Cyanogenic glucosides Ch 12 GLEADOW-galley proofs

') Ct. V t(I' +\1&[ ,'c, i to. I CHAPTER 12 CYANOGENIC GLYCOSIDES ROSLYN GLEADOvi*, NA.l'WA BJARNIIOLT**, KIRSTEN J0RGENSEN**, JEr-.'NIFER Fox**,REBECCA MILLER'" INTRODUCTION Cyanogenic glycosides are found in many types of organisms (plants, L insects, fungi and some micro-organisms) (Nahrstedt, 1985). They consists ｾＬ＠ of an®:ydl'oxynitrile stabilised by a glycosidic linkage to a sugar moiety 1979) and are classifiedu-as-per their preClll'SOr amino acids and their sugar moiety, which is usually glucose (Fig. 12,1), When the glycoside is hydrolysed, the aglycone structure becomes unstable and cyanide is released in what is primarily a herbivore defence response (Gleadow and Woodrow, 2002a). Cyanide is highly toxic to all aerobic organisms, because X it binds to the haem group of the cytochrome oxidase located ink!. the mitochonlli'ia, the final step in oxidative respiration. Organisms avoid autotoxicity by the spatial ｳｾ｡ｲｴｩｯｮ＠ of the stable cyanogenic glycoside >< and the specific ､･ｧｲ｡ｴｩｶｾｬｵ｣ｯｳＬ＠ either at tissue or organelle l{ level\ When the cyanogenic glycoside and a suitable(b)lucosidase ai'e brought together, for example when plants or insects are crushed or chewed, then cyanide is evolved (M¢lIer and Conn, 1980; Fig. 12,2). ,J('( I Iy" I X(Conn, CYANOGENlC GLYCOSIDES • no. ｌｾＬ｜＠ ＶｾＮ＠ ｣ｾ＠ Cyanogenic glycosides can be d@lel.nHled either directly or indirectly. Direct methods include extraction and separation using liquid cm'omatography followed by mass spectroscopy or post column cleavage (Brimer and Dalgaard, ＱＹＮｾｩＡ［＠ Bjarnholt ef ai" 2008a), Indirect methods rely on the degradation ｯｦＢ｣ｙｾｨｏｧ･ｮｩ＠ glycoside and then measuring the degradative products (cyanide or ｧｬｵ｣ｯｳ･Ｚｾ｡ｫｩｲ＠ and Mpller, 1989; ｾ＠ H,,, \ I Vy')'i \ I /\ \ I V'·f",L (' A'" ＧＱＩｾｊｑｉ＠ i ._.J 0 'AlltbJ" r"",'!",t ｦｾｻｬｐＭＬ＠ /) 10 * School of Biological Sciences, Monash University, Victoria, 3800 Australia. Fax: +61"3"9905 1460" E-mail: ros.gleadow@sal.monash.edlhaU Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvel, DK-1871 Frederiksberg C, Denmark 'HSchool of Botany, The University of .Melbourne, Vic. 3100 Australia /<, Phone: ＫＶＱＭＳＢＹＰＵｾＷ＠ 'X. ** t{t\ 1M t'f b tt/l ct, Soil Allelochemicals 284 OH 1I0-( ＱｉｾＺｳＬｶ＠ ｏｾ＠ -0 Oll CII, on ＱｉｾＰＴｎ＠ CHs CN Iif04 0 ?N II Q 1" CII, (a) Linamarin (b) Lotaustralin (2m 1\IW = 247 gfmo) 1\fW = 261gfmol '\ ) I ＱｉＰｾ＠ II 011 CN 011 110 ( c) Prunasin (2R) ]"IW = 295 g/mol lvk ＱｉｾｏｯＧ［ｬ＠ Ir \- ,. X "1 '., (.,' 1\ VII f" 1\ (t,/ }\,\) W·j Id ＨＢｾ＠ (' (' f) Ｌ｜ｾ＠ ＧＭｾ＠ 1'f'!t',,:_'_Cf ) J f\. X >< ex "I '-) (Of t'.,., . t' \ ＨＩｾＬ＠ (d) Amygdalin (2R) MW = 457 g/mol 1" h Gleadow and Woodrow, 2002h; Miller et at., ＲＰｾ＠ This can be done in a highly quantitative manner in closed systems ｯｲｾｵ｡ｬｩｴｶ･ｹＬ＠ using test papers (Brinker and Seigler, 1989). If the animal or plant tissue contains specific(]:glucosidases, cyanide starts to evolve immediately upon maceration. If the tissue lacks ｴｨｾｬｵ｣ｯｳｩ､｡･Ｌ＠ then exogenous enzyme can be added to the medium to ensure conversion of cyanogenic glycosides to cyanide. HaC '. CN ｌｾｴ｡ＬＱＴｮ＠ (isoleucine), ＨｾｌｐＱＧｗｬ｡ｳｩｮ＠ (phenyla!aninc). ＨｾＩ＠ amygdalin (diglycoslde related to.,Qrunastn), and (e) Dhurrm (tyrosrnm. (Source: HaIkier ef al., 1988). -< glucose 0- glucose ',>) J) Fig. 12.1. Common cyanogenic glucosidcs (a) Linamarin (leucine), (b) 1(:\ I ;.\'1" \ tY hI')! ｴｊＨＧｾＢ＠ r ｾＧ｜＠ OH I" ｾ＠ ('(-'\ j) OH (e) Dhurrin(2S0 1\IW = 311 glmol 110 110-(·0 ｉｏＭｾｯＧ＠ ｾ＠ ｜ｾ＠ P ( ({}{ (,(> K ()<, ｨ｜ｊＨｾ＠ ｾ＠ \ t· ><, ( Ｌｾｴ｜Ｂ＠ eN ｾ＠ H2C Cyanogenic glycoside Fig. 12.2. u)!'li (Y(4W,)< lO! Ljn j:)V';!r"''< \ • )f p-glucosidase -< OH Ii ft HaC 0 ;II eN (Hydroxynitrile ,(1)ydroxynilrile lyase) H3C -.!J + HeN Aetonc or aldehyde Degradation of ｣ｹ｡ｮｯｧ･ｾｩ＠ glycosides into HeN and aglycones is a hvo step process. The first is catalyscd ｢ｙｬｴＩｾｵ｣ｯｳｩＬ･Ｎ＠ The second is spontaneous at low pH but is accclcl'atedl5y ｷｨ･ｾＱｙＭｹ､ｲｯｸｮｩｴｬ＠ lyase is present (Adapted from Poulton, 1988). Experiment 1. Screening of Field Samples Using Test Papers Feigl-Anger test papers anow a quick semi-quantitative estimate of the / \ cyanogenic status of a samplerI1l&is. pru·ticularly useful when screening a large number of samples or when testing materials in the field. The reaction is based on colour conversion of the copper reagent to bluepurple in the presence of cyanide. :x, I ｏｖＯＧｾｦ＠ ('b. 'h)f ｉＧｾ＠ ｾ＠ (1 1< (I 1'(;" \ '0 285 Cyanogenic Glycosides Materials and Equipment' Fresh plant or animal tissue; mortar and pestle. sand or liquid Nitrogen (to facilitate grinding); vials that can be sealed (e.g. 10 ml centrifuge tubes); Pasteur pipettes; filter papers (e.g. Whatman no. 541), oven (50·C); watch glass; beaker (100 ml); tweezers and retort stand for handling papers; access to fume hood; ice; heating block; silica gel and brown glass jar for storing papers; laboratory gloves and protective clothing, a Petri dish. Reagents \Vater; tetra base (4-4'-methylene-bis-N ,N-dimethyl-aniline; Sigma 40H2635); copper ethyl acetate; pure ethanol (HG). The cOfJper ethyl acetate can be simply made from cupric sulphate (4% w/v)in ethyl ;. acetoacetate (18% w/v) and sodium acetate (18% w/v) accordmg to the procedure below (W. Foley, personal communication). Procedure 'f)o 10}(> '''rkll I.'. A '. ' ( ｾＢ＠ , (If),: ('1)\'\'\)' «.\'!)k'll ii I"U+..ＢＬｾＯｴ＠ \ \1\,\ ti'l A. i" tV I , t ) Preparation of copper ethyl acetate reagent (i) Weigh 4 g cupric sulphate into a beaker and add 100 ml distilled water to make a 4% (w/v) solution. (ii) [Gently heat 30 ml cupric sulphate (4% w/v) with 10 ml ethyl acetoacetate (18% w/v) and 20 ml sodium acetate (18% w/v) for 10 min in a small beaker on a hot plate in the fume hood. (iii) Cool the mixture in the beaker by placing it on ice. (iv) Collect the crystals that form using a small spatula. (v) Redissolve the crystals in 2 ml ethanol in a watch glass. (vi) Recrystalise the copperBthyl acetate crystals by placing the watch glass in an oven at 56' ｾ＠ and dry overnight. '" "1\,, ,I r;,t',c'i'c.,!··Q, A (i) '. (ii) (iii) (iv) (v) x >, , i'2. e ()\t( (P L,\! ｜ＡＩＬＱｾ＠ (vi) (vii) I YI,'p( I t· I ()It\l.() I y Preparation of Feigl·Anger test papers X Dissolve 0.5 g coppelxeagent in 50 ml chloroform. Dissolve 0.5 g tetrabase in 50 ml chloroform. Make up a solution containing equal volumes of copper ethyl acetate and tetrabase in a beaker in a fume hood (e.g. mixing 50 ml of each). Tip solution into an open dish such as a Pebi dish. Using gloves and !\ilQlzers, dip filter paper in solution and then hang)( to air dry onlretort stand. Once dry, cut the papers into strips (approx 1 cm wide and 4 em long) with scissors. Store paper strips in a brown glass jar (or a jar covered with aluminium foil). ＱｬＧｳｦｩｾｴ＠ a small amount of silica gel or other Soil Allelochemicals desiccant ｾ＠ the jar. Shelf life is extended beyond one year, ifpapers are stored at 4°C. Using the Test Papers (i) Crush or finely grind samples in a mortar and pestle using a small (ii) (iii) (iv) volume of liquid Nitrogen or some sand as an abrasive (Refer Observation i). Quickly place the tissue in the base of a tube, moisten with water or buffer (Refer Observation ii). Fold one end of a test paper over the top edge of the vial and hold in place by screwing on the lid. It is important to do this quickly because once the plant tissue is crushed, the cyanogenic glycosides will start to break down and release cyanide. Take care not to let the papers get wet or to actually touch the tissue. Papers turn purple in the presence of cyanide. Record the colour of the test paper after 0.5 h, 2 hand 24 h on a scale of 1 to 5 (Refer Observations iii and iv). Observations (i) Cyanide is volatile at quickly and on ice. ＲｦｾＩＰ＠ generally these steps are performed ' ｾ＠ (ii) The water used to moisten the plant tissue can be replaced with )luffer (0.1 M citrate or phosphate buffer, pH 5.5-6.8). Non-specific (\!')lucosidases can be added to the buffer to test for presence of cyanogenic glycoside in the absence of endogenous degradative glucosidases in the tissue being tested (Refer Experiments 3-5). (iii) The speed and intensity of the colour change is an indication of the concentration of cyanogenic glycosides. (iv) Readings should not be extended beyond 24 h because of the risk of false positive tests through the action of cyanogenic bacteria (Brinker and Seigler, 1989). Precautions /\ I (C<\ !x){ (,\ -f-'l}•. \ 1ｾ＠ , ( I t I . \.. <. VI d /\ '('" " Always wear gloves when handling the test papers as chloroform is toxic and tetrabaslk is classified as a carcinogen. Papers should be prepared in a fume hood.'{l..aboratory coat should be worn when preparing reagents. XBefore handlihg liquid Nitrogen, ensure you are familiar with ｾｳ｡ｦ･ｴｹ＠ information and recommended personal protective equipment adVIsed by your commercial provider. Experiment 2. Quantitative Measurement of Cyanide Evolved from the Fresh Tissue Principle ､Ｖｨｴｾｮ＠ of cyanogenic glycosides can be measured after hydrolysis and 287 Cyanogenic Glycosides trapping the resultant HeN in a well containing a NaOH solution, in a sealed chamber (Brinker and Seigler, 1989). The amount of NaCN trapped fJ\, \ '\1) ＨＧｾＢｊＩ＠ h) t t() fQ , q;, ＩＧｬＢｾｲ＠ NaOH is then measured using Konig colour reactions (Lambert et al., ｾＮ＠ X 'i'975). Thiocyanate (SCN·) ...ｉＢ｣ＨＧｈｬ･｡ｾｩｮＮｴｨｳ＠ assay and may cause false Ｈｾｪｙ｜ｊＬＧ＠ ,\:, t:\h' 111.-') X positives (Bak et al., 1999), but its contents is negligible in cyanogenic rich plants (Gleadow, 1999). In the past, this colour reaction was done using relatively large volumes (i.e. 5 ml) of 0.1 M NaOH in test tubes (Brinker and Seigler, 1989; Gleadow ef al., 1998; Ballhom ef ai., 2008) c-l'!') 1.0 11' t'11J {)} :; \'\ but the method can also be performed on a smallel,,"cale using titre plates , (Woodrow et al., 2002; Goodger et al., 2006; Webber ef ai., 2007; Gleadow ef al., 2009a; 2009b). The procedure below is presented in two parts: (i) Degradation of cyanogenic glycosides and trapping of the resultant HCN and (in Colour reaction and spectrometric measurement of cyanide, ii, . \}Q \ \ v ｾＮ＾＠ 1\ (, ｾ＠ I ·,PC'I'.!) ··'··-1. '.' I (0 '0 >< () Ｎｾｊ＠ ｬＩＨｲｻｾ＠ .' ＨＬｰ［｜ｾＮ＠ ｾＮＭＬ＠ (:{Z"9; "c t.. "}[1 Ｈｾ＠ Ｎｬｩｾｊ＠ Itvv',I,hhfQ/,(\f t )\,\ Preparation: Tissue corer (dia..t to fit the chosen vials, 0.5 cm diameter is suggested); balance; small plastic tubes (e.g. 200 III Eppendorf tubes); SCiSSOl'S; mortar and pestle (optional); pH meter, liquid Nitrogen or dry ice. For incubation procedure: micropipettes; vials th&t can be racks . "'" X Xsealed (e.g. 5 ml GC vials; Refer Observation(1)j)incubator for storing small tubes; test tube rack to hold GC vials. . ｾ＠ II U.'.ｾ＠ ".\ t,\·'J, • ｾＩＯ［Ｎｒ･｡ｧｬｴｳ＠ ('vi' ,,\' ,.11'0','\ t) \' ,/,0. . vV-" \' t" "I' \ J, ,( (! Ｌｾ＠ '.'-.\. '\ (.,V.:\W) 1 Materials and ｅｱｵｩｰｭ･ｬｴｾ＠ (t! iCHV1 J J Sample N) b ｾ｜＠ Reagents Preparation: Magnetic stiTrer; vortex mixer (optional); Schott bottles for storing reagents; fume hood. For colour determination: 96 well titre plates (or 10 ml test tubes or centrifuge tubes); spectrophotometer to read 595 nm (or a plate reader if using titre plates). Nt-.D P CA \'((/..10 \-)v" ,(;"Of' (',:,( 1.)1) ｉｴ｜｟ｾＩ＠ . ｾ＠ , Ｈ｜ＮｬＧｾｶｴＬｩＢｏＯ＠ I ｨｻＧＯ､ｬｩＨＬｴ｣｜ＺＭｾＩ＠ Ｎｰｾ｜ｊ･＠ ｬ｜ｦＱｑＬＨ［ｻ｣ＧＩｾｩ＠ N", ｴＮｊＩＯＨｬｽＧ［ｌＬ｟ｉ｜ｦＢＭﾷﾣｾ＠ NaOH (1 M and 0.1 M solutions): Dlssolve 40 g NaOH 1ll 1 L ,hstilled water. " TIlls can be stored for 1 yeaI' and diluted to make 0.1 M as ｾＧ･ｱｵｩｲ､［＠ Citrate buffer (0.1 M, pH 5.5-6.5): For 1000 ml of 0.1 M pH 5.5 cltrate buffer add 232.5 ml of 0.1 M citric acid to 767.5 ml of 0.1 M sodium citrate (Refer Observation ii);(b)glucosidase from almond emulsin Qi'>y-glucoside , glucohydl'olase; EC 3.2.1.21; Sigma) or similar (Refer below to choose X X appropriate types oJ(!,Cglucosidase): weigh 0.02 g ol(h'glucosidase into a 50 ml centrifuge tube (01' equivalent) and making up to 50 ml with 0.1 M citrate X buffer (pH 5.5) (Refer .observation iii). This can be stored at -20 "C for long telm storage. Working solutions can be stored at 4 °C for 1 week; (! X X. ') 00101 () , {) Reagent A: Weigh 2.5 g succinimide and 0.25 g N-chlorosuccinimide into It may X a beaker. Dissolve in 30 ml distilled H 2 0 ｾＬ｡ｫｩｮＡｦＢｯｬＧＭｶｲｴ･ｸｧ＠ require mixing on a magnetic stirrer for 30 min with a mild heat (approx 50 "C\to fully ､ｩｳｯｬｶｾ［＠ Make up to 500 ml in a volumetric flask with X distilfed H 20 and store in a Schott bottle 01' ･ｱｵｩｶ｡ｬｮｾ＠ with the >, ｬｩ､ｾｲｔｨｳ＠ solution is stable for 4 weeks at 4 °C; ｎｾ｣ｨｬｯｲｳｵｩｮｭ､･Ｌ＠ X sllccinimide; ＢｾＨ＾＠ .. 'J rl'< Ｈｾｉ＠ Ｈｾ［Ｎ＠ ｉＮｾ＠ C\c/'/<' ＨＮＢＧｬｨｾ＠ ｾＮ｜ＬＧｻ｣ｴＡ＠ j< . ' .. Q "1;"" . " ; 1 ,/,,). , 1, ti V\ f' ｾ＠ I >1 ;.\ ' ' _it. \ (I ' .,. 1,.1r tic. J 288 \ ｾ＠ Ｋ｜ｾＬｴｬＩ＠ f><. Soil Allelochemicals Reagent B: In a fume hood. mix 3 g barbituric acid with 30 ml pyridine until dissolved. Add water to bring tbe total volume to 100 m!. Ensure complete dissolution 'Rnd"",vortex>. if necessaly. Wrap in aluminium foil because reagent B is light sensitive and store at 4°C. This solution is stable for a maximum of 10 days if stored conectly; §Odium cyanide ' (NaCN) for standard solutions; 0.5 M acetic ｡｣ｩｾＺｊ＠ Procedure I L Ｎｾ＠ Sample Preparation and Incubation (i) Cut off the lids of Eppendorf tubes and place them in a safe place (e.g. a labelled Petri dish or vial). (ii) Place tubes in a suitable rack and pipette 100 VI 1 M NaOH into each vial. (iii) Sample plant tissue using the tissue corer and place about 0.01 g of it into Gas Chromatograph vials. Alternatively place a known mass of soil or other tissue into the vials (Refer ObservatiorQ-1) (iv) Add 1 ml 0.1 M citrate buffer to the Gas Chromatograph vial to cover the plant tissue (Refer Observations ii and iii) and insert an inner vial containing 1 M NaOH (Fig. 12.3). Then, seal the whole apparatus. (v) Tissue is lysed using the n'eeze/thaw method by placing the vials (in racks) on dry ice (or liquid Nitrogen) until frozen, then removing vials to thaw at room temperature. Repeat twice to ensure complete tissue disruption (Refer Observation ii). (vi) Incubate at room temperatw'e for 1 h and then transfer vials to the incubator (37°C) overnight to allow conversion of the glycoside to HCN, and tlt"",fo. trapping of all the volatile HCN by the NaOH (approx. 15 h) (Refer Observation iv). (vii) After incubation remove the inner vials, label and reseal using the lids put aside earlier. These can then be stored at 4 "C for 1 week until analysed or at -20°C for long-term storage. (viii) The concentration of HeN in the NaOH is detmmined using a colour reaction, as described below. Colour Reaction and Spectrometric Measurement of Cyanide (i) Make up reagents A and B (see Observation v). (ii) Take 50 pI aliquot from the inner NaOH vial and add 450 pI distilled water to make a 0.1 ｾｍ＠ NaOH solution. IVlix for 5 s using a vortex mixer and place in a plastic rack (see Observation 6). (iii) Pipette 50 VI in duplicate from each diluted sample into a 96 well titre plate. (iv) Neutralise the solution by adding 50 pI of 0.5 M acetic acid to each well. Mix well using the shake function on the plate reader. 289 Cyanogenic Glycosides Airtight lid Ｍｩｾ＠ Fig. 12.3. Appamtus to trap cyanide evolved from the degradation of cyanogenic glycosides. The cyanogenic glycosides are broken down in a two step process (Refer Fig. 12.2), releasing hydrogen cyanide (HeN). HeN-4e' ｜Ｇｯｬ｡ｴｩＱ･ＭＢＸｾｮＦﾷｧｶＤｦｐｲｭ｣､ｳｵＮ＠ Alternative X buffers can be used (Refer Experiments 2 and 3) and different enzymes or leaf extracts <Refer Experiments 3-5). If buffer is used \"it.hout exogenous enzyme, then the evolution of cyanide is dependent on \ ｾＱＬｙＧｩ＠ rl {) (j to / j n I / ( ｾＨｴＱｈＬ､ＺｩＧ＠Ｂ ｴｾｬ＠ HHl ＧＩｾｃｰＭｬＯｽ＠ ,1\'<::ct",L\(' DOV1A·p,1I ＿ｎｯ｜ｩｾＩＧＭＨＢ＠ ' ) ) Add 125 pI succirrimide/chlorosuccinimide reagent (reagent A). Mix) well using the plate reader. Add 50 pI pyridine-barbitmic acid reagent (reagent B). Mix well using the plate reader. lncubate at 22 °C or room temperatme for 15 min (see Observation 7). Read absorbance at 595 run using a plate reader (see ObservationS). \}) (v) (vi) (vii) (viii) (0' Preparation of Calibration Curve (i) (ii) (iii) Make up a stock solution of200 mM NaCN in 0.1 M NaOH solution follmving appropriate safety procedures. Store in small volumes (1-2 rnl) at -20 °C (see also Table 1 in Experiment 7). Create a dilution ｳ･ｲｾ＠ by taking 0-150 mL of the stock solution and making up to 1000 pI with 0.1 M NaOH. Store working solutiops at 4°C for 1 week. Use in the same way as the samples in the titre plates. Always include a series of standards on each titre plate. Observations (i) " ｾＩ＠ ｾＩ＠ /'\ (ii) (iii) '\ ) f )( The size of vials is dependent on sample size. The mass of sample required can be adjusted by trial and error. This method relies on the presence of endogenou(b'jlucosidase. If the necessary enzymes are not present, cyanide wl11 not be evolved (see Experiments 3 and 4). Brinker and Seigler (19S9) recommend 0.1 mM pH 6.S phosphate buffer but the pH optimum for the cyanogerri{bj'lucosidase from Eucalyptus ciadDcalyx, for example is around 5.7 (Fox)200S). Soil Allelochemicals 290 ｾ［＠ Lt5' (' ＬｾｊＢ＠ ,,< ｊｾｲｧ･ｮｳ＠ ef al. (2005) use 1 mM Tricine buffer (pH 7.8). Other methods use 0.1 M citrate buffer (pH 5.5) as this is within the range for the(b)lucosidase and acidic enough to ensure that the hydrox0;itrile will spontaneously degrade to HeN antl..tbe ｦｯｲｮ｡ｴｩｾＧｏｙ･ｈｇｊｉ＠ (e.g. Woodrow ef 01., 2002; Webber ef al., 2007; Gleadow ef al., 2009a). (iv) A simple time-course experiment can be devised to determine the optimum incubation time for particular tissue or soil types. Incubation should be kept to less ｴｨ｡ｮｦｾＴｊＩ＠ to ensure that there is no HeN production by contaminating organisms. (v) Quantitative measurement also can be performed using Merck Spectroquant cyanide detection kit (Gleadow et at., 1998; Gleadow and Woodrow 2000a, Ballhorn ef 01., 2008). Follow the procedure as outlined in the instruction leaflet that comes with the kit. The assay is not as accurate but it is fast and it avoids the need to prepare reagents A and B. (vi) For accuracy, the absorbance should be between 0.1 and 1.0. It may be necessary to dilute samples before putting them into the titre plates to get them ,vithin this range. It is important that the dilutions are made with 0.1 M NaOH. (vii) The intensity of the colour varies with the temperature and dw'ation of incubation. For comparisons between plates it is therefore important to keep these exactly the same. It is best to include a series of NaCN or KCN solutions on each plate. (viii) Many plate readers come with a 595 nm ｦｩｬｾｲ＠ installed for use in protein analyses. We have tested this and foUnd that the assay is just as accurate if the absorbance is measured at 595 nm, or at 585 nm (as recommended by Brinker and Seigler, 1989). Precautions 1 )' \o1,{) / \"' ｾＨＩＡＯｬｽｉｃＧ＠ f'J) OV)'",>: /4('1\) (,(", h" l\k'/l/bo4 ｦｽＧｲＢ｜Ｈｊｾ＠ 'lkL ｾｽＬｩＧＩ＠ Pyridine is toxic and has an ｯｦ･ｾｩｶ＠ odour. Cyanide is highly toxic and its in:btdution. can be very lethal. Only trained personnel should prepare standard solutions and they shoul never be alone. Always handle cyanide cold and in a fume hood, wear gloves and safety glasses. Cyanide stocks should be stored in small quantities in a locked box or cupboard according to recognised occupational health and safety procedm'es. Early symptoms of cyanide poisoning are dizziness and headaches. If someone complains of this immediately give them fresh air. Cyanide detoxification kits should always be available. Experiment 3. Preparation of Commercial Glucosidase for Incubation Assays Principle Cyanogenesis is the natural evolution of cyanide from cyanogenic glycosides f')( ＬＧｮｾﾥＱ｜ＨＩ＠ 291 Cyanogenic Glycosides p ｾ＠ ｾｉ＠ and the degree of cyanogenesis is dependent on the activity of endogenous cyanogenicc:h"";glucosidases (see Experiment 2; Nahl'stedt, 1985). If the amount of evolved cyanide is used to determine the total concentration of cyanogenic glycosides (see Experiment 2; Figure 3), then it is essential that the specific ､･ｧｲ｡ｴｩｶｾｬｵ｣ｯｳＨＩ＠ are present. This may require >( X the addition of exogenou(b':.:'glucosidase. A pl'eliIp.inary experiment using X replicate samples with and without exogenou{b':glucosidase should be conducted to determine the concentration of enzyme required and the appropriate incubation time. If the cyanogenic glycoside is known, it may )< be possible to purchastf'...e.:glucosidase commercially. The tlu'ee most common commercially available enzymes (1). almond emulsin, (ii). fungal pectinase and (iii). mollusc salivary enzyme (Dahler ef al., 1995; Gleadow ef al., 1998; Miller ef al., 2006b) are suitable for use in cyanogenic glycosides ｡ｳＬｹＧｾ＠ ｜ｾ＠ described in Experiment 2. Almond emulsin will break down X. ｴｾ｣ｹ｡ｮｯｧ･ｩ＠ glycosides but not all (Brinker and Seigler, 1989). "'< Other SOUl'ces of exogenous hydrolytic enzymes can be prepared from )it( plant tissue in"JaboPatOl"Y, as indicated in Experiments 4 and 5. ><- .x Reagents Almond emulsin from Prunus ｡ｭＩｾ､ｬｵｳ＼Ｖ＿ｄＭｧ｣ｯｩ･＠ glucohydI'olase, EC 3.2.1.21); Fungal pectinase from Rhizopus sp. (Macerase-Pectinase, E.C. 3.2.1.15; 4,4120lfalbiochem, Calbiochem-Novabiochem Corp., CA, USA); Mollus(bfolucUl'onidase (10 mg Illr\ also available from Sigma); 0.1 M citrate 01' phosphate buffer pH 5.5 (see Experiment 2). Procedure (i) (ii) (iii) ." I I I ('0.(> ,', +1"1 t\A <J (l, Weigh 0.25 g of selected enzyme into a 50 ml f'a-lcqn tube (see Observations 1 and 2). Make up to 50 ml using buffer. Prepare the incubation device as depicted in Experiment 2, adding sufficient enzyme-buffer solution to cover the tissue (approx 1 Ill]) instead of buffer alone and inse!·t an inner vial containing 1 M NaOH (Fig. 3) (see Observation@}) Then, continue the procedUl'e as described for colour reaction and spectrometric measurement of cyanide in Experiment 2 (Fig. 12.3). Observations (i) (ii) (iii) For almond emulsin this makes a solution of approximately 1.12 units mr1. Experiments to determine the required concentration can be made by adding different concentrations of enzyme and incubating for 10, 15 and 20 h (Gleadow, 1999). Although there is no direct relationship between the amount of tissue and buffer (or buffer plus enzyme) it is recommended that 292 I AY.(J 'i?J ｜Ｉｏｈｾ＠ ＧｾＵ＠ on!y enough buffer to cover the plant tissue be added. This is because cyanide is highly soluble in water and ｾｭ｡ｹ＠ increase the required I L' I '. .incubation time, I V CJ t:,('f 11,;:"1 \1/.1\--- \ " X ｾ＠ re. cI l 't,f·e.l) ｾＢ＠ L \. of' Soil Allelochemicals Experiment 4. Prepal'ation of Crude Protein Extract to Supply Degradative Enzyme from Plant Tissue q c: ui h:>J)\"e >- 0\. > ,1:,.. ｾＺＨＩ＠ Principle Ｍｴｾｑ＠ " " I I I 'S1..... "rt-<:) ＢｾＬＧ＠ -' " " n f C--'<IA:MY-\v'",,Y'-"'3 Ws. ·ey, Ｇｲｹｾ｣ＭＫ＼＿＠ J Inj.absence oflommercial!h;glucosidase, enzyme can be eblained-ia. \ )<..a crude protein exh'!H?ROn J made from ｬ･｡ｶｳＮｦｴＶｵｾｨＴＱｓｇｮＭｩ｣｢ｯ＠ )<. ｡ｾ＠ (e.g. Brinker and Seigler, 1989; Gleadow and Woodrow, 2000b; Gleadow et oZ., 2003; Miller et oZ., 2006a; Webber et oZ., 2007). The procedure below is based on Delgado et oZ. (1994) as modified by Gleadow l<. I 7.0'*) et al. ＨＱＹＸＩｾｯｲ＠ work with Eucalyptus leaves. 1;>' Materials and Equipment Balance, volumetric flasks (200 In!, 1000 mI); variable micropipettes (100 to 1000 )11 and 20-200 )11); mortar and pestle, muslin or Miracloth (Calbiochem, San Diego, USA) for filtering; ｣･ｮｨｾｦｵｧ＠ capable of 27,000 £ dialysis cassette (Slide-A-Lyzer 3.5K, MWCO 3500, Pierce, Rockford, IL, USA); dialysis tubing or any other type of desalting column can be used instead of the cassette. Reagents C.:> 0.1 M citrate buffer (Refer Experiment 2), EDTA-Na2' (disodiuIllethylenediaminetetraacetic acid),(Q;)nercaptoethano] (also known as B:ME or b-met), polyvinylpynolidone (PVPP), 'I\veen 80 (polyoxyethylene-sorbitan lllonooleate), commercial proteinase inhibitor cocktail kit containing 0.2 Illg mr' AESBSF [4-(2-Aminoethyl)-benzenesulfonyl fluoride], 0.5 )1g mI' 'Ieupeptin, O.G]! Illr' pepstatin A and 2.5 mg mrl EDTA-Na2 (or similar), methanol, ammonium sulphate [(NH4l2S04 ]; liquid Nitrogen. X Procedure Preparation of Extraction Buffer (i) (ii) X , I (iii) (iv) Make up the proteinase inhibitor cocktail mix according to the suppliers instructions. Store at -20°C in 10 Ill] aliquots until needed. Weigh 74.4 g EDTA-Na2 and make up to 200 IllI with distilled water in a volumetric flask to give a stock solution of 1 M. Weigh 2 g PVPP into 100 IllI Schott bottle. Add 50 ml 0.1 M citrate buffer, 1 ml EDTA-Na2 stock, 100 mlej;:) mercaptoethanol, 50 Ill} proteinase inhibitor cocktail and 20 ).11 'I\veen 80 and make up to 100 mI with 0.1 M citrate buffer (Refer ObservationQ!) . ,,() 293 Cyanogenic Glycosides Crude Protein Preparation Using the Extraction Buffer Place fl·eeze·dJ:ied (5 g) or fresh (20 g) tissue in a mortal' and pestle and grind to a fine powder in liquid Nitrogen. (ii) Wait a few minutes until temperature returns to about 4 °c. (iii) Add 20 ml of protein extraction buffer and grind. (iv) Filter the mixture tlu'ough muslin or Miracloth into centrifuge tubes and then centrifuge (20 min at 27,DOOg) to remove remaining tissue. (v) Decant the supernatant into vials. Fractionate the supernatant by adding solid (NH4)2S04 to a final concentration of 90% (w/v). Mix well until all the ammonium sulphate is dissolved. (vi) Centrifuge again, 20 min at 27,000 g. Discard supernatant. (vii) Resuspend the precipitated protein pellet in a minimum amount of buffer (0.1 M citrate buffer, pH 5.5). In (viii) Desalt the protein extraction using a dialysis cassette ｡ｾｳｴ＠ 0.1 M citrate buffer (pH 5.5). (ix) Incubate aliquots of the crude enzyme preparation to verifY that no cyanogenic glycoside has been ･ｘｾＧ｡｣ｴ､＠ with the protein X J preparation,){ｲ［ｾｦｴＮＭ {:', v' NrfLh'1 r,;L,) IJ (x) Test for activity against denatured plant tissue extract over a 24 h at 37°C to determine the minimum quantity of crude enzyme needed for complete hydrolysis of cyanogenic glycosides, (i) IV, >< "YI ｑｊｬ＼ｶｾＮａＧＩ｜＠ the.... A ru \ Observations x X i\-. '<. If known, use a buffer in ｾｰｲｯｩ｡ｴ･＠ range, Optimum pH may vary for specifi<\!2glucosidases, Tll'e optimum for cassava linamarase is above pH 7 (Refer Experiment 5, ｊｾｲｧ･ｮｳｯ＠ et al., 2005). The optimum for the prunasin hydrolase fi'om Eucalyptus cladocalyx is pH 5.7 (Fox, 2008). The method, described in Experiment 2, is dependent on evolved HeN and should be done under acidic conritions (e.g. pH 6), If more basic buffers are required, then the single \vial method should be used (see Experiment 7). r Precautions Mercaptoethanol, including any buffer containing it, is toxic and should be handled with care (gloves, lab coat, safety glasses) and only used in a fume hood. Experiment 5. Purification of Linamarase from Cassava Latex Principle )< Cassava latex is lich in linamarase ｡ｮｾｹ､ｲｯｸｩｴｬ･＠ lyase (lVIcMahon et al., 1995) which are active against cyanogenic glycosides derived from Soil Allelochemicals 294 x aliphatic amino acids. Linamarase can be pm'tially purified, for further used in incubation assays, Ｎ｡ＦｩＧｆｏｾｇｓ･､Ｍｉﾥｴｨ＠ procedure below (J¢rgensen ef al., 2005). Materials and Equipment Volumetric flasks for preparing stock solutions, 25 m} glass cylinder, ice, balance, aluminium foil, razor blades, two pairs of forceps, magnetic stirrer, funnel and filter paper (e.g. Buchner funnel), Erlenmeyer flask (50 ml), Parafilm (or other sealing material), small Eppendorf tubes. Reagents 0.1 M sodium phosphate buffer pH 8.0. Prepare 1000 ml of 0.2 M phosphate buffer by mixing 53 ml 0.2 M Na,HP04 (35.6 g in 1000 ml distilled water) with 947 ml 0.2 M NaH,P04 (27.6 g in 1000 ml water). Dilute by half with water for 0.1 M (Refer Observatior(.lD . •L Procedure (i) (ii) (iii) (iv) (v) (vi) (vii) X Thoroughly rinse the cassava plant with water. l\1easure 2 m1 buffer into the cylinder. Weigh the cylinder containing the buffer. Place the cylinder plus buffer on ice and use the aluminium foil as a lid. Cut the stem of the cassava plant using the razor blade. As it is cut, latex oozes out. Remove the latex with the forceps and scrape it off carefully into the buffer in the cylinder. Repeat with other stems until several microlitres are collected. The aim is to collect about 1 pI latex per 1000 pI buffer. Re-weigh the cylinder ,vith the buffer and latex and calculate the volwne of latex. Leave the latex solution at room temperature and stir for 30 min , ｾＬＮ＠ X (viii) (ix) (x) (xi) Filter the solution using a Buchner funnel. ｉｾ･ｰ＠ at 4 'C (Refer Observation 2). Collect in a glass Erlenmeyer flask. • Leave solution in the flask overnight at 4 °C, sealed with parafilm. Take aliquots (about 1 ml) of the linamarase solution using the Eppendorf tubes and freeze in liquid Nitrogen. Store at -80 °C. When thawed, the enzyme is slable for at least 1 week at 4 'C. Observations (i) (ii) Use glass and not plastic when making up phosphate solutions. An ordinary glass funnel can be used for filtration but it ,viII take a long time. Cyanogenic Glycosides 295 Experiment 6. Measurement of Evolved Cyanide Using Dried Tissue Principle This experiment is the same as Experiment 2, except that dried tissue is used instead of fresh tissue. Additional Equipment and Reagents Liquid Nitrogen, freeze-drier or drying oven (approx 50°C), ball mill grinders, desiccator. A simple desiccator can be made by placing dry samples into plastic zip-lock bag containing a small packet of silica gel. Procedure For Freeze Dlying: Wrap tissue in aluminium-foil, snap freeze in liquid Nitrogen and place in the freeze drier. It is important that the vacuum on the freeze-drier begins to draw down immediately as cells are lysed during the freezing process (Refer Observation@ • C For Oven Dlyillg: Place tissue in paper bags arranged loosely on shelves at 50°C for 48 h or until there is no further loss of mass (Refer Observation 2). Oven dried tissue should be cooled in a desiccator to avoid up-take 'of moisture. d'ildQ ((;) Sh"",V\ M t)VP" ｃｾＺ＠ i ｃＭｾＧｑ｜＼Ｌｊｦｨｻ＠ . 0 . () ,. ｾ｜＠ Grinding: Samples need to be ｧｉｾｕｮ､ＮＢｴｑＺ＠ h9mogenous "fine" powder. The tissue should be very finely ( 0.5 ｭｾ､ＧＭＢｯｮｳｩＸｴ･ｬｹ＠ gI'olmd}jn a mortar and pestle to facilitate th Ｇｾ｣ｯｭｰｬ･ｴ＠ release of the cyanoge£i"ic glycosides from the cells. Small bal£i,nill grinders can be made using dental amalgam mixers (e.g. Gleadow et 01., 2003). Insert leaf tissue into an empty plastic amalgam vial, with two tungsten balls. Grind for 10 s. Repeat. Larger samples can be ground using high quality coffeestyle grinders (e.g. IKA Labortechnic AlO microgrinder, Janke and Kunkel GmbH Co, KG, Germany) although they are not suitable for grass leaves which have parallel venation. The IKA grinder has the additional advantage of keeping the tissue cooled. Cyanogenic glycosides are stable in the ground tissue for several years if samples are stored in a desiccator. f'M'y"2J' X IJ n... \ ｾ＠ _I Q\.1 CA'· ,·Q1 I::> . X Incubation: Samples (0.01-0.02 g) are weighed into vials. Add buffer, ｾ＠ ｾ＠ ｾ＠ NaOH and proceed as for Experiment 2 (Refer Fig. ＱＲＮＳＩｾ＠ oven dried material, it is essential to add the appropriate ･ｸｯｧｮｵｾＩＱ｣ｳｩ､｡＠ as the enzyme is destroyed by the heat (Refer Experlments 3-5). Cyanide trapped into the NaOH wells is analysed as for Experiment 2. Observations (i) Typically, plant tissue is fully dried (Iyophilised) after 48 h on the 296 (ii) Soil Allelochemicals freeze drier. This can be checked by making replicate samples and removing and weighing them at intervals over 72 h until there is no further loss of mass. If the bags are tightly packed then the samples will take longer to dry. It is possible that cyanogenic glycosides could be degraded during that time. Experiment 7. Measurement of Evolved Cyanide Using a Single Vial Principle x X 'J)eJ(,J) SjX"l ( ,€, /'Q p,",,, ｜ｾ＠ vv;V, ＧｍＢｾｊ＠ ｢ｾ｜ｉｬ＼Ｇ＠ I In Experiments 2 and 6, cyanogenic glycosides are measured by trapping the evolved cyanide in a NaOH well. This requires an incubation time of approximately 15 h at 37 ·C. to allow enough time for all the cyanide to diffuse from th(b)glucosidase solution to the inner vial. The long incubation time can be circumvented, if all the steps are can'ied out in 1989; Jorgensen ef one vial. with no N aOH trap (l(Hallrier and ｍｾｬ･ｲＬ＠ al., 2005). The ､ｩｳ｡ｬｾＧｗｽＮｧ･＠ is that the absorption spectrum ofthe Konig reaction products ｣｡ｾ＠ ｯｶ･ｲｬ｡ｰｾ＠ the absorption spectra of other plant metabolites (i.e. phenolics, anthocyanins and chlorophylls). This interference can be minimised by scanning across a range of wavelengths and calculating the absorbance using integration of the peak. This method is ideal for a quick result as it does not require overnight incubation, but it is less accurate. Additional Equipment and Reagents 1.5 ml Eppendorf tubes, scanning spectrophotometer, Reagents A and B from Experiment 2, micropipettes; centrifuge (if ground samples are used), 6 M NaOH (weigh 60 g NaOH and make up to 250 ml with water), 0.2 M potassium cyanide standards (KCN) (make by weighing 0.027 g into a 50 rnl ｧｲ｡ｾｴ･､＠ vial and making up to 50 ml with 0.1 M NaOH), Refer Observatio,,-lJJ 0.5 M acetic acid. Procedure I' l L Sample Analysis (i) Prepare samples for analysis (e.g. collect fresh leaf discs, weigh dried material or an extract, as for Experiments 2 and 7). (ii) Place samples in small vials, such as 1.5 ml Eppendorf tubes. (iii) Add 180 "I buffer (Experiment 2) and seal. (iv) Freeze in liquid Nitrogen. )<,y (v) While frozen open lid and add ｾｩｬＩ､･ｧｲ｡ｴＮＢＨ＠ b)cJucosidase enzyme (Refer Expenments 3·6), Refer ｏ｢ｳ･ｲｶ｡ｨｯｾ＠ . )\ (vi) Defrost samples and mix. (vii) Incubate at 28 ·C for 1·2 h. I 297 \.cyanogelliC Glycosides ｾ｜Ｏｉ＠ oJ J }. rll/I'06l '(viii) During the incubati0'h prepare standards needed for building a calibration curve in Eppendorftubes according to Table 12.1. Note that the buffer is 50 IllM. To make this, dilute the sallle buffer used in step (iii) with water to give 50 mM. Table 12.1. Volume of NaOH and KeN stock solutions required to make a series of standard solutions (0-20 pmoles) suitable for use in colour reactions in single vial cyanogenic glycoside determinations. The final figure is micromoles per 240 pt The concentration of solutions therefore range from 0 to 83.3 }.ll\I. 0< fA X X ｒ･｡ｧｮｴｾＩ＠ 6MNaOH 0.2MKCN 50 m1\1 Buffer (ix) ,,,A/v "AA. x »< Ld ｌＮｾ＠ >< ＯｾｌＮＢＬ＠ (x) (xi) (xii) (xiii) (xiv) (xv) • , I lLL (xvi) I III 0 2 40 0 200 40 10 190 KCN Ｈｩ［ｾｯｬ･ｳＩ＠ 4 40 20 180 6 8 16 20 40 30 170 40 40 160 40 80 120 40 100 100 After incubation} freeze samples in liquid Nitrogen and add 40 II 6 M NaOH (this stops the catalysis of the cyanogenic glycoside). Thaw and incubate a further 20 min at room temperature. Add buffer to make up to 420 ml. Add 5oJl,cetic acid to standards and samples. ＢｐＺ＼ｾ＠ Add ＲＰｑＱｉｊｾ･｡ｧｮｴ＠ A to standards and samples. Add ＲＰＨＡｾ･｡ｧｮｴｳ＠ B to standards and samples. Ir ground tissue is used, centrifuge for 2 min at 27,000 g at this point (Refer ObservatiOl(2JJ Pipette 200 ).11 of sample into a microtitre plate, incubate for 10 min at room temperature, Refer Observation<[) Measure the absorbance of samples and standards at 595 nm mid 650 nm. Subtract the absorbance at 650 run fi'om the absorbance at 595 nm)1.Il\' . d compare the samples to the standard curve, Refer Observati6lt. ｾ＠ Observations (i) v' /'\ ;"';, >< (ii) ｾＮ＠ (iii) To minimise handling KeN, weigh approximately the correct amount into the tube and note the mass. Then add appropriate volume to make a 0.2 M solution. Always make up cyanide standards in basic solutions (in which it is stable and not volatile at 1'00m temperature). ( Noiv,,·;,c,.( I'U jHVNHAJ.'O,·\' (;,., /1.( I\J I /li'<lj.r ;) '/ (\ ) If the vials are kept fi'ozen,'there will be no loss of cyanide fi'om ｴｨｾＬ＠ ｶｩ｡Ｑｾｷｨ･ｮ＠ opened to add/the valious reagents. Ifwhole leaf discs or extracts m·e used, then the aliquot for analysis is taken from the solution. If a ground extract is used, it must be centrifuged fil·st to remove the particulate matter and then an aliquot can be taken from the supernatant. 298 (iv) Soil Allelochemicals To fully adjust for inteIierence, plates should be scanned from 500 to 650 mn and the total absorbance of each curve compared. For further details, see Jorgensen et al. (2005). Experiment 8. Methanolic Extraction and Quantification of Cyanogenic G1ycosides from Plant Tissue Principle In this procedure, cyanogenic glycosides are extracted with methanol from tissue or soil and directly measured by LC-MS (Nielsen et al., 2002; Jorgensen et 01.,2005; Bjarnholt et 01., 2008b). Quantitative extraction of cyanogenic glycosides from plant tissue is best :,0, ,ttained by boiling in 85 % methanol. Boiling ensures denaturing ｯＨｦｩＧｾｧｬｵ｣ｳ､｡･Ｌ＠ which avoid degradation of cyanogenic glycosides and a]so'helps to disrupt the plant cells. Aqueous methanol facilitates protein denaturing, cell wall disruption and ensures solubility of the glycosides. The procedure can be used with fresh as wen as dried tissue, and the extracts can be used for either quantitative measurements or purification of cyanogenic compounds. Thin fresh leaves or leaf discs can be extracted directly, while thicker tissue should be ground in liquid Nitrogen to ensure complete extraction. Dry tissues should be ground to a fine powder. Weighing u'ozen tissue or leaf discs for quantitative analysis can be time consuming and may lead to significant loss of cyanogenic glycosides before extraction. An alternative is to weigh the extraction tubes before and after adding the test material. "R, Materials and Equipments Water bath for boiling, ice, 2 rul Eppendorf tubes with Safe-Lock and lid clamp or 1.5 ml microfuge tubes with screw cap and O-ring, floater for submerging extraction tubes in water bath, balance, lyophiliser. Reagents Liquid Nitrogen; 85 % methanol; Procedure x I n',"f\' ｾ＠ ｾ＠ ｾＩｻ｜ｴｽＯ＠ t: , l, (i) (ii) >< (iii) (iv) (v) Grind samples in liquid Nitrogen and store at -80 "C until 1..(,) 11l"< ｻＮｾＧＨ＠ extraction. Add 500 ]11 85 % methanol to extraction tubes and weigh, Refer Observation,' 1)) Coolon ice. Heat water bath to more than 100 "C to ensure rapid heating of extl'action tubes and solution. Add the material to be analysed to the tubes and weigh again. >- Cyanogenic Glycosides • L, 1)91·" \\ "ft,·o \', V (vi) )< l( rV VI (vii) (viii) (ix) ( '< ｾＨＩｉＮ｜Ｇﾫ［ｯＬｖｽｲ＼＠ t /' Ｎｾ＠ (':> ｾ･｡ｬ＠ 299 Subtract ｷ･ａｾｨｴｳ＠ to obtain mass of added plant tissue (Refer Observation Q)I tubes with a lid clamp or a screw cap and ｰｬ｡ｾ･＠ them immediately in a boiling water bath (Refer Observation(Z)to@.)) 6ft Boil immediately for 2-10 min (Refer Observati04'S) ｡ｮｾＮ＠ Coolon ice before removing lid clamp or unscrewing cap. Use for quantification (Experiment 2 ｾ＠.o. 7), or identification (Experiment 10 or 11). Refer Observatio',1 7) Qllalltificatiol! . , '. I ' I·' . I / I - .. / / , / r.... ｾｴ｜ｨｶｖＧＬ＠ .. \\( ＧＬ｜ｬＨｖｙｾｴｊＯ｛＠ｲ Ct{Vl "),{!-, c L)n'Q, ,Jlj r,{/I,\t! Ｇｾ､ＨｊｉｪＬ＠ t l:jrH >( 'V(\ .I4'ah.•b&done'eitheIL b}Ldeterminingthe intact cyanogenic glycosides with GC, HPLC or LC-MS as described below (Experiments 10 and 11, X Observation 4-6), or the HeN can be released with exogenou@lucosidase as described above (Experiments 2, 7). For the latter, make sure that the methaI}gLconcentl'ation is low enough to avoid inhibition of the activity '<, of ｴｨ･ＨＳ［ｾＡ｜Ｉｵ｣ｯｳｩ､｡＠ employed (Refer Experiments 3-5). Observations (i) /'. (ii) " )" (iii) X X. (iv) (v) The amount of tissue used depends on its cyanogenic glycoside concentration, e.g. 50-200 pg tissue or 1-21eafdis\s (diarn=8 mm) ｰｾｲ＠ 500 }II 85 % ml1thanol. . . pl. ｾ＠ '" .1J(thetubesare.not.tight.enough,\lkigh ｴｨｬ［Ｇｮ＿ｾｦｲ＠ adding the plant material as quickly as possible\ ｾ･ｲ＠ boiling and cooling, clear the extraction solution by spinnirlg andlor filtering or remove intact leaves and then lyophilise entire solution. Redissolve in a known volume 85% methanol. This can also be used to concentrate samples. In case of dhurrll (Fig. 12.1), a sma)) quantity can be expected to be lost during boiling because th<I'PllYdroxy group is unstable (Mao and Anderson 1965). A mOI'e geritle way of extracting is to submit plant tissue to three cycles of snap-fmezing in liquid Nitrogen and thawing, in 85 % methanol. Bjarnholt (unpublished data) suggests that for dhurrll, the boiling method maybe quantitatively superior because ｴｨ＼ｾ［ｧｬｵ｣ｯｳｩ､｡･＠ may not be completely deactivated (allowing degradation) or tissue disruption may not be complete when using the freeze method (01' both). High temperature extraction methods increase the risk of racemisation, resulting in epimeric changes. particularly in aqueous 01' alkaline conditions (Nahrstedt, 1981). This does not matter for quantification, but it may be important for structural elucidation (G1eadow et al., 2003; Miller et al., 2006c). Boiling must begin quickly. Thawing or decaying tissue and disrupted cells of leaf disks will quickly lose cyanogenic glycosides as a result of a-glycosidase activity, if not immediately boiled. 0i/ 300 (vi) (vii) x Soil Allelochemicals The length of time required for full extraction by boiling varies. This can be determined by performing a time course study and measuring the effectiveness (Nielsen et at., 2002; Franks et at., 2005; Jprgensen ef al., 2005; Kristensen ef al., 2005; Bjarnholt ef al., 2008b; Morant ef al., 2008; Sanchez-Perez ef 01.,2008). If lipids are interfering with the analysis, the methanolic extract can be lyophilised, redissolved in water and then defatted by extracting three (or more) times with one volume ｯｦＢｬＱｾＧＪＬｰｃＮ＠ (GJeadow ef 01., 2003; Goodger ef 01.,2004; Miller ef al., 2006a). Precautions Boiling should be done in fume hood as toxic methanol may escape the extraction tubes. Experiment 9. Extraction of Cyanogenic Glycosides from Soil Using Boiling Methanol Principle hlt\tb .e- ｾ＠ ,...-, X>t /;:. .'Sl .ｾ＠ \"1). Cyanogenij; glycosides can easily be extracted {rom soil with boiling Xmethano1<lRefer Experiment 8 for plant materia). However, this is only suitable for "high" concentrations 0:[ cyanogenic glycosides, in the range [Of mmol kg-I. ｾａ｡Ｑｴ･ｲｮｩｶ＠ method\for lower ｣ｯｮ･ｴｲ｡ｩｾｵｳｧ＠ ABE . (Accelerated Solvent Extraction) ana SPE (Solid Phase Extl'action), is beyond the scope of this chapter. For details refer to Bjarnholt ef al. "(2008a). Materials and Reagents Flasks for boiling with tightly fitting lids, e.g. Duran blue caps (size: at least 100 ml for extracting in 25 ml solvent), water bath, ice, balance, micropipettes, Eppendorf tubes, centrifuge, cellulose syringe filters (0.45 pm). ｃｨ･ｭｾｩ｣｡ｬｳ＠ 85% methanol Procedllre (i) (ii) Ｈ＼ａｾｦｴＮ＠ : I' X (iii) (iv) w";k\) .th.II)') (, (v) (vi) Weigh 5 g soil and add to an extraction flask. methanol. Seal. .' Add 25 ml Ｘｾ＠ Boil for 5 min in a water bath (Refer Observation(1)) Coolon ice before unscrewing the tube cap. Take an aliquot (1m]) and clear an aliquot by centrifuging at 27,000 for 10 min. j' Filter the supernatant through 0.45 ).lm filter, e.g. regenerated cellulose syringe filter. -':f h 0/0)..,.",' ｻＧＮｾＫＬｏｬ＠ 301 Cyanogenic Glycosides ,, (vii) ｾｌ＠ Analyse using LC-MS (Experiment 10) or HPLC analysis (Experiment 11) (Refer ｏ｢ｳ･ｲｶ｡ｴｩｯｾ＠ Observations (i) (ii) Preheat the water bath to boiling to ensuxe that tubes are heated quickly. High temperature extraction methods increase the risk of racemisation, resulting in epimeric changes, particularly in aqueous or alkaline conditions (Nahrstedt, 1981). This does not matter for quantification, but it may for structural elucidation (Gleadow et aZ., 2003; Miller ef al., 2006c). Experiment 10. Identification and Measurement of Cyanogenic Glycosides Using LC·MS Principle c, " Q (1:)f t ｾＬ＠ YV) \. n./\(" I' {. 'I·. k).,,' 't_ ｾＮＺＩ＠ LC-MS (Liquid Chromatography· Mass Spectrometry) is useful for analysis of all cyanogenic glycosides from organic matter or soil, but it plays a particularly important role in determining the aliphatic cyanogenic Xglycosides (e.g. linamarin and lotaustralin) which cannot be detected\by UV absorption (Refer Experiment 11). Besides, LC-MS provides structu\.'al information needed for accurate identification of cyanogenic compounds. While GC-MS (Gas Chromatography-MS) analysis of cyanogenic glycosides requires dehydration of plant samples and derivatilation of the compounds (e.g. Gleadow et al., 2003), LC-MS analysis only requires a previous filtering to avoid clogging of columns. There are a number of examples where LC·MS has been applied to the analysis of cyanogenic compounds (e.g. Hansen et aZ., 2003; Franks et aZ., 2005; Bjarnholt et aZ., 2008.b; Sanchez-Perez et aZ.) 2008). LC separation is carried out on reversed phase columns with mobile phases consisting of water and methanol/acetonitIile plus formidacetic acid; ionisation is obtained with electrospray, and the cyanogenic ｧｬｹ｣ｯｳｩ､･｟ｾ＠ are detected in positive mode as Na-adducts (Table 12.2; ObservatiOl(l)) Table 12.2. List of common cyanogenic glycosides, their molecular masses and the ml z-values of their diagnostic peaks in LC':MS (see Experiment 10). Compound (g mol· Linamarin Lotaustralill Prunasin Amygdalin* Dhurrin Diagnostic mJz-values Molecular mass 1) 247.25 261.27 295.29 457.43 311.29 [M+Naj' [M·HCN+Naj' 270 284 318 480 334 243 257 291 453 307 * ｛ｳｵｧ｡ｲＭｈＲＰＫｎ｝ｾ＠ 185 185 185 347 185 * Since amygdalin is a di-glucoside, "sugar" refers to cellobiose minus two water molecules, whereas for the rest of compounds the diagnostic peak originates fl:om glucose minus one water molecule. Soil Allelochemicals 302 Reagents Liquid Nitrogen, 85% Methanol, balance, ice. l\1illipore water, Reagent A: 0.1% (v/v) HCOOH (formic acid) and 50 mM sodium chloride (2.92 g NaCI pel' 1000 m]); and B, 0.1% (v/v) HCOOH and 80% (v/v) acetonitrile (MeCN); cyanogenic glycoside standards (0-200 pM in 20% methano]). A 200 pM stock solution of amygdalin (Sigma A8320) is made by dissolving 91.48 mgin 1 ml20% methanol. A 200 pM stock solution ofDhl'min (MW=311.29) is made by dissolving 62.2 mg in 1 ml 20% methanol. ｾ＠ " I 1, I Equipment ｾ＠ tubes with additional cap sealers; ｭｩ｣ｻｾＺｰ［･ｴｳ＠ hot plate 01' saucepan; ｭｩ｣ｲｯｾ･ｮｴｦｵｧ［＠ filter titre plates (membrane size :::: 0.45 pm) with fitted lids; centrifuge that can be used for titre plates; micro-pipettes with filter tips (0.45 pm); small scintillation vials (1 ml capacity, e.g. from Bonnett); glass inserts for the scintillation vials (50 pl). HPllOO LC (01' equivalent) coupled to a Bruker Esquire-LC ion trap mass spectrometer (Bruker Instruments, Billelica, MA); XTen'a MS C18 column (Waters, Milford, MA) ,vith internal diameter of 2.1 mm, length 100 rum, >( particle size 2.5 pm) (Refer Observation 2). For analysis of data use the Bruker Daltonics DataAnalysis or other proprietary software. ("",D, (" Q j>1\lil \1)4'\ ", \ ,r II,,,) ('t,r /'\ ｾｉｅｰ･ｮ､ｯｲｦ＠ ( \'\t,'l, ,.\ \'i\''\)\ \ "1) Ｈ｜ｦＢｾ＠ p <, «)h \ ,-'",1 ｉ｜［ＧＢｾ＠ '<." electric t , .' ,, t, I ,) Procedure (i) Grind plant material in liquid Nitrogen until it is a fine powder Do not let the plant matelial thaw (powder may be stored at ＭＸｾｃＩＮ＠ out. (ii) Add 500 pI of 85% methanol to 2 ml Eppendorftube. Record weight of Eppendorf tube containing methanol. (iii) Add few scoops of gl'Ound plant 01' soil material (appl'Ox. 150 mg to 200 mg). Seal Eppendorftube with stoppel' and boil for 3 min. Cool on ice. (iv) (vii) (viii) Remove cap stopper and weigh Eppendorf tube again in order to obtain the net weight of matelial added. Centrifuge for 3 min at 27,000 g. Remove supernatant. and store in -1 ml scintillation vials. Extract may be stored aCiIQ °C while completing subsequent extractions. Before running the extract on LC-MS, dilute it to 20% methanol and filter through 0.45 11m filter membrane. Fol' it pipette 10 pI sample into a micrp:),ttre filter plate and add 40 pI H 2 0. Covel' ,vith the plate lid. Indicate which wells have been used on the lid so that any remaining empty wells can be used another time. Centrifuge the covered plates for 5 min at 18,000 g. Transfer 30 111 to the glass insert tube in 1 ml scintillation vials and replace the lids. Samples can be stored overnight at 4°C, before running on the LC-MS. Cyanogenic Glycosides 303 (ix) '"II I Make up standard solutions of dhurrin (or other cyanogenic glycosides) in 20% methanol. Transfer 30 VI into the glass insert tube using filter pipettes that have been placed in the 1 ml scintillation vials and put the lids on. (x) Arrange vials on the autosampler with a series of four standards every 40 vials. (xi) Start the LC-MS with a flow rate of 0.2 ml min" using Reagents A and B 3% (Vl.\::l; (Refer Observation(3)) ," (xii) Inject sample onto the column. The volume can be 3 to 15 pI, but note the volume for later calculations. (xiii) Use following gradient pl'ogranune: 0 -2 min, iS9.Crat!c 3% (v/v) AI B; 11 -3()lmin, linear gradient 3% to 50% (v/v) B;i30 -3& min, linear gradient 50% to 100% (v/v) B; and 35, -50 min, isocratic 100% (v/v) B. and total ion (Refer ObservatiOli 4)) (xiv) Run the mass spectrometer in positive ion mode and record the ｾﾻｮ･ｴ＠ and specific [M-HCN+Nat adduct ions (Refer Observations V n-ltnd:5).')Note the retention times. Compare the retention times and fi'agmentation pattern with authentic standards to verifY the I I identification of cyanogenic glycoside or nitrile (Refer Observations vI V II (6).nd 7;)Table 12.2). (xv) Analyse results using Bruker Daltonics DataAnalysis software. (xvi) Check that the intensity of the maximum absorbance peaks of the samples is between 105 and 107 . Ifit is too high, use a higher dilution (still"esulting in 20% methanol) and repeat. (xvii) Select the 'integrate' option from the tool bar. This will automatically number the peaks and list them in a table at the bottom, along with the retention time and the area of the peak. (xviii) Cut and paste the peaks of interest into a data spread sheet (e.g. MC Excel). Calculate a standard curve to convert area to concentration of cyanogenic glycosides. Knowing the injection volume, the dilution factor and the extraction volume, the total amount (mass) of cyanogenic glycosides in the initial sample can be calculated (Refer Observation viii). Observations (i) (ii) The plant extract alone can supply the ions for formation of these adducts (Franks et al., 2005), but if NaCI is added to the LC mobile phase, peak areas are sufficiently reproducible to facilitate quantification (Bjarnholt et aI., 2008a; ｊｾｲｧ･ｮｳ＠ et al., 2005; Morant et al., 2008). Column characteristics are those used by Nielson et al. (2002). Analysis of the extremely polar small aliphatic cyanogenic glycosides requires suitable LC column material. Here we describe the Waters X-Tena RP C18. Columns such as Phenomenex Synergy Soil Allelochemicals 304 v v (iii) (iv) (v) (vi) (vii) (viii) Ｏｾｰｲ＿ＱＩｮｵｴｩｯｳＬ｟Ｚ｣＠ Fusion RP or Agilent Zorbax SB-CI8 would also be suitable. Columns of various length (e.g. 50-150 mm) and particle sizes (1.72.5 pm) can be used but this will affect retention times (Refer also Observation ｾＮ＠ A flow rate of 0.2-0.3 ml min- 1 can be used but this will affect the retention time (Refer ｏ｢ｳ･ｲｶ｡ｴｩｯｮｾＵＩ＠ .. Other gradient programs can be used depending on the separation required (Nielsen et al., 2002, Forslund et aZ., 2004). With ion trap or triple quadmpole MS instruments it is possible to distinguish cyanogenic glycosides from other compounds in a complex mixture such as plant or soil extracts. These instruments Can provide MSfMS-spectra, i.e. fragmentation patterns of the ions in the electrospray. Fragmentation of Na-adducts of cyanogenic glycosides leads to two diagnostic ions, ODe originating from a neutral loss ofHCN from the molecular adduction ([M-HCN+Nat), and one originating from the Na-adducts of the dehydrated SUgill'S resulting from cleavage of the glycosidic bonds (e.g. [glc-H20+Na]+) (Hansen et al., 2003; Franks ef aZ., 2005; Thorsoe ef aZ., 2005; Bjarnholt ef aI., 2008b). Refer to Table 12.2 for masses of molecular ions and diagnostic fragment ions of the compounds from Fig.12.1. Examples of retention time for cyanogenic and nitrile glucosides using this configuration are as follows: linamarin, 4.5 min; dhurrin, 5.5 min; rhodiocyanoside D, 8.5 min; rhodiocyanoside A, 9.5 min; and lotaustralin, 14.3 min. This is the simplest and most basic analysis of the data. The Bruker software is very powerful and can be used to make many other calculations and interpretations beyond the scope of this protocol. Other LC-MS will come with their own data analysis software. See the relevant operating manuals for details. 1) .............. ｾＮ］＠ 1'/ I IV 'r () ｾｬ＼ＺＢ＠ b' J) ｾｯ＠ dD lYD Vi? i ,t"j? v, t", {J (J ｾｬ｜ＮＡ｟ｻＧｕｉｃＬＨＹｴＱ＠ k) Ｇﾫ｜ＨＬｻＡｾＩ＠ ................................................... ).. \..... <,., " '. ' ｨｖｾＱ＠ 'tf ('1''1' ＢＬｾ＠ i\\,'.,\f\ t\-\\!,) \,t(,t)l',., -"\ '" -,-, (' f· (\';,?-' \I ()(f"ikJl(l Experiment 11. Isolation of Cyanogenic Glycosides Using HPLC () and HP·TLC Principle v\t)"')". ｘ｣ｨ｡ｲｴ･ｬｾｩｯｮ＠ Several clU'Olnatographic techniques have been used to fractionate and purity the cyanogenic glycosides. Many reviews provide information about the range and details of HPLC colwnns, and solvent systems used for fractionation by HPLC and HP-TLC (e.g., Seigler, 1975; Brimer, 1988; Brinker and Seigler, 1992). Therefore, we have only provided simple examples of application of HPLC and HP-TLC in purification and preliminary of cyanogenic compounds. HPLC is useful for the LUI'\\" fA" ' \ 'I,A.\ Ill'" "r '·,d) " Cyanogenic Glycosides ＬｾＴ＠ 305 quantitative and qualitative analysis of both cyanogens and then' dedvatives. The majodty of reseoo'chers have used ｒＬＭｃｬｾＩｯｵｭｮｳ＠ which appear to be suitable for most classes of cyanogens. Visualisation reagents used in TLC can vary but they usually detect cyanogenic glycosides based on evolved cyanide (e.g. sandwich method using FA papers ｡ｮ､｝ｾｬｵ｣ｯｳｩ･＠ spray) or their carbohydrate moiety (e.g. orcinol; Miller ef al., 2006c). Depending on the yield of cyanogenic glycoside in the e"loract, both HPI,C and ｈｐｾｔｌｃ＠ are valuable preparative techniques for the isolation of sufficient pure cyanogenic compounds for structure elucidation (i.e. by mrn). Materials alld Equipment In c,'J( ,,0\' \>'< HPLC system and an approPliate column (e.g. Phenomenex Luna C18 column 250 ｭｾｏ＠ ｭｴｲｬｾＵ＠ lllin particle size); solid phase extraction (SPE) cartridges (e.g. Alltech maxi-clean CIS cartridges); luer lock ｳＩｾｩｮｧ･＠ or peIistaltic pump; membrane filteI's (e.g. ｐｔｆｅｾ＠ 45 p.m pore size); access Nitrogen manifold, or to a vacuum desiccating system (e.g. ｬｹｯｰｨｩｦＬｾＢ＠ speed vacuum concentrator). Aluminium-backed Silica gel 60 HP-TLC plates; an oven (up to 110 °C); spray device for detection reagents; TLC tank; filter paperlhlotting paper; single edged razor blades; ruler. Reagents Methanol for partial puIification by SPE and various (e.g. chloroform, methanol, acetonihile, ethyl acetate) for fractionation, depending on likely cyanogens; buffer, 1 M NaOH.®lucosidase enzyme preparation and reagents for cyanide quantification (Refer Experiments 2 and 3); orcinol spray reagent (20 mg mrl orcinol monohydrate in ethanol: conc. H 2S04 : H 20; 75:10:5, v/v) Procedure Fractionation by HPLC The crude methanol extract can be cleaned up and the cyanogenic compounds partially purified by elution t1lJ:ough a SPE cartlidge. (i) (ii) ｾ＠ (iii) "'<' (iv) Condition the cartlidge by linsing with methanol (5 mIl and then H 20 (5 ml) using either a peristaltic pump or by hand using a syringe. TIns primes the cartridge using positive pressure. Load 1 ml of extract resuspended in H 2 0 onto the primed cartridge. Elute at 1 ml min- 1 using a step methanol gradient (0%, 10%, 20%, 40%, 60%, 100% MeOH in H 2 0). Collect fractions of 10 ml and concentrate them l.n vacuo. ｑｵ｡ｮｴｩｶｾｬｹ＠ assay an aliquot for cyanogenic glycosides with addition ｯＨ＿ｾｧｬｵ｣ｳｩ､｡･＠ enzyme preparation. Filter a resuspended aliquot of the eluant with highest cyanogen content through a membrane filter plioI' to injection on the HPLC column. Soil AHelochemicals 306 Nil \ \' Fractionate the eluant isocratically by reverse phase HPLC e.g. g 10% MeCN-H2 0 (1 ml min-') through a Phenomenex Luna ><, 1.2!§,jolumn (250 ffimX 4.6 mlll<' 5 mm particle size). (vi) Collect fractions, each one of 1 ml, concentrate these in vacuo, resuspend in buffer, add enzyme and determine the presence of >e. cyanogenic glycosides using th" quantitative assayl\ ( (-" 1 pO ( i (V\ (v) ｗＮｪＩＧＺｦｴ｜ｾ＠ CI'(;. 1\' X tvA. 0\4 X ｾＮＧｗＺＡＩｬ＠ ｾＬ＠ Procedure Fractiollation by TLC (i) (iD • l (iii) ＨｾａＧｩｙｦ｜＠ (iv) \ ＢＩｬｾＯ､ＧＬＮ＠ ｜ＩｮｬｾＧ＠ 1 1(,;. { ' } ) 0 \.jl\O !<3i',I( (I (v) (vi) (vii) Carefully rule up the TLC plate, including 2 mm markers up each side for subsequent scraping. . Resuspend an aliquot of the partially purified cyanogens, following the SPE clean up step. Load in a volatile solvent to reduce bleeding and for dlying of the sample (e.g. chloroform:methanol, 2:1, v/v). Use a hail' dryer to accelerate drying. Initially load at least 2 lanes - 1 for visualisation with spray reagent, and 1 for analysis Qf silica fractions and desorption of cyanogens (Refer Observatio(l)) Add approx. 150 ml solvent mixture (e.g. CHCI 3 :MeOH:H2 0, 65:35:4, v/v; Miller ef 01., 2006c) to TLC tank, with blotting paper soaked in the solvent against the side of the tank to ensure an even atmosphere within the tank. Place the dr'y TLC plate into the tank in the fume hood, seal with glass lid, and run until a couple of cm Ii'om the top of the plate (mark solvent Ii'ont); allow plate to dry. II. Cut plate and spray 1 lane with orcinol spray reagent (in fumeft-0od), develop at 110 'C for 5 min. Once dry, precisely scrape 2 mm sections from unsprayed lane directly into a glass vial. Add buffer and enzyme and assay quantitatively for evolved HCN. Sonication and mixing will help desorb the cyanogens from the silica. The purity of fl'actions can be assessed by analytical TLC, some cyanogenic bands may need subsequent fractionation in alternative solvent solutions to increase purity (Refer Observation 2). Observations (i) (ii) Extracts can be derivatised using Tri-Sil® Reagent (HMDA:TMCS:Pyr'idine, 2:1:10) (Pierce, Rockford, Illinois, U.S.A.), warmed to 70 °C, and allowed to stand at room temperature for 10 min (e.g. Gleadow et al., 2003) or using fundamental acid methanolysis and TMSi (trimethylsilyl) derivatisation techniques (e.g. McConville et 01., 1990; Ralton and McConville, 1998). Even if a single cyanogenic peak is resolved by HPLC, he aware 0 ,\ I· ').'j I Cyanogenic Glycosides 307 that epimers (e.g. S-sambunigrin and R-prunasin) will co-elute, and their occurrence must be investigated. The chirality of the cyanogenic compounds can be resolved by NMR (Goodger and Woodrow, 2002; Milleret al., 2004), and also by GC-MS of the TMS ethers of the cyanogenic glycosides (Seigler, 1975; Brinker and Seigler, 1992; Gleadow et al., 2003; Miller et al., 2006c). Precalltions Prepare the solvent systems perform TLC analysis and dry developed TLC plates in a fumehood. ApproPliate gloves and protective equipment should be used for solvent handling. Orcinol spray reagent (containing conc. H 2S04 ) is VeJY corrosive. It should be prepared and used in the fumehood. SUGGESTED READINGS Bak, S., Olsen, C.E., Petersen, B.L. and Ｑ｜ｉｾｬ｣ｲＬ＠ B.L. and Halkier, B.A. 1999. Metabolic engineering of p-hydroxybenzylglucosinolate in Arabidopsis by expression of the cyanogenic CYP79A1 fiUlU Sorghum bicolor. Plant Journal 20: 663-71. Ballhorn, D.J., Kautz, S., Lion, U. and Heil, M. 2008. n'adc-offs between direct and indirect defences of lima bean (Phaseolus lwwtllS). Journal of Ecology 96: 971980. Bjarnholt, N., Laegdsmand, M., Hansen, H.C.B., Jacobsen, O.H. and ｍｾｬ･ｲＬ＠ B.L. 2008a. Leaching of cyanogenic glucosides and cyanide from white clover green manure. Chemospliere 72: 897-904. Bjarnholt, N., Rook, F., 1\'1otawia, M.S., Jorgensen, K, Olsen, C.E., Jaroszewski, J.W., Bak, S. and ｍｾｬ･ｲＬ＠ B.L. 2008b. Diversification of an ancient theme: hydroxynitrile glucosides. PhytochemistlY 69: 1507-16. Brimer, L. 1988. Determination of cyanide and cyanogenic compounds in biological systems In: Cyanide compounds in Biology (Eels., Evered, E.H.S. and Harnett, S.), pp. 177-200. John Wiley & Sons, Chichester, USA. Brimer, L. and Dalgaard, L. 198,!. Cyanogenic glycosides and cyanohydrins in plant tissues: Qualitative and quantitative determination by enzymatic post-column cleavage and electrochemical detection, after separation by high-performance liquid chromatography. Journal of Chromatography 303: 77-88. Brinker, A.l'.L and Seigler, D.S. 1989. Methods for the detection and quantitative determination of cyanide in plant materials. PhytochemIcal Bulletin 21: 24-31. Brinker, A.M. and Seigler, D.S. 1992. Determination of cyanide and cyanogenic glycosides from plants. III: Plant Toxin Analysis. (Eds., Linskens, H.F. and Jackson, J.F.), pp. 359-381. Springer-Verlag, Berlin. Conn, E.E. 1979. Cyanogenic glycosides. In: Biochemistl)' of Nutritioll (Eds., Jukes, T.R. and Neuberger, A.). Intematiollal Review of Biochemistl)' 27: 21-43. Dahler, J.M., McConchie, C.A. and Turnbull, C.G.N.1995. Quantification of cyanogenic glycosides in seedlings ofthree Macadamia (Proteaceae) species. Australian Journal of Botany 43: 619-628. Delgado, E., Mitchell, R.AC., Parry, M.A.J., Driscoll, S.P., Mitchell, V.J. and Lawlor, D.W. 1994. Interacting effects of CO2 concentration temperature and nitrogen supply on the photosynthesis and composition of winter wheat leaves. Plallt, Cell and Environment 17: 1207-1213. 308 x Soil Allelochemicals Egan, S.V., Yeah, H.H. and Bradbury, J.H, 1998. Simple picrate paper kit for determination of cyanogenic potential of cassava flour. Journal of Science of Food and Agriculture 76: 39-48. Fox, J.L. 2008. Purification (wd Characteri.sation ofihe CYUIIOgClll\b'.gulcosidase from Eucalyptus clue/ocalyx'. Ph.D. Thesis, The School of Botany, ｔｨｾ＠ University of l\Ielbourne. Australia. Franks, T.K., Hayasaka, Y., Choimes, S. and van Heeswijck, R. 2005. Cyanogenic glucosides in grapevine: Polymorphism, identification and developmental patterns. Phytochemistry 66: 165-73. Gleadow, R.l\L 1999. ResourceAllocatioli in Eucalyptusc1adocalyx F.l\IueU. PhD Thesis, School of Botany. The University of ]o,'Ielbourne, Australia. Gleadow, RM. and Woodrow, I.E. 2000a. Temporal and spatial variation in cyanogenic glycosides in Eucalyptus dadocalyx. Tree Physiology 20: 591-198. Gleadow, R.M. and ｗｾＮ＠ ,'ow, I.E. 2000b. Polymorphism in cyanogenic glycoside content and cyanogenic ｾｴｯＯｧｬｵ｣ｳｩ､｡･＠ activity in natural populations of Eucalyptus dadocalyx. Australian Journal of Plant Physiology 27: 693-699. Gleadow, R.:M. and Woodrow, I.E. 2002a. Constraints on the effectiveness of cyanogenic glycosides in herbivore defence. Journal ofClwmical Ecology 28: 1301-1313. Gleadow, RI\L and Woodrow, I.E. 2002b. Defence chemistry of Eucalyptus dadocalyx seedlings is affected by water supply. Tree Physiology 22: 939-945. Gleadow, R,1o.L, Foley, W.J. and Woodrow, I.E. 1998. Enhanced CO 2 alters the relationship between photosynthesis and defence in cyanogenic Eucalyptus cl(f(/ocalyx F. MucH. Plant, Cell and Environment 21: 12-22. Gleadow, RM., Vecchies, A.C. and Woodrow, I.E. 2003. Cyanogenic Eucalyptus nobilis is polymorphic for both prunasin and specific b·glucosidases. Phytochemistry 63: 699·704. Gleadow, RM., Edwards, E. and Evans, J.R 2009a. Changes in nutritional value of cyanogenic Trifolium repens at elevated CO 2 , JOllrnal Chemical Ecology 35: 476487. Gleadow, RM., Evans, J,R, :McCaffrey, S. and Cavagnaro, T.R. 2009b. Growth and nutritive value of cassava (Manihot esculenta Cranz,) are reduced when grown at elevated CO2 . Plant Biology 11: 76·82. Goodger, J.Q.D. and Woodrow, I.E. 2002, Cyanogenic polymorphism as an indicator of genetic diversity in the rare species Eucalyptus yarraellsis (Myrtaccae), Functional Plant Biology 29: 1445-1452. Goodger, J.Q.D., Capon, RJ. and Woodrow, I,E. 2002. Cyanogenic polymorphism in Eucalyptus polyanthemos Schauer subsp vestita L. Johnson andIC. Rill (Myrlaceae), Biochemical Systematics and Ecology 80: 617·630, Goodger, J.Q.D., Gleadow, RM. and Woodrow, I.E. 2006, Growth cost and ontogenetic exprcssion patterns of defence in cyanogenic Eucalyptus spp. Trees 20: 757-765. Halkier, B.A and Moller, B.L. 1989. Biosynthesis of the cyanogenic glucoside dhul"l'in in seedlings of Sorghum bicolor (L.) IHoench and partial purification of the enzy"Ille system involved. Plant Physiology 90: 1552-1559, Halkier, B.A., Scheller, R.N. and Moller, B.L, 1988. The hiosynthctic pathway and the enzyme system involved, III: Cyanide Compounds in Biology (Eds., Evered, E,H,S. and Harnett, S.), pp. 49-66. John Wiley and Sons, Chichester, USA. Hansen, KS., Kristensen, C., Tattersall, D.B., Jones, P.R, Olsen, C.E" Bak, S. and 1o.1011el', B.L, 2003. The in vitro substrate regiospecificity of recombinant UGT85Bl, the cyanohydrin glucosyltransferase from Sorghum bicolor, Phytochemistry 64: 143-51. 309 Cyanogenic Glycosides Jurgensen, K, Bak, S., Busk, P.K, S!1rensen, C., Olsen, C.E., Puonti-Kaerlas, J. and Alpllcr. B.L. 2005. Cassava plants with a depleted cyanogenic glucoside content in leaves and tubers. Distribution of cyanogenic glucosidcs, their site of synthesis and transport, and blockage of the biosynthesis by RNA interference technology. Plant Physiology 139: 363-74. Kristensen, C., h.forant., 1\1., Olsen, C.E. Ekstrom, C.T., Galbraith, D.W., l\Ipllcr. BL. and Bak, S. 2005.l\'Ietabolic engineering ofdhurrin in transgenicArabidopsis plants with marginal inadvertent effects on the metabolomc and transcriptomc. Proceedings, National Academy of Sciences of United Stotes of America 102: 177984. Lambert., J.F,S., Ramasamy, J. and Paukstelis, J.V. 1975. Stable reagents for the colorimetric determination of cyanide by modified Konig reactions. Analytical Chemistry 47: 916-918. lo.1ao, C.H. and Anderson, L. 1965. CY('!pogencsis in Sorghum vulgare. 2. 1o.Iechanism of X alkaline hydrolysis of ､ｨｵｲｩｮﾫｾＬＭＹｹｯｸｭ｡･ｬｴ＠ glucoside), Journal of Organic Chemistry 30: 603-607. ' , :McCollville, 1I.J., Thomas-Oates, J.E., Ferguson, l\lA.J. and Homans, S.W. 1990. Structure of the lipophosphoglycan from Leishmania major. The Journal of Biological Chemistry 265: 19611-19623. l\Icl\Iahon, J.M., White, \V.L.B. and Sayre, R.T. 1995. Cyanogenesis in cassava (Mani/wt esculenta Crantz). Journal of Experimental Botany 46: 731-741. Miller, R.E., Gleadow, RM. and Woodrow, I.E. 2004. Cyanogenesis in t.ropical Pnmus tumeriana: charactelisation, variation and responses to Imv light. Functional Plant Biology 31: 491-503 . .Miller, RE., Stewart., 1\'1., Capon, RJ. and Woodrow, I.E. 2006a. A galloylated cyanogenic glycoside from tllC Australian rainforest endemic Elaeocal'pus seriopetalus. Phytochemistry 67: 1365·1371. Miller, R.E., Jensen, R. and Woodrow, I.E. 2006b. Frequency of cyanogenesis in Australian tropical rainforests. Annals of Botany 97: 1017-1044. I\Hller, R.E., McConville, M.J. and Woodrow, I.E. 2006c. Cyanogenic glycosides from the rare Australian endemic rainforest tree Clerodem/rum grayi (Lamiaceae). Phytochemistry 67:43-51. Mpller, B.L. and Conn, E.E. 1980. The biosynthesis of cyanogenic glucosides in higher plants. Channeling ofintermediates in dhurl'in biosynthesis by a microsomal system from Sorghum bicolor (L.) Moench. Journal of Biological Chemistry 255: 30493056. Morant, A. V., Kragh, M.E., Kjrel'gaard, C,H., Jprgensen, K, Paquette, S.M" Pioh'owski, 1\1., Imberty, A., Bjarnholt, N" Olsen, C,E., :Mpller, B.L, and Bak, S. 2008, Cyanogenic b·glucosidases in Lotusjaponicus, Plant Physiology 147: 1-20. Nalu'stedt, A. 1981. Isolation and structure elucidation of cyanogenic glycosides, 111: Cyanide in Biology, (Eds" Vennesland, B., Conn, E.E., Knowles, C.J., Westley, J. and Wissing, F,) pp, 145-181, Academic Press, New York. , IVYP{)-'y9 1QJ(»/I 'Q ＬＢＭｾ＠ I I Nahrstedt, A. 1985. Cyanogenic compounds as protecting agents for organisms, Plant Systematics and Evolution 150: 35-47. Nielsen, Ie.A., Olsen, C.E., Pontoppidan, K. and I\Ipllel', B.L. 2002, Leucine-derived cyano glucosides in barley, Plant Physiology 129: 1066-1075. rPoulton, J,E. 1988. Localisation and catabolism of cyanogenic glycosides. III: Cyanide Compounds in Biology (Eds" Evered, D. and Harnett, S,), pp. 67-91. John Wiley'& Sons, Chichester. O'"[)onnl'li ) 1'-/, {rAI·" Ｒａｾ＠ tl oC !"b '( Ｈｾ｜Ｌ＠ .\q \ '\ \' Ov, ,J ':1 \( t', \ , ! ｾ＠ I t i \ I 310 Soil Allelochemicals Ralton, J,E. and l\'icCollville, l\LJ. 1998. Delineation of three pathways of glycosylphosphatidylinositol biosynthesis in Leishmania mexicQllu- Precursors from different pathways are assembled on distinct pools of phosphatidylinositol and undergo fatty acid remodeling. Journal of Biological Chemistry 273: 4245-4257. Sanchez-Perez, R., Jorgensen, K.. Olsen, e.E., Dicenta, F. and Moller, B.L. 2008. Bitterness in almonds (Prunus dulcis (lI.Hller) D. A. Webb,), Plant Physiology 146: 1040-52. Seigler, D.S. 1975. Isolation and characterization of naturally occurring cyanogenic compounds. Phytochemistry 14: 9-29, Thorsoe, K.8., Bak, S., Olsen, C.E., Imbedy. A., Breton, C. and Moller, B.L. 2005. Determination of catalytic key amino acids and UDP sugar donor specificity of the cyanohydrin glycosyltransferase UGT85Bl from Sorghum bicolor. l\Iolecular modeling substantiated by site-specific mutagenesis and biochemical analyses. Plant Physiology 139: 664-73. Webber, B.L., Miller, R.E. and Woodrow, I.E. 2007. Constitutive polymorphic cyanogenesis in the Australian rainforest tree, Ryparosa kurrallgii (Achariaceae). Phytochemistry 68: 2068-2074. Woodrow, I.E., Slocum, D. and Gleadow, R.l\I. 2002. Influence of water stress on cyanogenic capacity in Eucalyptus cladocalyx. Functional Plant Biology 29: 103110.