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Cyanogen Metabolism in Cassava Roots: Impact on Protein Synthesis and Root Development

Cyanogen Metabolism in Cassava Roots: Impact on Protein Synthesis and Root Development fpls-08-00220 February 22, 2017 Time: 15:3 # 1 ORIGINAL RESEARCH published: 24 February 2017 doi: 10.3389/fpls.2017.00220 Cyanogen Metabolism in Cassava Roots: Impact on Protein Synthesis and Root Development 1 2 1,3 Tawanda Zidenga , Dimuth Siritunga and Richard T. Sayre 1 2 Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USA, Department of Biology, University of Puerto Rico, Mayaguez, PR, USA, New Mexico Consortium, Los Alamos, NM, USA Cassava (Manihot esculenta Crantz), a staple crop for millions of sub-Saharan Africans, contains high levels of cyanogenic glycosides which protect it against herbivory. However, cyanogens have also been proposed to play a role in nitrogen transport from leaves to roots. Consistent with this hypothesis, analyses of the distribution and activities of enzymes involved in cyanide metabolism provides evidence for cyanide assimilation, derived from linamarin, into amino acids in cassava roots. Both b-cyanoalanine synthase (CAS) and nitrilase (NIT), two enzymes involved in cyanide assimilation to produce asparagine, were observed to have higher activities in roots compared to leaves, consistent with their proposed role in reduced nitrogen assimilation. In addition, rhodanese activity was not detected in cassava roots, indicating that this competing means for cyanide metabolism was not a factor in cyanide detoxification. In contrast, Edited by: leaves had sufficient rhodanese activity to compete with cyanide assimilation into Henrik Toft Simonsen, Technical University of Denmark, amino acids. Using transgenic low cyanogen plants, it was shown that reducing root Denmark cyanogen levels is associated with elevated root nitrate reductase activity, presumably to Reviewed by: compensate for the loss of reduced nitrogen from cyanogens. Finally, we overexpressed Roslyn Gleadow, Arabidopsis CAS and NIT4 genes in cassava roots to study the feasibility of enhancing Monash University, Australia Kirsten Jørgensen, root cyanide assimilation into protein. Optimal overexpression of CAS and NIT4 resulted University of Copenhagen, Denmark in up to a 50% increase in root total amino acids and a 9% increase in root *Correspondence: protein accumulation. However, plant growth and morphology was altered in plants Tawanda Zidenga tawanda@lanl.gov overexpressing these enzymes, demonstrating a complex interaction between cyanide metabolism and hormonal regulation of plant growth. Specialty section: This article was submitted to Keywords: cassava, linamarin, cyanide, cyanogen, b-cyanoalanine synthase, nitrilase, auxin, ethylene Plant Biotechnology, a section of the journal Frontiers in Plant Science INTRODUCTION Received: 08 November 2016 Accepted: 06 February 2017 Cyanide is ubiquitous in nature and as such most eukaryotic organisms have developed Published: 24 February 2017 mechanisms for its detoxification. For example, all plants produce some level of cyanide as a Citation: byproduct of the ethylene biosynthesis (Peiser et al., 1984; Dong et al., 1992). Additionally, Zidenga T, Siritunga D and Sayre RT some plants are highly cyanogenic including; cassava, bitter almonds, and rubber (Poulton, 1990; (2017) Cyanogen Metabolism Gleadow and Møller, 2014). The leaves and roots of cassava plants may accumulate between in Cassava Roots: Impact on Protein 200 and 1,300 mg CN equivalents/kg dry weight (Siritunga and Sayre, 2004). Generation of free Synthesis and Root Development. cyanide from cyanogenic compounds would obviously have toxic consequences, but generally Front. Plant Sci. 8:220. doi: 10.3389/fpls.2017.00220 does not happen in intact plants due to the physical separation of the cyanogenic compounds Frontiers in Plant Science | www.frontiersin.org 1 February 2017 | Volume 8 | Article 220 fpls-08-00220 February 22, 2017 Time: 15:3 # 2 Zidenga et al. Cyanogen Metabolism in Cassava Roots from the enzymes that degrade them (Kojima et al., 1979; detoxification pathway results in the assimilation of cyanide into Poulton, 1990; McMahon et al., 1995; Maruyama et al., 2001). amino acid biosynthesis pathways and is present in all higher In cassava, the cyanogenic glycoside linamarin, is stored in plants thus far examined (Figure 1; Blumenthal et al., 1968; Miller vacuoles while its corresponding b-glucosidase, linamarase, and Conn, 1980). is localized to the cell wall and laticifers (Mkpong et al., In this study, we use a combination of biochemical 1990; McMahon et al., 1995; Elias et al., 1997a). Tissue assays and transgene expression studies to demonstrate that disruption, e.g., during mechanical damage, initiates hydrolysis cyanide metabolism in roots most likely occurs via the CAS of linamarin by the generalized b-glucosidase, linamarase, pathway to produce amino acids. Furthermore, we show no to produce acetone cyanohydrin. Acetone cyanohydrin can competing rhodanese activity in roots, consistent with CAS spontaneously decompose to yield cyanide and acetone at mediated assimilation of cyanide into amino acids. Furthermore, pH > 5.0 or temperatures > 35 C, or is broken down by the overexpression of CAS and nitrilase was shown to lead to elevated enzyme hydroxynitrile lyase (HNL), which is expressed only in free amino acid pool sizes and root protein content under optimal cassava leaves and stems and not in roots (White et al., 1998). enzyme expression levels, indicating that these enzymes facilitate The cassava tuberous root, the main consumed part of the cyanide assimilation. These results provide insights into the role plant, is actually a true root, not a tuber, and thus cannot be used for cyanogenic glycosides in nitrogen metabolism in the roots, to vegetatively propagate the plant (Alves, 2002). Chronic, low- as well as possibilities for redirecting root linamarin toward level dietary cyanide exposure associated with the consumption protein production. Finally, alterations in cyanide assimilation of improperly processed cassava can lead to several health and other metabolic pathways associated with overexpressing disorders, including tropical ataxic neuropathy, characterized by CAS and NIT4 was shown to dramatically alter plant growth optic atrophy and an inability to coordinate muscle movements, and morphology. These results demonstrate a potential complex and a paralytic disorder known as Konzo (Osuntokun, 1981; interaction between cyanide and ethylene and auxin metabolism. Rosling, 1994; Tylleskar, 1994; Adamolekun, 2010). Individuals with these disorders typically have very low concentrations of MATERIALS AND METHODS sulfur amino acids in the blood (available sulfur is preferentially used in cyanide detoxification by rhodanese) and elevated Cassava Cultivars levels of plasma thiocyanate (Shibamoto and Bjeldanes, 1993; Cassava cultivar Manihot Columbia 2215 (MCol 2215) was used Adamolekun, 2010). for initial assays of leaf and root enzyme activity. Transformation For obvious health impact reasons, several efforts have been work was done using cultivar TMS 60444, selected due to ease directed toward lowering the cyanogens (a group of nitrile- of transformation. Comparative enzyme assays with transgenic containing plant secondary compounds that release hydrogen cyanide through enzymatic activity) in cassava. The major lines were performed using TMS 60444 as the wild-type control. Transgenic lines previously generated for low cyanogenesis cyanogens in cassava are linamarin and acetone cyanohydrin. Transgenic cassava lines having less than 1% of normal root (by selectively inhibiting cyanogenic glycoside synthesis in the leaves), Cab1-1, Cab1-2, and Cab1-3 used MCol 2215 as the cyanogen levels have been generated by inhibiting linamarin background (Siritunga and Sayre, 2003). biosynthesis in leaves (Siritunga and Sayre, 2003). However, these plants could not grow without ammonia and generally produced smaller tuberous roots compared to wild-type plants. It was Tissue Culture Propagation of Plant hypothesized that the poor performance of these plants was due Material to the suppression of cyanogen synthesis, an important source Cassava plants were propagated in vitro on the Murashige of reduced nitrogen for roots. A second approach to reduce and Skoog (MS) basal medium (Murashige and Skoog, 1962) cyanogen levels in cassava foods was to overexpress HNL to supplemented with Gamborg vitamins (Gamborg et al., 1968) accelerate the conversion of acetone cyanohydrin into cyanide, and 2% (w/v) sucrose. In vitro plants were propagated in growth which is then volatilized during processing (Siritunga et al., 2004; incubators at 28 C with a photoperiod of 16 h of light and Narayanan et al., 2011). Both acetone cyanohydrin and linamarin 8 h of darkness. Micropropagation of plant materials was done contribute to cyanide toxicity in poorly processed cassava foods, once every 5–8 weeks depending on the requirements of specific whereas cyanide does not due to its volatilization. Significantly, experiments (Siritunga and Sayre, 2003; Ihemere et al., 2006; by increasing root nitrogen sink strength by overexpressing HNL, Zidenga et al., 2012). it was observed that there was a 50–75% reduction in root steady- state linamarin levels suggesting that linamarin provided reduced Total Protein Extraction and Analysis nitrogen for protein synthesis. Total protein was measured using the Bradford assay according It had been proposed that some fraction of the cyanogens to the supplier’s (Invitrogen ) instructions. Protein was extracted transported from leave to roots were metabolized to generate free from root and leaf tissue of cassava using 50 mM Tris-HCl (pH cyanide which would then either be detoxified by the enzyme 8.5), 5 mM dithiothreitol and 1 mM EDTA. Extraction buffer was rhodanese (cyanide: thiosulfate sulfurtransferase) or assimilated used at a ratio of 5 mL of buffer per gram fresh tissue. Leaves into amino acids by CAS and nitrilase (Siritunga and Sayre, were ground in liquid nitrogen to a fine powder before adding 2007; Figure 1). CAS catalyzes the reaction between cyanide and cysteine to form b-cyanoalanine and hydrogen sulfide. This www.invitrogen.com Frontiers in Plant Science | www.frontiersin.org 2 February 2017 | Volume 8 | Article 220 fpls-08-00220 February 22, 2017 Time: 15:3 # 3 Zidenga et al. Cyanogen Metabolism in Cassava Roots FIGURE 1 | Model of cyanide assimilation in plants. CAS converts cyanide released from the cyanogen linamarin, to cyanoalanine plus hydrogen sulfide in the presence of cysteine. Cyanoalanine is converted by a nitrilase to asparagine, which is then converted to aspartate and ammonia by asparaginase. In this way, cyanide nitrogen can be incorporated into the free amino acid pool of the plant. An alternative pathway (red arrow) can potentially result in cyanide detoxified to thiocyanate by rhodanese. the buffer. Tuberous roots were blended together in the buffer in sulfide (Na S) was estimated from the absorbance using a the Magic Bullet MB1001 blender (Homeland Houseware LLC) standard curve prepared by adding 0.1 mL 30 mM ferric chloride for 30 s at 4 C. The ground extract was passed through four in 1.2 N HCl per mL of total reaction mixture followed by 0.1 mL layers of cheesecloth and centrifuged at 21000 g for 10 min. of 20 mM N,N dimethyl-p-phenylenediamine sulfate in 7.2 N The supernatant was used as the crude extract and measured for HCl to 1 mL of known concentrations of Na S. protein using the Bradford reagent with bovine serum albumin (BSA) as the standard. Rhodanese Activity Rhodanese activity was assayed as described by Wang and Volini Activity of b-Cyanoalanine Synthase (1968) with modifications. Cassava plant tissue was ground (CAS) in Cassava Tissue in liquid nitrogen using a motor and pestle and extracted in b-Cyanoalanine synthase activity was determined using 200 mM sodium phosphate buffer pH 7.8, 5.0 mM DTT, 5.0 mM the method described by Goudey et al. (1989) with some PMSF, 1.0 mM EDTA, 1.0 mM Na thiosulfate (to keep the modifications. Cassava tissue was ground in liquid nitrogen enzyme in a stable rhodanese-sulfur intermediate), 5 mM KCl using a motor and pestle and extracted in a buffer containing and 2% w/v polyvynilpolypyrrolidone (PVP). The homogenate 50 mM Tris-HCl, 5.0 mM dithiothreitol (DTT), 5.0 mM was passed through four layers of cheesecloth to remove debris phenylmethylsulfonyl fluoride (PMSF) and 1.0 mM EDTA at pH and centrifuged at 21000 g for 5 min at 4 C. The supernatant 8.0. The extract was filtered through four layers of cheesecloth was used in subsequent assays. To start the reaction, about 100– to remove debris. To 500 mL of substrate solution (10 mM 200 mg of protein was added to 0.5 mL of 50 mM NaCN and L-cysteine and 10 mM NaCN in 50 mM Tris buffer pH 8.0), 50 mM of Na thiosulfate in 200 mM sodium phosphate buffer and 100–200 mg of crude protein extract was added to make a (pH 7.8) to a total volume of 1.0 mL. The reaction was incubated total reaction volume of 1.0 mL. The optimum concentration at 30 C for 10 min and stopped by adding 0.5 mL 15% (v/v) of enzyme used was determined after testing different amounts formaldehyde. Absorbance at 460 nm was measured after adding of wild-type plant enzyme extract. The reaction was carried 2.5 ml of ferric nitrate reagent. The reagent was prepared by out for 10 min and stopped by adding 0.1 mL of 30 mM ferric adding 20 mL nitric acid (65%) to 60 mL of water, dissolving chloride in 1.2 N HCl followed by 0.1 mL of 20 mM N,N 10 g ferric nitrate: 9 H O and making up to a final volume of dimethyl-p-phenylenediamine sulfate in 7.2 N HCl. Absorbance 100 mL. The reaction was blanked using boiled (inactive) enzyme was measured at 640 nm after 10 min. Concentration of sodium extract. The standard curve used to estimate the concentration of Frontiers in Plant Science | www.frontiersin.org 3 February 2017 | Volume 8 | Article 220 fpls-08-00220 February 22, 2017 Time: 15:3 # 4 Zidenga et al. Cyanogen Metabolism in Cassava Roots thiocyanate was prepared using a range of known concentrations Transformation of Cassava with the of thiocyanate in the same volume as the reaction. b-Cyanoalanine Synthase and Nitrilase Genes Determination of Nitrilase Activity Construct Design Nitrilase activity was determined as described by Piotrowski et al. Constructs were assembled as previously described in Zidenga (2001) with some modifications. Cassava tissue was homogenized et al. (2012). A modified pBI121 plasmid with a 1.2 kb Solanum in an extraction buffer containing 50 mM Tris-HCl (pH 8.5), tuberosum class I patatin promoter was used for both constructs 2.0 mM EDTA, 8.0 mM cysteine, 2% (w/v) PVP plus and minus (Ihemere, 2002; Siritunga and Sayre, 2003). The CAS and NIT4 (for plant protein quantification) 0.1% (w/v) BSA. Tuberous (TAIR: At5g22300) genes of Arabidopsis were received from the greenhouse roots were homogenized in a blender for 5 s  2 s, Arabidopsis Biological Research Center in the pUNI51 vector while in vitro plant material was homogenized by grinding with and cloned into SmaI and SstI restriction sites of the modified liquid nitrogen in a motor and pestle. In all cases, the homogenate pBI121 binary plasmid (Figure 3A). was filtered through four layers of cheesecloth and centrifuged for 5 min at 22000 g. Approximately 400 mg of plant protein was Cassava Transformation used in the subsequent enzyme assay. Enzyme extracts were pre- Somatic embryogenesis, co-cultivation with Agrobacterium and warmed at 37 C for 2 min before being incubated with substrate plant regeneration were carried out as described by Zidenga (10 mM cyanoalanine in 50 mM Tris-HCl, pH 8.5 and 1.0 mM et al. (2012) while cassava transformation was done following DTT) for 10 min at 37 C. The total reaction volume was 1.0 mL. the method described by Taylor et al. (1996) with modifications The reaction was stopped by adding 100 mL of tricarboxylic (Zidenga et al., 2012). acid and centrifuged at 22000 g for 2 min. To 500 mL of the supernatant, 1.0 mL of Nessler’s reagent (Sigma-Aldrich ) was RT-PCR Analysis of Transgenic Plants added. The samples were incubated at room temperature for RNA was isolated from 100 mg of cassava roots using the Qiagen 10 min to allow color development. For blank samples, TCA RNeasy Plant Mini kit (Qiagen, Inc., Valencia, CA, USA). To was added at time 0. Absorbance was read at 480 nm and the quantify RNA, absorbance was measured at 260 and 280 nm amount of ammonia produced was estimated using a standard (Sambrook et al., 1989). Concentrations of RNA were calculated curve. based on absorbance at 260 nm. RNA purity was judged based on the 260/280 ratio where pure RNA has a value of 2. Prior to cDNA Nitrate Reductase Activity synthesis, the RNA was treated to remove DNA contamination Nitrate reductase was assayed using the method described using the Promega DNAse treatment (Promega Corporation, by the Nitrate Elimination, Co., Inc. (NECi ) with some Madison, WI, USA). About 2–10 mg of RNA was used for modifications. Crude protein was extracted from the ground cDNA synthesis using the Qscript cDNA kit (Quanta Biosciences, tissue using an extraction buffer containing 100 mM 3-(N - Gaithersburg, MD, USA). morpholino)propanesulfonic acid (MOPS) pH 7.5, 1.0 mM The cDNA was used to check for the expression of the EDTA and 10 mM L-cysteine. PVPP [1% (w/v)] was added to transgene by RT-PCR. For CAS, the forward primer was the grinding mixture during extraction. Four mL of extraction CATGCTATCACAGGCAATGG while the reverse primer was buffer was used per gram fresh weight plant tissue. The GCCAAATGTTTG AACGATCGG. For NIT4 the forward homogenate was passed through four layers of cheesecloth and primer was GCACTTGAGGGTGGATGTTT and the reverse centrifuged at 21000 g for 5 min at 4 C. The supernatant was was GCCAAATGTTTG AACGATCGG. For tubulin control, used in subsequent assays. Approximately 100–200 mg of the the primers TubF (TATATGGCC AAGTGCGATCCTCGACA) extracted protein was added to 800 mL of substrate solution and TubR (TTACTCTTCATAATCCTTCTCAAGGG) were used (30 mM potassium nitrate in 100 mM MOPS, pH 7.5). The as positive controls for the PCR reaction. The PCR reaction reaction was started by adding 100 mL of 25 mM NADH TM conditions were based on Choice Taq DNA polymerase from and stopped after 10 min by adding 100 mL of 100 mM zinc Denville Scientific, Inc. acetate. After centrifuging at 22000 g for 2 min, 500 mL of the supernatant was add to a fresh 1.5 mL tube. To this, Plant Growth in the Greenhouse 500 mL (an equal volume to the volume of supernatant) Greenhouse plants were grown as described by Zidenga et al. was added of each of the color development reagents [1% (2012). (w/v) sulfanilamide in 1.5 N HCl and 0.02% N -(napththyl)- ethylenediaminehydrochloride]. Samples were left at room Measurement of Yield Parameters temperature for 10–20 min to allow full color development. Plants were grown in the greenhouse in rectangular trays with Absorbance was read at 540 nm. Nitrite concentration was only six plants per tray. Greenhouse grown plants were harvested estimated using a standard curve prepared by diluting known after 4–8 months of growth and fresh weight measurements were concentrations of nitrite in 500 mL and adding the color taken on all the tuberous roots. development reagents. 2 4 www.sigmaaldrich.com/ http://abrc.osu.edu/ 3 5 www.nitrate.com www.densci.com Frontiers in Plant Science | www.frontiersin.org 4 February 2017 | Volume 8 | Article 220 fpls-08-00220 February 22, 2017 Time: 15:3 # 5 Zidenga et al. Cyanogen Metabolism in Cassava Roots not been firmly established (Chew, 1973; Miller and Conn, Free and Hydrolyzed Amino Acid 1980; Hatzfeld and Saito, 2000). Relevant to this observation Extraction and Analysis it has been demonstrated that rhodanese activity does not Free amino acid extraction was based on the method by Hacham correlate with cyanogenic potential (Miller and Conn, 1980). et al. (2002). Approximately 150 mg of tissue was ground in liquid It has been suggested, however, that thiocyanate, derived nitrogen and homogenized by motor pestle with 600 mL of water: from rhodanese activity could be hydrolyzed by a thiocyanate chloroform: methanol (3:5:12 v/v). After centrifugation at 21000 g hydrolase to generate ammonia and carbonyl sulfide in for 2 min, the supernatant was collected and the residue was re- plants, but this activity has yet to be confirmed (Yu et al., extracted with 600 mL of water: chloroform: methanol followed 2012). by centrifugation. Supernatants from the first and second To determine whether rhodanese plays a role in cyanide extraction were combined in a 2 mL tube. 300 mL of chloroform detoxification in cassava, we measured rhodanese activity and 450 mL of water were added followed by centrifugation in leaves and tuberous roots of the cassava. The average at 21000 g for 2 min. The upper water: methanol phase was rhodanese activity detected in cassava leaves was relatively collected and transferred to a fresh tube, dried by speed-vac low at 4.19 mmol/mg protein/min (Figure 2A). Significantly, and dissolved in 200 mL of water. Detection of free amino we detected no rhodanese activity in cassava tuberous roots acids was performed by the Proteomics and Mass Spectrometry (Figure 2A). Since cyanogen accumulation occurs in roots, Facility at the Donald Danforth Plant Science Center using the the lack of rhodanese activity in these tissues suggests that AccQTag system. Protein hydrolysis was carried out as described rhodanese is not involved in cyanide metabolism in roots. As by Narayanan et al. (2011). Samples were hydrolyzed for 24 h previously discussed, linamarin is produced in the leaves and at 116 C in 6 N HCl containing 0.5% (v/v) phenol, dried and transported to the roots where it presumably provides reduced resuspended in 20 mM HCl before derivatization with the AccQ- nitrogen for protein synthesis (Bediako et al., 1981; Ramanujam tag reagent and subsequent separation by ACQUITY UPLC and Indira, 1984; Siritunga and Sayre, 2003; Siritunga et al., System (Waters, Milford, MA, USA) according to manufacturer’s 2004). Since no rhodanese activity was detected in cassava instructions. roots, we hypothesized that cyanide released from linamarin breakdown is preferentially assimilated via CAS. To test this Analysis of IAA hypothesis, we analyzed CAS activity in cassava tuberous roots Indole acetic acid (IAA) analysis was carried out using an and leaves. If cyanogens are a source of reduced nitrogen for LC–MS/MS analysis method developed and performed by the protein synthesis in roots, we would expect that CAS activity Proteomics and Mass Spectrometry Facility at the Donald (unlike rhodanese activity) would be higher in cassava roots Danforth Plant Science Center. The method is similar to Chen than leaves. In cassava tuberous roots, average CAS activity et al. (2009), but modified to include additional plant hormone was 13.7 mmol HS/mg protein/min, compared to 5.1 in leaves, species. an approximately threefold higher CAS activity in roots than leaves (Figure 2B). In contrast, in potato, a non-cyanogenic Statistical Analysis plant, leaf CAS activity (0.04 mmol H S per mg protein Statistical analysis was carried out using GraphPad Prism per min) was substantially lower than in cassava and twofold software package . Student’s t-test and one-way ANOVA with greater than potato tuber activity (0.02 mmol H S per mg Dunnett’s Multiple Comparison test for comparing multiple lines protein per min). These rates in potato correlate well with with the control were used. All analyses for significant differences levels required for CN detoxification associated with ethylene were performed at P 0.05. biosynthesis (Maruyama et al., 2001). Similar differences (3X) in CAS activity between cassava roots and leaves had previously been reported by Nambisan and Sundaresan (1994) and Elias RESULTS et al. (1997b). If cyanide assimilation occurs through the CAS, then it would Cyanide Metabolism in Cassava Roots be expected that additional enzymes in the cyanogen assimilation Occurs via b-Cyanoalanine Synthase pathway would have enzymatic activities commensurate with CAS activity. Thus, we assayed nitrilase activity, the enzyme Cyanide is detoxified in plants either through condensation involved in the conversion of cyanoalanine, the product of with cysteine, catalyzed by CAS, or via condensation with CAS activity, into aspartate, asparagine, and ammonia. The thiosulfate, derived from sulfur metabolism, and catalyzed by average nitrilase activity in cassava leaves was 12.8 mmol the enzyme rhodanese (Figure 1; Hatzfeld and Saito, 2000). ammonia/mg protein per min while that in the roots was Cyanide detoxification by rhodanese is prominent in mammals where the thiocyanate is excreted in urine (Shibamoto and 16.4 mmol ammonia/mg protein per min (Figure 2C). These apparent nitrilase activity levels were similar to those observed Bjeldanes, 1993). The use of thiosulfate as a cyanide antidote is based on the rhodanese activity (Shibamoto and Bjeldanes, for CAS in cassava roots. In addition, root nitrilase activity was about 1.3 times (or 30%) greater than in leaves. These 1993; Shepherd and Velez, 2008). In plants, however, the observations are consistent with root cyanide assimilation by relationship between rhodanese and cyanide metabolism has CAS and nitrilase leading to amino acid synthesis (Siritunga and http://www.graphpad.com/prism/Prism.htm Sayre, 2003). Frontiers in Plant Science | www.frontiersin.org 5 February 2017 | Volume 8 | Article 220 fpls-08-00220 February 22, 2017 Time: 15:3 # 6 Zidenga et al. Cyanogen Metabolism in Cassava Roots FIGURE 2 | Cyanide metabolizing enzymes (except rhodanese) in cassava have higher activities in roots compared to leaves. (A) Activities of rhodanese (in mmol thiocyanate/mg protein/minute) in tuberous roots and leaves of 8 months old cassava plants grown under glasshouse conditions. (B) CAS activity (in mmol hydrogen sulfide (H S) per mg protein per minute) in 8 months old cassava (C). Nitrilase (cyanoalanine hydratase) enzyme activities (in mmol ammonia/mg protein/min) in roots and leaves of in vitro cassava plants at 5 weeks. (D) Analysis of nitrate reductase activity (mmol nitrite/mg protein/min) in wild-type (WT) and transgenic low cyanogen (Cab1-1, Cab1-2, and Cab1-3; Siritunga and Sayre, 2003) lines. The assay was conducted on 5 weeks old in vitro plants. The results of all experiments are the averages from biological four trials. Statistical analysis was done by one-way ANOVA with Dunnett’s Multiple Comparison Test. Asterisks indicate significant difference at P  0.05. FIGURE 3 | (A) Gene cassette used in transforming cassava with b-cyanoalanine synthase (CAS) and nitrilase genes. The construct was assembled in a modified pBI121 plasmid where the gene of interest (GOI) was driven by a class I patatin promoter for root-specific expression. (B) CAS transcripts in wild-type (WT) and transgenic (PCAS1-4) cassava lines as detected by RT-PCR. RNA was extracted from 100 mg of 5 week-old in vitro cassava roots. RT-PCR was performed using primers for the CAS insert with tubulin as the internal control. (C) In vitro growth comparison of transgenic CAS-overexpressing plants (PCAS1-4) and wild-type (TMS 60444) plants after 3 weeks. (D) Expression of CAS increased the activity of the enzyme in cassava roots. The activity of CAS was correlated to reduced growth and root development (C,E). (E) Root development in in vitro transgenic CAS plants grown in MS medium for 3 weeks. Data are averages of n D 20. Statistical analysis was done by one-way ANOVA with Dunnett’s Multiple Comparison Test. All transgenics were significantly different from wild-type at P  0.05. Frontiers in Plant Science | www.frontiersin.org 6 February 2017 | Volume 8 | Article 220 fpls-08-00220 February 22, 2017 Time: 15:3 # 7 Zidenga et al. Cyanogen Metabolism in Cassava Roots in transgenic PCAS plants (with the exception of PCAS4, the Compensation for Reduced Cyanogens line with the enzyme activity closest to wild-type) relative to in Low Cyanide Transgenic Plants and wild-type plants. The transgenic plants exhibiting the highest the Impact of Elevating Cyanide CAS activity, had the lowest fresh weight. These data suggest Assimilatory Enzymes on Amino Acid that overexpression of CAS impairs growth and development in and Protein Levels cassava plants. To determine if CAS overexpression impacted amino acid Previously, transgenic plants having low root cyanogen levels pool sizes in cassava roots, total free amino acids and those were generated by suppressing leaf linamarin biosynthesis obtained from hydrolyzed proteins were assessed. There was through antisense expression of CYP79D1/D2 genes encoding a significant difference (at P  0.05) in total amino acids two cytochrome P450s that catalyze the first dedicated step in between wild-type and transgenic plants (Table 1). In addition, cyanogenic glycoside synthesis, resulting in a 99% reduction total root protein content was increased up to 9.3% in CAS in root linamarin levels relative to wild-type plants (Andersen transgenic plants relative to wild-type (Table 2) as were total et al., 2000; Siritunga and Sayre, 2003). These results confirmed that cyanogenic glycosides were transported from leaves to free and protein amino acids including most notably arginine, aspartate, and glutamate in the PCAS1 transformant, which had roots, as demonstrated also in rubber tree (Selmar et al., 1988). We have used the transgenic low root linamarin the lowest increases in CAS activity (Table 1). Since aspartate, glutamate, and arginine are entry points for reduced nitrogen plants as tools to study cyanide metabolism in cassava. As suggested by biochemical assays described above, cyanide assimilation and transfer, respectively, into amino acids it is not unexpected that their levels would be increased by enhancing assimilation via CAS allows for entry of cyanide into amino cyanide assimilation into amino acids by CAS. acid synthesis. To determine if disruption of linamarin metabolism impacted the activity of select enzymes involved in nitrogen metabolism, we compared root nitrate reductase TABLE 1 | Hydrolyzed and free amino acid content of 4 months-old activity between low (transgenic) and high cyanogen cassava transgenic and wild-type cassava tuberous roots in pmole/mg dry weight. plants. Nitrate reductase activity is highly regulated in plants, is up-regulated in plants with reduced nitrogen availability, Amino acid WT PCAS1 PCAS2 and repressed in plants with sufficient ammonia (Campbell, CyA 1.30  0.05 1.49  0.19 1.40  0.08 1999). Wild-type cassava roots were observed to have an His 0.56  0.17 0.7  0.22 0.7  0.2 average nitrate reductase activity of 1.78 mmol nitrite/mg Ser 1.63  0.4 2.08  0.42 2.19  0.28 protein/min while low cyanogen (Cab1, Figure 2D) lines a ab Arg 1.30  0.21 2.68  0.54 1.83  0.25 had nitrate reductase rates ranging from 4.5 to 5 mmol Gly 4.01  0.18 4.87  1.01 5.56  0.45 nitrite/mg protein/minute, or three times higher than wild-type a a Asp 6.56  0.78 9.12  0.92 8.43  0.86 (Figure 2D). These data suggest that when cyanogen synthesis MetS 1.64  0.25 1.74  0.3 1.86  0.11 is reduced, other root-based nitrogen assimilation pathways a a Glu 7.27  0.35 9.76  1.07 9.92  0.88 compensate. Thr 1.62  0.32 1.98  0.51 2.25  0.15 Thus, we hypothesized that enhancing cyanide assimilation Ala 3.93  0.28 4.49  1 4.96  0.34 via overexpressing enzymes in the CAS pathway (Figure 1) could Pro 2.05  0.17 2.41  0.56 2.67  0.3 result in elevated root total amino acids or protein levels. Lys 3.01  0.16 3.62  0.69 3.87  0.19 Four transgenic lines were generated overexpressing CAS Val 2.78  0.19 3.32  0.74 3.64  0.28 as confirmed by RT-PCR (Figures 3A,B). To determine if Ile 2.01  0.11 2.35  0.48 2.58  0.17 CAS overexpression resulted in increased enzyme activity, CAS Leu 3.0  0.16 3.51  0.73 3.89  0.26 enzyme activity was assessed. Root CAS activity in transgenic Phe 1.62  0.09 1.78  0.32 1.86  0.14 plants was elevated as much as twofold relative to wild-type roots a a Total 44.3 55.9 57.6 (Figure 3D). Unexpectedly, however, we observed reduced recovery of Superscript ‘a’ indicates significant difference from wild-type at P  0.05. Superscript ‘b’ indicates significant difference between the two transgenic lines transgenic plants overexpressing CAS relative to transgenics at P 0.05. Values are standard deviation. CyA, cysteic acid (hydrolysis product expressing other genes of interest in cassava roots (e.g., Siritunga of cysteine); Asp, AspCAsn (due to hydrolysis of Asn to Asp); Glu, GluCGln (due to and Sayre, 2003; Ihemere et al., 2006; Zidenga et al., 2012), hydrolysis of Gln to Glu). suggesting negative effects of CAS overexpression. Of the recovered CAS transgenic lines, the lines with higher CAS activity TABLE 2 | Total protein comparison in 4 months-old wild-type (WT) and showed more stunted growth (Figure 3C). Analysis of transgenic transgenic cassava tuberous roots. PCAS plants indicated poor root development compared to wild- Plant line WT PCAS1 PCAS2 type plants (Figure 3E), especially during the first 4 weeks of growth. Wild-type plants had an average of 4 roots per plant, a a b Total protein 16.73  0.56 17.36  0.24 18.28  0.17 while PCAS transgenics ranged from 1 to 3 roots per plant. (mg/mg dry weight) Root development was poorest in PCAS2 and PCAS3. Poor root Same letter superscript indicates no significant difference at P  0.05. Different development was also associated with reduced fresh weight and letter superscripts (a/b) indicate significant difference. Measurements were poor growth. There was a 2 to 4-fold decrease in fresh weight performed in biological triplicates. Values are  standard deviation. Frontiers in Plant Science | www.frontiersin.org 7 February 2017 | Volume 8 | Article 220 fpls-08-00220 February 22, 2017 Time: 15:3 # 8 Zidenga et al. Cyanogen Metabolism in Cassava Roots As previously described, we observed elevated nitrilase activity et al., 2001). The branching phenotype in our transgenic plants is in cassava roots relative to leaves. Thus, we hypothesized that similar to that observed upon decapitation of apical meristems NIT overexpression would increase assimilation of cyanide into which removes the inhibition of lateral bud growth (apical amino acids (Figure 1). Transformation of cassava plants with dominance) resulting in increased branching. Decapitation the Arabidopsis NIT4 gene was carried out as described in Section results in reduced IAA levels, the most common bioactive form “Materials and Methods.” Transgenic lines were confirmed by of auxin (Ferguson and Beveridge, 2009). However, while a RT-PCR (Figure 4A). To determine whether overexpression of significant amount of root auxin is derived from the shoot, it is NIT4 in cassava roots increased nitrilase activity, enzyme assays now known that roots are also sites of auxin biosynthesis (Ross were carried out as previously described. Three transgenic lines et al., 2006). were used for this analysis. Total nitrilase activity in transgenic Since nitrilases are also known to be involved in auxin lines was increased as much as fourfold relative to wild-type biosynthesis, we hypothesized that NIT4 overexpression affected plants (Figure 4B). Interestingly, free amino acid pool sizes auxin metabolism in cassava tuberous roots leading to the increased as much as 50% in plants with NIT activities less than increased branching phenotype. To test this hypothesis, we 3X wild-type levels but dropped to as much as 50% of wild- measured IAA concentrations in cassava tuberous roots of type levels in plants having fourfold increases in NIT activity 4 months-old greenhouse grown wild-type and NIT4 transgenic (Figure 4E). These results suggest that NIT4 may have pleiotropic plants. Transgenic cassava plants expressing NIT4 had up to effects with super-elevated NIT4 activity having negative impacts 50% less root IAA compared to wild-type plants (Figure 4C). on plant metabolism. To assess whether there were additional Wild-type roots, with16 ng/g fresh weight, had twice the level phenotypic effects of NIT4 overexpression, we assessed their of free IAA as the highest NIT4 expressing transgenic plants. performance in the greenhouse. Interestingly, PNIT plants IAA concentrations also decreased in the leaves, with PNIT2 displayed an increased branching phenotype compared to wild- having 50% less than wild-type levels (Figure 4C). The observed type plants (data not shown). In addition, in the early stages reduction in IAA concentration is consistent with the increased of growth (up to 8 weeks) greenhouse-grown PNIT plants root branching but is inconsistent with the enhanced root NIT4 tended to have more fibrous root development compared to activity if NIT4 is expected to impact IAA levels (O’Reilly and wild-type plants. These phenotypic traits mimicked potential Turner, 2003). The role of nitrilases in auxin biosynthesis is still morphological responses associated with alterations in ethylene not clearly defined (Ljung et al., 2002; Mano and Nemoto, 2012). or auxin levels. It is known that auxin promotes cell division in All four Arabidopsis nitrilases have been shown to convert indole- root pericycle cells, which leads to lateral root formation, but 3-acetonitrile (IAN) to the plant hormone IAA, but NIT4 appears inhibits cell division in lateral meristems of the shoot, resulting in to be mainly involved in cyanide metabolism (Bartling et al., 1992, the inhibition of lateral bud growth, or apical dominance (Rogg 1994; Schmidt et al., 1996; Normanly et al., 1997; Piotrowski et al., FIGURE 4 | (A) NIT4 transcript abundance by RT-PCR. RNA was extracted from 100 mg of 5 week-old in vitro cassava roots. RT-PCR was performed using primers for the NIT4 insert while tubulin primers were used for the control. (B) Expression of Nitrilase increases cyanoalanine hydrolase activity in cassava roots. Rates of conversion of cyanoalanine to ammonia were determined for n D 4. (C) IAA analysis in 4 months-old greenhouse-grown wild-type and transgenic cassava tuberous roots and leaves showing decreased IAA in transgenic plants overexpressing NIT4. (D) Storage root fresh weight per plant in WT and transgenic NIT4 (PNIT2, 4, and 6) lines. (E) Total free amino acid analysis in wild-type and NIT4 transgenic lines. The data are averages of three biological trials. Frontiers in Plant Science | www.frontiersin.org 8 February 2017 | Volume 8 | Article 220 fpls-08-00220 February 22, 2017 Time: 15:3 # 9 Zidenga et al. Cyanogen Metabolism in Cassava Roots 2001; Ljung et al., 2002). In our experiments, overexpression of CAS activity and 1.3-times higher nitrilase activity in cassava NIT4, while increasing cyanide assimilation into amino acids, was roots than in shoots, consistent with cyanide assimilation by associated with reduced IAA levels. It is not immediately clear CAS. As a corollary to this hypothesis, it would be predicted how NIT4 overexpression in roots may have decreased IAA levels, that competing cyanide assimilation pathways that do not lead to however, we observed additional phenotypic impacts of NIT4 amino acid synthesis would have low activity in cassava roots. It overexpression including reduced storage root yield in transgenic was observed that rhodanese activity was not detected in cassava lines having the highest nitrilase activity (Figures 4B,D). Finally, roots. Thus, there is no apparent competing pathway for cyanide elevated total free amino acids were only observed in NIT4 assimilation in cassava roots. There was, however, no significant transgenics expressing the lowest levels of increased total NIT difference in apparent rhodanese and CAS catalytic turnover activity relative to wild-type (Figures 4D,E). Plants with the activity in cassava leaves suggesting that a substantial portion of highest NIT activity had the lowest total free amino acid pool CN produced in damaged leaves may be detoxified by rhodanese sizes. Thus, the regulation of amino acid accumulation by NIT is (Bediako et al., 1981; Ramanujam and Indira, 1984; Siritunga and complicated with only a narrow window of enhanced NIT activity Sayre, 2003; Siritunga et al., 2004). yielding enhanced amino acid accumulation. If cyanogens are a significant source of reduced nitrogen in plants, a reduction in linamarin synthesis would be expected to impact nitrogen assimilation. Using previously generated low cyanogen plants (Siritunga and Sayre, 2003), we observed DISCUSSION elevated nitrate reductase activity in low cyanogen plants relative Cyanogenic plants produce sufficient levels of cyanogens to wild-type plants suggesting that loss (99%) of cyanogens is to provide protection against a variety of herbivores and compensated by increased nitrate reductase activity in roots. pathogens (Nahrstedt, 1985; Jones, 1998). It is this function of However, elevated root nitrate reductase activity in cassava cyanogens that has received the greatest attention since it can plants with reduced root linamarin is not sufficient to support potentially impact human health. In addition to their defensive plant growth in the absence of supplemental ammonia, further role, cyanogenic glycosides have been proposed to function supporting the central role of linamarin turnover in cassava roots as transportable forms of reduced nitrogen in some plants as a source of reduced nitrogen for amino acid and protein including; rubber tree, cassava and sorghum (Selmar et al., 1988; synthesis (Siritunga and Sayre, 2004). Poulton, 1990; Siritunga and Sayre, 2007; Gleadow and Møller, Over 60% of the reduced nitrogen in stem phloem exudates 2014). Cyanogenic plants can allocate a substantial amount of cassava is in the form of linamarin (Calatayud and Le of nitrogen to cyanogenic glycoside accumulation. Eucalyptus Ru, 1996). Thus, reducing the cyanogenic potential of cassava cladocalyx allocates up to 20% of leaf nitrogen to accumulation of presents a challenge; while toxic to humans, it has an the cyanogenic glycoside, prunasin (Gleadow et al., 1998). Since important role in primary nitrogen assimilation. Redirecting cyanogen nitrogen is fully reduced, it does not require additional cyanogen metabolism toward amino acid and protein synthesis, reduction steps to be assimilated into amino acids. To convert particularly HNL synthesis in roots to be a nitrogen sink as cyanogen nitrogen into amine nitrogen requires the release and well as to simultaneously accelerate residual cyanogen (acetone rapid assimilation of cyanide from cyanogens. It is assumed that cyanohydrin) turnover during processing, is therefore a more generalized b-glucosidases generate cyanohydrins which then viable option for reducing steady-state linamarin pool sizes, spontaneously decompose to yield cyanide. Cyanide assimilation elevating protein content and the nutritional status of cassava would then occur via CAS (Figure 1) allowing cyanogens to roots than blocking linamarin synthesis to reduce cyanide provide reduced nitrogen for protein synthesis (Nartey, 1969; toxicity. Regardless, addressing cyanogen toxicity in cassava roots Selmar et al., 1988; Siritunga et al., 2004; Ebbs et al., 2010). while supporting active cyanide assimilation into protein remains To test this hypothesis, we assessed both the activity of a complex challenge. CAS and nitrilase, leading to aspartate and asparagine synthesis, To determine if cyanogen assimilation into proteins could in cassava roots. In addition, we assessed alternate nitrogen be enhanced by elevating cyanide assimilating enzymes, we assimilation pathways in plants engineered to have very low overexpressed CAS and NIT4. Overexpression of CAS in cassava cyanogen levels. The discovery of genes encoding the cytochrome roots successfully lead to increased CAS activity, elevated total P450s (CYP79D1 and CYP79D2) that catalyze the first-dedicated amino acid pool sizes and increased protein content (C9% step in linamarin synthesis (Andersen et al., 2000; Siritunga and relative to wild-type) consistent with elevated CAS activity Sayre, 2003; Siritunga et al., 2004) made it possible to design enhancing cyanide assimilation into amino acids. However, a transgenic approach to reduce cyanogens in cassava. Cassava there were unintended consequences of CAS overexpression. lines in which linamarin biosynthesis was inhibited in the roots CAS overexpression was associated with poor root development had wild-type linamarin levels in the roots while those in which and reduced total fresh weight. At present the mechanism linamarin biosynthesis was inhibited in the leaves had a 99% by which plant growth is altered in CAS overexpressors reduction of root linamarin levels (Siritunga and Sayre, 2003). is unknown. However, free cyanide has been implicated in This provided confirmation of the leaf as the primary source plant growth regulation (Smith and Arteca, 2000; Siegien for root linamarin. We hypothesized that enzymes involved in and Bogatek, 2006; Garcia et al., 2010; Xu et al., 2012). cyanide assimilation would have preferentially higher activities Recently, Garcia et al. (2010) have shown that mitochondrial in the roots compared to the leaves. We detected 3-times higher CAS activity is essential for maintaining low cyanide levels Frontiers in Plant Science | www.frontiersin.org 9 February 2017 | Volume 8 | Article 220 fpls-08-00220 February 22, 2017 Time: 15:3 # 10 Zidenga et al. Cyanogen Metabolism in Cassava Roots essential for root hair development. CAS Arabidopsis mutants 1994; Schmidt et al., 1996). Arabidopsis NIT4, however, has which accumulate elevated cyanide levels were shown to be been reported to not only to have high substrate specificity for defective in root-hair development. This phenotype was rescued cyanoalanine, but also to not recognize IAN as a substrate in by addition of hydroxocobalamin, a cyanide antidote (Garcia the production of IAA (Piotrowski et al., 2001; O’Reilly and et al., 2010). It appears from our studies and studies discussed Turner, 2003). However, the overexpression of NIT4 in our above that a threshold level of cyanide may be required for studies lead to reduced IAA levels, contrary to expectations, proper root development. Reductions in cyanide levels (as suggesting that cyanoalanine turnover by NIT4 may indirectly expected in transgenic lines with the highest CAS activity) impact IAA synthesis. At present the mechanism by which as well as elevated cyanogen levels (Arabidopsis CAS mutant) cyanoalanine or cyanide impacts IAA synthesis is not known. appear to both negatively impact root development. One possible However, cyanide is known to stimulate ethylene synthesis means by which cyanide may impact root development is which in turn stimulates IAA synthesis and transport (Smith through regulation of ethylene, and as discussed below IAA and Arteca, 2000; Normanly, 2007). The complex interplay production. Previously, Smith and Arteca (2000) have shown between ethylene and IAA in regulating plant development that low levels (1 mm) of cyanide enhance transcription of with altered cyanide levels may account for impaired root 1-aminocyclopropane-1-carboxylic-acid synthase, the enzyme growth in transgenic plants inconsistent with reduction in root which mediates the first-dedicated step in ethylene biosynthesis. IAA levels but consistent with a potential increase in ethylene Overexpression of Arabidopsis NIT4 in cassava roots was levels. shown to increase nitrilase activity and alter amino acid pool sizes. However, transgenic plants with the highest NIT activity (>3X wild-type rates) had the lowest total amino acids (50% AUTHOR CONTRIBUTIONS lower than wild-type), while transgenics having less than a threefold increase in NIT activity had as much as a 50% RS, DS, and TZ were all involved in study design, data acquisition increase in total amino acids relative to wild-type. These results and analysis, as well as manuscript draft and revision. suggest that there is a complex interplay between NIT enzyme activity and phenotypic response. This is best illustrated by the observed impact of elevated NIT4 activity on cassava growth and FUNDING development. 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Nat. 10.1101/cshperspect.a001594 Biotechnol. 14, 726–730. doi: 10.1038/nbt0696- 726 Frontiers in Plant Science | www.frontiersin.org 11 February 2017 | Volume 8 | Article 220 fpls-08-00220 February 22, 2017 Time: 15:3 # 12 Zidenga et al. Cyanogen Metabolism in Cassava Roots Tylleskar, T. (1994). The association between cassava and the paralytic disease Zidenga, T., Leyva-Guerrero, E., Moon, H., Siritunga, D., and Sayre, R. T. (2012). Konzo. Acta Hortic. 375, 331–339. doi: 10.17660/ActaHortic.1994.375.33 Extending cassava root shelf life via reduction of reactive oxygen species Wang, S., and Volini, M. (1968). Studies on the active site of rhodanese. J. Biol. production. Plant Physiol. 159, 1396–1407. doi: 10.1104/pp.112.200345 Chem. 243, 5465–5470. White, W. L. B., Arias-Garzon, D. I., McMahon, J. M., and Sayre, R. T. (1998). Conflict of Interest Statement: The authors declare that the research was The role of hydroxynitrile lyase in root cyanide production. Plant Physiol. 116, conducted in the absence of any commercial or financial relationships that could 1219–1225. doi: doi:10.1104/pp.116.4.1219 be construed as a potential conflict of interest. Xu, F., Zhang, D.-W., Zhu, F., Tang, H., Lv, X., Cheng, J., et al. (2012). A novel role for cyanide in the control of cucumber (Cucumis sativus L.) seedlings Copyright © 2017 Zidenga, Siritunga and Sayre. This is an open-access article response to environmental stress. Plant. Cell Environ. 35, 1983–1997. doi: 10. distributed under the terms of the Creative Commons Attribution License (CC BY). 1111/j.1365- 3040.2012.02531.x The use, distribution or reproduction in other forums is permitted, provided the Yu, X. Z., Zhang, F. Z., and Li, F. (2012). Phytotoxicity of thiocyanate to rice original author(s) or licensor are credited and that the original publication in this seedlings. Bull. Environ. Contam. Toxicol. 88, 703–706. doi: 10.1007/s00128- journal is cited, in accordance with accepted academic practice. No use, distribution 012- 0545- 7 or reproduction is permitted which does not comply with these terms. Frontiers in Plant Science | www.frontiersin.org 12 February 2017 | Volume 8 | Article 220 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Frontiers in Plant Science Pubmed Central

Cyanogen Metabolism in Cassava Roots: Impact on Protein Synthesis and Root Development

Frontiers in Plant Science , Volume 8 – Feb 24, 2017

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Copyright © 2017 Zidenga, Siritunga and Sayre.
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fpls-08-00220 February 22, 2017 Time: 15:3 # 1 ORIGINAL RESEARCH published: 24 February 2017 doi: 10.3389/fpls.2017.00220 Cyanogen Metabolism in Cassava Roots: Impact on Protein Synthesis and Root Development 1 2 1,3 Tawanda Zidenga , Dimuth Siritunga and Richard T. Sayre 1 2 Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USA, Department of Biology, University of Puerto Rico, Mayaguez, PR, USA, New Mexico Consortium, Los Alamos, NM, USA Cassava (Manihot esculenta Crantz), a staple crop for millions of sub-Saharan Africans, contains high levels of cyanogenic glycosides which protect it against herbivory. However, cyanogens have also been proposed to play a role in nitrogen transport from leaves to roots. Consistent with this hypothesis, analyses of the distribution and activities of enzymes involved in cyanide metabolism provides evidence for cyanide assimilation, derived from linamarin, into amino acids in cassava roots. Both b-cyanoalanine synthase (CAS) and nitrilase (NIT), two enzymes involved in cyanide assimilation to produce asparagine, were observed to have higher activities in roots compared to leaves, consistent with their proposed role in reduced nitrogen assimilation. In addition, rhodanese activity was not detected in cassava roots, indicating that this competing means for cyanide metabolism was not a factor in cyanide detoxification. In contrast, Edited by: leaves had sufficient rhodanese activity to compete with cyanide assimilation into Henrik Toft Simonsen, Technical University of Denmark, amino acids. Using transgenic low cyanogen plants, it was shown that reducing root Denmark cyanogen levels is associated with elevated root nitrate reductase activity, presumably to Reviewed by: compensate for the loss of reduced nitrogen from cyanogens. Finally, we overexpressed Roslyn Gleadow, Arabidopsis CAS and NIT4 genes in cassava roots to study the feasibility of enhancing Monash University, Australia Kirsten Jørgensen, root cyanide assimilation into protein. Optimal overexpression of CAS and NIT4 resulted University of Copenhagen, Denmark in up to a 50% increase in root total amino acids and a 9% increase in root *Correspondence: protein accumulation. However, plant growth and morphology was altered in plants Tawanda Zidenga tawanda@lanl.gov overexpressing these enzymes, demonstrating a complex interaction between cyanide metabolism and hormonal regulation of plant growth. Specialty section: This article was submitted to Keywords: cassava, linamarin, cyanide, cyanogen, b-cyanoalanine synthase, nitrilase, auxin, ethylene Plant Biotechnology, a section of the journal Frontiers in Plant Science INTRODUCTION Received: 08 November 2016 Accepted: 06 February 2017 Cyanide is ubiquitous in nature and as such most eukaryotic organisms have developed Published: 24 February 2017 mechanisms for its detoxification. For example, all plants produce some level of cyanide as a Citation: byproduct of the ethylene biosynthesis (Peiser et al., 1984; Dong et al., 1992). Additionally, Zidenga T, Siritunga D and Sayre RT some plants are highly cyanogenic including; cassava, bitter almonds, and rubber (Poulton, 1990; (2017) Cyanogen Metabolism Gleadow and Møller, 2014). The leaves and roots of cassava plants may accumulate between in Cassava Roots: Impact on Protein 200 and 1,300 mg CN equivalents/kg dry weight (Siritunga and Sayre, 2004). Generation of free Synthesis and Root Development. cyanide from cyanogenic compounds would obviously have toxic consequences, but generally Front. Plant Sci. 8:220. doi: 10.3389/fpls.2017.00220 does not happen in intact plants due to the physical separation of the cyanogenic compounds Frontiers in Plant Science | www.frontiersin.org 1 February 2017 | Volume 8 | Article 220 fpls-08-00220 February 22, 2017 Time: 15:3 # 2 Zidenga et al. Cyanogen Metabolism in Cassava Roots from the enzymes that degrade them (Kojima et al., 1979; detoxification pathway results in the assimilation of cyanide into Poulton, 1990; McMahon et al., 1995; Maruyama et al., 2001). amino acid biosynthesis pathways and is present in all higher In cassava, the cyanogenic glycoside linamarin, is stored in plants thus far examined (Figure 1; Blumenthal et al., 1968; Miller vacuoles while its corresponding b-glucosidase, linamarase, and Conn, 1980). is localized to the cell wall and laticifers (Mkpong et al., In this study, we use a combination of biochemical 1990; McMahon et al., 1995; Elias et al., 1997a). Tissue assays and transgene expression studies to demonstrate that disruption, e.g., during mechanical damage, initiates hydrolysis cyanide metabolism in roots most likely occurs via the CAS of linamarin by the generalized b-glucosidase, linamarase, pathway to produce amino acids. Furthermore, we show no to produce acetone cyanohydrin. Acetone cyanohydrin can competing rhodanese activity in roots, consistent with CAS spontaneously decompose to yield cyanide and acetone at mediated assimilation of cyanide into amino acids. Furthermore, pH > 5.0 or temperatures > 35 C, or is broken down by the overexpression of CAS and nitrilase was shown to lead to elevated enzyme hydroxynitrile lyase (HNL), which is expressed only in free amino acid pool sizes and root protein content under optimal cassava leaves and stems and not in roots (White et al., 1998). enzyme expression levels, indicating that these enzymes facilitate The cassava tuberous root, the main consumed part of the cyanide assimilation. These results provide insights into the role plant, is actually a true root, not a tuber, and thus cannot be used for cyanogenic glycosides in nitrogen metabolism in the roots, to vegetatively propagate the plant (Alves, 2002). Chronic, low- as well as possibilities for redirecting root linamarin toward level dietary cyanide exposure associated with the consumption protein production. Finally, alterations in cyanide assimilation of improperly processed cassava can lead to several health and other metabolic pathways associated with overexpressing disorders, including tropical ataxic neuropathy, characterized by CAS and NIT4 was shown to dramatically alter plant growth optic atrophy and an inability to coordinate muscle movements, and morphology. These results demonstrate a potential complex and a paralytic disorder known as Konzo (Osuntokun, 1981; interaction between cyanide and ethylene and auxin metabolism. Rosling, 1994; Tylleskar, 1994; Adamolekun, 2010). Individuals with these disorders typically have very low concentrations of MATERIALS AND METHODS sulfur amino acids in the blood (available sulfur is preferentially used in cyanide detoxification by rhodanese) and elevated Cassava Cultivars levels of plasma thiocyanate (Shibamoto and Bjeldanes, 1993; Cassava cultivar Manihot Columbia 2215 (MCol 2215) was used Adamolekun, 2010). for initial assays of leaf and root enzyme activity. Transformation For obvious health impact reasons, several efforts have been work was done using cultivar TMS 60444, selected due to ease directed toward lowering the cyanogens (a group of nitrile- of transformation. Comparative enzyme assays with transgenic containing plant secondary compounds that release hydrogen cyanide through enzymatic activity) in cassava. The major lines were performed using TMS 60444 as the wild-type control. Transgenic lines previously generated for low cyanogenesis cyanogens in cassava are linamarin and acetone cyanohydrin. Transgenic cassava lines having less than 1% of normal root (by selectively inhibiting cyanogenic glycoside synthesis in the leaves), Cab1-1, Cab1-2, and Cab1-3 used MCol 2215 as the cyanogen levels have been generated by inhibiting linamarin background (Siritunga and Sayre, 2003). biosynthesis in leaves (Siritunga and Sayre, 2003). However, these plants could not grow without ammonia and generally produced smaller tuberous roots compared to wild-type plants. It was Tissue Culture Propagation of Plant hypothesized that the poor performance of these plants was due Material to the suppression of cyanogen synthesis, an important source Cassava plants were propagated in vitro on the Murashige of reduced nitrogen for roots. A second approach to reduce and Skoog (MS) basal medium (Murashige and Skoog, 1962) cyanogen levels in cassava foods was to overexpress HNL to supplemented with Gamborg vitamins (Gamborg et al., 1968) accelerate the conversion of acetone cyanohydrin into cyanide, and 2% (w/v) sucrose. In vitro plants were propagated in growth which is then volatilized during processing (Siritunga et al., 2004; incubators at 28 C with a photoperiod of 16 h of light and Narayanan et al., 2011). Both acetone cyanohydrin and linamarin 8 h of darkness. Micropropagation of plant materials was done contribute to cyanide toxicity in poorly processed cassava foods, once every 5–8 weeks depending on the requirements of specific whereas cyanide does not due to its volatilization. Significantly, experiments (Siritunga and Sayre, 2003; Ihemere et al., 2006; by increasing root nitrogen sink strength by overexpressing HNL, Zidenga et al., 2012). it was observed that there was a 50–75% reduction in root steady- state linamarin levels suggesting that linamarin provided reduced Total Protein Extraction and Analysis nitrogen for protein synthesis. Total protein was measured using the Bradford assay according It had been proposed that some fraction of the cyanogens to the supplier’s (Invitrogen ) instructions. Protein was extracted transported from leave to roots were metabolized to generate free from root and leaf tissue of cassava using 50 mM Tris-HCl (pH cyanide which would then either be detoxified by the enzyme 8.5), 5 mM dithiothreitol and 1 mM EDTA. Extraction buffer was rhodanese (cyanide: thiosulfate sulfurtransferase) or assimilated used at a ratio of 5 mL of buffer per gram fresh tissue. Leaves into amino acids by CAS and nitrilase (Siritunga and Sayre, were ground in liquid nitrogen to a fine powder before adding 2007; Figure 1). CAS catalyzes the reaction between cyanide and cysteine to form b-cyanoalanine and hydrogen sulfide. This www.invitrogen.com Frontiers in Plant Science | www.frontiersin.org 2 February 2017 | Volume 8 | Article 220 fpls-08-00220 February 22, 2017 Time: 15:3 # 3 Zidenga et al. Cyanogen Metabolism in Cassava Roots FIGURE 1 | Model of cyanide assimilation in plants. CAS converts cyanide released from the cyanogen linamarin, to cyanoalanine plus hydrogen sulfide in the presence of cysteine. Cyanoalanine is converted by a nitrilase to asparagine, which is then converted to aspartate and ammonia by asparaginase. In this way, cyanide nitrogen can be incorporated into the free amino acid pool of the plant. An alternative pathway (red arrow) can potentially result in cyanide detoxified to thiocyanate by rhodanese. the buffer. Tuberous roots were blended together in the buffer in sulfide (Na S) was estimated from the absorbance using a the Magic Bullet MB1001 blender (Homeland Houseware LLC) standard curve prepared by adding 0.1 mL 30 mM ferric chloride for 30 s at 4 C. The ground extract was passed through four in 1.2 N HCl per mL of total reaction mixture followed by 0.1 mL layers of cheesecloth and centrifuged at 21000 g for 10 min. of 20 mM N,N dimethyl-p-phenylenediamine sulfate in 7.2 N The supernatant was used as the crude extract and measured for HCl to 1 mL of known concentrations of Na S. protein using the Bradford reagent with bovine serum albumin (BSA) as the standard. Rhodanese Activity Rhodanese activity was assayed as described by Wang and Volini Activity of b-Cyanoalanine Synthase (1968) with modifications. Cassava plant tissue was ground (CAS) in Cassava Tissue in liquid nitrogen using a motor and pestle and extracted in b-Cyanoalanine synthase activity was determined using 200 mM sodium phosphate buffer pH 7.8, 5.0 mM DTT, 5.0 mM the method described by Goudey et al. (1989) with some PMSF, 1.0 mM EDTA, 1.0 mM Na thiosulfate (to keep the modifications. Cassava tissue was ground in liquid nitrogen enzyme in a stable rhodanese-sulfur intermediate), 5 mM KCl using a motor and pestle and extracted in a buffer containing and 2% w/v polyvynilpolypyrrolidone (PVP). The homogenate 50 mM Tris-HCl, 5.0 mM dithiothreitol (DTT), 5.0 mM was passed through four layers of cheesecloth to remove debris phenylmethylsulfonyl fluoride (PMSF) and 1.0 mM EDTA at pH and centrifuged at 21000 g for 5 min at 4 C. The supernatant 8.0. The extract was filtered through four layers of cheesecloth was used in subsequent assays. To start the reaction, about 100– to remove debris. To 500 mL of substrate solution (10 mM 200 mg of protein was added to 0.5 mL of 50 mM NaCN and L-cysteine and 10 mM NaCN in 50 mM Tris buffer pH 8.0), 50 mM of Na thiosulfate in 200 mM sodium phosphate buffer and 100–200 mg of crude protein extract was added to make a (pH 7.8) to a total volume of 1.0 mL. The reaction was incubated total reaction volume of 1.0 mL. The optimum concentration at 30 C for 10 min and stopped by adding 0.5 mL 15% (v/v) of enzyme used was determined after testing different amounts formaldehyde. Absorbance at 460 nm was measured after adding of wild-type plant enzyme extract. The reaction was carried 2.5 ml of ferric nitrate reagent. The reagent was prepared by out for 10 min and stopped by adding 0.1 mL of 30 mM ferric adding 20 mL nitric acid (65%) to 60 mL of water, dissolving chloride in 1.2 N HCl followed by 0.1 mL of 20 mM N,N 10 g ferric nitrate: 9 H O and making up to a final volume of dimethyl-p-phenylenediamine sulfate in 7.2 N HCl. Absorbance 100 mL. The reaction was blanked using boiled (inactive) enzyme was measured at 640 nm after 10 min. Concentration of sodium extract. The standard curve used to estimate the concentration of Frontiers in Plant Science | www.frontiersin.org 3 February 2017 | Volume 8 | Article 220 fpls-08-00220 February 22, 2017 Time: 15:3 # 4 Zidenga et al. Cyanogen Metabolism in Cassava Roots thiocyanate was prepared using a range of known concentrations Transformation of Cassava with the of thiocyanate in the same volume as the reaction. b-Cyanoalanine Synthase and Nitrilase Genes Determination of Nitrilase Activity Construct Design Nitrilase activity was determined as described by Piotrowski et al. Constructs were assembled as previously described in Zidenga (2001) with some modifications. Cassava tissue was homogenized et al. (2012). A modified pBI121 plasmid with a 1.2 kb Solanum in an extraction buffer containing 50 mM Tris-HCl (pH 8.5), tuberosum class I patatin promoter was used for both constructs 2.0 mM EDTA, 8.0 mM cysteine, 2% (w/v) PVP plus and minus (Ihemere, 2002; Siritunga and Sayre, 2003). The CAS and NIT4 (for plant protein quantification) 0.1% (w/v) BSA. Tuberous (TAIR: At5g22300) genes of Arabidopsis were received from the greenhouse roots were homogenized in a blender for 5 s  2 s, Arabidopsis Biological Research Center in the pUNI51 vector while in vitro plant material was homogenized by grinding with and cloned into SmaI and SstI restriction sites of the modified liquid nitrogen in a motor and pestle. In all cases, the homogenate pBI121 binary plasmid (Figure 3A). was filtered through four layers of cheesecloth and centrifuged for 5 min at 22000 g. Approximately 400 mg of plant protein was Cassava Transformation used in the subsequent enzyme assay. Enzyme extracts were pre- Somatic embryogenesis, co-cultivation with Agrobacterium and warmed at 37 C for 2 min before being incubated with substrate plant regeneration were carried out as described by Zidenga (10 mM cyanoalanine in 50 mM Tris-HCl, pH 8.5 and 1.0 mM et al. (2012) while cassava transformation was done following DTT) for 10 min at 37 C. The total reaction volume was 1.0 mL. the method described by Taylor et al. (1996) with modifications The reaction was stopped by adding 100 mL of tricarboxylic (Zidenga et al., 2012). acid and centrifuged at 22000 g for 2 min. To 500 mL of the supernatant, 1.0 mL of Nessler’s reagent (Sigma-Aldrich ) was RT-PCR Analysis of Transgenic Plants added. The samples were incubated at room temperature for RNA was isolated from 100 mg of cassava roots using the Qiagen 10 min to allow color development. For blank samples, TCA RNeasy Plant Mini kit (Qiagen, Inc., Valencia, CA, USA). To was added at time 0. Absorbance was read at 480 nm and the quantify RNA, absorbance was measured at 260 and 280 nm amount of ammonia produced was estimated using a standard (Sambrook et al., 1989). Concentrations of RNA were calculated curve. based on absorbance at 260 nm. RNA purity was judged based on the 260/280 ratio where pure RNA has a value of 2. Prior to cDNA Nitrate Reductase Activity synthesis, the RNA was treated to remove DNA contamination Nitrate reductase was assayed using the method described using the Promega DNAse treatment (Promega Corporation, by the Nitrate Elimination, Co., Inc. (NECi ) with some Madison, WI, USA). About 2–10 mg of RNA was used for modifications. Crude protein was extracted from the ground cDNA synthesis using the Qscript cDNA kit (Quanta Biosciences, tissue using an extraction buffer containing 100 mM 3-(N - Gaithersburg, MD, USA). morpholino)propanesulfonic acid (MOPS) pH 7.5, 1.0 mM The cDNA was used to check for the expression of the EDTA and 10 mM L-cysteine. PVPP [1% (w/v)] was added to transgene by RT-PCR. For CAS, the forward primer was the grinding mixture during extraction. Four mL of extraction CATGCTATCACAGGCAATGG while the reverse primer was buffer was used per gram fresh weight plant tissue. The GCCAAATGTTTG AACGATCGG. For NIT4 the forward homogenate was passed through four layers of cheesecloth and primer was GCACTTGAGGGTGGATGTTT and the reverse centrifuged at 21000 g for 5 min at 4 C. The supernatant was was GCCAAATGTTTG AACGATCGG. For tubulin control, used in subsequent assays. Approximately 100–200 mg of the the primers TubF (TATATGGCC AAGTGCGATCCTCGACA) extracted protein was added to 800 mL of substrate solution and TubR (TTACTCTTCATAATCCTTCTCAAGGG) were used (30 mM potassium nitrate in 100 mM MOPS, pH 7.5). The as positive controls for the PCR reaction. The PCR reaction reaction was started by adding 100 mL of 25 mM NADH TM conditions were based on Choice Taq DNA polymerase from and stopped after 10 min by adding 100 mL of 100 mM zinc Denville Scientific, Inc. acetate. After centrifuging at 22000 g for 2 min, 500 mL of the supernatant was add to a fresh 1.5 mL tube. To this, Plant Growth in the Greenhouse 500 mL (an equal volume to the volume of supernatant) Greenhouse plants were grown as described by Zidenga et al. was added of each of the color development reagents [1% (2012). (w/v) sulfanilamide in 1.5 N HCl and 0.02% N -(napththyl)- ethylenediaminehydrochloride]. Samples were left at room Measurement of Yield Parameters temperature for 10–20 min to allow full color development. Plants were grown in the greenhouse in rectangular trays with Absorbance was read at 540 nm. Nitrite concentration was only six plants per tray. Greenhouse grown plants were harvested estimated using a standard curve prepared by diluting known after 4–8 months of growth and fresh weight measurements were concentrations of nitrite in 500 mL and adding the color taken on all the tuberous roots. development reagents. 2 4 www.sigmaaldrich.com/ http://abrc.osu.edu/ 3 5 www.nitrate.com www.densci.com Frontiers in Plant Science | www.frontiersin.org 4 February 2017 | Volume 8 | Article 220 fpls-08-00220 February 22, 2017 Time: 15:3 # 5 Zidenga et al. Cyanogen Metabolism in Cassava Roots not been firmly established (Chew, 1973; Miller and Conn, Free and Hydrolyzed Amino Acid 1980; Hatzfeld and Saito, 2000). Relevant to this observation Extraction and Analysis it has been demonstrated that rhodanese activity does not Free amino acid extraction was based on the method by Hacham correlate with cyanogenic potential (Miller and Conn, 1980). et al. (2002). Approximately 150 mg of tissue was ground in liquid It has been suggested, however, that thiocyanate, derived nitrogen and homogenized by motor pestle with 600 mL of water: from rhodanese activity could be hydrolyzed by a thiocyanate chloroform: methanol (3:5:12 v/v). After centrifugation at 21000 g hydrolase to generate ammonia and carbonyl sulfide in for 2 min, the supernatant was collected and the residue was re- plants, but this activity has yet to be confirmed (Yu et al., extracted with 600 mL of water: chloroform: methanol followed 2012). by centrifugation. Supernatants from the first and second To determine whether rhodanese plays a role in cyanide extraction were combined in a 2 mL tube. 300 mL of chloroform detoxification in cassava, we measured rhodanese activity and 450 mL of water were added followed by centrifugation in leaves and tuberous roots of the cassava. The average at 21000 g for 2 min. The upper water: methanol phase was rhodanese activity detected in cassava leaves was relatively collected and transferred to a fresh tube, dried by speed-vac low at 4.19 mmol/mg protein/min (Figure 2A). Significantly, and dissolved in 200 mL of water. Detection of free amino we detected no rhodanese activity in cassava tuberous roots acids was performed by the Proteomics and Mass Spectrometry (Figure 2A). Since cyanogen accumulation occurs in roots, Facility at the Donald Danforth Plant Science Center using the the lack of rhodanese activity in these tissues suggests that AccQTag system. Protein hydrolysis was carried out as described rhodanese is not involved in cyanide metabolism in roots. As by Narayanan et al. (2011). Samples were hydrolyzed for 24 h previously discussed, linamarin is produced in the leaves and at 116 C in 6 N HCl containing 0.5% (v/v) phenol, dried and transported to the roots where it presumably provides reduced resuspended in 20 mM HCl before derivatization with the AccQ- nitrogen for protein synthesis (Bediako et al., 1981; Ramanujam tag reagent and subsequent separation by ACQUITY UPLC and Indira, 1984; Siritunga and Sayre, 2003; Siritunga et al., System (Waters, Milford, MA, USA) according to manufacturer’s 2004). Since no rhodanese activity was detected in cassava instructions. roots, we hypothesized that cyanide released from linamarin breakdown is preferentially assimilated via CAS. To test this Analysis of IAA hypothesis, we analyzed CAS activity in cassava tuberous roots Indole acetic acid (IAA) analysis was carried out using an and leaves. If cyanogens are a source of reduced nitrogen for LC–MS/MS analysis method developed and performed by the protein synthesis in roots, we would expect that CAS activity Proteomics and Mass Spectrometry Facility at the Donald (unlike rhodanese activity) would be higher in cassava roots Danforth Plant Science Center. The method is similar to Chen than leaves. In cassava tuberous roots, average CAS activity et al. (2009), but modified to include additional plant hormone was 13.7 mmol HS/mg protein/min, compared to 5.1 in leaves, species. an approximately threefold higher CAS activity in roots than leaves (Figure 2B). In contrast, in potato, a non-cyanogenic Statistical Analysis plant, leaf CAS activity (0.04 mmol H S per mg protein Statistical analysis was carried out using GraphPad Prism per min) was substantially lower than in cassava and twofold software package . Student’s t-test and one-way ANOVA with greater than potato tuber activity (0.02 mmol H S per mg Dunnett’s Multiple Comparison test for comparing multiple lines protein per min). These rates in potato correlate well with with the control were used. All analyses for significant differences levels required for CN detoxification associated with ethylene were performed at P 0.05. biosynthesis (Maruyama et al., 2001). Similar differences (3X) in CAS activity between cassava roots and leaves had previously been reported by Nambisan and Sundaresan (1994) and Elias RESULTS et al. (1997b). If cyanide assimilation occurs through the CAS, then it would Cyanide Metabolism in Cassava Roots be expected that additional enzymes in the cyanogen assimilation Occurs via b-Cyanoalanine Synthase pathway would have enzymatic activities commensurate with CAS activity. Thus, we assayed nitrilase activity, the enzyme Cyanide is detoxified in plants either through condensation involved in the conversion of cyanoalanine, the product of with cysteine, catalyzed by CAS, or via condensation with CAS activity, into aspartate, asparagine, and ammonia. The thiosulfate, derived from sulfur metabolism, and catalyzed by average nitrilase activity in cassava leaves was 12.8 mmol the enzyme rhodanese (Figure 1; Hatzfeld and Saito, 2000). ammonia/mg protein per min while that in the roots was Cyanide detoxification by rhodanese is prominent in mammals where the thiocyanate is excreted in urine (Shibamoto and 16.4 mmol ammonia/mg protein per min (Figure 2C). These apparent nitrilase activity levels were similar to those observed Bjeldanes, 1993). The use of thiosulfate as a cyanide antidote is based on the rhodanese activity (Shibamoto and Bjeldanes, for CAS in cassava roots. In addition, root nitrilase activity was about 1.3 times (or 30%) greater than in leaves. These 1993; Shepherd and Velez, 2008). In plants, however, the observations are consistent with root cyanide assimilation by relationship between rhodanese and cyanide metabolism has CAS and nitrilase leading to amino acid synthesis (Siritunga and http://www.graphpad.com/prism/Prism.htm Sayre, 2003). Frontiers in Plant Science | www.frontiersin.org 5 February 2017 | Volume 8 | Article 220 fpls-08-00220 February 22, 2017 Time: 15:3 # 6 Zidenga et al. Cyanogen Metabolism in Cassava Roots FIGURE 2 | Cyanide metabolizing enzymes (except rhodanese) in cassava have higher activities in roots compared to leaves. (A) Activities of rhodanese (in mmol thiocyanate/mg protein/minute) in tuberous roots and leaves of 8 months old cassava plants grown under glasshouse conditions. (B) CAS activity (in mmol hydrogen sulfide (H S) per mg protein per minute) in 8 months old cassava (C). Nitrilase (cyanoalanine hydratase) enzyme activities (in mmol ammonia/mg protein/min) in roots and leaves of in vitro cassava plants at 5 weeks. (D) Analysis of nitrate reductase activity (mmol nitrite/mg protein/min) in wild-type (WT) and transgenic low cyanogen (Cab1-1, Cab1-2, and Cab1-3; Siritunga and Sayre, 2003) lines. The assay was conducted on 5 weeks old in vitro plants. The results of all experiments are the averages from biological four trials. Statistical analysis was done by one-way ANOVA with Dunnett’s Multiple Comparison Test. Asterisks indicate significant difference at P  0.05. FIGURE 3 | (A) Gene cassette used in transforming cassava with b-cyanoalanine synthase (CAS) and nitrilase genes. The construct was assembled in a modified pBI121 plasmid where the gene of interest (GOI) was driven by a class I patatin promoter for root-specific expression. (B) CAS transcripts in wild-type (WT) and transgenic (PCAS1-4) cassava lines as detected by RT-PCR. RNA was extracted from 100 mg of 5 week-old in vitro cassava roots. RT-PCR was performed using primers for the CAS insert with tubulin as the internal control. (C) In vitro growth comparison of transgenic CAS-overexpressing plants (PCAS1-4) and wild-type (TMS 60444) plants after 3 weeks. (D) Expression of CAS increased the activity of the enzyme in cassava roots. The activity of CAS was correlated to reduced growth and root development (C,E). (E) Root development in in vitro transgenic CAS plants grown in MS medium for 3 weeks. Data are averages of n D 20. Statistical analysis was done by one-way ANOVA with Dunnett’s Multiple Comparison Test. All transgenics were significantly different from wild-type at P  0.05. Frontiers in Plant Science | www.frontiersin.org 6 February 2017 | Volume 8 | Article 220 fpls-08-00220 February 22, 2017 Time: 15:3 # 7 Zidenga et al. Cyanogen Metabolism in Cassava Roots in transgenic PCAS plants (with the exception of PCAS4, the Compensation for Reduced Cyanogens line with the enzyme activity closest to wild-type) relative to in Low Cyanide Transgenic Plants and wild-type plants. The transgenic plants exhibiting the highest the Impact of Elevating Cyanide CAS activity, had the lowest fresh weight. These data suggest Assimilatory Enzymes on Amino Acid that overexpression of CAS impairs growth and development in and Protein Levels cassava plants. To determine if CAS overexpression impacted amino acid Previously, transgenic plants having low root cyanogen levels pool sizes in cassava roots, total free amino acids and those were generated by suppressing leaf linamarin biosynthesis obtained from hydrolyzed proteins were assessed. There was through antisense expression of CYP79D1/D2 genes encoding a significant difference (at P  0.05) in total amino acids two cytochrome P450s that catalyze the first dedicated step in between wild-type and transgenic plants (Table 1). In addition, cyanogenic glycoside synthesis, resulting in a 99% reduction total root protein content was increased up to 9.3% in CAS in root linamarin levels relative to wild-type plants (Andersen transgenic plants relative to wild-type (Table 2) as were total et al., 2000; Siritunga and Sayre, 2003). These results confirmed that cyanogenic glycosides were transported from leaves to free and protein amino acids including most notably arginine, aspartate, and glutamate in the PCAS1 transformant, which had roots, as demonstrated also in rubber tree (Selmar et al., 1988). We have used the transgenic low root linamarin the lowest increases in CAS activity (Table 1). Since aspartate, glutamate, and arginine are entry points for reduced nitrogen plants as tools to study cyanide metabolism in cassava. As suggested by biochemical assays described above, cyanide assimilation and transfer, respectively, into amino acids it is not unexpected that their levels would be increased by enhancing assimilation via CAS allows for entry of cyanide into amino cyanide assimilation into amino acids by CAS. acid synthesis. To determine if disruption of linamarin metabolism impacted the activity of select enzymes involved in nitrogen metabolism, we compared root nitrate reductase TABLE 1 | Hydrolyzed and free amino acid content of 4 months-old activity between low (transgenic) and high cyanogen cassava transgenic and wild-type cassava tuberous roots in pmole/mg dry weight. plants. Nitrate reductase activity is highly regulated in plants, is up-regulated in plants with reduced nitrogen availability, Amino acid WT PCAS1 PCAS2 and repressed in plants with sufficient ammonia (Campbell, CyA 1.30  0.05 1.49  0.19 1.40  0.08 1999). Wild-type cassava roots were observed to have an His 0.56  0.17 0.7  0.22 0.7  0.2 average nitrate reductase activity of 1.78 mmol nitrite/mg Ser 1.63  0.4 2.08  0.42 2.19  0.28 protein/min while low cyanogen (Cab1, Figure 2D) lines a ab Arg 1.30  0.21 2.68  0.54 1.83  0.25 had nitrate reductase rates ranging from 4.5 to 5 mmol Gly 4.01  0.18 4.87  1.01 5.56  0.45 nitrite/mg protein/minute, or three times higher than wild-type a a Asp 6.56  0.78 9.12  0.92 8.43  0.86 (Figure 2D). These data suggest that when cyanogen synthesis MetS 1.64  0.25 1.74  0.3 1.86  0.11 is reduced, other root-based nitrogen assimilation pathways a a Glu 7.27  0.35 9.76  1.07 9.92  0.88 compensate. Thr 1.62  0.32 1.98  0.51 2.25  0.15 Thus, we hypothesized that enhancing cyanide assimilation Ala 3.93  0.28 4.49  1 4.96  0.34 via overexpressing enzymes in the CAS pathway (Figure 1) could Pro 2.05  0.17 2.41  0.56 2.67  0.3 result in elevated root total amino acids or protein levels. Lys 3.01  0.16 3.62  0.69 3.87  0.19 Four transgenic lines were generated overexpressing CAS Val 2.78  0.19 3.32  0.74 3.64  0.28 as confirmed by RT-PCR (Figures 3A,B). To determine if Ile 2.01  0.11 2.35  0.48 2.58  0.17 CAS overexpression resulted in increased enzyme activity, CAS Leu 3.0  0.16 3.51  0.73 3.89  0.26 enzyme activity was assessed. Root CAS activity in transgenic Phe 1.62  0.09 1.78  0.32 1.86  0.14 plants was elevated as much as twofold relative to wild-type roots a a Total 44.3 55.9 57.6 (Figure 3D). Unexpectedly, however, we observed reduced recovery of Superscript ‘a’ indicates significant difference from wild-type at P  0.05. Superscript ‘b’ indicates significant difference between the two transgenic lines transgenic plants overexpressing CAS relative to transgenics at P 0.05. Values are standard deviation. CyA, cysteic acid (hydrolysis product expressing other genes of interest in cassava roots (e.g., Siritunga of cysteine); Asp, AspCAsn (due to hydrolysis of Asn to Asp); Glu, GluCGln (due to and Sayre, 2003; Ihemere et al., 2006; Zidenga et al., 2012), hydrolysis of Gln to Glu). suggesting negative effects of CAS overexpression. Of the recovered CAS transgenic lines, the lines with higher CAS activity TABLE 2 | Total protein comparison in 4 months-old wild-type (WT) and showed more stunted growth (Figure 3C). Analysis of transgenic transgenic cassava tuberous roots. PCAS plants indicated poor root development compared to wild- Plant line WT PCAS1 PCAS2 type plants (Figure 3E), especially during the first 4 weeks of growth. Wild-type plants had an average of 4 roots per plant, a a b Total protein 16.73  0.56 17.36  0.24 18.28  0.17 while PCAS transgenics ranged from 1 to 3 roots per plant. (mg/mg dry weight) Root development was poorest in PCAS2 and PCAS3. Poor root Same letter superscript indicates no significant difference at P  0.05. Different development was also associated with reduced fresh weight and letter superscripts (a/b) indicate significant difference. Measurements were poor growth. There was a 2 to 4-fold decrease in fresh weight performed in biological triplicates. Values are  standard deviation. Frontiers in Plant Science | www.frontiersin.org 7 February 2017 | Volume 8 | Article 220 fpls-08-00220 February 22, 2017 Time: 15:3 # 8 Zidenga et al. Cyanogen Metabolism in Cassava Roots As previously described, we observed elevated nitrilase activity et al., 2001). The branching phenotype in our transgenic plants is in cassava roots relative to leaves. Thus, we hypothesized that similar to that observed upon decapitation of apical meristems NIT overexpression would increase assimilation of cyanide into which removes the inhibition of lateral bud growth (apical amino acids (Figure 1). Transformation of cassava plants with dominance) resulting in increased branching. Decapitation the Arabidopsis NIT4 gene was carried out as described in Section results in reduced IAA levels, the most common bioactive form “Materials and Methods.” Transgenic lines were confirmed by of auxin (Ferguson and Beveridge, 2009). However, while a RT-PCR (Figure 4A). To determine whether overexpression of significant amount of root auxin is derived from the shoot, it is NIT4 in cassava roots increased nitrilase activity, enzyme assays now known that roots are also sites of auxin biosynthesis (Ross were carried out as previously described. Three transgenic lines et al., 2006). were used for this analysis. Total nitrilase activity in transgenic Since nitrilases are also known to be involved in auxin lines was increased as much as fourfold relative to wild-type biosynthesis, we hypothesized that NIT4 overexpression affected plants (Figure 4B). Interestingly, free amino acid pool sizes auxin metabolism in cassava tuberous roots leading to the increased as much as 50% in plants with NIT activities less than increased branching phenotype. To test this hypothesis, we 3X wild-type levels but dropped to as much as 50% of wild- measured IAA concentrations in cassava tuberous roots of type levels in plants having fourfold increases in NIT activity 4 months-old greenhouse grown wild-type and NIT4 transgenic (Figure 4E). These results suggest that NIT4 may have pleiotropic plants. Transgenic cassava plants expressing NIT4 had up to effects with super-elevated NIT4 activity having negative impacts 50% less root IAA compared to wild-type plants (Figure 4C). on plant metabolism. To assess whether there were additional Wild-type roots, with16 ng/g fresh weight, had twice the level phenotypic effects of NIT4 overexpression, we assessed their of free IAA as the highest NIT4 expressing transgenic plants. performance in the greenhouse. Interestingly, PNIT plants IAA concentrations also decreased in the leaves, with PNIT2 displayed an increased branching phenotype compared to wild- having 50% less than wild-type levels (Figure 4C). The observed type plants (data not shown). In addition, in the early stages reduction in IAA concentration is consistent with the increased of growth (up to 8 weeks) greenhouse-grown PNIT plants root branching but is inconsistent with the enhanced root NIT4 tended to have more fibrous root development compared to activity if NIT4 is expected to impact IAA levels (O’Reilly and wild-type plants. These phenotypic traits mimicked potential Turner, 2003). The role of nitrilases in auxin biosynthesis is still morphological responses associated with alterations in ethylene not clearly defined (Ljung et al., 2002; Mano and Nemoto, 2012). or auxin levels. It is known that auxin promotes cell division in All four Arabidopsis nitrilases have been shown to convert indole- root pericycle cells, which leads to lateral root formation, but 3-acetonitrile (IAN) to the plant hormone IAA, but NIT4 appears inhibits cell division in lateral meristems of the shoot, resulting in to be mainly involved in cyanide metabolism (Bartling et al., 1992, the inhibition of lateral bud growth, or apical dominance (Rogg 1994; Schmidt et al., 1996; Normanly et al., 1997; Piotrowski et al., FIGURE 4 | (A) NIT4 transcript abundance by RT-PCR. RNA was extracted from 100 mg of 5 week-old in vitro cassava roots. RT-PCR was performed using primers for the NIT4 insert while tubulin primers were used for the control. (B) Expression of Nitrilase increases cyanoalanine hydrolase activity in cassava roots. Rates of conversion of cyanoalanine to ammonia were determined for n D 4. (C) IAA analysis in 4 months-old greenhouse-grown wild-type and transgenic cassava tuberous roots and leaves showing decreased IAA in transgenic plants overexpressing NIT4. (D) Storage root fresh weight per plant in WT and transgenic NIT4 (PNIT2, 4, and 6) lines. (E) Total free amino acid analysis in wild-type and NIT4 transgenic lines. The data are averages of three biological trials. Frontiers in Plant Science | www.frontiersin.org 8 February 2017 | Volume 8 | Article 220 fpls-08-00220 February 22, 2017 Time: 15:3 # 9 Zidenga et al. Cyanogen Metabolism in Cassava Roots 2001; Ljung et al., 2002). In our experiments, overexpression of CAS activity and 1.3-times higher nitrilase activity in cassava NIT4, while increasing cyanide assimilation into amino acids, was roots than in shoots, consistent with cyanide assimilation by associated with reduced IAA levels. It is not immediately clear CAS. As a corollary to this hypothesis, it would be predicted how NIT4 overexpression in roots may have decreased IAA levels, that competing cyanide assimilation pathways that do not lead to however, we observed additional phenotypic impacts of NIT4 amino acid synthesis would have low activity in cassava roots. It overexpression including reduced storage root yield in transgenic was observed that rhodanese activity was not detected in cassava lines having the highest nitrilase activity (Figures 4B,D). Finally, roots. Thus, there is no apparent competing pathway for cyanide elevated total free amino acids were only observed in NIT4 assimilation in cassava roots. There was, however, no significant transgenics expressing the lowest levels of increased total NIT difference in apparent rhodanese and CAS catalytic turnover activity relative to wild-type (Figures 4D,E). Plants with the activity in cassava leaves suggesting that a substantial portion of highest NIT activity had the lowest total free amino acid pool CN produced in damaged leaves may be detoxified by rhodanese sizes. Thus, the regulation of amino acid accumulation by NIT is (Bediako et al., 1981; Ramanujam and Indira, 1984; Siritunga and complicated with only a narrow window of enhanced NIT activity Sayre, 2003; Siritunga et al., 2004). yielding enhanced amino acid accumulation. If cyanogens are a significant source of reduced nitrogen in plants, a reduction in linamarin synthesis would be expected to impact nitrogen assimilation. Using previously generated low cyanogen plants (Siritunga and Sayre, 2003), we observed DISCUSSION elevated nitrate reductase activity in low cyanogen plants relative Cyanogenic plants produce sufficient levels of cyanogens to wild-type plants suggesting that loss (99%) of cyanogens is to provide protection against a variety of herbivores and compensated by increased nitrate reductase activity in roots. pathogens (Nahrstedt, 1985; Jones, 1998). It is this function of However, elevated root nitrate reductase activity in cassava cyanogens that has received the greatest attention since it can plants with reduced root linamarin is not sufficient to support potentially impact human health. In addition to their defensive plant growth in the absence of supplemental ammonia, further role, cyanogenic glycosides have been proposed to function supporting the central role of linamarin turnover in cassava roots as transportable forms of reduced nitrogen in some plants as a source of reduced nitrogen for amino acid and protein including; rubber tree, cassava and sorghum (Selmar et al., 1988; synthesis (Siritunga and Sayre, 2004). Poulton, 1990; Siritunga and Sayre, 2007; Gleadow and Møller, Over 60% of the reduced nitrogen in stem phloem exudates 2014). Cyanogenic plants can allocate a substantial amount of cassava is in the form of linamarin (Calatayud and Le of nitrogen to cyanogenic glycoside accumulation. Eucalyptus Ru, 1996). Thus, reducing the cyanogenic potential of cassava cladocalyx allocates up to 20% of leaf nitrogen to accumulation of presents a challenge; while toxic to humans, it has an the cyanogenic glycoside, prunasin (Gleadow et al., 1998). Since important role in primary nitrogen assimilation. Redirecting cyanogen nitrogen is fully reduced, it does not require additional cyanogen metabolism toward amino acid and protein synthesis, reduction steps to be assimilated into amino acids. To convert particularly HNL synthesis in roots to be a nitrogen sink as cyanogen nitrogen into amine nitrogen requires the release and well as to simultaneously accelerate residual cyanogen (acetone rapid assimilation of cyanide from cyanogens. It is assumed that cyanohydrin) turnover during processing, is therefore a more generalized b-glucosidases generate cyanohydrins which then viable option for reducing steady-state linamarin pool sizes, spontaneously decompose to yield cyanide. Cyanide assimilation elevating protein content and the nutritional status of cassava would then occur via CAS (Figure 1) allowing cyanogens to roots than blocking linamarin synthesis to reduce cyanide provide reduced nitrogen for protein synthesis (Nartey, 1969; toxicity. Regardless, addressing cyanogen toxicity in cassava roots Selmar et al., 1988; Siritunga et al., 2004; Ebbs et al., 2010). while supporting active cyanide assimilation into protein remains To test this hypothesis, we assessed both the activity of a complex challenge. CAS and nitrilase, leading to aspartate and asparagine synthesis, To determine if cyanogen assimilation into proteins could in cassava roots. In addition, we assessed alternate nitrogen be enhanced by elevating cyanide assimilating enzymes, we assimilation pathways in plants engineered to have very low overexpressed CAS and NIT4. Overexpression of CAS in cassava cyanogen levels. The discovery of genes encoding the cytochrome roots successfully lead to increased CAS activity, elevated total P450s (CYP79D1 and CYP79D2) that catalyze the first-dedicated amino acid pool sizes and increased protein content (C9% step in linamarin synthesis (Andersen et al., 2000; Siritunga and relative to wild-type) consistent with elevated CAS activity Sayre, 2003; Siritunga et al., 2004) made it possible to design enhancing cyanide assimilation into amino acids. However, a transgenic approach to reduce cyanogens in cassava. Cassava there were unintended consequences of CAS overexpression. lines in which linamarin biosynthesis was inhibited in the roots CAS overexpression was associated with poor root development had wild-type linamarin levels in the roots while those in which and reduced total fresh weight. At present the mechanism linamarin biosynthesis was inhibited in the leaves had a 99% by which plant growth is altered in CAS overexpressors reduction of root linamarin levels (Siritunga and Sayre, 2003). is unknown. However, free cyanide has been implicated in This provided confirmation of the leaf as the primary source plant growth regulation (Smith and Arteca, 2000; Siegien for root linamarin. We hypothesized that enzymes involved in and Bogatek, 2006; Garcia et al., 2010; Xu et al., 2012). cyanide assimilation would have preferentially higher activities Recently, Garcia et al. (2010) have shown that mitochondrial in the roots compared to the leaves. We detected 3-times higher CAS activity is essential for maintaining low cyanide levels Frontiers in Plant Science | www.frontiersin.org 9 February 2017 | Volume 8 | Article 220 fpls-08-00220 February 22, 2017 Time: 15:3 # 10 Zidenga et al. Cyanogen Metabolism in Cassava Roots essential for root hair development. CAS Arabidopsis mutants 1994; Schmidt et al., 1996). Arabidopsis NIT4, however, has which accumulate elevated cyanide levels were shown to be been reported to not only to have high substrate specificity for defective in root-hair development. This phenotype was rescued cyanoalanine, but also to not recognize IAN as a substrate in by addition of hydroxocobalamin, a cyanide antidote (Garcia the production of IAA (Piotrowski et al., 2001; O’Reilly and et al., 2010). It appears from our studies and studies discussed Turner, 2003). However, the overexpression of NIT4 in our above that a threshold level of cyanide may be required for studies lead to reduced IAA levels, contrary to expectations, proper root development. Reductions in cyanide levels (as suggesting that cyanoalanine turnover by NIT4 may indirectly expected in transgenic lines with the highest CAS activity) impact IAA synthesis. At present the mechanism by which as well as elevated cyanogen levels (Arabidopsis CAS mutant) cyanoalanine or cyanide impacts IAA synthesis is not known. appear to both negatively impact root development. One possible However, cyanide is known to stimulate ethylene synthesis means by which cyanide may impact root development is which in turn stimulates IAA synthesis and transport (Smith through regulation of ethylene, and as discussed below IAA and Arteca, 2000; Normanly, 2007). The complex interplay production. Previously, Smith and Arteca (2000) have shown between ethylene and IAA in regulating plant development that low levels (1 mm) of cyanide enhance transcription of with altered cyanide levels may account for impaired root 1-aminocyclopropane-1-carboxylic-acid synthase, the enzyme growth in transgenic plants inconsistent with reduction in root which mediates the first-dedicated step in ethylene biosynthesis. IAA levels but consistent with a potential increase in ethylene Overexpression of Arabidopsis NIT4 in cassava roots was levels. shown to increase nitrilase activity and alter amino acid pool sizes. However, transgenic plants with the highest NIT activity (>3X wild-type rates) had the lowest total amino acids (50% AUTHOR CONTRIBUTIONS lower than wild-type), while transgenics having less than a threefold increase in NIT activity had as much as a 50% RS, DS, and TZ were all involved in study design, data acquisition increase in total amino acids relative to wild-type. These results and analysis, as well as manuscript draft and revision. suggest that there is a complex interplay between NIT enzyme activity and phenotypic response. This is best illustrated by the observed impact of elevated NIT4 activity on cassava growth and FUNDING development. 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Plant Physiol. 116, conducted in the absence of any commercial or financial relationships that could 1219–1225. doi: doi:10.1104/pp.116.4.1219 be construed as a potential conflict of interest. Xu, F., Zhang, D.-W., Zhu, F., Tang, H., Lv, X., Cheng, J., et al. (2012). A novel role for cyanide in the control of cucumber (Cucumis sativus L.) seedlings Copyright © 2017 Zidenga, Siritunga and Sayre. This is an open-access article response to environmental stress. Plant. Cell Environ. 35, 1983–1997. doi: 10. distributed under the terms of the Creative Commons Attribution License (CC BY). 1111/j.1365- 3040.2012.02531.x The use, distribution or reproduction in other forums is permitted, provided the Yu, X. Z., Zhang, F. Z., and Li, F. (2012). Phytotoxicity of thiocyanate to rice original author(s) or licensor are credited and that the original publication in this seedlings. Bull. Environ. Contam. Toxicol. 88, 703–706. doi: 10.1007/s00128- journal is cited, in accordance with accepted academic practice. No use, distribution 012- 0545- 7 or reproduction is permitted which does not comply with these terms. Frontiers in Plant Science | www.frontiersin.org 12 February 2017 | Volume 8 | Article 220

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