC and GL2 expression, thereby influencing the cell-fate determination of root hairs. Additionally, calcium signaling appeared to be involved in PROPEP2/Pep2-induced root hair growth. These findings shed light on the function of Peps in root hair development.

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Ran GTPases play essential roles in plant growth and development. Our previous studies revealed the nuclear localization of DlRan3A and DlRan3B proteins and proposed their functional redundancy and distinction in Dimocarpus longan somatic embryogenesis, hormone, and abiotic stress responses. To further explore the possible roles of DlRan3A and DlRan3B, gene expression analysis by qPCR showed that their transcripts were both more abundant in the early embryo and pulp in longan. Heterologous expression of DlRan3A driven by its own previously cloned promoter led to stunted growth, increased root hair density, abnormal fruits, bigger seeds, and enhanced abiotic stress tolerance. Conversely, constitutive promoter CaMV 35S (35S)-driven expression of DlRan3A, 35S, or DlRan3B promoter-controlled expression of DlRan3B did not induce the alterations in growth phenotype, while they rendered different hypersensitivities to abiotic stresses. Based on the transcriptome profiling of longan Ran overexpression in tobacco plants, we propose new mechanisms of the Ran-mediated regulation of genes associated with cell wall biosynthesis and expansion. Also, the transgenic plants expressing DlRan3A or DlRan3B genes controlled by 35S or by their own promoter all exhibited altered mRNA levels of stress-related and transcription factor genes. Moreover, DlRan3A overexpressors were more tolerant to salinity, osmotic, and heat stresses, accompanied by upregulation of oxidation-related genes, possibly involving the Ran-RBOH-CIPK network. Analysis of a subset of selected genes from the Ran transcriptome identified possible cold stress-related roles of brassinosteroid (BR)-responsive genes. The marked presence of genes related to cell wall biosynthesis and expansion, hormone, and defense responses highlighted their close regulatory association with Ran.

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Cereal plants form complex networks with their associated microbiome in the soil environment. A complex system including variations of numerous parameters of soil properties and host traits shapes the dynamics of cereal microbiota under drought. These multifaceted interactions can greatly affect carbon and nutrient cycling in soil and offer the potential to increase plant growth and fitness under drought conditions. Despite growing recognition of the importance of plant microbiota to agroecosystem functioning, harnessing the cereal root microbiota remains a significant challenge due to interacting and synergistic effects between root traits, soil properties, agricultural practices, and drought-related features. A better mechanistic understanding of root-soil-microbiota associations could lead to the development of novel strategies to improve cereal production under drought. In this review, we discuss the root-soil-microbiota interactions for improving the soil environment and host fitness under drought and suggest a roadmap for harnessing the benefits of these interactions for drought-resilient cereals. These methods include conservative trait-based approaches for the selection and breeding of plant genetic resources and manipulation of the soil environments.

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Polyploidization is a major event driving plant evolution and domestication. However, how reshaped epigenetic modifications coordinate gene transcription to generate phenotypic variations during wheat polyploidization is currently elusive. Here, we profiled transcriptomes and DNA methylomes of two diploid wheat accessions (SlSl and AA) and their synthetic allotetraploid wheat line (SlSlAA), which displayed elongated root hair and improved root capability for nitrate uptake and assimilation after tetraploidization. Globally decreased DNA methylation levels with a reduced difference between subgenomes were observed in the roots of SlSlAA. DNA methylation changes in first exon showed strong connections with altered transcription during tetraploidization. Homoeolog-specific transcription was associated with biased DNA methylation as shaped by homoeologous sequence variation. The hypomethylated promoters showed significantly enriched binding sites for MYB, which may affect gene transcription in response to root hair growth. Two master regulators in root hair elongation pathway, AlCPC and TuRSL4, exhibited upregulated transcription levels accompanied by hypomethylation in promoter, which may contribute to the elongated root hair. The upregulated nitrate transporter genes, including NPFs and NRTs, also are significantly associated with hypomethylation, indicating an epigenetic-incorporated regulation manner in improving nitrogen use efficiency. Collectively, these results provided new insights into epigenetic changes in response to crop polyploidization and underscored the importance of epigenetic regulation in improving crop traits.

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Tube-like outgrowths from root epidermal cells, known as root hairs, enhance water and nutrient absorption, facilitate microbial interactions, and contribute to plant anchorage by expanding the root surface area. Genetically regulated and strongly influenced by environmental conditions, longer root hairs generally enhance water and nutrient absorption, correlating with increased stress resistance. Wheat, a globally predominant crop pivotal for human nutrition, necessitates the identification of long root hair genotypes and their regulatory genes to enhance nutrient capture and yield potential. This study focused on 261 wheat samples of diverse genotypes during germination, revealing noticeable disparities in the length of the root hair among the genotypes. Notably, two long root hair genotypes (W106 and W136) and two short root hair genotypes (W90 and W100) were identified. Transcriptome sequencing resulted in the development of 12 root cDNA libraries, unveiling 1180 shared differentially expressed genes (DEGs). Further analyses, including GO function annotation, KEGG enrichment, MapMan metabolic pathway analysis, and protein-protein interaction (PPI) network prediction, underscored the upregulation of root hair length regulatory genes in the long root hair genotypes. These included genes are associated with GA and BA hormone signaling pathways, FRS/FRF and bHLH transcription factors, phenylpropanoid, lignin, lignan secondary metabolic pathways, the peroxidase gene for maintaining ROS steady state, and the ankyrin gene with diverse biological functions. This study contributes valuable insights into modulating the length of wheat root hair and identifies candidate genes for the genetic improvement of wheat root traits.

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Root hair, single-celled tubular structures originating from the epidermis, plays a vital role in the uptake of nutrients from the soil by increasing the root surface area. Therefore, optimizing root hair growth is crucial for plants to survive in fluctuating environments. Root hair length is determined by the action of various plant hormones, among which the roles of auxin and ethylene have been extensively studied. However, evidence for the involvement of cytokinins has remained elusive. We recently reported that the cytokinin-activated B-type response regulators, ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1) and ARR12 directly upregulate the expression of ROOT HAIR DEFECTIVE 6-LIKE 4 (RSL4), which encodes a key transcription factor that controls root hair elongation. However, depending on the nutrient availability, it is unknown whether the ARR1/12-RSL4 pathway controls root hair elongation. This study shows that phosphate deficiency induced the expression of RSL4 and increased the root hair length through ARR1/12, though the transcript and protein levels of ARR1/12 did not change. These results indicate that cytokinins, together with other hormones, regulate root hair growth under phosphate starvation conditions.

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Root hairs are crucial in the uptake of essential nutrients and water in plants. This study showed that a zinc finger protein, GIS3 is involved in root hair growth in Arabidopsis. The loss-of-function gis3 and GIS3 RNAi transgenic line exhibited a significant reduction in root hairs compared to the wild type. The application of 1-aminocyclopropane-1-carboxylic acid (ACC), an exogenous ethylene precursor, and 6-benzyl amino purine (BA), a synthetic cytokinin, significantly restored the percentage of hair cells in the epidermis in gis3 and induced GIS3 expression in the wild type. More importantly, molecular and genetic studies revealed that GIS3 acts upstream of ROOT HAIR DEFECTIVE 2 (RHD2) and RHD4 by binding to their promoters. Furthermore, exogenous ACC and BA application significantly induced the expression of RHD2 and RHD4, while root hair phenotype of rhd2-1, rhd4-1, and rhd4-3 was insensitive to ACC and BA treatment. We can therefore conclude that GIS3 modulates root hair development by directly regulating RHD2 and RHD4 expression through ethylene and cytokinin signals in Arabidopsis.

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