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.     

Hormones act downstream of TTG and GL2 to promote root hair outgrowth during epidermis development in the Arabidopsis root.

The Arabidopsis root produces a position-dependent pattern of hair-bearing and hairless cell types during epidermis development. Five loci (TRANSPARENT TESTA GLABRA [TTG], GLABRA2 [GL2], ROOT HAIR DEFECTIVE6 [RHD6], CONSTITUTIVE TRIPLE RESPONSE1 [CTR1], and AUXIN RESISTANT2 [AXR2]) and the plant hormones ethylene and auxin have been reported to affect the production of root hair and hairless cells in the Arabidopsis root. In this study, genetic, molecular, and physiological tests were employed to define the roles of these loci and hormones. Epistasis tests and reporter gene studies indicated that the hairless cell-promoting genes TTG and GL2 are likely to act early to negatively regulate the ethylene and auxin pathways. Studies of the developmental timing of the hormone effects indicated that ethylene and auxin pathways promote root hair outgrowth after cell-type differentiation has been initiated. The genetic analysis of ethylene-and auxin-related mutations showed that root hair formation is influenced by a network of hormone pathways, including a partially redundant ethylene signaling pathway. A model is proposed in which the patterning of root epidermal cells in Arabidopsis is regulated by the cell position-dependent action of the TTG/GL2 pathway, and the ethylene and auxin hormone pathways act to promote root hair outgrowth at a relatively late stage of differentiation.     

Effects of secondary mutation in det2-1 on root growth and development in Arabidopsis

Brassinosteroids (BRs) are plant steroidal hormones that regulate a wide range of developmental processes. Most BR mutants display impaired growth and responses to developmental and environmental stimuli. Here, we found a BR-deficient mutant det2-1 that displayed exceedingly short roots and agravitropic growth, which were not present in other BR mutants. By back-crossing det2-1 with the wild type, we isolated a secondary mutation named det2-1 phenotype modifier 1 (dpm1) and demonstrated that those aberrant phenotypes in the original det2-1 were independent of the BR deficiency. Phenotypic analysis showed that impaired root growth of dpm1 appeared in BR-deficient condition, but not in a normal condition. In the light condition, the mutant showed enhanced shoot growth which was suppressed in the det2-1 background. Starch granules in the columella cells of the root tip were highly accumulated and expanded in dpm1. Agravitropic roots and the expanded starch granules of dpm1 could not be recovered by BR. Taken together, these results suggest that DPM1 is required for gravitropic growth, and that its functions on root and shoot growth are BR-dependent.     

Actin organization and root hair development are disrupted by ethanol-induced overexpression of Arabidopsis actin interacting protein 1 (AIP1)

* Actin organization and dynamics are essential for cell division, growth and cytoplasmic streaming. Here we analyse the effects of the overexpression of Actin Interacting Protein 1 (AIP1) on Arabidopsis development. * Arabidopsis plants were transformed with an ethanol-inducible AIP1 construct and the characteristics of these plants were analysed after induction. * When AIP1 was increased to approx. 90% above wild-type values, root hair development and actin organization in all cell types examined were disrupted. * Our data demonstrate that AIP1 is a key regulator of actin organization and that its regulation is essential for normal plant cell morphogenesis.     

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 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.     

Arabidopsis VILLIN4 is involved in root hair growth through regulating actin organization in a Ca2+-dependent manner

? Villin is one of the major actin filament bundling proteins in plants. The function of Arabidopsis VILLINs (AtVLNs) is still poorly understood in living cells. In this report, the biochemical activity and cellular function of AtVLN4 were examined. ? The biochemical property of AtVLN4 was characterized by co-sedimentation assays, fluorescence microscopy and spectroscopy of pyrene fluorescence. The in vivo function of AtVLN4 was analysed by ectopically expressing it in tobacco pollen and examining the phenotypes of its T-DNA insertional plants. ? Recombinant AtVLN4 protein exhibited multiple activities on actin, including actin filament bundling, calcium (Ca(2+))-dependent filament severing and barbed end capping. Expression of AtVLN4 in tobacco pollen induced the formation of supernumerary actin cables and reduced pollen tube growth. Loss of function of AtVLN4 resulted in slowing of root hair growth, alteration in cytoplasmic streaming routes and rate, and reduction of both axial and apical actin bundles. ? Our results demonstrated that AtVLN4 is involved in root hair growth through regulating actin organization in a Ca(2+)-dependent manner.     

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.     

Auxin, actin and growth of the Arabidopsis thaliana primary root

To understand how auxin regulates root growth, we quantified cell division and elemental elongation, and examined actin organization in the primary root of Arabidopsis thaliana. In treatments for 48 h that inhibited root elongation rate by 50%, we find that auxins and auxin-transport inhibitors can be divided into two classes based on their effects on cell division, elongation and actin organization. Indole acetic acid (IAA), 1-naphthalene acetic acid (NAA) and tri-iodobenzoic acid (TIBA) inhibit root growth primarily through reducing the length of the growth zone rather than the maximal rate of elemental elongation and they do not reduce cell production rate. These three compounds have little effect on the extent of filamentous actin, as imaged in living cells or by chemical fixation and immuno-cytochemistry, but tend to increase actin bundling. In contrast, 2,4-dichlorophenoxy-acetic acid (2,4-D) and naphthylphthalamic acid (NPA) inhibit root growth primarily by reducing cell production rate. These compounds remove actin and slow down cytoplasmic streaming, but do not lead to mislocalization of the auxin-efflux proteins, PIN1 or PIN2. The effects of 2,4-D and NPA were mimicked by the actin inhibitor, latrunculin B. The effects of these compounds on actin were also elicited by a 2 h treatment at higher concentration but were not seen in two mutants, eir1-1 and aux1-7, with deficient auxin transport. Our results show that IAA regulates the size of the root elongation zone whereas 2,4-D affects cell production and actin-dependent processes; and, further, that elemental elongation and localization of PINs are appreciably independent of actin.     

Arabidopsis VILLIN2 and VILLIN3 act redundantly in sclerenchyma development via bundling of actin filaments

The organization of the actin cytoskeleton has been implicated in sclerenchyma development. However, the molecular mechanisms linking the actin cytoskeleton to this process remain poorly understood. In particular, there have been no studies showing that direct genetic manipulation of the actin cytoskeleton affects sclerenchyma development. Villins belong to the villin/gelsolin/fragmin superfamily and are versatile actin-modifying proteins. Several recent studies have implicated villins in tip growth of single cells, but how villins act in multicellular plant development remains largely unknown. Here, we found that two closely related villin isovariants from Arabidopsis, VLN2 and VLN3, act redundantly in sclerenchyma development. Detailed analysis of cross-sections from inflorescence stems of vln2 vln3 double mutant plants revealed a reduction in stem size and in the number of vascular bundles; however, no defects in synthesis of the secondary cell wall were detected. Surprisingly, the vln2 vln3 double mutation did not affect cell elongation of inter-fascicular fibers. Biochemical analyses showed that recombinant VLN2 was able to cap, sever and bundle actin filaments, similar to VLN3. Consistent with these biochemical activities, loss of function of VLN2 and VLN3 resulted in a decrease in the amount of F-actin and actin bundles in plant cells. Collectively, our findings demonstrate that VLN2 and VLN3 act redundantly in sclerenchyma development via bundling of actin filaments.     

A mutation in MRH2 kinesin enhances the root hair tip growth defect caused by constitutively activated ROP2 small GTPase in Arabidopsis

Root hair tip growth provides a unique model system for the study of plant cell polarity. Transgenic plants expressing constitutively active (CA) forms of ROP (Rho-of-plants) GTPases have been shown to cause the disruption of root hair polarity likely as a result of the alteration of actin filaments (AF) and microtubules (MT) organization. Towards understanding the mechanism by which ROP controls the cytoskeletal organization during root hair tip growth, we have screened for CA-rop2 suppressors or enhancers using CA1-1, a transgenic line that expresses CA-rop2 and shows only mild disruption of tip growth. Here, we report the characterization of a CA-rop2 enhancer (cae1-1 CA1-1) that exhibits bulbous root hairs. The cae1-1 mutation on its own caused a waving and branching root hair phenotype. CAE1 encodes the root hair growth-related, ARM domain-containing kinesin-like protein MRH2 (and thus cae1-1 was renamed to mrh2-3). Cortical MT displayed fragmentation and random orientation in mrh2 root hairs. Consistently, the MT-stabilizing drug taxol could partially rescue the wavy root hair phenotype of mrh2-3, and the MT-depolymerizing drug Oryzalin slightly enhanced the root hair tip growth defect in CA1-1. Interestingly, the addition of the actin-depolymerizing drug Latrunculin B further enhanced the Oryzalin effect. This indicates that the cross-talk of MT and AF organization is important for the mrh2-3 CA1-1 phenotype. Although we did not observe an apparent effect of the MRH2 mutation in AF organization, we found that mrh2-3 root hair growth was more sensitive to Latrunculin B. Moreover, an ARM domain-containing MRH2 fragment could bind to the polymerized actin in vitro. Therefore, our genetic analyses, together with cell biological and pharmacological evidence, suggest that the plant-specific kinesin-related protein MRH2 is an important component that controls MT organization and is likely involved in the ROP2 GTPase-controlled coordination of AF and MT during polarized growth of root hairs.     

The role of EMF1 in regulating the vegetative and reproductive transition in Arabidopsis thaliana (Brassicaceae)

To understand the genetic regulation of vegetative to reproductive transition in higher plants, further characterization of the Arabidopsis mutant embryonic flower1, emf1, was conducted. Using three flowering symptoms, we showed that emf1 mutants could only grow reproductive and not rosette shoots under five different growth conditions. The mutant embryos did not produce the typical tunica–corpus shoot apical structures at the heart-, torpedo-, and mature stages. The divergent shoot apical development during mutant and wild-type embryogenesis indicated that the wild-type EMF1 gene was expressed in early embryogenesis. Mutations in the EMF1 gene affected the embryonic shoot apical development and caused the germinating embryo and regenerating callus to grow inflorescence, instead of rosette, shoots. Our results support the hypothesis that the EMF1 gene regulates the switch between vegetative and reproductive growth in Arabidopsis.     

KAI2 promotes Arabidopsis root hair elongation at low external phosphate by controlling local accumulation of AUX1 and PIN2

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Sustained root culture for generation and vegetative propagation of transgenic Arabidopsis thaliana

Summary Excised roots of wild-type and nitrate-reductase deficient mutant Arabidopsis thaliana (L.) Heynh. can be propagated as sustained root cultures in liquid medium. Culture initiation from a single seedling required a two-day indoleacetic acid treatment at 0.05 mg/l concentration. Indoleacetic acid facilitated subculture but was not essential for sustained growth. This procedure has allowed the clonal propagation of roots derived from individual wildtype and mutant seedlings for more than 21 months. The cultured roots retained their shoot regeneration ability; however, a controlled desiccation treatment was required to restore it to the level of freshly excised roots. The chromosome number remained diploid and no evidence for the accumulation of recessive mutations was observed. The cultured roots are competent for Agrobacterium-mediated transformation. The sustained root culture technology allowed the maintenance of transgenic tissues in which expression of a dominant, seed-lethal gene (seed-specific pea vicilin promoter fused to diphtheria toxin A chain gene) precluded generative propagation.On leave from State of South Carolina Governor's School for Science and Mathematics, Hartsville, SC 29550