Cell-type-specific transcriptomics reveals that root hairs and endodermal barriers play important roles in beneficial plant-rhizobacterium interactions

Growth-and health-promoting bacteria can boost crop productivity in a sustainable way.Pseudomonas simiae WCS417is sucha bacterium that efficiently colonizes roots,modifiesthe architecture of the root systemto increase its size,and induces systemic resistance to make plants more resistant to pests and pathogens.Our previous work suggested that WCS417-induced phenotypes are controlled by root cell-type-specific mechanisms.However,it remains unclear how WCS417 affects these mechanisms.In this study,we transcriptionally profiled five Arabidopsis thaliana root cell types following WCS417 colonization.We found that the cortex and endodermis have the most differentially expressed genes,even though they are not in direct contact with this epiphytic bacterium.Many of these genes are associated with reduced cell wall biogenesis,and mutant analysis suggests that this downregulation facilitates WCS417-driven root architectural changes.Furthermore,we observed elevated expression of suberin biosynthesis genes and increased deposition of suberin in the endodermis of WCS417-colonized roots.Using an endodermal barrier mutant,we showed the importance of endodermal barrier integrity for optimal plant-beneficial bacterium association.Comparison of the transcriptome profiles in the two epidermal cell types that are in direct contact with WcS417-trichoblasts that form root hairs and atrichoblasts that do not-implies a difference in potential for defense gene activation.While both cell types respond to WCS417,trichoblasts displayed both higher basal and WCS417-dependent activation of defense-related genes compared with atrichoblasts.This suggests that root hairs may activate root immunity,a hypothesis that is supported by differential immune responses in root hair mutants.Taken together,these results highlight the strength of cell-type-specific transcriptional profiling to uncover"masked"biological mechanisms underlying beneficial plant-microbe associations.

Plasticity of wheat seedling responses to K^(+) deficiency highlighted by integrated phenotyping of roots and root hairs over the whole root system

The availability in the soil of potassium(K^(+)),a poorly mobile macronutrient required in large quantities for plant growth,is generally suboptimal for crop production in the absence of fertilization,making improvement of the ability of crops to adapt to K^(+)deficiency stress a major issue.Increasing the uptake capacity of the root system is among the main strategies to achieve this goal.Here,we report an integrative approach to examine the effect of K^(+)deficiency on the development of young plant entire root system,including root hairs which are known to provide a significant contribution to the uptake of poorly mobile nutrients such as K^(+),in two genetically distant wheat varieties.A rhizobox-type methodology was developed to obtain highly-resolved images of root and root hairs,allowing to describe global root and root hair traits over the whole root system via image analysis procedures.The two wheat varieties responded differently to the K^(+)shortage:Escandia,a wheat ancestor,reduced shoot biomass in condition of K^(+)shortage and substantially increased the surface area of its root system,specifically by increasing the total root hair area.Oued Zenati,a landrace,conversely appeared unresponsive to the K^(+)shortage but was shown to constitutively express,independently of the external K^(+)availability,favorable traits to cope with reduced K^(+)availability,among which a high total root hair area.Thus,valuable information on root system adaptation to K^(+)deficiency was provided by global analyses including root hairs,which should also be relevant for other nutrient stresses.

Three CNGC Family Members, CNGC5, CNGC6, and CNGC9, Are Required for Constitutive Growth of Arabidopsis Root Hairs as Ca2+-Permeable Channels

The genetic identities of Ca2+ channels in root hair (RH) tips essential for constitutive RH growth have remained elusive for decades. Here, we report the identification and characterization of three cyclicnucleotide-gated channel (CNGC) family members, CNGC5, CNGC6, and CNGC9, as Ca2+ channels essential for constitutive RH growth in Arabidopsis. We found that the cngc5-1cngc6-2cngc9-1 triple mutant(designated shrh1) showed significantly shorter and branching RH phenotypes as compared with thewild type. The defective RH growth phenotype of shrh1 could be rescued by either the expression ofCNGC5, CNGC6, or CNGC9 single gene or by the supply of high external Ca2+, but could not be rescuedby external K+ supply. Cytosolic Ca2+ imaging and patch-clamp data in HEK293T cells showed that thesethree CNGCs all function as Ca2+-permeable channels. Cytosolic Ca2+ imaging in growing RHs furthershowed that the Ca2+ gradients and their oscillation in RH tips were dramatically attenuated in shrh1compared with those in the wild type. Phenotypic analysis revealed that these three CNGCs are Ca2+ channels essential for constitutive RH growth, with different roles in RHs from the conditional player CNGC14.Moreover, we found that these three CNGCs are involved in auxin signaling in RHs. Taken together, ourstudy identified CNGC5, CNGC6, and CNGC9 as three key Ca2+ channels essential for constitutive RHgrowth and auxin signaling in Arabidopsis.

Effect of CO_2 Elevation on Root Growth and Its Relationship with Indole Acetic Acid and Ethylene in Tomato Seedlings

A hydroponic experiment was carried out to study the effect of elevated carbon dioxide(CO2) on root growth of tomato seedlings.Compared with the control(350 μL L-1),CO2 enrichment(800 μL L-1) significantly increased the dry matter of both shoot and root,the ratio of root to shoot,total root length,root surface area,root diameter,root volume,and root tip numbers,which are important for forming a strong root system.The elevated CO2 treatment also significantly improved root hair development and elongation,thus enhancing nutrient uptake.Increased indole acetic acid concentration in plant tissues and ethylene release in the elevated CO2 treatment might have resulted in root growth enhancement and root hair development and elongation.

Induction of Root Hair Growth in a Phosphorus-Buffered Culture Solution

A system to control the release of phosphate in water was successfully established, based on solubility product of [Ca^2＋] and [PO4^3-] using tricalcium phosphate as P source in the hydroponic solution, and adding CaCl2 for supplementing extra Ca^2＋. The system, similar to soil solutions, was a P nutrient buffer solution with very low bioavailable P. The buffer solution induced the roots of both monocotyledon and dicotyledon species to grow abundant root hairs, 3 mm in maximum length. The monocotyledons were corn （Zea mays L.） （var. Yellow Rose）, wheat （Triticum aestivum L.） （var. Yanzhong 144）, Triticale secale L. （vat. Jingsong 5）, and ryegrass （Lolium rigidum L.） （var. Ruanni）, and the dicotyledons were Arabidopsis thaliana L. （var. Columbia）, white clover （Trifolium repens） （var. Kopu）, Lotus （Lotus peduncucatus Cav. Luliginosus Schkuhr） （var. Grasslands Maku）. For these species we proved that the root environment controls the induction of root hair formation. However, the hydroponic buffer solution failed to induce root hairs on the roots of onion （Allium cepa L.）. Other investigators have concluded that corn does not form root hairs in hydroponics, but abundant long root hairs on corn were induced by this buffer system. The roots with abundant long root hairs are called ＂hedgehog roots＂ because they have hairs everywhere just like a hedgehog.

Actin-depolymerizing factors 8 and 11 promote root hair elongation at high pH

A root hair is a polarly elongated single-celled structure that derives from a root epidermal cell and func-tions in uptake of water and nutrients from the surrounding environment.Previous reports have demon-strated that short periods of high pH inhibit root hair extension;but the effects of long-term high-pH treat-ment on root hair growth are still unclear.Here,we report that the duration of root hair elongation is signicantly prolonged with increasing external pH,which counteracts the effect of decreasing root hair elongation rate and ultimately produces longer root hairs,whereas loss of actin-depolymerizing factor 8 and 11(ADF8/11)function causes shortening of root hair length at high pH(pH 7.4).Accumulation of ADF8/11 at the tips of root hairs is inhibited by high pH,and increasing environmental pH affects the actinlament(F-actin)meshwork at the root hair tip.At high pH,the tip-focused F-actin meshwork is absent in root hairs of the adf8/11 mutant,actinlaments are disordered at the adf8/11 root hair tips,and actin turn-over is attenuated.Secretory and recycling vesicles do not aggregate in the apical region of adf8/11 root hairs at high pH.Together,our results suggest that,under long-term exposure to high extracellular pH,ADF8/11 may establish and maintain the tip-focused F-actin meshwork to regulate polar trafcking of secretory/recycling vesicles at the root hair tips,thereby promoting root hair elongation.

Phosphorus Uptake from Rhizosphere Soil by Two Wheat Cultivars

Inorganic soil phosphorus extractable with sodium bicarbonate (NaHCO3-Pi), soil pH and root hairs length and density in the rhizosphere of two winter wheat cultivars (Triticum aestivum L. cv. Shichun,Sleipner) grown on a high pH Chinese silt loam (52.7 mg NaHCO3-Pi kg-1) and a Danish sandy loam (43.4mg NaHCO3-Pi kg-1) were studied to assess how these wheat cultivars differed in phoephorus uptake.The rhizosphere soil pH of two wheat cultivars grown on the two soils were fairly unchanged with increasing distance from the root surface. However the root hairs of Shichun were 2.1 times longer than those of Sleipner. Root surface area (RSA) of Shichun increased by 192% due to root hairs whereas root hairs of Sleipner increased RSA by 68% only. Hence the root system of Shichun was in contact with more soil than that of Sleipner, even though Sleipner had a longer root. Grown at the lower pH and level of NaHCO3-Pi in the Danish soil Shichun absorbed more inorganic phosphorus than Sleipner whereas at the higher pH and level of NaHCO3-Pi in the Chinese soil there was no phosphorus uptake difference between the two wheat cultivars.

FERONIA-mediated TIR1/AFB2 oxidation stimulates auxin signaling in Arabidopsis

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.

The lncRNA APOLO interacts with the transcription factor WRKY42 to trigger root hair cell expansion in response to cold

Plant long noncoding RNAs(lncRNAs)have emerged as important regulators of chromatin dynamics,impacting on transcriptional programs leading to different developmental outputs.The lncRNA AUXIN-REGULATED PROMOTER LOOP(APOLO)directly recognizes multiple independent loci across the Arabidopsis genome and modulates their three-dimensional chromatin conformation,leading to transcriptional shifts.Here,we show that APOLO recognizes the locus encoding the root hair(RH)master regulator ROOT HAIR DEFECTIVE 6(RHD6)and controls RHD6 transcriptional activity,leading to cold-enhanced RH elongation through the consequent activation of the transcription factor gene RHD6-like RSL4.Furthermore,we demonstrate that APOLO interacts with the transcription factor WRKY42 and modulates its binding to the RHD6 promoter.WRKY42 is required for the activation of RHD6 by low temperatures and WRKY42 deregulation impairs cold-induced RH expansion.Collectively,our results indicate that a novel ribonucleoprotein complex with APOLO and WRKY42 forms a regulatory hub to activate RHD6 by shaping its epigenetic environment and integrate signals governing RH growth and development.

The RALF1-FERONIA Complex Phosphorylates eIF4E1 to Promote Protein Synthesis and Polar Root Hair Growth

The molecular links between extracellular signals and the regulation of localized protein synthesis in plant cells are poorly understood.Here,we show that in Arabidopsis thaliana,the extracellular peptide RALF1 and its receptor,the FERONIA receptor kinase,promote root hair(RH)tip growth by modulating protein synthesis.We found that RALF1 promotes FERONIA-mediated phosphorylation of elF4E1,a eukaryotic translation initiation factor that plays a crucial role in the control of mRNA translation rate.Phosphorylated elF4E1 increases mRNA affinity and modulates mRNA translation and,thus,protein synthesis.The mRNAs targeted by the RALF1-FERONIA-elF4E1 module include ROP2 and RSL4,which are important regulators of RH cell polarity and growth.RALF1 and FERONIA are expressed in a polar manner in RHs,which facilitate elF4E1 polar丨ocalization and thus may control local f?OP2 translation.Moreover,we demonstrated that high-level accumulation of RSL4 exerts negative-feedback regulation of RALF1 expression by directly binding the RALF1 gene promoter,determining the final RH size.Our study reveals that the link between RALF1-FERONIA signaling and protein synthesis constitutes a novel component regulating cell expansion in these polar growing cells.

Ubiquitin-Specific Protease 14 （UBP14） Is Involved in Root Responses to Phosphate Deficiency in Arabidopsis

A mutant isolated from a screen of EMS-mutagenized Arabidopsis lines, per1, showed normal root hair development under control conditions but displayed an inhibited root hair elongation phenotype upon Pi deficiency. Additionally, the per1 mutant exhibited a pleiotropic phenotype under control conditions, resembling Pi-deficient plants in several aspects. Inhibition of root hair elongation upon growth on low Pi media was reverted by treatment with the Pi analog phosphite, suggesting that the mutant phenotype is not caused by a lack of Pi. Reciprocal grafting experiments revealed that the mutant rootstock is sufficient to cause the phenotype. Complementation analyses showed that the PER1 gene encodes an ubiquitin-specific protease, UBP14. The mutation caused a synonymous substitution in the 12th exon of this gene, resulting in a lower abundance of the UBP14 protein, probably as a consequence of reduced translation efficiency. Transcriptional profiling of per1 and wild-type plants subjected to short-term Pi starvation revealed genes that may be important for the signaling of Pi deficiency. We conclude that UBP14 function is crucial for adapting root development to the prevailing local availability of phosphate.

Complex Regulation of Prolyl-4-Hydroxylases Impacts Root Hair Expansion

Root hairs are single cells that develop by tip growth, a process shared with pollen tubes, axons, and fungal hyphae. However, structural plant cell walls impose constraints to accomplish tip growth. In addition to polysaccharides, plant cell walls are composed of hydroxyproline-rich glycoproteins （HRGPs）, which include several groups of O-glycoproteins, including extensins （EXTs）. Proline hydroxylation, an early post-translational modification （PTM） of HRGPs catalyzed by prolyl 4-hydroxylases （P4Hs）, defines their subsequent O-glycosylation sites. In this work, our genetic analyses prove that P4H5, and to a lesser extent P4H2 and P4H13, are pivotal for root hair tip growth. Second, we demonstrate that P4H5 has in vitro preferred specificity for EXT substrates rather than for other HRGPs. Third, by P4H promoter and protein swapping approaches, we show that P4H2 and P4H13 have interchangeable functions but cannot replace P4H5. These three P4Hs are shown to be targeted to the secretory pathway, where P4H5 forms dimers with P4H2 and P4H13. Finally, we explore the impact of deficient proline hydroxylation on the cell wall architec- ture. Taken together, our results support a model in which correct peptidyl-proline hydroxylation on EXTs, and possibly in other HRGPs, is required for proper cell wall self-assembly and hence root hair elongation in Arabidopsis thaliana.

Brassinosteroids Regulate Root Growth, Development, and Symbiosis

Brassinosteroids （BRs） are natural plant hormones critical for growth and development. BR-deficient or signaling mutants show significantly shortened root phenotypes. But for a long time, it was thought that these phenotypes were solely caused by reduced root cell elongation in the mutants. Functions of BRs in regulating root development have been largely neglected. Recent detailed analyses, however, revealed that BRs are not only involved in root cell elongation but are also involved in many aspects of root development, such as maintenance of meristem size, root hair formation, lateral root initiation, gravitropic response, mycorrhiza formation, and nodulation in legume species. In this review, current findings on the functions of BRs in mediating root growth, development, and symbiosis are discussed.

MARIS plays important roles in Arabidopsis pollen tube and root hair growth

In flowering plants, male gametes are delivered to female gametes for double fertilization through pollen tubes. Therefore, pollen tube growth is crucial for double fertilization. Despite its importance to sexual reproduction, genetic mechanisms of pollen tube growth remain poorly understood. In this study, we characterized the receptor-like cytoplasmic protein kinase （RLCK） gene, MARLS （MRI） that plays critical roles in pollen tube growth. MRI is preferentially expressed in pollen grains, pollen tubes and roots. Mutation in MRI by a Ds insertion led to a burst of pollen tubes after pollen germination. Pollen-rescue assay by pollen and pollen tubespecific expression of MRI in the mri-4 mutant showed that loss of MRI function also severely affected root hair elongation. MRI protein interacted with the protein kinase OXIDATIVE SIGNAL INDUCIBLEI （OXII） in the in vitro and in vivo assays, which functions in plant defence and root hair development, and was phosphorylated by OXII in vitro. Our results suggest that MRI plays important roles in pollen tube growth and may function in root hair elongation through interaction with OXII.

Primary root and root hair development regulation by OsAUX4 and its participation in the phosphate starvation response

Among the five members of AUX1/LAX genes coding for auxin carriers in rice,only OsAUX1 and OsAUX3 have been reported.To understand the function of the other AUX1/LAX genes,two independent alleles of osaux4 mutants,osaux4-1 and osaux4-2,were constructed using the CRISPR/Cas9 editing system.Homozygous osaux4-1 or osaux4-2 exhibited shorter primary root(PR)and longer root hair(RH)compared to the wild-type Dongjin(WT/DJ),and lost response to indoleacetic acid(IAA)treatment.OsAUX4 is intensively expressed in roots and localized on the plasma membrane,suggesting that OsAUX4 might function in the regulation of root development.The decreased meristem cell division activity and the downregulated expression of cell cycle genes in root apices of osaux4 mutants supported the hypothesis that OsAUX4 positively regulates PR elongation.OsAUX4 is expressed in RH,and osaux4 mutants showing longer RH compared to WT/DJ implies that OsAUX4 negatively regulates RH development.Furthermore,osaux4 mutants are insensitive to Pi starvation(-Pi)and OsAUX4 effects on the-Pi response is associated with altered expression levels of Pi starvation-regulated genes,and auxin distribution/contents.This study revealed that OsAUX4 not only regulates PR and RH development but also plays a regulatory role in crosstalk between auxin and-Pi signaling.

Cloning of the Full-length cDNA of the Wheat Involved in Salt Stress：Root Hair Defective 3 Gene （RHD3）

: The full-length cDNA of the wheat (Triticum aestivum L.) root hair defective 3 gene (RHD3) has been cloned from the salt-tolerant hybrid wheat variety Shanrong No. 3 (Za3) using the mRNA differential display and 5’rapid amplification of cDNA ends (RACE) methods. Analysis of the amino acid sequence deduced from the wheat RHD3, gene shows that two conservative GTP-binding motifs, namely GXXXXGKS and DXXG, in eukaryotes also exist at the N-terminal of wheat RHD3. In addition, an 18 amino acid residue transmembrane domain, namely FYLAVMFVVFLVGKAIWV, exists at positions 701—718 of the C-terminal of the deduced protein of wheat RHD3 obtained, but this domain is absent in another three proteins aligned, including rice RHD3, Arabidopsis RHD3, and yeast homologue SEY1. Northern blot revealed that transcription of the wheat RHD3, gene is down-regulated in both the salt-tolerant line and in JN177 under saline stress. A possible stress-responsive mechanism for this gene is discussed.

Receptor-like protein kinases:Key regulators controlling root hair development in Arabidopsis thaliana

Root hairs are tubular outgrowths specifically differentiated from epidermal cells in a differentiation zone. The formation of root hairs greatly increases the surface area of a root and maximizes its ability to absorb water and inorganic nutrients essential for plant growth and development. Root hair development is strictly regulated by intracellular and intercellular signal communications. Cell surface-localized receptor-like protein kinases （P, LKs） have been shown to be important components in these cellular processes, tn this review, the functions of a number of key P, LKs in regulating Arabidopsis root hair development are discussed, especially those involved in root epidermal cell fate determination and root hair tip growth.

Arabidopsis SYP121 acts as an ROP2 effector in the regulation of root hair tip growth

Tip growth is an extreme form of polarized cell expansion that occurs in all eukaryotic kingdoms to generate highly elongated tubular cells with specialized functions, including fungal hyphae, animal neurons, plant pollen tubes, and root hairs (RHs). RHs are tubular structures that protrude from the root epidermis to facilitate water and nutrient uptake, microbial interactions, and plant anchorage. RH tip growth requires polarized vesicle targeting and active exocytosis at apical growth sites. However, how apical exocytosis is spatially and temporally controlled during tip growth remains elusive. Here, we report that the Qa-Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) SYP121 acts as an effector of Rho of Plants 2 (ROP2), mediating the regulation of RH tip growth. We show that active ROP2 promotes SYP121 targeting to the apical plasma membrane. Moreover, ROP2 directly interacts with SYP121 and promotes the interaction between SYP121 and the R-SNARE VAMP722 to form a SNARE complex, probably by facilitating the release of the Sec1/Munc18 protein SEC11, which suppresses the function of SYP121. Thus, the ROP2-SYP121 pathway facilitates exocytic trafficking during RH tip growth. Our study uncovers a direct link between an ROP GTPase and vesicular trafficking and a new mechanism for the control of apical exocytosis, whereby ROP GTPase signaling spatially regulates SNARE complex assembly and the polar distribution of a Q-SNARE.

Signaling network controlling ROP-mediated tip growth in Arabidopsis and beyond

Cell polarity operates across a broad range of spatial and temporal scales and is essential for specific biological functions of polarized cells.Tip growth is a special type of polarization in which a single and unique polarization site is established and maintained,as for the growth of root hairs and pollen tubes in plants.Extensive studies in past decades have demonstrated that the spatiotemporal localization and activity of Rho of Plants(ROPs),the only class of Rho GTPases in plants,are critical for tip growth.ROPs are switched on or off by different factors to initiate dynamic intracellular activities,leading to tip growth.Recent studies have also uncovered several feedback modules for ROP signaling.In this review,we summarize recent progress on ROP signaling in tip growth,focusing on molecular mechanisms that underlie the dynamic distribution and activity of ROPs in Arabidopsis.We also highlight feedback modules that control ROPmediated tip growth and provide a perspective for building a complex ROP signaling network.Finally,we provide an evolutionary perspective for ROP-mediated tip growth in Physcomitrella patens and during plant–rhizobia interaction.

Pathways for Epidermal Cell Differentiation via the Homeobox Gene GLABRA2:Update on the Roles of the Classic Regulator

Recent plant development studies have identified regulatory pathways for epidermal cell differentiation in Arabidopsis thafiana. Interestingly, some of such pathways contain transcriptional networks with a common structure in which the homeobox gene GLABLA2 （GL2） is downstream of the transactivation complex consisting of MYB, bHLH, and WD40 proteins. Here, we review the role of GL2 as an output device of the conserved network, and update the knowledge of epidermal cell differentiation pathways downstream of GL2. Despite the consistent position of GL2 within the network, its role in epidermal tissues varies; in the root epidermis, GL2 promotes non-hair cell differentiation after cell pattern formation, whereas inthe leaf epidermis, it is likely to be involved in both pattern formation and differentiation of trichomes. GL2 expression levels act as quantitative factors for initiation of cell differentiation in the root and leaf epidermis; the quantity of hairless cells in non-root hair cell files is reduced by g12 mutations in a semi-dominant manner, and entopically additive expression of GL2 and a heterozygous g12 mutation increase and decrease the number of trichomes, respectively. Although few direct target genes have been identified, evidence from genetic and expression analyses suggests that GL2 directly regulates genes with various hierarchies in epidermal cell differentiation pathways.