OsCSLD1, a cellulose synthase-like D1 gene, is required for root hair morphogenesis in rice

Root hairs are long tubular outgrowths that form on the surface of specialized epidermal cells. They are required for nutrient and water uptake and interact with the soil microflora. Here we show that the Oryza sativa cellulose synthase-like D1 (OsCSLD1) gene is required for root hair development, as rice (Oryza sativa) mutants that lack OsCSLD1 function develop abnormal root hairs. In these mutants, while hair development is initiated normally, the hairs elongate less than the wild-type hairs and they have kinks and swellings along their length. Because the csld1 mutants develop the same density and number of root hairs along their seminal root as the wild-type plants, we propose that OsCSLD1 function is required for hair elongation but not initiation. Both gene trap expression pattern and in situ hybridization analyses indicate that OsCSLD1 is expressed in only root hair cells. Furthermore, OsCSLD1 is the only member of the four rice CSLD genes that shows root-specific expression. Given that the Arabidopsis (Arabidopsis thaliana) gene KOJAK/AtCSLD3 is required for root hair elongation and is expressed in the root hair, it appears that OsCSLD1 may be the functional ortholog of KOJAK/AtCSLD3 and that these two genes represent the root hair-specific members of this family of proteins. Thus, at least part of the mechanism of root hair morphogenesis in Arabidopsis is conserved in rice.     

AtCSLD3, a cellulose synthase-like gene important for root hair growth in arabidopsis

A member of the cellulose synthase-like (subfamily D) gene family of Arabidopsis, AtCSLD3, has been identified by T-DNA tagging. The analysis of the corresponding mutant, csld3-1, showed that the AtCSLD3 gene plays a role in root hair growth in plants. Root hairs grow in phases: First a bulge is formed and then the root hair elongates by polarized growth, the so-called "tip growth." In the mutant, root hairs were initiated at the correct position and grew into a bulge, but their elongation was severely reduced. The tips of the csld3-1 root hairs easily leaked cytoplasm, indicating that the tensile strength of the cell wall had changed at the site of the tip. Based on the mutant phenotype and the functional conservation between CSLD3 and the genuine cellulose synthase proteins, we hypothesized that the CSLD3 protein is essential for the synthesis of polymers for the fast-growing primary cell wall at the root hair tip. The distinct mutant phenotype and the ubiquitous expression pattern indicate that the CSLD3 gene product is only limiting at the zone of the root hair tip, suggesting particular physical properties of the cell wall at this specific site of the root hair cell.     

Root hair-specific disruption of cellulose and xyloglucan in <Emphasis Type="Italic">AtCSLD3</Emphasis> mutants,and factors affecting the post-rupture resumption of mutant root hair growth

The glycosyl transferase encoded by the cellulose synthase-like gene CSLD3/KJK/RHD7 (At3g03050) is required for cell wall integrity during root hair formation in Arabidopsis thaliana but it remains unclear whether it contributes to the synthesis of cellulose or hemicellulose. We identified two new alleles, root hair-defective (rhd) 7-1 and rhd7-4, which affect the C-terminal end of the encoded protein. Like root hairs in the previously characterized kjk-2 putative null mutant, rhd7-1 and rhd7-4 hairs rupture before tip growth but, depending on the growth medium and temperature, hairs are able to survive rupture and initiate tip growth, indicating that these alleles retain some function. At 21°C, the rhd7 tip-growing root hairs continued to rupture but at 5oC, rupture was inhibited, resulting in long, wild type-like root hairs. At both temperatures, the expression of another root hair-specific CSLD gene, CSLD2, was increased in the rhd7-4 mutant but reduced in the kjk-2 mutant, suggesting that CSLD2 expression is CSLD3-dependent, and that CSLD2 could partially compensate for CSLD3 defects to prevent rupture at 5°C. Using a fluorescent brightener (FB 28) to detect cell wall (1 → 4)-β-glucans (primarily cellulose) and CCRC-M1 antibody to detect fucosylated xyloglucans revealed a patchy distribution of both in the mutant root hair cell walls. Cell wall thickness varied, and immunogold electron microscopy indicated that xyloglucan distribution was altered throughout the root hair cell walls. These cell wall defects indicate that CSLD3 is required for the normal organization of both cellulose and xyloglucan in root hair cell walls.     

Genetic Control of Root Hair Development in Arabidopsis thaliana

Visual examination of roots from 12,000 mutagenized Arabidopsis seedlings has led to the identification of more than 40 mutants impaired in root hair morphogenesis. Mutants from four phenotypic classes have been characterized in detail, and genetic tests show that these result from single nuclear recessive mutations in four different genes designated RHD1, RHD2, RHD3, and RHD4. The phenotypic analysis of the mutants and homozygous double mutants has led to a proposed model for root hair development and the stages at which the genes are normally required. The RHD1 gene product appears to be necessary for proper initiation of root hairs, whereas the RHD2, RHD3, and RHD4 gene products are required for normal hair elongation. These results demonstrate that root hair development in Arabidopsis is amenable to genetic dissection and should prove to be a useful model system to study the molecular mechanisms governing cell differentiation in plants.     

The efficiency of Arabidopsis thaliana (Brassicaceae) root hairs in phosphorus acquisition

Arabidopsis thaliana root hairs grow longer and denser in response to low-phosphorus availability. In addition, plants with the root hair response acquire more phosphorus than mutants that have root hairs that do not respond to phosphorus limiting conditions. The purpose of this experiment was to determine the efficiency of root hairs in phosphorus acquisition at high- and low-phosphorus availability. Root hair growth, root growth, root respiration, plant phosphorus uptake, and plant phosphorus content of 3-wk-old wild-type Arabidopsis (WS) were compared to two root hair mutants (rhd6 and rhd2) under high (54 mmol/m) and low (0.4 mmol/m) phosphorus availability. A cost-benefit analysis was constructed from the measurements to determine root hair efficiency. Under high-phosphorus availability, root hairs did not have an effect on any of the parameters measured. Under low-phosphorus availability, wild-type Arabidopsis had greater total root surface area, shoot biomass, phosphorus per root length, and specific phosphorus uptake. The cost-benefit analysis shows that under low phosphorus, wild-type roots acquire more phosphorus for every unit of carbon respired or unit of phosphorus invested into the roots than the mutants. We conclude that the response of root hairs to low-phosphorus availability is an efficient strategy for phosphorus acquisition.     

KINKY POLLEN encodes a SABRE-like protein required for tip growth in Arabidopsis and conserved among eukaryotes

In higher plants, pollen tubes and root hairs share an ancient growth process named tip growth. We have isolated three allelic Arabidopsis mutant lines showing kinky-shaped pollen tubes and, when homozygous, showing shorter and thicker root hairs. The ultrastructure of pollen tubes in these kinky pollen (kip) mutants is similar to that of the wild type; however, time-lapse studies suggest that aberrant pollen tube shape is caused by periodic growth arrests alternated with phases of tube axis reorientation. The KIP gene encodes a protein of 2587 amino acids that is predicted to be targeted to the secretory pathway. KIP mRNA was detected in all organs investigated but was most abundant in pollen and roots. KIP has putative homologues in many eukaryotes, including mammals and yeast, and is similar to the Arabidopsis SABRE gene, whose mutation causes a dwarf phenotype. The phenotype of the kip/sab double mutant suggests related functions for both genes, however, the KIP protein is mostly required for tip-growth.     

A class I ADP-ribosylation factor GTPase-activating protein is critical for maintaining directional root hair growth in Arabidopsis

Membrane trafficking and cytoskeletal dynamics are important cellular processes that drive tip growth in root hairs. These processes interact with a multitude of signaling pathways that allow for the efficient transfer of information to specify the direction in which tip growth occurs. Here, we show that AGD1, a class I ADP ribosylation factor GTPase-activating protein, is important for maintaining straight growth in Arabidopsis (Arabidopsis thaliana) root hairs, since mutations in the AGD1 gene resulted in wavy root hair growth. Live cell imaging of growing agd1 root hairs revealed bundles of endoplasmic microtubules and actin filaments extending into the extreme tip. The wavy phenotype and pattern of cytoskeletal distribution in root hairs of agd1 partially resembled that of mutants in an armadillo repeat-containing kinesin (ARK1). Root hairs of double agd1 ark1 mutants were more severely deformed compared with single mutants. Organelle trafficking as revealed by a fluorescent Golgi marker was slightly inhibited, and Golgi stacks frequently protruded into the extreme root hair apex of agd1 mutants. Transient expression of green fluorescent protein-AGD1 in tobacco (Nicotiana tabacum) epidermal cells labeled punctate bodies that partially colocalized with the endocytic marker FM4-64, while ARK1-yellow fluorescent protein associated with microtubules. Brefeldin A rescued the phenotype of agd1, indicating that the altered activity of an AGD1-dependent ADP ribosylation factor contributes to the defective growth, organelle trafficking, and cytoskeletal organization of agd1 root hairs. We propose that AGD1, a regulator of membrane trafficking, and ARK1, a microtubule motor, are components of converging signaling pathways that affect cytoskeletal organization to specify growth orientation in Arabidopsis root hairs.     

AKT1 and TRH1 are required during root hair elongation in Arabidopsis

TRH1 is a member of the AtKT/AtKUP/AtHAK family of potassium carriers that is required for root hair elongation and AKT1 is an inward rectifying potassium channel expressed in the root epidermis, endodermis and cortex of Arabidopsis thaliana. Plants homozygous for the trh1-1 mutation form short root hairs. The Trh1(-) phenotype cannot be suppressed by growing plants homozygous for the trh1-1 mutation in the presence of high external KCl concentration. This indicates an absolute requirement for TRH1 in root hair tip growth. Plants homozygous for the akt1-1 mutation develop longer root hairs than the wild type when grown in 0 mM external potassium, but develop shorter hairs than the wild type when grown in higher concentrations [>10 mM] of potassium. These data indicate that both TRH1 and AKT1 are active in the root hair over a wide range of external potassium concentrations, but suggest they have different functions in the growing hair cell.     

The COW1 locus of arabidopsis acts after RHD2, and in parallel with RHD3 and TIP1, to determine the shape, rate of elongation, and number of root hairs produced from each site of hair formation.

Two recessive mutant alleles at CAN OF WORMS1 (COW1), a new locus involved in root hair morphogenesis, have been identified in Arabidopsis thaliana L. Heynh. Root hairs on Cow1- mutants are short and wide and occasionally formed as pairs at a single site of hair formation. The COW1 locus maps to chromosome 4. Root hairs on Cow1- plants form in the usual positions, suggesting that the phenotype is not the result of abnormal positional signals. Root hairs on Cow1- roots begin hair formation normally, forming a small bulge, or root hair initiation site, of normal size and shape and in the usual position on the hair-forming cell. However, when Cow1- root hairs start to elongate by tip growth, abnormalities in the shape and elongation rate of the hairs become apparent. Genetic evidence from double-mutant analysis of cow1-1 and other loci involved in root hair development supports our conclusion that COW1 is required during root hair elongation.     

OsSNDP1, a Sec14-nodulin domain-containing protein, plays a critical role in root hair elongation in rice

Rice is cultivated in water-logged paddy lands. Thus, rice root hairs on the epidermal layers are exposed to a different redox status of nitrogen species, organic acids, and metal ions than root hairs growing in drained soil. To identify genes that play an important role in root hair growth, a forward genetics approach was used to screen for short-root-hair mutants. A short-root-hair mutant was identified and isolated by using map-based cloning and sequencing. The mutation arose from a single amino acid substitution of OsSNDP1 (Oryza sativa Sec14-nodulin domain protein), which shows high sequence homology with Arabidopsis COW1/AtSFH1 and encodes a phosphatidylinositol transfer protein (PITP). By performing complementation assays with Atsfh1 mutants, we demonstrated that OsSNDP1 is involved in growth of root hairs. Cryo-scanning electron microscopy was utilized to further characterize the effect of the Ossndp1 mutation on root hair morphology. Aberrant morphogenesis was detected in root hair elongation and maturation zones. Many root hairs were branched and showed irregular shapes due to bulged nodes. Many epidermal cells also produced dome-shaped root hairs, which indicated that root hair elongation ceased at an early stage. These studies showed that PITP-mediated phospholipid signaling and metabolism is critical for root hair elongation in rice.     

SOS4, a pyridoxal kinase gene,is required for root hair development in Arabidopsis

Root hair development in plants is controlled by many genetic, hormonal, and environmental factors. A number of genes have been shown to be important for root hair formation. Arabidopsis salt overly sensitive 4 mutants were originally identified by screening for NaCl-hypersensitive growth. The SOS4 (Salt Overly Sensitive 4) gene was recently isolated by map-based cloning and shown to encode a pyridoxal (PL) kinase involved in the production of PL-5-phosphate, which is an important cofactor for various enzymes and a ligand for certain ion transporters. The root growth of sos4 mutants is slower than that of the wild type. Microscopic observations revealed that sos4 mutants do not have root hairs in the maturation zone. The sos4 mutations block the initiation of most root hairs, and impair the tip growth of those that are initiated. The root hairless phenotype of sos4 mutants was complemented by the wild-type SOS4 gene. SOS4 promoter-beta-glucuronidase analysis showed that SOS4 is expressed in the root hair and other hair-like structures. Consistent with SOS4 function as a PL kinase, in vitro application of pyridoxine and pyridoxamine, but not PL, partially rescued the root hair defect in sos4 mutants. 1-Aminocyclopropane-1-carboxylic acid and 2,4-dichlorophenoxyacetic acid treatments promoted root hair formation in both wild-type and sos4 plants, indicating that genetically SOS4 functions upstream of ethylene and auxin in root hair development. The possible role of SOS4 in ethylene and auxin biosynthesis is discussed.     

The Arabidopsis Rop2 GTPase is a positive regulator of both root hair initiation and tip growth

Root hairs provide a model system for the study of cell polarity. We examined the possibility that one or more members of the distinct plant subfamily of RHO monomeric GTPases, termed Rop, may function as molecular switches regulating root hair growth. Specific Rops are known to control polar growth in pollen tubes. Overexpressing Rop2 (Rop2 OX) resulted in a strong root hair phenotype, whereas overexpressing Rop7 appeared to inhibit root hair tip growth. Overexpressing Rops from other phylogenetic subgroups of Rop did not give a root hair phenotype. We confirmed that Rop2 was expressed throughout hair development. Rop2 OX and constitutively active GTP-bound rop2 (CA-rop2) led to additional and misplaced hairs on the cell surface as well as longer hairs. Furthermore, CA-rop2 depolarized root hair tip growth, whereas Rop2 OX resulted in hairs with multiple tips. Dominant negative GDP-bound Rop2 reduced the number of hair-forming sites and led to shorter and wavy hairs. Green fluorescent protein-Rop2 localized to the future site of hair formation well before swelling formation and to the tip throughout hair development. We conclude that the Arabidopsis Rop2 GTPase acts as a positive regulatory switch in the earliest visible stage in hair development, swelling formation, and in tip growth.     

The Arabidopsis Phosphatidylinositol Phosphate 5-Kinase PIP5K3 is a key regulator of root hair tip growth

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] functions as a site-specific signal on membranes to promote cytoskeletal reorganization and membrane trafficking. Localization of PtdIns(4,5)P2 to apices of growing root hairs and pollen tubes suggests that it plays an important role in tip growth. However, its regulation and mode of action remain unclear. We found that Arabidopsis thaliana PIP5K3 (for Phosphatidylinositol Phosphate 5-Kinase 3) encodes a phosphatidylinositol 4-phosphate 5-kinase, a key enzyme producing PtdIns(4,5)P2, that is preferentially expressed in growing root hairs. T-DNA insertion mutations that substantially reduced the expression of PIP5K3 caused significantly shorter root hairs than in the wild type. By contrast, overexpression caused longer root hairs and multiple protruding sites on a single trichoblast. A yellow fluorescent protein (YFP) fusion of PIP5K3, driven by the PIP5K3 promoter, complemented the short-root-hair phenotype. PIP5K3-YFP localized to the plasma membrane and cytoplasmic space of elongating root hair apices, to growing root hair bulges, and, notably, to sites about to form root hair bulges. The signal was greatest in rapidly growing root hairs and quickly disappeared when elongation ceased. These results provide evidence that PIP5K3 is involved in localizing PtdIns(4,5)P2 to the elongating root hair apex and is a key regulator of the machinery that initiates and promotes root hair tip growth.     

Plant growth and phosphorus accumulation of wild type and two root hair mutants of Arabidopsis thaliana (Brassicaceae)

Arabidopsis thaliana root hairs grow longer and denser in response to low-phosphorus availability. We tested the hypothesis that wild-type Arabidopsis would acquire more phosphorus under phosphorus-limiting conditions than mutants that do not have the root hair response. The growth and phosphorus acquisition of wild-type Arabidopsis (WS) were compared to two root hair mutants (rhd6 and rhd2) under eight phosphorus treatments ranging from 0.4 mmol/m to 54 mmol/m phosphorus. At the lowest phosphorus treatment, all plants were small and showed severe phosphorus stress symptoms. At 1.5 mmol/m phosphorus, WS plants had greater shoot biomass, absolute growth rate, total phosphorus, and specific phosphorus absorption than the two root hair mutants. At the highest phosphorus treatment, there was no difference between genotypes in any of the parameters measured. We conclude that the response of increased root hair growth under low phosphorus availability in Arabidopsis is important in increasing phosphorus acquisition under phosphorus-limiting conditions.     

AGD1, a class 1 ARF-GAP, acts in common signaling pathways with phosphoinositide metabolism and the actin cytoskeleton in controlling Arabidopsis root hair polarity

The Arabidopsis thaliana AGD1 gene encodes a class 1 adenosine diphosphate ribosylation factor‐gtpase‐activating protein (ARF‐GAP). Previously, we found that agd1 mutants have root hairs that exhibit wavy growth and have two tips that originate from a single initiation point. To gain new insights into how AGD1 modulates root hair polarity we analyzed double mutants of agd1 and other loci involved in root hair development, and evaluated dynamics of various components of root hair tip growth in agd1 by live cell microscopy. Because AGD1 contains a phosphoinositide (PI) binding pleckstrin homology (PH) domain, we focused on genetic interactions between agd1 and root hair mutants altered in PI metabolism. Rhd4, which is knocked‐out in a gene encoding a phosphatidylinositol‐4‐phosphate (PI‐4P) phosphatase, was epistatic to agd1. In contrast, mutations to PIP5K3 and COW1, which encode a type B phosphatidylinositol‐4‐phosphate 5‐kinase 3 and a phosphatidylinositol transfer protein, respectively, enhanced the root hair defects of agd1. Enhanced root hair defects were also observed in double mutants to AGD1 and ACT2, a root hair‐expressed vegetative actin isoform. Consistent with our double‐mutant studies, targeting of tip growth components involved in PI signaling (PI‐4P), secretion (RABA4b) and actin regulation (ROP2), were altered in agd1 root hairs. Furthermore, tip cytosolic calcium ([Ca²⁺]_cyt) oscillations were disrupted in root hairs of agd1. Taken together, our results indicate that AGD1 links PI signaling to cytoskeletal‐, [Ca²⁺]_cyt?, ROP2‐, and RABA4b‐mediated root hair development.     

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.     

RPA, a class II ARFGAP protein, activates ARF1 and U5 and plays a role in root hair development in Arabidopsis

The polar growth of plant cells depends on the secretion of a large amount of membrane and cell wall materials at the growing tip to sustain rapid growth. Small GTP-binding proteins, such as Rho-related GTPases from plants and ADP-ribosylation factors (ARFs), have been shown to play important roles in polar growth via regulating intracellular membrane trafficking. To investigate the role of membrane trafficking in plant development, a Dissociation insertion line that disrupted a putative ARF GTPase-activating protein (ARFGAP) gene, AT2G35210, was identified in Arabidopsis (Arabidopsis thaliana). Phenotypic analysis showed that the mutant seedlings developed isotropically expanded, short, and branched root hairs. Pollen germination in vitro indicated that the pollen tube growth rate was slightly affected in the mutant. AT2G35210 is specifically expressed in roots, pollen grains, and pollen tubes; therefore, it is designated as ROOT AND POLLEN ARFGAP (RPA). RPA encodes a protein with an N-terminal ARFGAP domain. Subcellular localization experiments showed that RPA is localized at the Golgi complexes via its 79 C-terminal amino acids. We further showed that RPA possesses ARF GTPase-activating activity and specifically activates Arabidopsis ARF1 and ARF1-like protein U5 in vitro. Furthermore, RPA complemented Saccharomyces cerevisiae glo3Delta gcs1Delta double mutant, which suggested that RPA functions as an ARFGAP during vesicle transport between the Golgi and the endoplasmic reticulum. Together, we demonstrated that RPA plays a role in root hair and pollen tube growth, most likely through the regulation of Arabidopsis ARF1 and ARF1-like protein U5 activity.     

The Arabidopsis root hair mutants der2-der9 are affected at different stages of root hair development

Root hairs are an excellent model system to study cell developmental processes as they are easily accessible, single-celled, long tubular extensions of root epidermal cells. In a genetic approach to identify loci important for root hair development, we have isolated eight der (deformed root hairs) mutants from an ethylmethanesulfonate (EMS)-mutagenized Arabidopsis population. The der lines represent five new loci involved in root hair development and show a variety of abnormalities in root hair morphology, indicating that different root hair developmental stages are affected. A double mutant analysis with the short root hair actin2 mutant der1-2 confirmed that the der mutants are disturbed at different time points of root hair formation. Auxin and ethylene are known to be important for trichoblast cell fate determination and root hair elongation. Here, we show that they are able to suppress the phenotype of two der mutants. As the auxin- and ethylene-responsive der mutants are affected at different stages of root hair formation, our results demonstrate that the function of auxin and ethylene is not limited to cell differentiation and root hair elongation but that the two hormones are effective throughout the whole root hair developmental process.