Beta-Cyanoalanine synthase action in root hair elongation is exerted at early steps of the root hair elongation pathway and is independent of direct cyanide inactivation of NADPH oxidase

Running head: Cyanide action in root hair elongation Corresponding author: Dr. I. García. Instituto de Bioquímica Vegetal y Fotosíntesis. Consejo Superior de Investigaciones Científicas and Universidad de Sevilla. Avenida Américo Vespucio, 49, 41092 Sevilla, Spain. Tel: + 34 954 489 500. Fax: +34 954 460 165. Email: [email protected] Subject area: growth and development This manuscript contains 4 black and white figures, 3 color figures, 2 tables, 7 supplementary figures and 1 supplementary video. © The Author 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: [email protected] Downloaded from https://academic.oup.com/pcp/advance-article-abstract/doi/10.1093/pcp/pcy047/4911869 by Centro de Información y Documentación Científica, [email protected] on 01 March 2018 2 ß-Cyanoalanine synthase action in root hair elongation is exerted at early steps of the root hair elongation pathway and is independent on direct cyanide inactivation of NADPH oxidase Running head: Cyanide action in root hair elongation Lucía Arenas-Alfonseca, Cecilia Gotor, Luis C. Romero and Irene García* Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, Avenida Américo Vespucio, 49, 41092 Sevilla, Spain * Email: [email protected]. Fax: +34 954 460 165 Abbreviations ACC: 1-aminocyclopropane-1-carboxylic acid bHLH: basic helix-loop-helix CAS: ß-cyanoalanine synthase COB: 2-hydroxocobalamin CT: cycle threshold FW: fresh weight GFP: green fluorescent protein H 2 DCFDA: 2',7'-dichlorodihydrofluorescein diacetate NBT: nitro blue tetrazolium ORF: open reading frame ROS: reactive oxygen species TF: transcription factor XTT: 3′-[1-[(phenylamino)-carbony]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfonic acid Downloaded from https://academic.oup.com/pcp/advance-article-abstract/doi/10.1093/pcp/pcy047/4911869 by Centro de Información y Documentación Científica, [email protected] on 01 March 2018 3 Abstract In Arabidopsis thaliana, cyanide is produced concomitantly with ethylene biosynthesis and is mainly detoxified by the ß-cyanoalanine synthase CAS-C1. In roots, CAS-C1 activity is essential to maintain a low level of cyanide for proper root hair development. Root hair elongation relies on polarized cell expansion at the growing tip, and we have observed that CAS-C1 locates in mitochondria and accumulates in root hair tips during root hair elongation, as shown by observing the fluorescence in plants transformed with the translational construct ProC1:CASC1-GFP, containing the complete CAS-C1 gene fused to GFP. Mutants in the SUPERCENTIPEDE (SCN1) gene, that regulate the NADPH oxidase RHD2/AtrbohC, are affected at the very early steps of the development of root hair that do not elongate and do not show a preferential localization of the GFP accumulation in the tips of the root hair primordia. Root hairs of mutants in CAS-C1 or RHD2/AtrbohC, which catalyzes the generation of ROS and the Ca 2+ gradient, correctly start to grow out but they do not elongate either. Genetic crosses between the cas-c1 mutant and scn1 or rhd2 mutants were performed and the detail phenotypic and molecular characterization of the double mutants demonstrate that scn1 mutation is epistatic to cas-c1 and cas-c1 is epistatic to rhd2 mutation, indicating that CAS-C1 acts in early steps of the root hair development process. Moreover, our results show that the role of CAS-C1 in root hair elongation is independent of H 2 O 2 production and of a direct NADPH oxidase inhibition by cyanide. Keywords Arabidopsis thaliana, ß-Cyanoalanine Synthase, Root Hair, Cyanide, SCN1, RHD2. Downloaded from https://academic.oup.com/pcp/advance-article-abstract/doi/10.1093/pcp/pcy047/4911869 by Centro de Información y Documentación Científica, [email protected] on 01 March 2018 4 Introduction Cyanide is extensively present in all organisms, including bacteria, fungi, insects and plants. However, cyanide is a toxic compound due to its high reactivity with keto compounds and Schiff bases and because it chelates diand trivalent metal ions in the prosthetic groups of metalloproteins (Donato et al. 2007). Mitochondria is the major target of cyanide, where it binds to the heme iron of cytochrome c oxidase, blocking the respiratory chain (Cooper and Brown 2008). In non-cyanogenic plant species such as A. thaliana, the main source of cyanide is the biosynthesis of the hormone ethylene, which is involved in regulating numerous developmental processes and responses to stress conditions (Bleecker and Kende 2000), and the biosynthesis of the phytoalexin camalexin, which is formed when A. thaliana plants are infected by a large variety of microorganisms (Glawischnig 2007). Therefore, under certain developmental or environmental conditions, plants produce significant amounts of cyanide that may be harmful to their cells, requiring detoxification. To keep cyanide below toxic concentrations, plants possess different metabolic pathways, the main pathway of which is that involving ß-cyanoalanine synthase (CAS) ((Machingura et al. 2016) and references therein). Arabidopsis plants express the mitochondrial ß-cyanoalanine synthase CAS-C1 (formerly CYS-C1, (Romero et al. 2014) (Watanabe et al. 2008)), which, together with the O-acetylserine(thiol)lyases (OASTLs), belongs to the family of ß-substituted alanine synthase enzymes. CAS is a pyridoxal phosphate-dependent enzyme that uses cysteine to detoxify cyanide by converting cyanide and cysteine in hydrogen sulfide (H 2 S) and ß-cyanoalanine. H 2 S also blocks the mitochondrial respiratory pathway and therefore needs to be detoxified by the true OASTL OAS-C, which incorporates H 2 S to O-acetylserine (OAS) to produce cysteine, which is recycled by CAS-C1 to detoxify cyanide, thus completing the cyanide detoxification cycle in mitochondria (Alvarez et al. 2012). ßCyanoalanine is converted to Asn, Asp and ammonia by NIT4 class nitrilases, thereby recycling the nitrogen for plant utilization (Piotrowski 2008). Cyanide at non-toxic levels has been suggested to perform regulatory roles in different physiological processes. In animal systems, for example, it has been hypothesized to act as a neuromodulator (Cipollone and Visca 2007). In plants, exogenously applied cyanide can act as a regulator of seed dormancy and germination (Bethke et al. 2006; Chivasa and Carr 1998; Cohn and Hughes 1986; Seo et al. 2011; Siegien and Bogatek 2006; Wong et al. 2002) and play a role in resistance to viral and fungal pathogens (Bethke et al. 2006; Chivasa and Carr 1998; Cohn and Hughes 1986; Seo et al. 2011; Siegien and Bogatek 2006; Wong et al. 2002). Our previous investigation on the CAS-C1 null mutant has provided insight into the role of the endogenously produced Downloaded from https://academic.oup.com/pcp/advance-article-abstract/doi/10.1093/pcp/pcy047/4911869 by Centro de Información y Documentación Científica, [email protected] on 01 March 2018 5 cyanide in Arabidopsis. Loss-of-function mutation of the CAS-C1 gene leads to a non-toxic increase in cyanide (Garcia et al. 2010) and an altered immune response, i.e., increased susceptibility to the necrotrophic fungus Botrytis cinerea and increased tolerance to the biotrophic pathogens Pseudomonas syringae pv. tomato DC3000 and beet curly top virus (Garcia et al. 2013). Because the null mutant exhibits an induced alternative oxidase respiration, reactive oxygen species (ROS) accumulation and salicylic acid-dependent pathway induction, it was hypothesized that cyanide might generate a mitochondrial signal, unknown to date, that could modulate the plant immune system (Garcia et al. 2014). The cas-c1 mutant also exhibits a root hairless phenotype, which is reverted either genetically by complementation with the corresponding CAS-C1 gene or chemically by the addition of the cyanide antidote hydroxocobalamin (Alvarez et al. 2012; Garcia et al. 2010). Transcriptional profiling of the casc1 mutant reveals that the genes encoding enzymes involved in cell wall rebuilding and root hair formation are underexpressed in the mutant, as are certain genes involved in ethylene signaling and metabolism (Garcia et al. 2010). Root hairs are tubular extensions of root epidermal cells produced in the differentiation zone of the root and confer the ability to absorb nutrients and water, interact with microbes, and physically anchor the plant to the soil. Due to the biological importance of these structures, which are also a model for studying tip growth in plants, the molecular mechanisms involved in the specification, differentiation, and physiology of root hairs in Arabidopsis have been extensively reviewed (Carol and Dolan 2002; Grierson et al. 2014; Ishida et al. 2008). Root hair specification is determined by position-dependent signaling and molecular feedback loops. Once an initiation site has been selected, cell polarity is established, and a small swelling forms. Root hair elongation relies on polarized cell expansion at the growing tip, which involves multiple integrated processes, including cell secretion, endomembrane trafficking, cytoskeletal organization, and cell wall modifications. Sustained root tip growth involves oscillations in extracellular pH, ROS and cytosolic calcium (Monshausen et al. 2007). Elongation is accompanied by generation of a tip-high calcium gradient that can be observed throughout the remainder of root hair growth (Dolan et al. 1994; Samaj et al. 2004; Schiefelbein et al. 1992; Wymer et al. 1997). Thus, ROS are required for the activation of calcium channels and calcium activates NADPH oxidase in a system of positive feedback that maintains cell polarity during root hair elongation (Gapper and Dolan 2006; Takeda et al. 2008). The genes involved in each of the phases of root hair development have been identified, including transcription factors, cell wall-modifying enzymes, protein from the secretion apparatus, and proteins related to Ca 2+ production, ROS generation and management, cytoskeleton, etc. (reviewed in (Carol and Dolan 2002; Grierson and Schiefelbein 2009)). Hormones also play a role in root hair development, principally auxin Downloaded from https://academic.oup.com/pcp/advance-article-abstract/doi/10.1093/pcp/pcy047/4911869 by Centro de Información y Documentación Científica, [email protected] on 01 March 2018 6 and ethylene ((Grierson et al. 2014) and references therein). The goal of this work is to further our understanding of the role of CAS-C1 and cyanide in root hair development. To this end, a cellular strategy was implemented to observe the spatiotemporal expression and subcellular localization of CAS-C1, with a particular focus on developing root hairs. Mutations in the SUPERCENTIPEDE1 (SCN1) gene affect very early steps of the root hair development, resulting in root hair bulges that do not elongate. The SCN1 protein is a Rho GTPase GDP dissociation inhibitor that functions as a negative regulator of Rho-related plant GTPases (ROPs). Throughout hair development, ROP GTPases ROP2, ROP4 and ROP6 localize to the earliest swelling of the basal region and tip. ROPs regulate the activity of the NADPH oxidase encoded by ROOT HAIR DEFECTIVE 2 (RHD2)/AtrbohC, which catalyzes the generation of ROS and participates in the generation of the Ca 2+ gradient at the tip of the root hair that drives root hair elongation (Foreman et al. 2003; Ishida et al. 2008; Jones et al. 2007). Indeed, SCN1 acts on the root hair initiation, swelling formation, transition to tip growth and tip growth steps of root hair formation (Foreman and Dolan 2001; Parker et al. 2000). In scn1 mutants, ROP2 is mislocalized, and supernumerary hair initiation sites are formed that do not elongate (Carol et al. 2005). Mutants on rhd2, on his part, are not able to produce superoxide anion and show abnormal short root hairs (Foreman et al. 2003). To demonstrate epistatic relationships in the root hair elongation process, genetic crosses between the cas-c1 mutant and scn1 or rhd2 mutants have been performed, and some clues regarding the underlying mechanisms are discussed. Results CAS-C1 localizes to mitochondria and accumulates in root hair tips Based on its sequence and the capacity of the N-terminal portion of the protein to direct the green fluorescent protein (GFP) to mitochondria in transient expression experiments (Yamaguchi et al. 2000), CAS-C1 has been classified as a mitochondrial protein. Aiming to better unravel the role of CAS-C1 in relation to its spatiotemporal expression, we constructed a promoter-genomic open reading frame (ORF)-GFP construct and transformed plants as described in the Materials and Methods section and schematized in Supplementary Fig. S1. We assumed that the intergenic PIP1-CAS-C1 region should contain all transcriptional regulatory signals for Downloaded from https://academic.oup.com/pcp/advance-article-abstract/doi/10.1093/pcp/pcy047/4911869 by Centro de Información y Documentación Científica, [email protected] on 01 March 2018 7 CAS-C1 expression and that the genomic CAS-C1 region, from +1 to the STOP codon, should contain the regulatory sequences for the subcellular location of the protein as well as possible additional transcriptional regulatory elements (Gutierrez-Alcala et al. 2005). A translational fusion was then obtained by joining 2676 bp of the genomic sequence containing 1442 bp of the complete intergenic PIP1-CAS-C1 region plus 1234 bp from the CAS-C1 ATG to the GAT before the STOP codon (Supplementary Fig. S1, www.arabidopsis.org), to the GFP gene carried on the pMDC110 Gateway vector. The plant transformation construct was named ProC1:CASC1-GFP. Transgenic plants were obtained in different backgrounds, the wild type (8 independent lines) and the cas-c1 (3 independent lines), scn1-1 (2 independent lines) and rhd2GK (6 independent lines) mutants, and analyzed by confocal microscopy for in vivo GFP detection. No relevant differences of intensity or localization of the fluorescence were observed among the different lines in any case, so one T3 line of each genotype was selected for further studies. The spatio-temporal expression of CAS-C1 was examined by observing the fluorescence in different tissues of wild type Arabidopsis plants transformed with the ProC1:CASC1-GFP construct. Root tissues presented a homogeneous dotted pattern of fluorescence in wild type background (Fig. 1A, D, G), which was consistent with a mitochondrial localization of the fusion protein. This was confirmed by co-localization of GFP fluorescence with the mitochondrion-specific dye Mitotracker Deep Red 633 (Supplementary Fig. S2). In the root, the signal was very intense, especially in the meristematic zone (Fig. 1A, B, C), likely due to the high mitochondria concentration in cells that are metabolically active. In the root hair, a strong fluorescence at the tip was observed in wild type background (Fig. 1D, G), following the apical growth characteristic of root hairs (Supplementary Video S1). This apical localization of the fluorescence was detected from the very beginning of root hair development, at the swelling region in the initiation stage to the tip of the well-formed root hair (Supplementary Video S1). These results were consistent with the proposed role of CAS-C1 in root hair formation by modulating the accumulation of cyanide present in the growing tip (Garcia et al. 2010), which would act as a repressor of this process from the initial steps. Interestingly, transformation of cas-c1 mutant plants with the ProC1:CASC1GFP construct reverted, although partially, the root hairless phenotype (Supplementary Fig. S3), showing the complemented line accumulation of GFP in the root hair tip as in wild type background (Supplementary Fig. S3 B, C). Furthermore, we observed that in the scn1-1 mutant, despite being unable to produce root hair primordia, the ProC1:CASC1-driven GFP fluorescence was localized in the mitochondria, yet in root hairs it did not show a preferential localization of accumulation at the tips of the root hair primordia (Fig. 1B, E, H). Therefore, the Downloaded from https://academic.oup.com/pcp/advance-article-abstract/doi/10.1093/pcp/pcy047/4911869 by Centro de Información y Documentación Científica, [email protected] on 01 March 2018 8 scn1-1 mutation does not affect the subcellular localization of CAS-C1 but rather to the polarized location of mitochondria at the tip of the developing hair. Similarly, in a T-DNA insertion root hair-specific NADPH oxidase rhd2 mutant, rhd2GK, the fluorescence was localized at the mitochondria as well, although it did not show a root hair tip specific localization (Fig. 1C, F, I). Therefore, our data were consistent with previous work showing that mitochondria are present at a high density in tip-growing cells and spatially associated with the initiation and elongation of the root hair bulge (Carol and Dolan 2002; Wang et al. 2010). Since cyanide is a potent inhibitor of the cytochrome respiration pathway and root hair elongation is a rapid cell expansion process with high cost of energy, we analyzed whether the increase in cyanide in cas-c1 mutants could have a detrimental effect because of his presumed inhibition of the energy source required for rapid tip growth. Previous report has shown that neither the respiration rate is diminished, nor the localization of mitochondria is altered within the root hair in the cas-c1 mutant compared to wild type (García et al 2010). However, we have further analyzed the levels of the main energy source, ATP, in wild type and cas-c1 mutants in order to ensure that the energy source is not lacking in the cas-c1 roots. Table 1 shows that ATP levels were indistinguishable in wild type and cas-c1 mutants, thus ruling out a deleterious effect of cyanide on the production of the energy source required for root hair growth. This result leads us to deepen the concept of cyanide and/or CAS as an independent signaling component in root hair development. cas-c1 mutation is hypostatic to the scn1-1 and epistatic to the rhd2-1 morphological phenotypes As CAS-C1 appears to be involved in the regulation of the root hair growth, we further examined the existence of a relationship between CAS-C1 and the root hair elongation pathway. To establish the genetic epistasis between them, genetic crosses were carried out between scn1-1 (Parker et al. 2000) or rhd2-1 (Foreman et al. 2003) and cas-c1 mutants. Double scn1-1 cas-c1 and rhd2-1 cas-c1 mutants were generated and confirmed by sequence and PCR analyses (Supplementary Fig. S4 and S5). Root pictures of wild type, parental mutants and the double scn1-1 cas-c1 and rhd2-1 cas-c1 mutants at 6-7 d after sowing were captured under a microscope (Fig. 2). The root hairs of the cas-c1 mutant correctly began to grow and developed small bulges (Fig. 2B, F), and scn1-1 displayed multiple sites of hair growth emerging from hair-forming cells (Fig. 2C) and rhd2-1 showed short malformed root hairs (Fig. 2G). The double scn1-1 cas-c1 mutant exhibited a phenotype that was indistinguishable from that of the scn1-1 simple mutant, showing multiple root hair primordia that did not elongate (Fig. 2D), whereas the double rhd2-1 cas-c1 mutant exhibited a phenotype identical to the cas-c1 Downloaded from https://academic.oup.com/pcp/advance-article-abstract/doi/10.1093/pcp/pcy047/4911869 by Centro de Información y Documentación Científica, [email protected] on 01 March 2018 9 simple mutant, i.e., small protuberances rather than short root hairs, more sparse than in the rhd2-1 mutant (Fig. 2H). This reveals a role of CAS-C1 in an intermediate point between scn1 and the NADPH oxidase action in the root hair elongation process. Addition of the ethylene donor ACC to the culture medium did not elongate root hair cells in any of the single cas-c1 or double rhd2-1 cas-c1 or scn1-1 cas-c1 mutants, demonstrating the independence of cas-c1 mutation and ethylene production (Supplementary Fig. S6), (Garcia et al. 2010). In addition, we took advantage of the use of the 2-hydroxocobalamin (COB) compound as an antidote for cyanide poisoning because it reacts with free cyanide, producing cyanocobalamin, or vitamin B12, which is not toxic (Borron et al., 2007; Hall et al., 2007). Addition of COB to the culture medium has no effect on the wildtype root but is able to partially complement the loss-of-hair phenotype of the cas-c1 mutant (Garcia et al. 2010). By comparing the phenotypes of COB-treated plants, we found that cas-c1 could partially recover the development of root hairs, as previously reported, but that neither the scn1-1 nor the double scn1-1 cas-c1 mutants showed even partial reversion of their loss of the root hair elongation phenotype (Fig. 3C versus 3G, 3D versus 3H). These findings suggest that cyanide accumulation as the result of cas-c1 loss-of-function is not involved in the scn1-1 phenotype. On the other hand, COB treatment was able to revert the hairless root phenotype of the double mutant rhd2-1 cas-c1 to the single rhd2-1 mutant (Fig. 3L versus 3P), but COB did not revert the rhd2-1 mutant phenotype (Fig. 3K versus 3O) under the conditions tested. The above observations were supported by further molecular data. Cyanide accumulation in the cas-c1 mutant has been described to repress several genes encoding enzymes involved in the formation of the root hair tip, mainly cell wall-related proteins including FLA6 and MRH5, an arabinogalactan protein and a glycerophosphoryl diester phosphodiesterase-like GPI-anchored protein respectively (Garcia et al. 2010). Root hair growth includes a variety of cellular components and compounds that work in concert (Carol and Dolan 2002); Mendrinna and Persson 2015). The basic helix-loop-helix (bHLH) transcription factor (TF) ROOT HAIR DEFECTIVE 6 RHD6 controls the initiation of the root hair (Grierson et al. 2014) whereas the bHLH TF RHD6-LIKE 4 RSL4 is a direct transcriptional target of RHD6, and it has been described to integrate the developmental program aiming to regulate the polar growth (Marzol et al., 2017, Vijayakumar et al., 2016). In order to deepen in the epistatic relationships between cas-c1 and scn1 or rhd2, real-time quantitative RT-PCR was conducted. Figure 4 shows that all genes analyzed were down-regulated in cas-c1, scn1-1 and rhd2-1 mutants when compared to wild type. When comparing the double scn1-1 cas-c1 mutant with the single scn1-1 mutant, we could appreciate that there were no further repression of RHD6, RSL4 or MHR5 and only a slight repression of FLA6 (Fig. 4A). This suggests that the cas-c1 mutation did not significantly affect the behavior of Downloaded from https://academic.oup.com/pcp/advance-article-abstract/doi/10.1093/pcp/pcy047/4911869 by Centro de Información y Documentación Científica, [email protected] on 01 March 2018 16 Root tissues were incubated with a 20 nM solution of MitoTracker Deep Red (Molecular Probes) for 10 min at room temperature. Samples were observed using a Leica HCX PLAN-APO 363 1.4 NA oil immersion objective with a Leica TCS SP2 spectral confocal microscope (Leica Microsystems). The dye was excited using a heliumneon laser at 644 nm, either in single confocal optical sections or in serial optical sections. Emitted light was collected through a triple dichroic beam splitter (TD 488/543/633) and detected after spectral separation in the 650 to 700 nm range (pseudocolored blue). Detection of ROS For detection of the superoxide anion, roots were stained with NBT (Nitrotetrazolium blue chloride, SigmaAldrich) as described previously (Garcia et al. 2010). Seedlings were incubated in 0.1 M Tris-HCl, 0.1 M NaCl, 0.05 M MgCl 2 , and 0.5 mg mL -1 NBT (pH 9.5) for 2 h at room temperature in the dark. After rinsing, roots were imaged under bright-field illumination under an Olympus BX50 microscope and images were taken using a Leica DFC300FX digital camera. For fluorimetric detection of H 2 O 2 , roots were incubated for 5 min with 10 mM H 2 DCFDA (Life Technologies) in the presence of 10 mM propidium iodide (Life Technologies) to visualize cell walls. The samples were observed using a TCS SP2 spectral confocal microscope (Leica Microsystems) with the following settings: excitation, 488 nm; emission, 500 to 550 nm for fluorescein detection and 600 to 650 nm for propidium iodide detection. Real-time RT-PCR Quantitative real-time RT-PCR was used to analyze the expression of the MHR5, FLA6, RHD6 and RSL4 genes. Total RNA was extracted from Arabidopsis leaves using the Qiagen RNeasy Plant Mini Kit. RNA was reverse transcribed using an oligo (dT) primer and Invitrogen Super-script First-Strand Synthesis System for RT-PCR following the manufacturer’s instructions. Gene-specific primers for each gene were designed using the Invitrogen Vector NTI Advance 10 software. The primer sequences were as follows: QFMRH5, 5´- GCTGCTTGCTGCTCAAATCC-3´ and QRMRH5, 5´-AATCCAGAGAATCCACCACG-3´ for the MRH5 gene; QFFLA6, 5´-CAAATCCAGCTCATGCTCTACC-3´ and QRFLA6, 5´-TCTTGTCCCGTAGCCTGAGTDownloaded from https://academic.oup.com/pcp/advance-article-abstract/doi/10.1093/pcp/pcy047/4911869 by Centro de Información y Documentación Científica, [email protected] on 01 March 2018 17 3´ for FLA6 gene; qRHD6-Fw, 5´- CCGGCTCAAGGAGGAAAA-3´ and qRHD6-R, 5´- CGAATTCCTGTCTCGTTGTGA-3´ for RHD6 gene; qRSL4-Fw, 5´- CAGATTAAGTTGTTGAGCTCGG-3´ and qRSL4-R, 5´- GAGACAAAAGGTTGTGATGGAA-3´ for RSL4 gene; qUbq10-F, 5´- GGCCTTGTATAATCCCTGATGAATAAG-3´ and qUbq10-R, 5´- AAAGAGATAACAGGAACGGAAACATAGT-3´ for the constitutive UBQ10 gene. Real-time PCR was performed using the Bio-Rad IQ SYBR Green Supermix. Signals were detected with a Bio-Rad iCYCLER according to the manufacturer’s instructions. The cycling profile consisted of 95°C for 10 min followed by 45 cycles of 95°C for 15 s and 60°C for 1 min. A melting curve from 60°C to 90°C was performed following PCR cycling. The expression levels of genes of interest were normalized to that of the constitutive UBQ10 gene by subtracting the cycle threshold (CT) value of UBQ10 from that of the gene of interest (∆CT) and calculated as 2 - ∆CT . The results shown are the means ± SD of at least three independent RNA samples. Determination of ATP The adenine triphosphate in the roots was extracted following the boiling water method (Yang et al. 2002). Briefly, 50 mg of root tissues were mixed with 100 µl of ice-cold distilled H 2 O, which was immediately heated in a boiling water bath for 10 min (Li et al. 2017). The boiled lysates were centrifuged at 15,000 g for 5 min at 4 ºC and the supernatants were collected for ATP measurement using an ATP Determination kit (A22066, ThermoFisher Scientific®), following the manufacturer’s protocol in a Thermo Scientific Varioskan® Flash. Cyanide determination by high-performance liquid chromatography (HPLC) A total of 100 mg of root tissue was homogenized in liquid nitrogen using a mortar and pestle and resuspended in cold borate-phosphate extraction buffer (2 mL g -1 fresh weight) containing 27 mM sodium borate and 47 mM potassium phosphate, pH 8.0. The homogenates were centrifuged at 15,000 g for 15 min at 4°C. Extracted cyanide was subsequently quantified by reverse-phase HPLC after derivatization with 2,3naphthalenedialdehyde to form a 1-cyano-2-alkyl-benz[f]isoindole derivative using previously described methods (Garcia et al. 2010; Lin et al. 2005). Determination of NADPH oxidase activity Downloaded from https://academic.oup.com/pcp/advance-article-abstract/doi/10.1093/pcp/pcy047/4911869 by Centro de Información y Documentación Científica, [email protected] on 01 March 2018 18 NADPH activity in root extracts was measured using a method adapted from a previous report (Kaundal et al. 2012). Frozen roots (50 mg) were homogenized in 1 ml of 50 mM HEPES buffer (pH 7.2) containing 0.25 M sucrose, 3 mM EDTA, 1 mM dithiotreitol (DTT), 3.6 mM L-cysteine, 0.1 mM MgCl 2 and 0.6% polyvinylpyrrolidone (PVP) with the addition of Complete Protease Inhibitor Cocktail Tablets (Sigma). The homogenate tissue was filtered through two layers of Miracloth and centrifuged at 10,000 x g for 45 min at 4ºC. The supernatant was centrifuged at 203,000 g for 60 min at 4ºC. The pellet was resuspended in 150 µl ice-cold 10 mM Tris-HCl (pH 7.4) and used for the enzyme assay. NADPH oxidase activity was assayed colorimetrically with XTT (3′-[1-[(phenylamino)-carbony]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfonic acid) sodium salt as a substrate. The reaction mixture contained 50 mM Tris-HCl buffer (pH 7.5), 0.5 mM XTT, 0.1 mM NADPH and 5 µg protein extract. The linear increase in absorption at 492 nm due to the formation of a yellow formazan was followed for 120 min (extinction coefficient of 21.6 mM -1 cm -1 ). Funding This work was supported in part by the European Regional Development Fund through Ministerio de Economía y Competitividad [grant MOLCYS, no. BIO2013-44648-P] and Agencia Estatal de Investigación [grant no. BIO2016-76633-P] L.A.-A. thanks the Ministerio de Economía y Competitividad for fellowship support through the program of Formación de Personal Investigador. Acknowledgments We thank Dr. Alicia Orea for confocal microscopy service and Inmaculada Moreno for technical assistance. 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(2013a) PFT1controlled ROS balance is critical for multiple stages of root hair development in Arabidopsis. Plant Signal. Behav. 8: e24066. Sundaravelpandian, K., Chandrika, N.N. and Schmidt, W. (2013b) PFT1, a transcriptional Mediator complex subunit, controls root hair differentiation through reactive oxygen species (ROS) distribution in Arabidopsis. New Phytol. 197: 151-161. Takeda, S., Gapper, C., Kaya, H., Bell, E., Kuchitsu, K. and Dolan, L. (2008) Local positive feedback regulation determines cell shape in root hair cells. Science 319: 1241-1244. Tsukagoshi, H., Busch, W. and Benfey, P.N. (2010) Transcriptional regulation of ROS controls transition from proliferation to differentiation in the root. Cell 143: 606-616. Wang, Y., Zhu, Y., Ling, Y., Zhang, H., Liu, P., Baluska, F., et al. (2010) Disruption of actin filaments induces mitochondrial Ca2+ release to the cytoplasm and [Ca2+]c changes in Arabidopsis root hairs. BMC Plant Biol. 10: 53. 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(2016) The LIKE SEX FOUR2 regulates root development by modulating reactive oxygen species homeostasis in Arabidopsis. Scientific reports 6: 28683. Downloaded from https://academic.oup.com/pcp/advance-article-abstract/doi/10.1093/pcp/pcy047/4911869 by Centro de Información y Documentación Científica, [email protected] on 01 March 2018 22 Tables Plant line Adenosine triphosphate (nM) Wild type 400.9 ± 118.6 cas-c1 427.6 ± 118.2 Table 1. Adenosine triphosphate levels in root extracts. The ATP level was determined in crude extracts prepared from the roots of wild type and cas-c1 mutant plants grown in solid MS medium supplemented with sucrose for 14 d. Values are means ± SD from three independent experiments. Plant line NADPH oxidase activity (mU/µg) wild type 23.23 ± 2.8 cas-c1 20.64 ± 0.6 wild type + KCN 0.1mM 21.09 ± 0.5 Table 2. NADPH oxidase activity level in root extracts. The activity was determined in crude extracts with or without 0.1 mM KCN treatment, prepared from the roots of wild type and mutants grown in solid MS medium supplemented with sucrose for 14 d. Values are means ± SD from five independent experiments. Legends to figures Figure 1. CAS-C1 expression and localization study. Representative GFP images of A,D,G, wild type, B,E,H, scn1-1 mutant and C,F,I, rhd2GK mutant transformed with ProC1:CASC1-GFP. Plants were grown in MS medium supplemented with sucrose for 2 days. Images are maximum projection of 20 optical sections. Figure 2. Root hair phenotypes of root hair formation mutants. (A, E) Wild type, (B, F) cas-c1, (C) scn1-1, (D) scn1-1 cas-c1, (G) rhd2-1, (H) rhd2-1 cas-c1 . Seedlings were grown 6-d on MS (A-D) or 7-d on MS pH4 (E-H) medium supplemented with sucrose in vertical plates. Representative images are shown. Bars = 0.5 mm. Figure 3. Hydroxocobalamin effect on root hair formation. (A-D) Root hair phenotype of wild type, cas-c1, scn1-1 and scn1-1 cas-c1 3-d-old plants, respectively, grown on MS sucrose vertical plates in the absence of hydroxocobalamin. (E-H) Root hair phenotype of wild type, cas-c1, scn1-1 and scn1-1cas-c1 3-d-old plants, respectively, grown on MS sucrose vertical plates in the presence of 5 mM hydroxocobalamin. (I-L) Root hair phenotype of wild type, cas-c1, rhd2-1 and rhd2-1 cas-c1 7-d-old plants, respectively, grown on MS pH4 Downloaded from https://academic.oup.com/pcp/advance-article-abstract/doi/10.1093/pcp/pcy047/4911869 by Centro de Información y Documentación Científica, [email protected] on 01 March 2018 23 sucrose vertical plates in the absence of 5 mM hydroxocobalamin. (M-P) Root hair phenotype of wild type, casc1, rhd2-1 and rhd2-1 cas-c1 7-d-old plants, respectively, grown on MS pH4 in the presence of 5 mM hydroxocobalamin. All the experiments were repeated at least three times, with similar results obtained each time. Bars = 0.5 mm. Figure 4. Root hair formation genes expression analysis. Real-time (RT)–PCR analysis of the expression of the RHD6, RSL4, FLA6 and MRH5 genes was performed in root extracts from 14-day-old wild-type, cas-c1, scn1-1 and scn1-1 cas-c1 mutants grown in MS pH 5,7 (A) or wild-type, cas-c1, rhd2-1 and rhd2-1 cas-c1 mutants grown in MS pH 4 (B). The transcript levels were normalized to the constitutive UBQ10 gene. Data shown are means ± SD of three independent experiments. Different letters above the bar indicate significant different mean (ANOVA test, P<0.01). Figure 5. Detection of superoxide in Arabidopsis roots. (A) Wild-type, (D) cas-c1, (B) scn1-1, (E) scn1-1 casc1, (C) rhd2-1 and (F) rhd2-1 cas-c1 seedlings were grown for 3 d on MS medium with sucrose. The roots were stained with NBT as described in Materials and Methods. Representative images are shown. All the experiments were repeated at least three times, with similar results obtained each time. Bars = 0.1 mm. Figure 6. Accumulation of H 2 O 2 in wild type and root hair mutants. H 2 O 2 was detected by H 2 DCFDA staining in 14-d-old roots of (A) wild-type, (D) cas-c1, (B) scn1-1, (E) scn1-1 cas-c1, (C) rhd2-1 and (D) rhd2-1 cas-c1 plants cultured on MS medium supplemented with 1% sucrose. All the experiments were repeated at least three times, with similar results obtained each time. Bars = 300 µm. Figure 7. Involvement of CAS-C1 and cyanide in the root hair growth. In an elongating root hair, SCN1 inhibits the ROPs proteins, which are essential for the NADPH oxidase RHD2 tip-localized action. RHD2 produces superoxide anion (stained with NBT) that is transformed to oxygen peroxide (visualized by H 2 DCFDA staining) by superoxide dismutase action. Both ROS are important for cell wall growth by breaking and rebuilding. Cyanide concentration, controlled by CAS-C1 and eliminated by COB, would act in a step between the SCN1 action and the ROS production by RHD2, establishing positive (arrows) or negative (blunt lines) relationships to hitherto unknown protein(s) or factor(s). Full lines indicate already established relationships, while dashed lines indicate the proposed sequence of action of CAS-C1 and cyanide in the root hair elongation process. Downloaded from https://academic.oup.com/pcp/advance-article-abstract/doi/10.1093/pcp/pcy047/4911869 by Centro de Información y Documentación Científica, [email protected] on 01 March 2018 24 Supplementary Fig. S1. Schematic representation of the CAS-C1 locus and flanking genes on chromosome 3. A, Diagram of the intergenic sequence between CAS-C1 and the adjacent genes on chromosome 3 of the Arabidopsis genome (www.arabidopsis.org). Yellow arrows indicate the direction of transcription of the different genes. Dark arrows indicate the designed oligonucleotide positions for ProC1:CASC1 sequence isolation. B, The nucleotide sequence of ProC1:CASC1, where ProC1 is the CAS-C1 promoter, and CASC1 is the CAS-C1 ORF. Colored letters indicate intron (purple), UTR (red) and exon (yellow) sequence. Blue shadowed letters show the translational start and stop codons. PIP-1 codes for a plasma membrane intrinsic protein and SYP-73 codes for a syntaxin of plants. Supplementary Fig. S2. Mitochondrial localization of CASC1-GFP (A) Maximum projection of 20 optical sections of 4-d-old root tissues from wild type plants transformed with ProC1:CASC1-GFP showing GFP fluorescence signal. (B) Fluorescence signal of the same tissues dyed with MitoTracker Deep Red 633 for 15 min, (C) Overlapping of (A) and (B) images showing co-localization of GFP fluorescence and stained mitochondria. Bars = 40 µm. Supplementary Fig. S3. Root phenotype of the cas-c1 and complemented mutant line. (A) Representative bright-field image of 4-day-old cas-c1 mutant plants is shown. (B-C) Representative bright-field and GFP fluorescence images, respectively, of 4-day-old cas-c1 mutant plants transformed with ProC1:CASC1-GFP are shown. Images are maximum projection of 20 optical sections. Bars = 50 µm Supplementary Fig. S4. Molecular characterization of the scn1-1 cas-c1 mutant. To identify individuals homozygous for SCN1 and CAS-C1 gene mutations, genomic DNA was extracted from leaves of wild-type and eight scn1-1 cas-c1 mutants grown in MS medium supplemented with sucrose (1%) and kanamycin (30 µg/mL) and transplanted to soil later. This DNA was subjected to the following: A, PCR genotyping using the primer pairs scn1-1 FW/scn1-1 REV and C1-F2/C1-R2, with UBQ10 amplification using UBQF1-UBQR1 as a positive control; B, sequencing genotyping using scn1-1 FW/scn1-1 REV primers. scn1-1 FW: 5´-TCAAAGAACATCTCGAGAAGGA-3´ scn1-1 REV: 5´-CACACAAACACACCTCCAATGT-3´ C1-F2: 5´-TGATGGGAATTGGCAGTGGAGGCAC-3´ C1-R2: 5´-AATTGCTTGCCACGGTGTTAGCTCCC-3´ Downloaded from https://academic.oup.com/pcp/advance-article-abstract/doi/10.1093/pcp/pcy047/4911869 by Centro de Información y Documentación Científica, [email protected] on 01 March 2018 25 UBQF1: 5’- GATCTTTGCCGGAAAACAATTGGAGGATGGT-3’ UBQR1: 5’- CGACTTGTCATTAGAAAGAAAGAGATAACAGG-3’ Supplementary Fig. S5. Molecular characterization of the rhd2-1 cas-c1 mutant. To identify individuals who were homozygous for RHD2 and CAS-C1 gene mutations, genomic DNA was extracted from leaves of wildtype, rhd2-1 mutant (as positive control) and five rhd2-1 cas-c1 mutants grown in MS medium supplemented with sucrose (1%) and kanamycin (30 µg/mL) and transplanted to soil later. This DNA was subjected to the following: A, PCR genotyping using the primer pairs rhd2-1 FW/rhd2-1 REV and C1-F2/C1-R2, with UBQ10 amplification using UBQF1-UBQR1 was used as a positive control; B, sequencing genotyping using rhd2-1 FW/rhd2-1 REV primers. rhd2-1 FW: 5´-TGAATGGTATGAACCAAACCGC-3´ rhd2-1 REV: 5´-GGCTTACACACCTGAAACAACA-3´ Supplementary Fig. S6. 1-Aminocyclopropane-1-carboxylic acid (ACC) effect on root hair formation. (A, C, E, G, I, K) Root hair phenotype of wild type, cas-c1, scn1-1, scn1-1cas-c1, rhd2-1 and rhd2-1 cas-c1 6-d-old plants, respectively, grown on MS sucrose vertical plates in the absence of ACC. (B, D, F, H, J, L) Root hair phenotype of wild type, cas-c1, scn1-1, scn1-1cas-c1, rhd2-1 and rhd2-1 cas-c1 6-d-old plants, respectively, grown on MS sucrose vertical plates in the presence of 50 µM ACC. Bars = 0.15 cm. Supplementary Fig. S7. Cyanide determination in root tissues. Fourteen-days old wild type, cas-c1, scn1-1, scn1-1 cas-c1, rhd2-1 and rhd2-1 cas-c1 mutant plants were grown on plates containing MS sucrose medium and then collected for cyanide content determination. Values are the means ± SD from five independent experiments. Increases of the media of the cyanide content in every couple of mutants are indicated at the top of the respective column pair. Asterisks indicate significant differences (ANOVA test, P<0,01) between wild type and cas-c1 and between pairs of single and double mutants. Supplementary Video S1. Root hair growth. ProC1:CASC1-GFP plants were grown for 2 d and fluorescence microscopy images were taken every 2 min over 2 h to analyze root hair formation in vivo. Bar = 25 µm. Downloaded from https://academic.oup.com/pcp/advance-article-abstract/doi/10.1093/pcp/pcy047/4911869 by Centro de Información y Documentación Científica, [email protected] on 01 March 2018 SCN1 CAS-C1 ROPs RHD2 O2.- H2O2 [CN-] COB NBT staining H2DCFDA staining Fig. 7. Involvement of CAS-C1 and cyanide in the root hair growth. In an elongating root hair, SCN1 inhibits the ROPs proteins, which are essential for the NADPH oxidase RHD2 tip-localized action. RHD2 produces superoxide anion (stained with NBT) that is transformed to oxygen peroxide (visualized by H2DCFDA staining) by superoxide dismutase action. Both ROS are important for cell wall growth by breaking and rebuilding. Cyanide concentration, controlled by CAS-C1 and eliminated by COB, would act in a step between the SCN1 action and the ROS production by RHD2, establishing positive (arrows) or negative (blunt lines) relationships to hitherto unknown protein(s) or factor(s). Full lines indicate already established relationships, while dashed lines indicate the proposed sequence of action of CAS-C1 and cyanide in the root hair elongation process. Page 32 of 32Plant & Cell Physiology Downloaded from https://academic.oup.com/pcp/advance-article-abstract/doi/10.1093/pcp/pcy047/4911869 by Centro de Información y Documentación Científica, [email protected] on 01 March 2018