Plant Biology ISSN 1435-8603
SHORT RESEARCH PAPER
Heterologous expression of mitochondria-targeted microbial nitrilase enzymes increases cyanide tolerance in Arabidopsis E. Molojwane1, N. Adams1, L. J. Sweetlove2 & R. A. Ingle1 1 Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, South Africa 2 Department of Plant Sciences, University of Oxford, Oxford, UK
Keywords Arabidopsis; bioremediation; cyanide; germination; mitochondria; nitrilase. Correspondence R. A. Ingle, Department of Molecular and Cell Biology, University of Cape Town, Rondebosch 7701, South Africa. E-mail: [email protected] Editor R. Mendel Received: 6 November 2014; Accepted: 20 February 2015
ABSTRACT Anthropogenic activities have resulted in cyanide (CN) contamination of both soil and water in many areas of the globe. While plants possess a detoxification pathway that serves to degrade endogenously generated CN, this system is readily overwhelmed, limiting the use of plants in bioremediation. Genetic engineering of additional CN degradation pathways in plants is one potential strategy to increase their tolerance to CN. Here we show that heterologous expression of microbial nitrilase enzymes targeted to the mitochondria increases CN tolerance in Arabidopsis. Root length in seedlings expressing either a CN dihydratase from Bacillus pumilis or a CN hydratase from Neurospora crassa was increased by 45% relative in wild-type plants in the presence of 50 lM KCN. We also demonstrate that in contrast to its strong inhibitory effects on seedling establishment, seed germination of the Col-0 ecotype of Arabidopsis is unaffected by CN.
doi:10.1111/plb.12323
INTRODUCTION The toxic effects of cyanide (CN) in biological systems result from coordinate bonding of the CN anion to metalloproteins. Although best known as a potent inhibitor of complex IV of the mitochondrial electron transport chain, where it binds to cytochrome c oxidase thus blocking electron transfer to oxygen, CN inhibits a wide range of enzymes including Rubisco (Wishnick & Lane 1969). CN occurs naturally in plants as a byproduct of ethylene biosynthesis (Grossmann 1996), and plants have a two-step detoxification pathway to prevent CN toxicity. The first step occurs in the mitochondrion, and is catalysed by b-cyanoalanine synthase (CAS), which adds CN to Cys to yield b-cyanoalanine that is exported to the cytosol and hydrolysed to Asn or Asp by b-cyanoalanine nitrilase (Piotrowski & Volmer 2006). While this pathway is sufficient to detoxify endogenously generated CN, it appears to have only limited capacity to cope with elevated exogenous levels of CN. Although increased CAS activity has been reported in response KCN treatment in a range of plant species (Ebbs et al. 2010), CN toxicity symptoms become apparent in plants at low external concentrations, suggesting that the detoxification system is easily overwhelmed. Toxicity symptoms in plants include inhibition of growth, reduced transpiration and chlorosis (Alstr€ om & Burns 1989; O’Leary et al. 2014). In contrast, exogenous CN has been shown to promote germination and break dormancy in the seeds of a wide range of plant species (Taylorson & Hendricks 1973), including the dormant C24 ecotype of Arabidopsis (Bethke et al. 2006). Indeed, a recent study has indicated that CN may act as a chemical signal to promote seed 922
germination in ecosystems subject to regular fire (Flematti et al. 2011). While CN occurs naturally in the environment, CN concentrations in some areas of the globe have been greatly increased due to anthropogenic activities. HCN is used in a range of synthetic processes including the manufacture of nylon, herbicides and plastic, as well as in hydrometallurgical gold mining (Dzombak et al. 2006). This latter industrial application has been responsible for substantial CN contamination of both soil and water through failure of tailing dams or leaking of hemp leaching pads (Dzombak et al. 2006; Donato et al. 2007). In light of this, there is considerable interest in developing strategies to allow bioremediation of such sites, including the use of plants. Initial attempts to increase CN tolerance in plants focused on augmenting the naturally occurring CN detoxification pathway. Arabidopsis thaliana plants expressing a nitrilase (pinA) from Pseudomonas fluorescens displayed modestly increased resistance to KCN (O’Leary et al. 2014). However, enhancement of the plant CN detoxification pathway may be limited by the demand imposed for Cys, and/or the toxicity of b-cyanoalanine and H2S produced by CAS activity, and it is thus worthwhile exploring alternative strategies for genetic engineering of increased CN tolerance. An array of nitrilase enzymes able to catalyse the hydrolysis of nitriles, including CN, have been characterised from microorganisms. We focused on two such enzymes, a CN hydratase from Neurospora crassa (CHT), which catalyses the conversion of CN to formamide, and a CN dihydratase from Bacillus pumilis (CynD), which hydrolyses CN to formate and ammonia (Meyers et al. 1993; Basile et al. 2008). As both enzymes catalyse the direct hydrolysis of CN, they neither require Cys nor
Plant Biology 17 (2015) 922–926 © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands
Molojwane, Adams, Sweetlove & Ingle
generate b-cyanoalanine or H2S. In addition, CynD and CHT are active across a wide pH range, and the recombinant proteins have previously been shown to be stable (Basile et al. 2008). A recent study has shown that heterologous expression of a plastid-targeted CN dihydratase from Pseudomonas stutzeri results in increased CN tolerance in Arabidopsis (Kebeish et al. 2015). Given that a primary site of CN toxicity is the mitochondrial cytochrome c oxidase, we generated transgenic Arabidopsis plants expressing either CHT or CynD with an N-terminal mitochondrial targeting peptide. Here we describe the effects of exogenous CN on germination and seedling establishment in the Col-0 ecotype of Arabidopsis, and demonstrate that transgenic seedlings expressing either mitochondriatargeted CHT or CynD have increased tolerance to CN. MATERIAL AND METHODS Plant growth conditions For growth on soil, Arabidopsis seeds were sown on a 1:1 mix of peat (Jiffy Products, Stange, Norway) and vermiculite and stratified for 48 h at 4 °C in the dark. For growth on halfstrength Murashige and Skoog (MS) plates, seeds were sterilised in 70% (v/v) EtOH for 5 min, air-dried and re-suspended in 0.1% (w/v) agar for 2 days at 4 °C prior to plating. Plants were grown under a long-day photoperiod (16-h light, 8-h dark) at 22 °C and 55% relative humidity, and cool white fluorescent light of 80–100 lmolm2s1. Generation of pFAST:atp-CynD and pFAST:atp-CHT expression constructs The mGFP region (encoding a GFP protein with an N-terminal mitochondrial targeting peptide from the Nicotiana plumbaginifolia b-ATPase gene) was amplified from pBINmgfp5-atpase (Logan & Leaver 2000) and cloned into the pENTR4 dual selection vector (Life Technologies, Carlsbad, CA, USA) via the BamHI/NotI sites in the MCS to yield pENTR4:atp-gfp. The CynD and CHT coding regions were amplified by PCR from plasmid DNA kindly provided by Trevor Sewell (University of Cape Town) using primers containing SpeI sites (forward primers) and NotI (reverse primers). Primers used were as follows: CynD 50 -GCACTAGTATGACAAGTATTTACCCAAAG TTTCG-30 and 50 -GCGCGGCCGCTTAAACTTTTTCTTCCAG TATACCATG-30 , and CHT 50 -GCACTAGTATGGTCCTTACCAAGTACAAGG-30 and 50 -GCGCGGCCGCTCACTTCTTTC CCTCCTTATC-30 . The GFP fragment of pENTR4:atp-gfp was released by SpeI/NotI digestion, and the CynD and CHT PCR products cloned into the plasmid via these same RE sites to yield pENTR4:atp-CynD and pENTR4:atp-CHT, respectively. DNA sequencing confirmed that the targeting peptide was inframe with the CynD and CHT ORFs. These vectors were then recombined with the GATEWAY destination vector pFASTG02 (Shimada et al. 2010) to yield pFAST:atp-CynD and pFAST:atp-CHT, with expression of the transgene driven by the 35S CaMV promoter. Generation and analysis of transgenic plants Arabidopsis plants (Col-0 ecotype) were transformed by floral dipping with pFAST:atp-CynD, pFAST:atp-CHT or the empty
Microbial nitrilase expression increases cyanide tolerance
pFAST-G02 vector. The resulting seedlings were sprayed with 0.015% (w/v) glufosinate ammonium (Bayer, Leverkusen, Germany) 7 and 10 days after germination to identify putative transgenic plants. Presence of the atp-CynD or atp-CHT transgene was confirmed by PCR on genomic DNA extracted from leaf material. Confirmed transgenic plants were then self-fertilised to obtain homozygous lines, and all analyses were carried out on T3 homozygous lines that displayed complete resistance to glufosinate ammonium. To measure transgene expression, cDNA was synthesised from 1 lg of DNAse-treated total RNA using Superscript III reverse transcriptase (Life Technologies), and quantitative PCR performed as described previously (Carstens et al. 2014). Relative expression of atp-CynD and atp-CHT was determined using the two standard curve method, with normalisation to Actin2 expression levels. Primers used were Actin2 (Carstens et al. 2014), atp-CynD 50 -AAGATGGCG GTTCTCTCTATTT-30 (200 nM) and 50 -CCGGCATCATACTT CCACTT-30 (200 nM) Ta 57 °C, and atp-CHT 50 -TCATTAAC GAAGCGGGTCAG-30 (200 nM) and 50 -CGCGGTACTTCTTC AGCATAG-30 (200 nM) Ta 59 °C. Formate and formamide tolerance assays Half-strength MS agar plates containing 0, 25, 50 or 75 lM formamide or formate were prepared by pipetting 200 ll 100 mM MES, pH 5.7 (control plates) or 200 ll of the appropriate dilution of a 18.75 mM formamide or potassium formate stock solution (in 100 mM MES, pH 5.7) for the final concentration required into the bottom of a 120 9 120 mm Petri dish, and adding 50 ml half-strength MS agar. Sterilised seed that had been stratified for 2 days at 4 °C was then transferred to these plates and root length determined at 7 days post-germination. CN tolerance assays The assay used was modified from that developed by O’Leary et al. (2014). Half-strength MS agar plates containing 0– 100 lM KCN were prepared by pipetting 62.5 ll 25 mM KOH (control plates) or 62.5 ll of the appropriate dilution of a 100 mM KCN stock solution (in 25 mM KOH) for the final concentration of KCN required into the bottom of a 120 9 120 mm Petri dish, and immediately adding 50 ml halfstrength MS agar pre-cooled to 42 °C (to minimise loss of CN as HCN). After transfer of stratified seed, the plates were wrapped once in Parafilm, and twice in electrical tape and placed vertically under standard growth conditions. Germination rate (measured as radicle protrusion through testa) was determined after 2 days, and root length and production of first true leaf scored after 7 days. RESULTS AND DISCUSSION Effect of CN on germination and seedling establishment in Arabidopsis While exogenous CN has previously been shown to inhibit root elongation in the Col-0 ecotype of Arabidopsis (O’Leary et al. 2014), its effects on Col-0 seed germination and seedling establishment have not been reported, to our knowledge. We thus monitored germination (scored as radicle protrusion
Plant Biology 17 (2015) 922–926 © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands
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Fig. 1. Cyanide inhibits seedling establishment but not germination in Arabidopsis. Wild-type seeds were sown on half-strength Murashige and Skoog plates containing 0, 25, 50 or 75 lM KCN and scored for germination after 2 days, and for two measures of seedling establishment (production of first true leaf and primary root length >10 mm) after 7 days. Data shown are the mean percentage of seeds/seedlings that reached these thresholds from three independent experiments (each with 40–60 seeds per KCN concentration) SEM.
Fig. 2. Cyanide inhibits root elongation in Arabidopsis. Relative primary root length of wild-type seedlings grown on half-strength Murashige and Skoog plates containing 0, 20, 40, 60, 80 or 100 lM KCN is shown. Relative root length was calculated by normalisation to the root length observed on control plates without KCN after 7 days. Values are means SEM from three independent experiments (each with ten seedlings per KCN concentration). Regression analysis revealed a significant (P < 0.0001) negative relationship between KCN concentration and root length.
Fig. 3. Exogenous formate and formamide have no effect on root elongation in Arabidopsis. Wild-type seedlings were germinated on plates containing 0, 25, 50 or 75 lM formate or formamide and primary root length determined after 7 days. Values shown are means SEM (with 15–18 seedlings per concentration). ANOVA revealed no significant effect of either formate or formamide on root elongation in seedlings.
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through the testa) and two measures of seedling establishment – the production of the first true leaf and the presence of a primary root >10 mm in length within 7 days – in seeds on plates supplemented with 0, 25, 50 or 75 lM KCN. Unsurprisingly, given the well-known stimulatory effect of CN on seed germination in a range of plant species, exogenous CN had no negative effect on germination, which averaged >96% across the KCN concentrations tested (Fig. 1). In contrast, a strong inhibitory effect of CN was observed on seedling establishment. While no significant difference in the percentage of plants producing a first true leaf was observed between seedlings in the presence of 0, 25 or 50 lM KCN, true leaf development was totally abolished by the presence of 75 lM KCN in the media. A more gradual effect of KCN on root elongation was observed; while 81% of seedlings had a primary root >10 mm in length at day 7 on control plates, this was reduced to 37% in seedlings on 25 lM KCN and 1% in the presence of 50 lM KCN. Given the more gradual effect of exogenous CN on root elongation than on development of the first true leaf in seedlings, we selected this treatment for further experimentation to develop an assay to measure KCN tolerance in Arabidopsis. Col-0 seeds were transferred to plates containing 0, 20, 40, 60, 80 or 100 lM KCN and primary root length determined after 7 days. A highly significant (P < 0.0001) negative relationship was observed between external KCN concentration and root elongation in wild-type seedlings (Fig. 2), with a calculated IC50 of approximately 56 lM KCN. These results are similar to those reported by O’Leary et al. (2014), where a 50% inhibition of root elongation was observed in the presence of between 20 and 50 lM KCN. In these experiments, 6day-old seedlings were transferred to half-strength MS plates containing KCN and root length determined after 5 days. There are several potential limitations with this approach, including potential damage to seedlings and/or bias in seedling selection for transfer, and loss of HCN when plate lids are removed for the transfer process. As we observed that exogenous CN does not inhibit germination of Col-0 seeds (Fig. 1), and seeds can be transferred more rapidly than can seedlings to plates containing KCN, we concluded that measurement of root length 7 days post-plating of stratified seeds on plates containing 50 lM KCN would be an appropriate assay for CN tolerance in transgenic plants expressing microbial nitrilase enzymes.
Plant Biology 17 (2015) 922–926 © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands
Molojwane, Adams, Sweetlove & Ingle
Tolerance of Arabidopsis to formate and formamide While heterologous expression of nitrilase enzymes able to hydrolyse CN is an attractive alternative to augmentation of the native CN detoxification pathway, the end products of the reactions catalysed by CHT and CynD must be considered. CHT converts CN to formamide, while CynD hydrolyses CN
Microbial nitrilase expression increases cyanide tolerance
to formate and ammonia (Basile et al. 2008). Formate is naturally present in plant tissues and plants contain a mitochondrial formate dehydrogenase (FDH) that converts formate to CO2 and H2O (Halliwell 1974). The capacity of plants to metabolise formamide is less clear. While two putative formamidase-encoding genes (At4g37550 and At4g37560) have been annotated in the Arabidopsis genome, no experimental validation of this putative function has been reported to date. It is possible that the reaction products of the CynD and CHT nitrilase enzymes might have direct negative (or positive) effects on biomass production in Arabidopsis, thereby confounding our measurements of CN tolerance using root elongation assays. To determine whether this was the case, we analysed the effects of exogenous formate and formamide on root elongation in seedlings. Seeds were sown on plates containing 0–75 lM formate or formamide and root length determined 7 days post-germination. No statistically significant effect of either compound was observed on root elongation in wild-type Arabidopsis seedlings (Fig. 3). It is not known whether the uptake of these two compounds differs in Arabidopsis, nor is it possible in this experiment to relate external concentrations to intracellular concentrations. However, given the 1:1 stochiometric relationship that exists between CN and formate/formamide produced by CynD and CHT, respectively, we concluded that our assay for CN tolerance (root elongation in the presence 50 lM KCN) was unlikely to be confounded by any direct effects of the formate/formamide produced on root elongation. Transgenic plants expressing CynD or CHT exhibit increased tolerance to CN
Fig. 4. Relative expression level of atp-CynD and atp-CHT in transgenic plants. Values shown are mean relative expression levels (normalised to Actin2 expression) in shoot tissue pooled from 2-week-old plants as determined by qPCR from two repeats SD. Transgenic lines used in the cyanide tolerance assays are indicated with ‘+’ symbol.
To determine whether heterologous expression of microbial nitrilase enzymes targeted to the plant mitochondria is a viable strategy to increase CN tolerance, we generated transgenic Arabidopsis plants expressing CynD (from B. pumilis) or CHT (from N. crassa) enzymes fused to a N-terminal mitochondrial targeting peptide. This 93 amino acid peptide (from the N. plumbaginifolia b-ATPase) has previously been shown to target GFP to the mitochondria in Arabidopsis (Logan &
Fig. 5. Transgenic plants expressing atp-CynD or atpCHT display increased tolerance to cyanide. Seeds were sown on plates containing 0 or 50 lM KCN and primary root length determined after 7 days. Mean root lengths (SEM, with 15–20 seedlings per line) significantly different from those observed in wild-type plants as determined by Student’s t-test are indicated by ****P < 0.0001. Identical results were obtained in a second independent experiment. Plant Biology 17 (2015) 922–926 © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands
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Microbial nitrilase expression increases cyanide tolerance
Leaver 2000). Quantitative PCR analysis of T3 plants from five independently transformed lines per construct revealed a range of atp-CynD and atp-CHT transgene expression levels (Fig. 4). For each construct we selected the two lines displaying the highest relative expression of the transgene for subsequent use in CN tolerance assays. Seeds were sown on plates containing either zero or 50 lM KCN and primary root length measured after 7 days under standard growth conditions (Fig. 5). In the absence of CN, no statistically significant difference was observed in primary root length between wild-type plants and transgenic plants transformed with the empty pFAST-G02 vector, pFAST:atpCynD or pFAST:atp-CHT. Thus expression of the atp-CynD and atp-CHT transgenes has no effect on seedling growth per se. However, in the presence of 50 lM KCN, root growth was significantly higher in both pFAST:atp-CynD lines tested (P < 0.0001) and in both pFAST:atp-CHT lines (P < 0.0001) compared to wild-type plants, with roots on average 45% longer in the transgenic lines, while no such effect was observed in plants transformed with the empty pFAST-G02 vector. Thus, heterologous expression of either of these microbial nitrilase enzymes is apparently sufficient to increase CN tolerance in Arabidopsis seedlings, and to a similar extent. The degree of increased tolerance to KCN observed here is similar to that reported in the two previous studies that have attempted to engineer increased CN tolerance in Arabidopsis. Seedlings expressing a cytosolic pinA nitrilase displayed a 35% increase in root length relative to control plants on plates containing 50 lM KCN (O’Leary et al. 2014), while a 56% increase REFERENCES Alstr€ om S., Burns R.G. (1989) Cyanide production by rhizobacteria as a possible mechanism of plant growth inhibition. Biology and Fertility of Soils, 7, 232–238. Basile L.J., Willson R.C., Sewell B.T., Benedik M.J. (2008) Genome mining of cyanide-degrading nitrilases from filamentous fungi. Applied Microbiology and Biotechnology, 80, 427–435. Bethke P.C., Libourel I.G., Reinohl V., Jones R.L. (2006) Sodium nitroprusside, cyanide, nitrite, and nitrate break Arabidopsis seed dormancy in a nitric oxide-dependent manner. Planta, 223, 805–812. Carstens M., McCrindle T.K., Adams N., Diener A., Guzha D.T., Murray S.L., Parker J.E., Denby K.J., Ingle R.A. (2014) Increased resistance to biotrophic pathogens in the Arabidopsis constitutive induced resistance 1 mutant is EDS1 and PAD4-dependent and modulated by environmental temperature. PLoS One, 9, e109853. Donato D.B., Nichols O., Possingham H., Moore M., Ricci P.F., Noller B.N. (2007) A critical review of the effects of gold cyanide-bearing tailings solutions on wildlife. Environment International, 33, 974–984. Dzombak D.A., Ghosh R.S., Chong-Wong G.M. (2006) Cyanide in water and soil: chemistry, risk and management. CRC Press, Boca Raton, FL, USA, p 616.
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was reported in plants co-expressing a plastid targeted CN dihydratase from P. stutzeri (and FDH) in the presence of KCN (Kebeish et al. 2015). Together, these results suggest that CN may have toxic effects in all three subcellular compartments, which can be ameliorated to some extent by heterologous expression of nitrilase enzymes. However, in all three studies root elongation in the transgenic lines in the presence of micromolar concentrations of KCN is lower than that observed in the same lines grown in the absence of CN, thus full CN tolerance has not been achieved. We suggest that a gene-stacking approach incorporating mitochondrial and/or plastid targeted nitrilases in combination with augmentation of the plant CN detoxification pathway would be a promising direction for future research, especially if undertaken in a plant species with higher natural tolerance to CN. Sorghum bicolor, which is tolerant to millimolar concentrations of KCN (Trapp et al. 2003), and for which transformation protocols have been developed, might be a suitable system in which to test this synthetic biology approach. ACKNOWLEDGEMENTS We thank Trevor Sewell (UCT) for providing plasmids containing the CynD and CHT ORFs, David Logan (Universite d’Angers) for the pBINmgfp5-atpase vector and Brendan O’Leary (University of Oxford) for advice on cyanide tolerance assays. This research was supported by the University of Cape Town, the Oppenheimer Fund (University of Oxford) and the Equity Development Program at UCT.
Ebbs S.D., Kosma D.K., Nielson E.H., Machingura M., Baker A.J., Woodrow I.E. (2010) Nitrogen supply and cyanide concentration influence the enrichment of nitrogen from cyanide in wheat (Triticum aestivum L.) and sorghum (Sorghum bicolor L.). Plant, Cell & Environment, 33, 1152–1160. Flematti G.R., Merritt D.J., Piggott M.J., Trengove R.D., Smith S.M., Dixon K.W., Ghisalberti E.L. (2011) Burning vegetation produces cyanohydrins that liberate cyanide and stimulate seed germination. Nature Communications, 2, 360. Grossmann K. (1996) A role for cyanide, derived from ethylene biosynthesis, in the development of stress symptoms. Physiologia Plantarum, 97, 772– 775. Halliwell B. (1974) Oxidation of formate by peroxisomes and mitochondria from spinach leaves. Biochemical Journal, 138, 77–85. Kebeish R., Aboelmy M., El-Naggar A., El-Ayouty Y., Peterhansel C. (2015) Simultaneous overexpression of cyanidase and formate dehydrogenase in Arabidopsis thaliana chloroplasts enhanced cyanide metabolism and cyanide tolerance. Environmental and Experimental Botany, 110, 19–26. Logan D.C., Leaver C.J. (2000) Mitochondria-targeted GFP highlights the heterogeneity of mitochondrial shape, size and movement within living plant cells. Journal of Experimental Botany, 51, 865–871.
Meyers P.R., Rawlings D.E., Woods D.R., Lindsey G.G. (1993) Isolation and characterization of a cyanide dihydratase from Bacillus pumilus C1. Journal of Bacteriology, 175, 6105–6112. O’Leary B., Preston G.M., Sweetlove L.J. (2014) Increased beta-cyanoalanine nitrilase activity improves cyanide tolerance and assimilation in Arabidopsis. Molecular Plant, 7, 231–243. Piotrowski M., Volmer J.J. (2006) Cyanide metabolism in higher plants: cyanoalanine hydratase is a NIT4 homolog. Plant Molecular Biology, 61, 111–122. Shimada T.L., Shimada T., Hara-Nishimura I. (2010) A rapid and non-destructive screenable marker, FAST, for identifying transformed seeds of Arabidopsis thaliana. The Plant Journal, 61, 519–528. Taylorson R.B., Hendricks S.B. (1973) Promotion of seed germination by cyanide. Plant Physiology, 52, 23–27. Trapp S., Larsen M., Pirandello A., Danquah-Boakye J. (2003) Feasibility of cyanide elimination using plants. The European Journal of Mineral Processing and Environmental Protection, 3, 128–137. Wishnick M., Lane M.D. (1969) Inhibition of ribulose diphosphate carboxylase by cyanide: inactive ternary complex of enzyme, ribulose diphosphate, and cyanide. Journal of Biological Chemistry, 244, 55–59.
Plant Biology 17 (2015) 922–926 © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands
SHORT RESEARCH PAPER
Heterologous expression of mitochondria-targeted microbial nitrilase enzymes increases cyanide tolerance in Arabidopsis E. Molojwane1, N. Adams1, L. J. Sweetlove2 & R. A. Ingle1 1 Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, South Africa 2 Department of Plant Sciences, University of Oxford, Oxford, UK
Keywords Arabidopsis; bioremediation; cyanide; germination; mitochondria; nitrilase. Correspondence R. A. Ingle, Department of Molecular and Cell Biology, University of Cape Town, Rondebosch 7701, South Africa. E-mail: [email protected] Editor R. Mendel Received: 6 November 2014; Accepted: 20 February 2015
ABSTRACT Anthropogenic activities have resulted in cyanide (CN) contamination of both soil and water in many areas of the globe. While plants possess a detoxification pathway that serves to degrade endogenously generated CN, this system is readily overwhelmed, limiting the use of plants in bioremediation. Genetic engineering of additional CN degradation pathways in plants is one potential strategy to increase their tolerance to CN. Here we show that heterologous expression of microbial nitrilase enzymes targeted to the mitochondria increases CN tolerance in Arabidopsis. Root length in seedlings expressing either a CN dihydratase from Bacillus pumilis or a CN hydratase from Neurospora crassa was increased by 45% relative in wild-type plants in the presence of 50 lM KCN. We also demonstrate that in contrast to its strong inhibitory effects on seedling establishment, seed germination of the Col-0 ecotype of Arabidopsis is unaffected by CN.
doi:10.1111/plb.12323
INTRODUCTION The toxic effects of cyanide (CN) in biological systems result from coordinate bonding of the CN anion to metalloproteins. Although best known as a potent inhibitor of complex IV of the mitochondrial electron transport chain, where it binds to cytochrome c oxidase thus blocking electron transfer to oxygen, CN inhibits a wide range of enzymes including Rubisco (Wishnick & Lane 1969). CN occurs naturally in plants as a byproduct of ethylene biosynthesis (Grossmann 1996), and plants have a two-step detoxification pathway to prevent CN toxicity. The first step occurs in the mitochondrion, and is catalysed by b-cyanoalanine synthase (CAS), which adds CN to Cys to yield b-cyanoalanine that is exported to the cytosol and hydrolysed to Asn or Asp by b-cyanoalanine nitrilase (Piotrowski & Volmer 2006). While this pathway is sufficient to detoxify endogenously generated CN, it appears to have only limited capacity to cope with elevated exogenous levels of CN. Although increased CAS activity has been reported in response KCN treatment in a range of plant species (Ebbs et al. 2010), CN toxicity symptoms become apparent in plants at low external concentrations, suggesting that the detoxification system is easily overwhelmed. Toxicity symptoms in plants include inhibition of growth, reduced transpiration and chlorosis (Alstr€ om & Burns 1989; O’Leary et al. 2014). In contrast, exogenous CN has been shown to promote germination and break dormancy in the seeds of a wide range of plant species (Taylorson & Hendricks 1973), including the dormant C24 ecotype of Arabidopsis (Bethke et al. 2006). Indeed, a recent study has indicated that CN may act as a chemical signal to promote seed 922
germination in ecosystems subject to regular fire (Flematti et al. 2011). While CN occurs naturally in the environment, CN concentrations in some areas of the globe have been greatly increased due to anthropogenic activities. HCN is used in a range of synthetic processes including the manufacture of nylon, herbicides and plastic, as well as in hydrometallurgical gold mining (Dzombak et al. 2006). This latter industrial application has been responsible for substantial CN contamination of both soil and water through failure of tailing dams or leaking of hemp leaching pads (Dzombak et al. 2006; Donato et al. 2007). In light of this, there is considerable interest in developing strategies to allow bioremediation of such sites, including the use of plants. Initial attempts to increase CN tolerance in plants focused on augmenting the naturally occurring CN detoxification pathway. Arabidopsis thaliana plants expressing a nitrilase (pinA) from Pseudomonas fluorescens displayed modestly increased resistance to KCN (O’Leary et al. 2014). However, enhancement of the plant CN detoxification pathway may be limited by the demand imposed for Cys, and/or the toxicity of b-cyanoalanine and H2S produced by CAS activity, and it is thus worthwhile exploring alternative strategies for genetic engineering of increased CN tolerance. An array of nitrilase enzymes able to catalyse the hydrolysis of nitriles, including CN, have been characterised from microorganisms. We focused on two such enzymes, a CN hydratase from Neurospora crassa (CHT), which catalyses the conversion of CN to formamide, and a CN dihydratase from Bacillus pumilis (CynD), which hydrolyses CN to formate and ammonia (Meyers et al. 1993; Basile et al. 2008). As both enzymes catalyse the direct hydrolysis of CN, they neither require Cys nor
Plant Biology 17 (2015) 922–926 © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands
Molojwane, Adams, Sweetlove & Ingle
generate b-cyanoalanine or H2S. In addition, CynD and CHT are active across a wide pH range, and the recombinant proteins have previously been shown to be stable (Basile et al. 2008). A recent study has shown that heterologous expression of a plastid-targeted CN dihydratase from Pseudomonas stutzeri results in increased CN tolerance in Arabidopsis (Kebeish et al. 2015). Given that a primary site of CN toxicity is the mitochondrial cytochrome c oxidase, we generated transgenic Arabidopsis plants expressing either CHT or CynD with an N-terminal mitochondrial targeting peptide. Here we describe the effects of exogenous CN on germination and seedling establishment in the Col-0 ecotype of Arabidopsis, and demonstrate that transgenic seedlings expressing either mitochondriatargeted CHT or CynD have increased tolerance to CN. MATERIAL AND METHODS Plant growth conditions For growth on soil, Arabidopsis seeds were sown on a 1:1 mix of peat (Jiffy Products, Stange, Norway) and vermiculite and stratified for 48 h at 4 °C in the dark. For growth on halfstrength Murashige and Skoog (MS) plates, seeds were sterilised in 70% (v/v) EtOH for 5 min, air-dried and re-suspended in 0.1% (w/v) agar for 2 days at 4 °C prior to plating. Plants were grown under a long-day photoperiod (16-h light, 8-h dark) at 22 °C and 55% relative humidity, and cool white fluorescent light of 80–100 lmolm2s1. Generation of pFAST:atp-CynD and pFAST:atp-CHT expression constructs The mGFP region (encoding a GFP protein with an N-terminal mitochondrial targeting peptide from the Nicotiana plumbaginifolia b-ATPase gene) was amplified from pBINmgfp5-atpase (Logan & Leaver 2000) and cloned into the pENTR4 dual selection vector (Life Technologies, Carlsbad, CA, USA) via the BamHI/NotI sites in the MCS to yield pENTR4:atp-gfp. The CynD and CHT coding regions were amplified by PCR from plasmid DNA kindly provided by Trevor Sewell (University of Cape Town) using primers containing SpeI sites (forward primers) and NotI (reverse primers). Primers used were as follows: CynD 50 -GCACTAGTATGACAAGTATTTACCCAAAG TTTCG-30 and 50 -GCGCGGCCGCTTAAACTTTTTCTTCCAG TATACCATG-30 , and CHT 50 -GCACTAGTATGGTCCTTACCAAGTACAAGG-30 and 50 -GCGCGGCCGCTCACTTCTTTC CCTCCTTATC-30 . The GFP fragment of pENTR4:atp-gfp was released by SpeI/NotI digestion, and the CynD and CHT PCR products cloned into the plasmid via these same RE sites to yield pENTR4:atp-CynD and pENTR4:atp-CHT, respectively. DNA sequencing confirmed that the targeting peptide was inframe with the CynD and CHT ORFs. These vectors were then recombined with the GATEWAY destination vector pFASTG02 (Shimada et al. 2010) to yield pFAST:atp-CynD and pFAST:atp-CHT, with expression of the transgene driven by the 35S CaMV promoter. Generation and analysis of transgenic plants Arabidopsis plants (Col-0 ecotype) were transformed by floral dipping with pFAST:atp-CynD, pFAST:atp-CHT or the empty
Microbial nitrilase expression increases cyanide tolerance
pFAST-G02 vector. The resulting seedlings were sprayed with 0.015% (w/v) glufosinate ammonium (Bayer, Leverkusen, Germany) 7 and 10 days after germination to identify putative transgenic plants. Presence of the atp-CynD or atp-CHT transgene was confirmed by PCR on genomic DNA extracted from leaf material. Confirmed transgenic plants were then self-fertilised to obtain homozygous lines, and all analyses were carried out on T3 homozygous lines that displayed complete resistance to glufosinate ammonium. To measure transgene expression, cDNA was synthesised from 1 lg of DNAse-treated total RNA using Superscript III reverse transcriptase (Life Technologies), and quantitative PCR performed as described previously (Carstens et al. 2014). Relative expression of atp-CynD and atp-CHT was determined using the two standard curve method, with normalisation to Actin2 expression levels. Primers used were Actin2 (Carstens et al. 2014), atp-CynD 50 -AAGATGGCG GTTCTCTCTATTT-30 (200 nM) and 50 -CCGGCATCATACTT CCACTT-30 (200 nM) Ta 57 °C, and atp-CHT 50 -TCATTAAC GAAGCGGGTCAG-30 (200 nM) and 50 -CGCGGTACTTCTTC AGCATAG-30 (200 nM) Ta 59 °C. Formate and formamide tolerance assays Half-strength MS agar plates containing 0, 25, 50 or 75 lM formamide or formate were prepared by pipetting 200 ll 100 mM MES, pH 5.7 (control plates) or 200 ll of the appropriate dilution of a 18.75 mM formamide or potassium formate stock solution (in 100 mM MES, pH 5.7) for the final concentration required into the bottom of a 120 9 120 mm Petri dish, and adding 50 ml half-strength MS agar. Sterilised seed that had been stratified for 2 days at 4 °C was then transferred to these plates and root length determined at 7 days post-germination. CN tolerance assays The assay used was modified from that developed by O’Leary et al. (2014). Half-strength MS agar plates containing 0– 100 lM KCN were prepared by pipetting 62.5 ll 25 mM KOH (control plates) or 62.5 ll of the appropriate dilution of a 100 mM KCN stock solution (in 25 mM KOH) for the final concentration of KCN required into the bottom of a 120 9 120 mm Petri dish, and immediately adding 50 ml halfstrength MS agar pre-cooled to 42 °C (to minimise loss of CN as HCN). After transfer of stratified seed, the plates were wrapped once in Parafilm, and twice in electrical tape and placed vertically under standard growth conditions. Germination rate (measured as radicle protrusion through testa) was determined after 2 days, and root length and production of first true leaf scored after 7 days. RESULTS AND DISCUSSION Effect of CN on germination and seedling establishment in Arabidopsis While exogenous CN has previously been shown to inhibit root elongation in the Col-0 ecotype of Arabidopsis (O’Leary et al. 2014), its effects on Col-0 seed germination and seedling establishment have not been reported, to our knowledge. We thus monitored germination (scored as radicle protrusion
Plant Biology 17 (2015) 922–926 © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands
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Fig. 1. Cyanide inhibits seedling establishment but not germination in Arabidopsis. Wild-type seeds were sown on half-strength Murashige and Skoog plates containing 0, 25, 50 or 75 lM KCN and scored for germination after 2 days, and for two measures of seedling establishment (production of first true leaf and primary root length >10 mm) after 7 days. Data shown are the mean percentage of seeds/seedlings that reached these thresholds from three independent experiments (each with 40–60 seeds per KCN concentration) SEM.
Fig. 2. Cyanide inhibits root elongation in Arabidopsis. Relative primary root length of wild-type seedlings grown on half-strength Murashige and Skoog plates containing 0, 20, 40, 60, 80 or 100 lM KCN is shown. Relative root length was calculated by normalisation to the root length observed on control plates without KCN after 7 days. Values are means SEM from three independent experiments (each with ten seedlings per KCN concentration). Regression analysis revealed a significant (P < 0.0001) negative relationship between KCN concentration and root length.
Fig. 3. Exogenous formate and formamide have no effect on root elongation in Arabidopsis. Wild-type seedlings were germinated on plates containing 0, 25, 50 or 75 lM formate or formamide and primary root length determined after 7 days. Values shown are means SEM (with 15–18 seedlings per concentration). ANOVA revealed no significant effect of either formate or formamide on root elongation in seedlings.
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through the testa) and two measures of seedling establishment – the production of the first true leaf and the presence of a primary root >10 mm in length within 7 days – in seeds on plates supplemented with 0, 25, 50 or 75 lM KCN. Unsurprisingly, given the well-known stimulatory effect of CN on seed germination in a range of plant species, exogenous CN had no negative effect on germination, which averaged >96% across the KCN concentrations tested (Fig. 1). In contrast, a strong inhibitory effect of CN was observed on seedling establishment. While no significant difference in the percentage of plants producing a first true leaf was observed between seedlings in the presence of 0, 25 or 50 lM KCN, true leaf development was totally abolished by the presence of 75 lM KCN in the media. A more gradual effect of KCN on root elongation was observed; while 81% of seedlings had a primary root >10 mm in length at day 7 on control plates, this was reduced to 37% in seedlings on 25 lM KCN and 1% in the presence of 50 lM KCN. Given the more gradual effect of exogenous CN on root elongation than on development of the first true leaf in seedlings, we selected this treatment for further experimentation to develop an assay to measure KCN tolerance in Arabidopsis. Col-0 seeds were transferred to plates containing 0, 20, 40, 60, 80 or 100 lM KCN and primary root length determined after 7 days. A highly significant (P < 0.0001) negative relationship was observed between external KCN concentration and root elongation in wild-type seedlings (Fig. 2), with a calculated IC50 of approximately 56 lM KCN. These results are similar to those reported by O’Leary et al. (2014), where a 50% inhibition of root elongation was observed in the presence of between 20 and 50 lM KCN. In these experiments, 6day-old seedlings were transferred to half-strength MS plates containing KCN and root length determined after 5 days. There are several potential limitations with this approach, including potential damage to seedlings and/or bias in seedling selection for transfer, and loss of HCN when plate lids are removed for the transfer process. As we observed that exogenous CN does not inhibit germination of Col-0 seeds (Fig. 1), and seeds can be transferred more rapidly than can seedlings to plates containing KCN, we concluded that measurement of root length 7 days post-plating of stratified seeds on plates containing 50 lM KCN would be an appropriate assay for CN tolerance in transgenic plants expressing microbial nitrilase enzymes.
Plant Biology 17 (2015) 922–926 © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands
Molojwane, Adams, Sweetlove & Ingle
Tolerance of Arabidopsis to formate and formamide While heterologous expression of nitrilase enzymes able to hydrolyse CN is an attractive alternative to augmentation of the native CN detoxification pathway, the end products of the reactions catalysed by CHT and CynD must be considered. CHT converts CN to formamide, while CynD hydrolyses CN
Microbial nitrilase expression increases cyanide tolerance
to formate and ammonia (Basile et al. 2008). Formate is naturally present in plant tissues and plants contain a mitochondrial formate dehydrogenase (FDH) that converts formate to CO2 and H2O (Halliwell 1974). The capacity of plants to metabolise formamide is less clear. While two putative formamidase-encoding genes (At4g37550 and At4g37560) have been annotated in the Arabidopsis genome, no experimental validation of this putative function has been reported to date. It is possible that the reaction products of the CynD and CHT nitrilase enzymes might have direct negative (or positive) effects on biomass production in Arabidopsis, thereby confounding our measurements of CN tolerance using root elongation assays. To determine whether this was the case, we analysed the effects of exogenous formate and formamide on root elongation in seedlings. Seeds were sown on plates containing 0–75 lM formate or formamide and root length determined 7 days post-germination. No statistically significant effect of either compound was observed on root elongation in wild-type Arabidopsis seedlings (Fig. 3). It is not known whether the uptake of these two compounds differs in Arabidopsis, nor is it possible in this experiment to relate external concentrations to intracellular concentrations. However, given the 1:1 stochiometric relationship that exists between CN and formate/formamide produced by CynD and CHT, respectively, we concluded that our assay for CN tolerance (root elongation in the presence 50 lM KCN) was unlikely to be confounded by any direct effects of the formate/formamide produced on root elongation. Transgenic plants expressing CynD or CHT exhibit increased tolerance to CN
Fig. 4. Relative expression level of atp-CynD and atp-CHT in transgenic plants. Values shown are mean relative expression levels (normalised to Actin2 expression) in shoot tissue pooled from 2-week-old plants as determined by qPCR from two repeats SD. Transgenic lines used in the cyanide tolerance assays are indicated with ‘+’ symbol.
To determine whether heterologous expression of microbial nitrilase enzymes targeted to the plant mitochondria is a viable strategy to increase CN tolerance, we generated transgenic Arabidopsis plants expressing CynD (from B. pumilis) or CHT (from N. crassa) enzymes fused to a N-terminal mitochondrial targeting peptide. This 93 amino acid peptide (from the N. plumbaginifolia b-ATPase) has previously been shown to target GFP to the mitochondria in Arabidopsis (Logan &
Fig. 5. Transgenic plants expressing atp-CynD or atpCHT display increased tolerance to cyanide. Seeds were sown on plates containing 0 or 50 lM KCN and primary root length determined after 7 days. Mean root lengths (SEM, with 15–20 seedlings per line) significantly different from those observed in wild-type plants as determined by Student’s t-test are indicated by ****P < 0.0001. Identical results were obtained in a second independent experiment. Plant Biology 17 (2015) 922–926 © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands
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Microbial nitrilase expression increases cyanide tolerance
Leaver 2000). Quantitative PCR analysis of T3 plants from five independently transformed lines per construct revealed a range of atp-CynD and atp-CHT transgene expression levels (Fig. 4). For each construct we selected the two lines displaying the highest relative expression of the transgene for subsequent use in CN tolerance assays. Seeds were sown on plates containing either zero or 50 lM KCN and primary root length measured after 7 days under standard growth conditions (Fig. 5). In the absence of CN, no statistically significant difference was observed in primary root length between wild-type plants and transgenic plants transformed with the empty pFAST-G02 vector, pFAST:atpCynD or pFAST:atp-CHT. Thus expression of the atp-CynD and atp-CHT transgenes has no effect on seedling growth per se. However, in the presence of 50 lM KCN, root growth was significantly higher in both pFAST:atp-CynD lines tested (P < 0.0001) and in both pFAST:atp-CHT lines (P < 0.0001) compared to wild-type plants, with roots on average 45% longer in the transgenic lines, while no such effect was observed in plants transformed with the empty pFAST-G02 vector. Thus, heterologous expression of either of these microbial nitrilase enzymes is apparently sufficient to increase CN tolerance in Arabidopsis seedlings, and to a similar extent. The degree of increased tolerance to KCN observed here is similar to that reported in the two previous studies that have attempted to engineer increased CN tolerance in Arabidopsis. Seedlings expressing a cytosolic pinA nitrilase displayed a 35% increase in root length relative to control plants on plates containing 50 lM KCN (O’Leary et al. 2014), while a 56% increase REFERENCES Alstr€ om S., Burns R.G. (1989) Cyanide production by rhizobacteria as a possible mechanism of plant growth inhibition. Biology and Fertility of Soils, 7, 232–238. Basile L.J., Willson R.C., Sewell B.T., Benedik M.J. (2008) Genome mining of cyanide-degrading nitrilases from filamentous fungi. Applied Microbiology and Biotechnology, 80, 427–435. Bethke P.C., Libourel I.G., Reinohl V., Jones R.L. (2006) Sodium nitroprusside, cyanide, nitrite, and nitrate break Arabidopsis seed dormancy in a nitric oxide-dependent manner. Planta, 223, 805–812. Carstens M., McCrindle T.K., Adams N., Diener A., Guzha D.T., Murray S.L., Parker J.E., Denby K.J., Ingle R.A. (2014) Increased resistance to biotrophic pathogens in the Arabidopsis constitutive induced resistance 1 mutant is EDS1 and PAD4-dependent and modulated by environmental temperature. PLoS One, 9, e109853. Donato D.B., Nichols O., Possingham H., Moore M., Ricci P.F., Noller B.N. (2007) A critical review of the effects of gold cyanide-bearing tailings solutions on wildlife. Environment International, 33, 974–984. Dzombak D.A., Ghosh R.S., Chong-Wong G.M. (2006) Cyanide in water and soil: chemistry, risk and management. CRC Press, Boca Raton, FL, USA, p 616.
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was reported in plants co-expressing a plastid targeted CN dihydratase from P. stutzeri (and FDH) in the presence of KCN (Kebeish et al. 2015). Together, these results suggest that CN may have toxic effects in all three subcellular compartments, which can be ameliorated to some extent by heterologous expression of nitrilase enzymes. However, in all three studies root elongation in the transgenic lines in the presence of micromolar concentrations of KCN is lower than that observed in the same lines grown in the absence of CN, thus full CN tolerance has not been achieved. We suggest that a gene-stacking approach incorporating mitochondrial and/or plastid targeted nitrilases in combination with augmentation of the plant CN detoxification pathway would be a promising direction for future research, especially if undertaken in a plant species with higher natural tolerance to CN. Sorghum bicolor, which is tolerant to millimolar concentrations of KCN (Trapp et al. 2003), and for which transformation protocols have been developed, might be a suitable system in which to test this synthetic biology approach. ACKNOWLEDGEMENTS We thank Trevor Sewell (UCT) for providing plasmids containing the CynD and CHT ORFs, David Logan (Universite d’Angers) for the pBINmgfp5-atpase vector and Brendan O’Leary (University of Oxford) for advice on cyanide tolerance assays. This research was supported by the University of Cape Town, the Oppenheimer Fund (University of Oxford) and the Equity Development Program at UCT.
Ebbs S.D., Kosma D.K., Nielson E.H., Machingura M., Baker A.J., Woodrow I.E. (2010) Nitrogen supply and cyanide concentration influence the enrichment of nitrogen from cyanide in wheat (Triticum aestivum L.) and sorghum (Sorghum bicolor L.). Plant, Cell & Environment, 33, 1152–1160. Flematti G.R., Merritt D.J., Piggott M.J., Trengove R.D., Smith S.M., Dixon K.W., Ghisalberti E.L. (2011) Burning vegetation produces cyanohydrins that liberate cyanide and stimulate seed germination. Nature Communications, 2, 360. Grossmann K. (1996) A role for cyanide, derived from ethylene biosynthesis, in the development of stress symptoms. Physiologia Plantarum, 97, 772– 775. Halliwell B. (1974) Oxidation of formate by peroxisomes and mitochondria from spinach leaves. Biochemical Journal, 138, 77–85. Kebeish R., Aboelmy M., El-Naggar A., El-Ayouty Y., Peterhansel C. (2015) Simultaneous overexpression of cyanidase and formate dehydrogenase in Arabidopsis thaliana chloroplasts enhanced cyanide metabolism and cyanide tolerance. Environmental and Experimental Botany, 110, 19–26. Logan D.C., Leaver C.J. (2000) Mitochondria-targeted GFP highlights the heterogeneity of mitochondrial shape, size and movement within living plant cells. Journal of Experimental Botany, 51, 865–871.
Meyers P.R., Rawlings D.E., Woods D.R., Lindsey G.G. (1993) Isolation and characterization of a cyanide dihydratase from Bacillus pumilus C1. Journal of Bacteriology, 175, 6105–6112. O’Leary B., Preston G.M., Sweetlove L.J. (2014) Increased beta-cyanoalanine nitrilase activity improves cyanide tolerance and assimilation in Arabidopsis. Molecular Plant, 7, 231–243. Piotrowski M., Volmer J.J. (2006) Cyanide metabolism in higher plants: cyanoalanine hydratase is a NIT4 homolog. Plant Molecular Biology, 61, 111–122. Shimada T.L., Shimada T., Hara-Nishimura I. (2010) A rapid and non-destructive screenable marker, FAST, for identifying transformed seeds of Arabidopsis thaliana. The Plant Journal, 61, 519–528. Taylorson R.B., Hendricks S.B. (1973) Promotion of seed germination by cyanide. Plant Physiology, 52, 23–27. Trapp S., Larsen M., Pirandello A., Danquah-Boakye J. (2003) Feasibility of cyanide elimination using plants. The European Journal of Mineral Processing and Environmental Protection, 3, 128–137. Wishnick M., Lane M.D. (1969) Inhibition of ribulose diphosphate carboxylase by cyanide: inactive ternary complex of enzyme, ribulose diphosphate, and cyanide. Journal of Biological Chemistry, 244, 55–59.
Plant Biology 17 (2015) 922–926 © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands
