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Journal of Experimental Botany, Vol. 63, No. 13, 2, pp. 2012 pp.695–709, 4875–4885, 2012 doi:10.1093/jxb/err313 doi:10.1093/jxb/ers166 Advance AdvanceAccess Accesspublication publication 412November, July, 20122011 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) RESEARCH PAPER Nitric oxide isoceanica essentialcadmium for vesicle formation and trafficking In Posidonia induces changes in DNA in Arabidopsis root hair growth methylation and chromatin patterning 1 2, M. C. Lombardo and L. LamattinaLeonardo * Maria Greco, Adriana Chiappetta, Bruno and Maria Beatrice Bitonti* 1 Departamento de Biología e Instituto de Investigaciones Biológicas, Universidad Nacional de MarBucci, del Plata, Consejo Nacional de Department of Ecology, University of Calabria, Laboratory of Plant Cyto-physiology, Ponte Pietro I-87036 Arcavacata di Rende, Investigaciones Cosenza, Italy Científicas y Técnicas, CC 1245, 7600 Mar del Plata, Argentina 2 Instituto de Investigaciones Biológicas, Universidad Nacional de Mar del Plata, Consejo Nacional de Investigaciones Científicas y * To whom correspondence should be addressed. E-mail: [email protected] Técnicas, CC 1245, 7600 Mar del Plata, Argentina * To whom should be2011; addressed. E-mail: [email protected] Received 29correspondence May 2011; Revised 8 July Accepted 18 August 2011 Received 8 December 2011; Revised 13 March 2012; Accepted 8 May 2012 Abstract In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent Abstract epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with Theeffect functions of nitric oxide (NO) in processes associated withDNA rootmethylation hair growthlevel in Arabidopsis were analysed. its on chromatin reconfiguration in Posidonia oceanica. and pattern were analysedNO in is located at high concentrations in the root hair cell files at any stage of development. NO is detected inside of Cd, the actively growing organs, under short- (6 h) and long- (2 d or 4 d) term and low (10 mM) and high (50 mM) doses of vacuole ina immature actively growing root hairs and, later, NO is localized in the cytoplasm when they become mature. through Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach, Experiments performed by depleting NO in Arabidopsis root hairs indicate that NO is required for endocytosis, vesicle respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase, formation, and trafficking and it is not involved in nucleus migration, vacuolar development, and transvacuolar strands. was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron The Arabidopsis G’4,3 mutant (double mutant nia1/nia2) is severely impaired in NO production and generates microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating smaller that de root hairs than the wild type (WT). Root hairs from the Arabidopsis G’4,3 mutant show altered vesicular trafficking novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of and are reminiscent NO-depleted root hairs from the Arabidopsis WT. Interestingly, normal formation interphase nuclei andofapoptotic figures were also observed after long-term treatment. The datavesicle demonstrate thatand Cd traffickingthe as DNA well as root hair growth is restored exogenous of NOa application in the Arabidopsis G’4,3changes mutant.are All perturbs methylation status through theby involvement specific methyltransferase. Such together, results firmly support the essential played byaNO in the Arabidopsis root-hair-growing chromatin. process. linked to these nuclear chromatin reconfiguration likely role to establish new balance of expressed/repressed Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants. Key words: Actin, Arabidopsis G’4,3 mutant, endocytosis, nitric oxide, root hair, tip growing, vesiculation. Key words: 5-Methylcytosine-antibody, cadmium-stress condition, chromatin reconfiguration, CHROMOMETHYLASE, DNA-methylation, Methylation- Sensitive Amplification Polymorphism (MSAP), Posidonia oceanica (L.) Delile. Introduction Root hairs are specialized root epidermal cells of higher plants Introduction whose functions are water absorption and anchorage. The charIn the Mediterranean ecosystem, thewith endemic acteristic polarized growthcoastal of root hairs is shared a few seagrass Posidonia oceanica (L.) Delile plays a relevant role other cells such as pollen tubes, moss protonemata, and funby ensuring primary production, water oxygenation gal hyphae. All of these cells show polar cell expansion and and provides some(Reiss animals, besides1979; counteracting have beenniches widely for studied and Herth, Heath and coastal erosion its widespread meadows (Ott, 1980; Geitmann, 2000;through Hepler et al., 2001). Piazzi et al., 1999; Alcoverro et al., that 2001). There also Tip growth depends on several processes include Ca2+isinflux, considerable evidence that P. oceanica plants are able to MAPK cascades, microtubular and actin arrangements, vacuolar absorb and accumulate metals from sediments (Sanchiz development, nucleus migration, and vesicular trafficking, among et al., 1990; Pergent-Martini, 1998;et al., Maserti etChytilova al., 2005)et al., thus others (Galway et al., 1997; Baluška 2000; influencing metal bioavailability in the marine ecosystem. 2000; Hepler et al., 2001; Ketelaar et al., 2002; Takahashi et al., For reason, this seagrass is widely considered to be 2003;this Šamaj et al., 2004a, b, 2006; Jones and Smirnoff, 2006). a metal bioindicator species (Maserti et al., 1988;arePergent At the time of root hair initiation, the trichoblasts extenet al., 1995; Lafabrie et al., 2007). Cd is of most sively vacuolated, the nucleus migrates from one a position in widespread metals intoward both the terrestrial andemerging marine the middle ofheavy the trichoblast site of the environments. hair and, eventually, it enters the growing hair (Klahre and Chua, 1999). Although not have essential growth, in terrestrial Several studies shownfor thatplant polarized plant cells display a plants, Cd is readily absorbed by roots and translocated into 2+ tip-focused gradient of cytosolic Ca (Yokota et al., 2000; Chen aerial organs while, in acquatic plants, it is directly taken up 2+ et al., 2008). External Ca is required for normal root-hair elonby leaves. In plants, Cd absorption induces complex changes 2+ gation. The Ca influx may be maintained by the continual fusion at the2+-channel-containing genetic, biochemical and at physiological levelsthewhich of Ca vesicles the tip, providing cytoultimately account for its toxicity (Valle and Ulmer, plasmic region with a random fine F-actin organization. The1972; actin Sanitz di Toppi and Gabrielli, 1999;vesicular Benavides et al., 2005; cytoskeleton organization and polar trafficking are Weber et al., 2006; Liutip-growth et al., 2008). The most obvious critical components of the mechanism in pollen tubes symptom of Cd toxicity is aet al., reduction plant growth due to (Hepler et al., 2001; Šamaj 2004b)inand root hairs (Šamaj an inhibition of photosynthesis, respiration, and nitrogen et al., 2006). In the root hair, the growing tip can be reached metabolism, as wellstreaming as a reduction water and andorganelles mineral because cytoplasmic transportsinvesicles uptake (Ouzonidou et al., 1997; Perfus-Barbeoch et al., from the root hair base to the tip and then back. This type of2000; cytoShukla al., 2003;isSobkowiak and Deckert,streaming 2003). (Hepler plasmic et streaming called reverse-fountain At the genetic in both animals and plants, Cd et al., 2001) wherelevel, the main direction of organelle transport can induce chromosomal aberrations, abnormalities in © 2011 The Author [2012]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. ª The Author(s). For Permissions, please article e-mail:distributed [email protected] This is an Open Access under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 4876 | Lombardo and Lamattina reverses 180 degrees before the cell tip is reached (Sieberer and Emons, 2000). When cytoplasm is transported back by F-actin, it forms a cytoplasmic strand that runs through the vacuole and is called the transvacuolar strand (Tominaga et al., 2000). In growing root hairs, new plasma membrane (PM) and cell wall components are incorporated continuously into the growing tips by polarized exocytosis (Galway et al., 1997). The growing process is driven by the co-ordinated trafficking of secretory vesicles (Ovečka et al., 2005); thus, the tip presents a remarkable vesiclerich cytoplasm (Miller et al., 1999). Root hairs severely affected in growth lacks this tip-localized cytoplasm and has a vacuole that extends into the root-hair tip (Miller et al., 1999; Jones and Smirnoff, 2006). Although there is considerable exocytosis at the tip of root hairs, there must also be active endocytosis to retrieve the excess membrane that is secreted (Picton and Steer, 1983; Derksen et al., 1995). The growing root hair is filled with a large central vacuole that enlarges as the cell expands (Galway et al., 1997). The RHD3 (ROOT HAIR DEFECTIVE3) gene encodes a putative GTP-binding protein required for appropriate cell enlargement in Arabidopsis (Wang et al., 1997). The Arabidopsis thaliana rhd3 mutant shows the importance of the vacuole in the development of root hairs because it produces short and wavy root hairs with an average volume less than one-third of the wild-type hairs, indicating abnormal cell expansion (Galway et al., 1997). Root hair growth is accompanied by the migration of the nucleus into the shank (Jones and Smirnoff, 2006), at a certain distance of the tip (Miller et al., 1997). In mature root hairs, the movement of the nucleus is bidirectional (Chytilova et al., 2000; Ketelaar et al., 2002). Chytilova et al. (2000) demonstrated that rapid and, in some cases, long-distance intracellular movement of nuclei within actively growing cells occurs. This fact has been widely observed, and the advantage for a cell to keep the nucleus in the vicinity of sites with highly required protein production like growing PM and new wall deposition is obvious (Ketelaar et al., 2002). Despite these observations, multiple root hairs showed that the requirement of nuclei migration is not absolute (Jones and Smirnoff, 2006). The actin cytoskeleton is responsible for different processes during root hair tip growth (Miller et al., 1999; Baluška et al., 2000), and microtubules are required to maintain the direction of the tip growth but not the growing process per se (Bibikova et al., 1999). Pharmacological experiments provide evidence that the maintenance of the nucleus in the vicinity of the growing root hair tip is a process that involves the sub-apical fine F-actin cytoskeleton, but microtubules are not implicated (Ketelaar et al., 2002). In root hair cells, actin is involved in vesicle trafficking; it mediates endocytosis, directs the vesicles into endosomal compartments, and brings secretory vesicles back to the PM (Geldner et al., 2001; Baluška et al., 2002; Kasprowicz et al., 2009). Many reports show that the actin cytoskeleton and vesicular trafficking are strongly associated processes in root hairs (Voigt et al., 2005; Šamaj et al., 2006; Galleta and Cooper, 2009). Nitric oxide (NO) is a bioactive molecule that is involved in numerous plant physiological processes such as the regulation of defence-related gene expression and programmed cell death, seed germination, stomatal closure, and root development, among others (Lamattina et al., 2003; Delledonne, 2005; Corpas et al., 2007; Besson-Bard et al., 2008; Wawer et al., 2010). Several works have demonstrated the involvement of NO in root development. It was shown that NO mediates the auxin response and cell wall biosynthesis in lateral root formation (Correa-Aragunde et al., 2004, 2008). NO also induces adventitious root development (Pagnussat et al., 2004) and modulates dynamic actin cytoskeleton and vesicle trafficking in root apices (Kasprowicz et al., 2009). NO was also shown to be implicated in Arabidopsis root hair formation in both the initiation and elongation processes (Lombardo et al., 2006). NO was also reported to be responsible for the S-nitrosylation of dynamin in animal cells (Wang et al., 2006). Dynamin is a GTPase that regulates endocytic vesicle budding from the PM, a central process in tip growth. Furthermore, it has also been shown that NO modulates the organization of actin filaments in tip growing cells like pollen tubes (Wang et al., 2009). In this work, it has been demonstrated that NO is required for some dynamin- and actin-regulated processes in root hair growth such as vesicle formation and release and fine F-actin formation in the cytoplasmic region and the exocytic route. Materials and methods Plant material and treatments Arabidopsis thaliana ecotype Col-0 and G’4,3 (double mutant nia1 and nia2, chlorate resistant due to very low levels of nitrate reductase activity, about 0.5% of the level in Columbia; Wilkinson and Crawford, 1991) seeds were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus). Seeds were surface-sterilized by immersion in 70% (v/v) ethanol for 5 min and 20% (v/v) bleach for 20 min, followed by three rinses in sterile water. Seeds were washed with sterilizing solution 30% (v/v) bleach with 20% (v/v) Triton X-100. Seeds were sown in Petri dishes with ATS medium: 0.6% (w/v) agar, 1% (w/v) sucrose, and mineral nutrients according to Wilson et al. (1990), kept for 24 h at 4 °C and then incubated in a chamber at 25 °C with a photoperiod of 14 h for 5 d (Col-0) and 8 d (mutant). Then, Col-0 seedlings were transferred to fresh medium with 500 µM of the NO scavenger 2-(4-carboxydiphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, (cPTIO), 200 µM of the NO donor S-nitroso N-acetyl penicillamine (SNAP) or nothing for 3 d. To see the reversion of the G’4,3 mutant phenotype, 8-d-old seedlings were transferred to fresh medium supplemented with 200 µM SNAP for 2 d. Roots were stained with Toluidine Blue O (TBO) and observed by light microscopy (LM). Pictures were taken with an Olympus SP-350 camera attached to the microscope. NO detection For the detection of NO, Arabidopsis seedlings were incubated in 10 µM of the cell-permeable fluorescent probe 3-amino, 4-aminomethyl-2,7-difluorofluorescein diacetate (DAF-FM DA, excitation at 490 nm, emission at 525 nm; Calbiochem, San Diego, CA, USA) in 20 mM HEPES-NaOH (pH 7.5) for 1 h. Thereafter, roots were washed three times with fresh buffer and examined with a Nikon Eclipse 200 (Tokyo, Japan) Fluorescence Microscope (FM) and a Nikon C1Confocal Laser Scanning Microscope (CLSM). Pictures were captured with a Nikon 900 camera. Fluorescence was quantified with ImageJ program and expressed in arbitrary units (AU). Vacuolar development and nucleus migration CPTIO-treated and -untreated seedlings were processed in accordance with LM or Transmission Electron Microscopy (TEM) protocols. Acetic Orcein (4%, w/v) treatment for 4 min was used to observe the nucleus Nitric oxide is essential for Arabidopsis root hair growth | 4877 position in LM and photographs were recorded with an Olympus SP/350 digital camera attached to the microscope. For the vacuolar position, samples were analysed by the Electron Microscopy Laboratory, Scientific Technologic Center (CCT-BB, Bahía Blanca, Argentina). Samples were from 0.5 cm above the root apices for each treatment. Explants were fixed in 2.5% (v/v) glutaraldehyde and processed according to Reynolds (1963): 80 kv, uranyl acetate for 2 min and lead for 40 s; longitudinal sections of 800 Å were mounted in a copper grid with a formvar film for observation. A JEOL 100 CXII (Tokio, Japan, 1983) TEM was used to observe and record the samples. A grid of small squares of 0.02 µm2 over pictures of 4.08 µm2 was used to assess the area covered with ER. Vesicle formation and vesicular trafficking FM4 64 endocytic tracer (excitation at 488 nm, emission at 760 nm) was used at a final concentration of 50 µM for studying vesicular trafficking. Seedlings were stained for either 5 s or 5 min according to the experiment. Root hair movies and pictures of samples were shot and recorded with a Nikon C1 CLSM. Fluorescence intensity was quantified with EZ-C1 3.9 Free Viewer software and expressed in AU. Unit areas of 0.25 µm2 were used to analyse changes in the fluorescent intensity. Cytoplasmic area and transvacuolar strands Root hair movies and pictures from Differential Interference Contrast (DIC) Microscopy were obtained with a Nikon C1 CLSM. The size of the cytoplasmic area was measured with EZ-C1 3.9 Free Viewer software. Transvacuolar strands were monitored through movies. Results The NO levels are higher in trichoblast than in atrichoblast In a previous work, it has been demonstrated that NO is implicated in the events leading to root hair formation in Arabidopsis thaliana (Lombardo et al., 2006). Arabidopsis roots were loaded with the fluorescent probe DAF-FM DA and observed. The detection of fluorescence indicates that NO is not homogeneously distributed in Arabidopsis roots. NO is located at higher concentrations in trichoblasts (Fig. 1A). Figure 1B shows that root hair cell files have nearly 50% more NO-dependent fluorescence than atrichoblasts. The subcellular localization of NO in growing root hairs was analysed by Confocal Laser Scanning Microscope (CLSM) in 8-d-old seedlings loaded with DAF-FM DA. In growing, immature root hairs, NO is inside the vacuole whereas, in mature root hairs, NO is localized outside the vacuole (Fig. 2). In mature root hairs, green fluorescence appeared as a punctate pattern in the cytoplasm. NO depletion induces abnormalities in root hair growth Since previous data showed that treatments with the NO scavenger cPTIO prevented the normal elongation of root hairs in a dose-dependent manner (Lombardo et al., 2006), the phenotype of root hairs grown in the presence of 500 µM cPTIO was analysed. Figure 3A shows DIC images of the different root hair forms found in Arabidopsis roots depleted in NO. Most root hairs appear sinuous and, in less proportion, thickness and ramified, dichotomously branched, and/or double. Figure 3B shows the percentage of the main root hair phenotypes formed when NO is depleted. Microscopic analysis also showed that, in some trichoblasts, root hair bulges were present but they did not elongate (not shown). Fig. 1. NO concentration is higher in Arabidopsis trichoblasts. Roots from 8-d-old Arabidopsis Col-0 seedlings were loaded with 10 µM DAF-FM DA probe and NO localization was detected by fluorescence microscopy. (A) Representative picture of fluorescent trichoblasts. (B) Roots were treated or not with 200 µM SNAP to show the presence of the fluorescent probe in trichoblasts and atrichoblasts. The bright green fluorescence corresponding to NO in trichoblast files, trichomes, and atrichoblasts is quantified in the lower pannel and expressed as arbitrary units (AU). The pictures are representative of at least three independent experiments. Arrows indicate trichoblasts (A) and trichoblasts files (B). Bar: 100 µm. Vacuolar development and nucleus migration are not affected by NO depletion Vacuolar development and nucleus migration are events involved in tip-growing processes and were analysed in NO-depleted root hairs. To know if NO is implicated in vacuolar development, root hair ultrastructure was observed with Transmission 4878 | Lombardo and Lamattina Fig. 2. NO changes its subcellular location during the growth of root hairs. Arabidopsis Col-0 roots were loaded with 10 µM DAFFM DA probe to detect intracellular NO localization. The images of root hairs in different stages of maturity were taken by CLSM. V, vacuole; N, nucleus; DIC, Differential Interference Contrast; Fl, Fluorescence. Bars: 20 µm. The inset in the mature root hair is shown to distinguish V clearly. The pictures are representative of at least three independent experiments. Electron Microscopy (TEM) in cPTIO-treated root hairs. Figure 4A shows the presence of completely enlarged vacuoles in both cPTIO-treated and untreated root hairs. Untreated root hairs show the normal size of mitochondria and rough endoplasmic reticulum (ER) with parallel cisterns, whereas cPTIO-treatment results in root hair cells with proportionally more abundant ER and an irregular disposition of cisterns, some of them being concentric (Fig. 4B). CPTIO-treated and untreated Arabidopsis roots were stained with the nuclear dye Acetic Orcein to reveal the position of nuclei. Figure 5 shows that root hairs had nuclei in the trichome vacuolar zone in both control and cPTIO-treated Arabidopsis roots. No nuclei were found outside the vacuolar zone in 100% of both control and cPTIO-treated root hairs indicating that NO is not essential for nucleus migration. manner to the tonoplast. Movies of two minutes in length were recorded and analysed. Figure 6 shows the dynamics of vesicle formation with five pictures taken from each movie. Fluorescence intensity was quantified with EZ-C1 3.9 Free Viewer software and expressed as arbitrary units (AU). Control root hairs show the permanent formation of vesicles as numerous red dots along the fluorescent line (Fig. 6A) and marks of high intensity in the same points (Fig. 6C). By contrast, cPTIOtreated root hairs present no changes in the fluorescent base line corresponding to the PM (Fig. 6B, C). In some cases, a few permanent red dots are detected in cPTIO-treated root hairs (Fig. 6B), indicating that the process of vesicle endocytosis is strongly prevented (Fig. 6C). A slight decrease in basal fluorescence can be seen after 2 min that might be due to bleaching processes (Fig. 6A, B). Vesicle formation is thoroughly affected by NO depletion The Arabidopsis mutant G’4,3 is defective in nitrate reductase activity and develops abnormal root hairs growth To observe the dynamic of endocytosis, control and cPTIOtreated root hairs were stained with the endocytosis-marker FM4 64 (amphiphilic styryl dye). The dye infiltrates the outer lipid bilayer of the PM and then moves in a temperature-dependent The root growing process was studied in the nia1/nia2 Arabidopsis mutant G’4,3. The NO content was analysed in the Nitric oxide is essential for Arabidopsis root hair growth | 4879 Fig. 3. NO depletion induces abnormalities in root hair morphology. Arabidopsis Col-0 seedlings were treated with 500 µM of the specific NO scavenger cPTIO. CLSM DIC images (A) show different forms of root hairs found in cPTIO-treated roots: sinuous (a), thickness and ramified (b), dichotomously branched (c), and double hair (d). Bar: 20 µm. (B) Percentages of root hair phenotypes found in cPTIO-treated Arabidopsis roots. G’4,3 mutant and compared with Col-0. Eigth-day-old seedlings were treated with the NO-sensitive fluorophore DAF-FM DA. Figure 7A and B indicate that the Arabidopsis G’4,3 mutant has a lower NO concentration than trichoblasts and trichomes from Col-0, suggesting the involvement of nitrate reductase in NO formation in root hairs. To verify that there is no preferential uptake of DAF FM by the G’4,3 mutant and/or differential distribution of the dye in subcellular compartments; seedlings were grown in a medium supplemented with the NO donor SNAP to observe if NO-derived fluorescence is ubiquitously located along the root hair. The results indicate that, in roots hairs treated with 200 µM SNAP, DAF-FM DA fluorescence observed in the vacuole and cytoplasm of both the G’4,3 mutant and the Arabidopsis WT (see Supplementary Fig. S1 at JXB online), is indistinguishable. Roots were then stained with TBO and observed with LM (Fig. 7C). The G’4,3 root hair length (225.5 ± 69 µm) is, on average, 46.8% lower than Col-0 (480 ± 92 µm). When G’4,3 seedlings are treated with the NO donor SNAP, the length of the root hair is restored to the values observed in WT root hairs (432.6 ± 52 µm). NO participates in the dynamic of endocytic route Root hairs from G’4,3 loaded with the endocytosis-maker FM4 64 for 5 s show basal fluorescence without red dots in the PM indicating that vesicle formation is dramatically impaired (Fig. 8A). Interestingly, the SNAP treatment results in increased vesicle formation in the G’4,3 mutant, indicating that exogenous NO application can rescue the mutant phenotype. Arabidopsis Col-0 seedlings treated or untreated with 500 µµM cPTIO, and Arabidopsis mutant G’4,3 were stained with FM4 64 for 5 min (long labelling). Long labelling allows Fig. 4. NO depletion does not affect vacuolar development but does ER organization. Eigth-day-old Arabidopsis Col-0 seedlings were treated with 500 µM of the NO scavenger cPTIO (cPTIO) or were untreated (Control). Root hairs from the growing root hair region (immature) were observed with TEM (A). On the top (Control and cPTIO), the insets show a transverse section of Arabidopsis roots observed with light licroscopy. The photomicrographs show the organization of the subcellular structures; ec1, epidermal cell 1; ec2, epidermal cell 2; ER, endoplasmic reticulum; m, mitochondrion; N, nucleus; n, nucleolus; T, trichome; V, vacuole. Bars: 10 µm and 0.5 µm (details). (B) The proportion of ER in the cytoplasm of control and cPTIO-treated root hairs was calculated and expressed as a percentage. fluorescence to be followed along the whole endocytic route. Figure 8B shows that Arabidopsis Col-0 root hairs have uniformly stained cytoplasm. However, the overexposure to FM4 64 results in the agglomeration of vesicles in cPTIO-treated Col-0 and G’4,3 root hairs, reminiscent of Brefeldin A-formed vesicles (Fig. 8B) (Ovečka et al., 2005; Kasprowicz et al., 2009). The agglomerations are caused by alterations of regular vesicle trafficking. 4880 | Lombardo and Lamattina Fig. 5. NO depletion does not alter nucleus migration in root hairs. Eigth-day-old Arabidopsis Col-0 seedlings treated with 500 µM of cPTIO (cPTIO) or untreated (Control) were stained with the nuclear dye Acetic Orcein and examined by light microscopy to reveal the position of the nuclei (arrows). I, Immature; M, mature. Bars: 50 µm. Cytoplasmic region but not transvacuolar strands is affected by NO depletion The effect of NO in the cytoplasmic region was studied. Movies from the root hairs of cPTIO-treated and untreated Arabidopsis Col-0 and G’4,3 mutant (see Supplementary Fig. S2B, A, and C, respectively, at JXB online) were analysed and indicate that the transvacuolar strands are unaltered when NO is depleted. Figure 9A shows a reduced cytoplasmic region in both the cPTIO-treated Col-0 and the G’4,3 mutant compared with the WT. Figure 9B shows that this area was reduced by 67% in the cPTIO-treated Col-0 and G’4,3 mutant compared with Col-0. Discussion Like all tip-growing cells, root hairs carry out the typical growth processes characterized by cell wall softening, vacuolar enlargement, vesicle trafficking, nucleus migration in concert with cytoplasmic streaming in a reverse fountain. In this work, it is reported that, in Arabidopsis root hairs, some of those processes rely on an adequate NO concentration. Vacuolar genesis and nucleus migration seems to proceed independently of the NO concentration in root hairs. By contrast, an altered morphology was detected in NO-depleted root hairs. From bulge to the mature root hair, NO depletion results in the Fig. 6. NO is required for vesicle formation during root hair growth. Root hairs from 8-d-old Arabidopsis Col-0 seedlings treated with 500 µM cPTIO (cPTIO) or untreated (Control) were loaded with the endocytosis-marker FM4 64 for 5 s. Films were shot with CLSM. Five pictures from the 2-min movies show the dynamics of vesicle formation. (A) Control root hair. (B) cPTIOtreated root hair. (C) Vesicle formation was quantified using EZ-C1 3.90 Free Viewer software. Changes in fluorescence intensity were recorded in a 0.25 µm2 area of the plasma membrane and quantified as arbitrary units (AU). Arrows denote places in which vesicle formation are detected. Bar: 5 µm. vacuole occupying part of the tip region, a reduced cytoplasmic area, and very limited vesicle trafficking. Preliminary studies based on pharmacological approaches showed that microtubules are not involved in positioning the nucleus but sub-apical fine F-actin between the nucleus and the Nitric oxide is essential for Arabidopsis root hair growth | 4881 Fig. 7. The Arabidopsis G’4,3 mutant defective in nitrate reductase shows low NO concentration and altered root hair growth. The Arabidopsis Col-0 and G’4,3 mutant were loaded with DAF-FM DA (A, B). (A) Immature trichomes from Col-0 and G’4,3 observed with FM, the fluorescence intensity was quantified with ImageJ and expressed in arbitrary units (AU) (right side). (B) Fluorescence of mature trichomes was registered with CLSM and the intensity was quantified with ImageJ and expressed in AU (right side). (C) G’4,3 root seedlings were treated with the 200 µM of the NO donor SNAP to analyse the reversion of the mutant phenotype. Root seedlings were observed with light microscopy. The length of the trichome is represented on the right side (n = 150). Bars: A, B, 20 µm; C, 100 µm. Figure represents the results of at least three independent experiments. root hair apex is required for maintaining the nuclear position with respect to the growing apex (Ketelaar et al., 2002). Villin is required for nucleate actin polymerization and cross-linking filaments (Hampton et al., 2008). Villin-like actin-binding proteins are expressed ubiquitously in Arabidopsis (Klahre et al., 2000). It was shown that the injection of an antibody against plant villin leads to actin filament unbundling and movement of the nucleus closer to the apex. Thus, the bundled actin at the tip side of the nucleus prevents the nucleus from approaching the apex (Ketelaar et al., 2002). It is shown here that NO is not required to upset nucleus migration in Arabidopsis root hairs and, thereby, it is speculated that villin-regulated processes are not NO-dependent during root hair tip growth. A large centrally positioned vacuole filled the basal regions of the Arabidopsis WT growing root hairs, with smaller vacuoles or finger-like extensions of the larger vacuole between the subapical and basal regions. In Arabidopsis, the RHD3 (Root Hair Defective 3) gene encodes a putative GTP-binding protein required for appropriate cell enlargement. The development of the root hairs in the atrhd3 mutant is similar to the wild-type regarding the position and migration of nuclei, and as the wild type, rhd3 hairs emerge at the apical ends of the trichoblasts but 4882 | Lombardo and Lamattina Fig. 8. NO is necessary for the dynamics of the endocytic route and vesicle formation in root hairs. (A) Root hairs from 8-d-old Arabidopsis Col-0, and G’4,3 mutant seedlings grown in the presence or absence of 200 µM SNAP were loaded with the endocytosis marker FM4 64 for 5 s and observed with CLSM. The arrows denote places in which vesicle formation are detected. (B) The roots from 8-d-old Arabidopsis Col-0 seedlings treated with 500 µM cPTIO (Col-0+cPTIO) or untreated (Col-0), and Arabidopsis G’4,3 mutant (G’4,3) were loaded with the endocytosis-marker FM4 64 for 5 min. Overexposure of the root hair to the FM4 64 probe provoked fully fluorescent root hairs due to its internalization. Arrows indicate the agglomeration of vesicles. Bar: 10 µm. display a striking reduction in vacuole size and a corresponding increase in the relative proportion of cytoplasm throughout hair development. Furthermore, the hairs from the mutant rdh3 differ in vesicle distribution during hair growth (Galway et al., 1997). The phenotype of root hairs in the atrhd3 mutant is, in some way, reminiscent of the cPTIO-treated WT root hairs. However, it is shown here that NO seems not to be involved in vacuolar genesis and this suggests that the phenotype generated by NO depletion does not exactly fit with the rhd3 phenotype. In this work, it is demonstrated that vesicle formation and release, as well as the exocytic route are severely affected by NO depletion in Arabidopsis root hairs. Previous work has shown that different actin-binding proteins (ABPs) are involved in these processes, for example, the Arp2/3 complex, profilin, and endocytic proteins such as clathrin and dynamin (Galleta and Cooper, 2009). It has been also reported that Rab GTPases play a role in the trafficking of vesicles during tip growth (Preuss et al., 2004, 2006; de Graaf et al., 2005). The endocytic marker FM4 64 shows that vesicle formation is not dynamic in the PM of cPTIOtreated Arabidopsis root hairs, indicating that vesicle release is affected. In animal cells, NO regulates endocytosis through S-nitrosylation of dynamin (Wang et al., 2006). It would be interesting to study if the same type of NO-mediated regulation of root hair endocytosis occurs through dynamin S-nitrosylation. Brefeldin A (BFA) is an inhibitor of vesicular trafficking that prevents vesicle formation in the exocytosis pathway (Robineau et al., 2000). Ovečka et al. (2005) used BFA to observe the fate of endocytic vesicles. They showed active endocytosis at the tip of growing root hairs and further trafficking of the endocytosed membranes through different putative endosomal compartments either back to the PM or towards the tonoplast. Ovečka et al. (2005) concluded that BFA does not affect the endocytic route but does inhibit exocytosis. It is demonstrated here that over-exposure to the FM4 64 probe reveals FM-positive vesicle agglomeration in root hairs from seedlings grown in the absence of low concentrations of NO. The NO depletion provoked BFAlike bodies in Arabidopsis root hairs indicating that the exocytic route is probably affected. ABPs are responsible for new F-actin strand formation or branching, active processes in both endocytosis and exocytosis. It is suggested that specific ABPs involved in exocytosis, as those required in vesicle cover or transport, should be affected by NO depletion. Our results indicate that transvacuolar strands are not affected by NO depletion. Villin is a calcium-controlled actin-modulating protein (Hesterberg and Weber, 1983). At low calcium concentrations, villin is an F-actin nucleating and cross-linking protein (Hampton et al., 2008). Villin is responsible of F-actin binding which produces transvacuolar strands and permit root hairs to generate the characteristic reverse streaming (Tominaga et al., 2000). A high Ca2+ concentration in the root hair tip is sufficient to inhibit villin (Yokota et al., 2000; Hepler et al., 2001) and to induce actin cytoskeleton disorganization at the apex. It is suggested that NO depletion keeps the Ca2+ concentration and gradient compatible with the occurrence of active villin and preserves transvacuolar strands at a low level. In this work, it is shown that NO is present in trichoblasts and trichomes; it is localized inside the vacuole in immature root hairs and in the cytosol when the root hair become mature. Granstedt and Huffaker (1982) reported the vacuole as a major nitrate/nitrite storage pool. Nitrate and nitrite are NO precursors at lower pHs like those found in vacuoles (Stöhr and Ullrich, 2002). Thus, it can be speculated that vacuoles modify nitrate/ nitrite accumulation and/or pH regulation for controlling NO generation during the root-hair-growing process. In addition, nitrate and nitrite are also substrates for NR activity leading to NO generation (Yamasaki et al., 1999). Interestingly, Stöhr (2007) has proposed the relevance of a nitrite:NO reductase (NI-NOR) activity that is bound to root-specific plasma membrane, and that the main pathway of NO formation in roots is nitrite-dependent. NO could also be associated with the regulation of the oscillating growth of the root hair as described by Monshausen et al. (2007). Overall, NO production and its intracellular localization seem to Nitric oxide is essential for Arabidopsis root hair growth | 4883 Fig. 9. NO depletion affects cytoplasmic tip region of root hairs. Root hairs from the growing region of 8-d-old Arabidopsis Col-0 seedlings treated with 500 µM cPTIO (Col-0+cPTIO) or untreated (Col-0), and the Arabidopsis G’4,3 mutant (G’4,3) were observed with CLSM. (A) DIC images showing transvacuolar currents (arrows) and the cytoplasmic area (arrowheads). Bars: 10 µm. (B) Length of cytoplasmic area. be tightly linked to the root hair growing process and arrest when attaining its mature size. Supplementary data Supplementary data can be found at JXB online. Supplementary Fig. S1. The fluorescent probe DAF FM DA is homogenously distributed in root hairs treated with 200 µM of the NO donor SNAP. Supplementary Fig. S2. Transvacuolar strands are not affected by NO depletion. Acknowledgements We thank Dr Beatriz G Galati for her kind help with the observations of TEM photographs and Dr Laura de la Canal for providing us with the fluorescent probe FM4 64. 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