synthesis of tertiary amine inhibitors  abamine was 
  synthesized according to published procedures 
  ( 31 , 
  34 ) . n - boc , o - benzyl - hydroxylamine was treated with nah in dmf , 
  followed by the appropriate benzyl or alkyl bromide 
  ( 35 ) . deprotection was carried 
  out by treatment with 1% trifluoroacetic acid in dichloromethane , to give the 
  n - substituted hydroxylamine . hydroxamic acid formation was carried 
  out using dcc ( 1.1 equiv . ) and 4-dimethylaminopyridine ( 0.2 equiv . ) and the 
  appropriate carboxylic acid , in dichloromethane . b1 , d12 , and d13 were 
  prepared by activation of the appropriate acid with methyl chloroformate and 
  triethylamine in thf , and reaction with hydroxylamine hydrochloride . the 
  intermediate for synthesis of b1 was synthesized from -ionone 
  ( 36 ) ; b1 was isolated as a 2:1 
  mixture of e / z isomers . spectroscopic data and yields for analogues d1-d13 , 
  f1 - 4 , and b1 are available as supplemental materials . table 1inhibition of recombinant leccd1a and lenced1 enzymes using in 
  vitro assaysenzyme assays , using e. coli cell extracts containing the 
  recombinant ccd , were initially carried out at 100 m inhibitor 
  concentration ; for compounds showing 95% inhibition of leccd1a at this 
  concentration , ic50 values were also determined . chemical structures of hydroxamic acid inhibitors are shown below , with x and 
  y given in the table . inhibition of recombinant leccd1a and lenced1 enzymes using in 
  vitro assays enzyme assays , using e. coli cell extracts containing the 
  recombinant ccd , were initially carried out at 100 m inhibitor 
  concentration ; for compounds showing 95% inhibition of leccd1a at this 
  concentration , ic50 values were also determined . chemical structures of hydroxamic acid inhibitors are shown below , with x and 
  y given in the table . 3 . a mean and range of two independent experiments . in vitro nced enzyme assay  we overexpressed lenced1 in 
  e. coli , as an n - terminal his6 fusion protein 
  ( supplemental materials ) . cell - free extract containing recombinant lenced1 was 
  prepared in 100 mm bis - tris buffer ( ph 6.7 ) . 15-l extract was 
  pre - activated by addition of iron ( ii ) sulfate ( 20 mm , 1 l ) and 
  ascorbic acid ( 20 mm , 1 l ) on ice for 2 min , prior to use . this 
  aliquot of enzyme was then added to an assay ( 150 l total volume ) 
  containing 100 mm bis - tris buffer ( ph 6.7 ) , 0.05% v / v triton x-100 , 
  1.0 mg ml catalase , and 3 g of 
  9-cis - neoxanthin . the enzyme 
  assay was incubated for 15 min in the dark at 20 c . water ( 700 l ) was 
  then added , and the products extracted with ethyl acetate ( 3  1 ml ) . the organic solvent was removed at reduced pressure , the residue was dissolved 
  in methanol ( 200 l ) , and then 100 l was injected onto a phenomenex 
  c18 reverse phase hplc column , and a gradient of 5 - 10% methanol in 
  acetonitrile/0.05% triethylamine was applied at 0.5 ml min over 
  20 min , detecting at 440 nm . nced inhibition assays contained 1 - 100 
  m inhibitor ; inhibition was calculated from the product 
  formation after 15 min , compared with a control assay with no inhibitor 
  present . retention times : 9-cis - neoxanthin , 10.2 min ; 
  c25 product , 6.5 min . in vitro leccd1a enzyme assay we overexpressed leccd1a in 
  e. coli , as a gst fusion protein ( supplemental materials ) . the in 
  vitro assay of leccd1a was based on reported methods 
  ( 37 ) , and was carried out in a 
  200-l total volume in a 96-well microtiter plate , with the signal detected 
  at 485 nm . to prepare substrate solution for each assay , 5 l of 4% ( w / v in 
  ethanol ) apo-8-carotenal ( sigma ) was mixed with 25 l of 4% ( w / v in 
  ethanol ) -octylglucoside ( sigma ) , the ethanol was then evaporated under 
  nitrogen , and the residue dissolved in 150 l of phosphate - buffered saline 
  buffer containing 10 mm sodium ascorbate by incubation at 20 c 
  for 30 min . 50 l of cell - free extract containing recombinant leccd1a was 
  added , and the reactions monitored over 30 min at 20 c . in vivo enzyme assays in e. coli  genes of interest 
  ( supplemental table s1 ) were cloned into the vector pet30c ( novagen ) fused 
  directly to the initial atg codon with no tag , or into pgex-4 t such that the 
  gene was fused to an n - terminal gst tag . all genes were full - length except 
  atccd7 , which had the chloroplast signal sequence removed ( supplemental 
  materials ) . this gene , when fused to gst in the pgex-4 t vector ( ge 
  healthcare ) , showed greater ccd activity than when expressed in a pet vector 
  without a tag . the 
  plasmids were transferred to the e. coli expression strain bl21(de3 ) , 
  harboring pac - beta ( 38 ) , and 
  therefore producing -carotene . for each inhibitor assay , 2.5 ml of lb medium with the appropriate 
  antibiotics ( 25 gml chloramphenicol and 50 
  gml kanamycin or 100 gml ampicillin ) and 2.5 
  m isopropyl-1-thio--d - galactopyranoside , was 
  prepared . inhibitors ( 0.1 m in 100% ethanol ) were added to the 
  media to a final concentration of 100 m . the media was then 
  inoculated with 0.25 ml of overnight culture ( grown at 37 c with the 
  appropriate antibiotics ) and incubated with shaking ( 200 rpm ) at 28 c for 
  16 h. 1 ml of culture was harvested by microcentrifugation and resuspended 
  thoroughly in 1 ml of ethanol containing 0.2% , 
  the extract was incubated at room temperature in the dark for 3 h , again 
  vortexed and then spun in a microcentrifuge for 5 min at 13,000 rpm . the amount of -carotene was 
  calculated using a standard curve generated from a dilution series of 
  -carotene ( sigma ) in ethanol with 0.2% triton x-100 ( supplemental fig . the relative inhibition was calculated by the equation : ( ci - 
  cc)/(cl - cc )  100 , where 
  ci is the carotenoid level with inhibitor and ccd present , 
  cc is the level with ccd but without the inhibitor and 
  cl is the level in a strain where lacz is expressed instead of the 
  ccd , and no inhibitor is present . thus the increase in -carotene due to 
  inhibition of the ccd ( ci - cc ) was expressed relative 
  to the maximum possible -carotene content when ccd is absent 
  ( cl - cc ) . growth of arabidopsis  wild - type ( ecotype col-0 ) and the 
  max3 - 9 mutant in the col-0 background 
  ( 21 ) were grown in double 
  magenta pots ( sigma ) on 30 ml of ats media 
  ( 39 ) containing 1% sucrose and 
  0.8% agar , supplemented , where stated , with 100 m inhibitor 
  from a 100 mm stock in ethanol . seeds were sterilized by immersion 
  for 1 min in 70% ethanol and 4 min in 50% household bleach before being washed 
  five times in distilled water . six seeds were placed in each magenta pot , they 
  were vernalized in the dark at 4 c for 2 days , and then placed in a 
  growth room ( 24 c , 16-h light period , 150 mol m 
  s photosynthetically active radiation ) for 45 days before being 
  photographed . axillary bud outgrowth assay in arabidopsis stem 
  sections  assays were performed essentially as described 
  ( 40 ) with the following 
  modifications . small petri dishes ( 50-mm diameter , 20-mm depth ) were filled 
  with 10 ml of ats ( 39 ) 
  supplemented with 1% agar and 1% sucrose . inhibitors and -naphthalene 
  acetic acid ( naa ) were added to the agar before pouring to give 100 
  m and 1 m , respectively . thus , when the central 
  strip was cut out of the agar , both the apical and basal media contained both 
  naa and inhibitor . any nodes in which the apical end had curled out of the 
  media or in which bud length was less than 2 mm at the end of the experiment 
  were discounted . measurement of the shoot length was performed every 24 h. for 
  each assay a logistic curve was fitted using genstat ( 10 edition , 
  vsn international ) with the fitcurve directive and the lag phase was 
  calculated by extrapolating the linear part of the curve and the initial 
  plateau ( see supplemental materials ) . analysis was by residual 
  maximum likelihood ( reml ) in genstat 10 . in both cases , an f - test showed that 
  overall the treatment effects were highly significant ( p < 0.001 ) . the maximum value of the least significant difference ( lsd ) was calculated by 
  multiplying the maximum standard error of differences by a t - value 
  ( p = 0.05 ) , and is presented on the graphs . there were 105 and 157 
  degrees of freedom for the lsds shown in figs . the maximum 
  lsd was used because individual lsds varied but if differences between means 
  were significant using the maximum values then they were also significantly 
  different at the individual value for any two selected means . nced was proposed to be a 
  dioxygenase ( 3 ) , with a 
  reaction mechanism involving a carbocation intermediate , followed by formation 
  of a dioxetane ring or a criegee rearrangement prior to cleavage 
  ( 41 ) ; such a mechanism was 
  supported by o labeling experiments with atccd1 
  ( 37 ) , and was the most likely 
  mechanism based on computational studies of the aco crystal structure 
  ( 42 ) . 3a for structure ) 
  is a reversible competitive inhibitor ( ki = 39 
  m ) of recombinant nced and that it inhibited abscisic acid 
  production in planta at 50 - 100 m concentration 
  ( 31 ) . abaminesg , with an 
  extended 3 carbon linker between the methyl ester and the nitrogen atom , was 
  subsequently developed with an improved activity ( ki = 
  18.5 m ) ( 32 ) . the precise mechanism of action of abamine is uncertain , but our hypothesis 
  was that the protonated amine mimics a carbocation intermediate in the 
  catalytic mechanism , with the oxygenated aromatic ring bound in place of the 
  hydroxy - cyclohexyl terminus of the carotenoid substrates 
  ( 41 ) , as shown in 
  fig . 3 . inhibition may be due 
  in part to chelation of the essential metal ion cofactor by the methyl ester 
  of abamine . however , a derivative of abamine , containing an acid group ( cooh ) 
  in place of the methyl ester , was not active 
  ( 32 ) , even though in theory 
  this should be more effective at binding the iron cofactor . protonated abamine ( a ) , a carotenoid 
  carbocation intermediate ( b ) , and a hydroxamic acid inhibitor 
  ( c ) are shown bound to the iron(ii ) cofactor of a ccd . protonated abamine ( a ) , a carotenoid 
  carbocation intermediate ( b ) , and a hydroxamic acid inhibitor 
  ( c ) are shown bound to the iron(ii ) cofactor of a ccd . hydroxamic acids are known to act as inhibitors of several different 
  classes of metalloenzymes , such as the matrix metalloproteases , by chelation 
  of the essential metal ion cofactor 
  ( 43 ) . therefore , hydroxamic 
  acid analogues were synthesized , in which the hydroxy - cyclohexyl terminus of 
  the carotenoid substrate was mimicked as above by an oxygenated aromatic ring , 
  and the hydroxamic acid functional group was positioned at variable distance 
  from the aromatic ring . thus , a collection of aryl - c3n analogues 
  ( d8-d13 ) , aryl - c2n analogues ( d1-d7 ) , and aryl - c1n 
  analogues ( f1-f4 ) was also synthesized 
  ( table 1 ) . the 4-fluorobenzyl 
  substituent , found to promote activity in the abamine series 
  ( 31 ) , was included in the 
  collection of hydroxamic acids . 2 , involves coupling of 
  the appropriate acid with a substituted o - benzyl hydroxylamine , 
  followed by deprotection . one hydroxamic acid containing a longer 
  c5 spacer from a cyclohexyl moiety ( b1 ) was also synthesized from 
  -ionone . a set of 18 hydroxamic acids was then used for inhibitor 
  screening ; numbering of chemical compounds is given in 
  table 1 . specificity of inhibition in vitro for tomato genes lenced1 and 
  leccd1a  to screen the inhibitors against enzymes which cleave 
  carotenoids at the 9,10 position , we used the recombinant tomato leccd1a 
  protein ( 44 ) , because this 
  type of enzyme can be studied using an in vitro colorimetric assay 
  with -apo-8-carotenal as substrate 
  ( 37 ) . to establish the 
  specificity of the inhibitors , they were also tested against the tomato 
  lenced1 recombinant protein which cleaves 9-cis carotenoids at the 
  11,12 position ( 45 ) for this 
  enzyme , the cleavage reaction was monitored by c18 reverse phase 
  hplc , using 9-cis - neoxanthin as substrate . as reported by 
  others ( 46 ) , each enzyme 
  activity was found to be unstable ( lifetime < 24 h ) toward storage or 
  purification ; therefore enzyme assays were carried out using recombinant 
  cell - free extract ( no cleavage activity was observed using e. coli 
  extract lacking the recombinant ccd gene ) . against lenced1 , several hydroxamic acids ( notably d8 , d7 , and d4 ) showed 
  1.5 - 2-fold higher inhibitory activity than the designated nced inhibitor , 
  abamine ( 31 ) , which in our 
  hands showed only 20% inhibition at 100 m concentration ( see 
  table 1 ) . against leccd1a , 
  potent inhibition was observed by all the aryl - c2n hydroxamic 
  acids , and certain other hydroxamic acids . 4-methoxyaryl hydroxamic acids were 
  effective inhibitors in each series , but the most potent inhibition was 
  observed with the 4-hydroxyaryl hydroxamic acids d1 , d2 , and d3 , which gave 
  ic50 values of 0.8 - 0.9 m . in contrast , abamine gave 
  an ic50 value of 210 m against leccd1a 
  ( table 1 ) . comparison of inhibition data for lenced1 and leccd1a shows that all the 
  active compounds show some selectivity toward leccd1a , with compounds d3 , f1 , 
  and f2 showing high levels of inhibition of leccd1a , and little or no 
  inhibition of lenced1 ( table 
  1 ) . in vivo activity of inhibitors applied to e. coli strains expressing 
  ccds colored e. coli strains that produce various 
  carotenoids can be constructed by expression of enzymes for carotenoid 
  synthesis ( 26 ) . upon 
  co - expression of the appropriate ccd , the bacteria lose their color due to 
  cleavage of the carotenoids to colorless products 
  ( 4 , 
  6 , 
  27 ) . this technique was 
  employed here to further explore the specificity of inhibitors , and to test 
  their activity in vivo . the level of carotenoid in each 
  ccd - expressing strain was compared with the level of the carotenoid in a 
  control strain producing -carotene but lacking any ccd gene . the 
  difference in the carotenoid levels gave a measure of ccd activity , and 
  inhibition of this activity was measured by addition of inhibitors to the 
  growing medium . the inhibitors were tested against four -carotene - producing e. 
  coli strains ( supplemental table s1 ) . three of the strains expressed 
  highly divergent ccds that cleave at the 9,10 position : atccd7 from 
  arabidopsis ( 21 ) and 
  mmbco2 from mouse ( 6 ) which 
  both cleave at a single site ( 9,10 or 9,10 but not both ) , and 
  the tomato enzyme leccd1a ( 44 ) 
  which cleaves at both sites in the same substrate molecule 
  ( 9,10/9,10 activity ) . the fourth strain expressed another mouse 
  ccd , mmbco1 ( 47 ) , which 
  cleaves centrally at 15,15. ourselves and other researchers 
  ( 48 ) have found that 
  expressing ccds ( and presumably other proteins ) can lead to loss of 
  carotenoids by nonspecific means . however , detection of cleavage products by 
  hplc in the e. coli cells and media confirmed that in all four 
  strains used here , ccd cleavage was the cause of carotenoid loss . nced 
  activity could not be studied in e. coli cells because the enzyme 
  required for production of the 9-cis carotenoid substrates of nced 
  has not yet been identified . we synthesized the genes cszcd 
  ( 9 ) and bolcd 
  ( 49 ) , expressed them in e. 
  coli , and looked for the reported 5,6 and 7,8 cleavage activities both 
  in vitro and in e. coli cells . however , we were not able to 
  detect activity , and so it was not possible to test inhibitors against the 5,6 
  and 7,8 cleavage specificities . the 
  strains were grown in the presence or absence of inhibitors ( 100 
  m ) for 16 h. this concentration of inhibitor was within the 
  linear range of the e. coli response ( see supplemental fig . the 
  relative inhibition of each class of ccd was determined by the increase in 
  -carotene accumulation in the presence of the inhibitor , a value of 0 
  would indicate -carotene levels equal to when no inhibitor was present , 
  and a value of 100 would equal the maximum level of -carotene as 
  observed in strains lacking a ccd ( see  experimental procedures  
  for equation ) . the floating 
  black bar represents the least significant difference ( p 
  < 0.05 ) for comparison of any two means . the 
  strains were grown in the presence or absence of inhibitors ( 100 
  m ) for 16 h. this concentration of inhibitor was within the 
  linear range of the e. coli response ( see supplemental fig . the 
  relative inhibition of each class of ccd was determined by the increase in 
  -carotene accumulation in the presence of the inhibitor , a value of 0 
  would indicate -carotene levels equal to when no inhibitor was present , 
  and a value of 100 would equal the maximum level of -carotene as 
  observed in strains lacking a ccd ( see  experimental procedures  
  for equation ) . 0.05 ) for comparison of any two means . figure 5.the effect of inhibitors on the outgrowth of buds from excised 
  arabidopsis nodes in the presence of 1 m 
  naa . the graph shows lag time before the commencement of bud outgrowth for 
  col-0 ( wt ) in the presence or absence of 100 m inhibitor . a 
  null mutant of atccd7 ( max3 ) was included without inhibitor 
  as control . values represent means from five independent experiments ; 
  n = 35 ( wt ) , n = 18 ( max3 ) , n = 14 - 16 ( wt 
  plus inhibitors ) . the floating black bar represents the least 
  significant difference for comparison of any two means , and asterisks 
  indicate values significantly different from the wt ( p < 
  0.05 ) . the effect of inhibitors on the outgrowth of buds from excised 
  arabidopsis nodes in the presence of 1 m 
  naa . the graph shows lag time before the commencement of bud outgrowth for 
  col-0 ( wt ) in the presence or absence of 100 m inhibitor . a 
  null mutant of atccd7 ( max3 ) was included without inhibitor 
  as control . values represent means from five independent experiments ; 
  n = 35 ( wt ) , n = 18 ( max3 ) , n = 14 - 16 ( wt 
  plus inhibitors ) . the floating black bar represents the least 
  significant difference for comparison of any two means , and asterisks 
  indicate values significantly different from the wt ( p < 
  0.05 ) . the compounds showed different patterns of inhibition against the three 
  9,10 enzymes ( fig . 4 ) mirrored the activity 
  observed in vitro ( table 
  1 ) : d5 and d6 exhibited relatively weaker inhibition activity than 
  the other d compounds and f3 exhibited virtually none . a different pattern was 
  obtained with atccd7 in the e. coli system 
  ( fig . 4 ) , with the compounds f1 
  and f2 , which exhibited good activity against leccd1a , showing poor 
  inhibition . in contrast f1 and f2 were the most effective compounds at 
  inhibiting mmbco2 ( fig . the 
  15,15 cleavage enzyme mmbco1 was not inhibited to any significant 
  extent by any of the compounds tested ( fig . stimulation of shoot branching in arabidopsis stem sections by 
  application of inhibitor  auxin inhibits the outgrowth of axillary 
  buds in wild - type arabidopsis plants . in the atccd7 and 
  atccd8 null mutants ( max3 and max4 , respectively ) 
  the response to auxin is reduced , presumably due to a block in formation of an 
  apocarotenoid hormone ( recently shown 
  ( 17 , 
  18 ) to be strigolactone or a 
  related compound ) that suppresses branching 
  ( 19 ) , and axillary buds extend 
  earlier , leading to formation of side branches . an in vitro assay was 
  previously developed in which the growth of axillary buds from isolated 
  sections of arabidopsis stem was used to assess max mutants 
  ( 40 ) . in such assays , it was 
  reported that bud outgrowth of the max4 - 1 mutant ( atccd8 ) 
  was 2 days earlier than for wild - type 
  ( 20 ) and a similar phenotype 
  is expected of the highly branched max3 - 9 mutant 
  ( 21 ) . we tested hydroxamic 
  acid inhibitors at 100 m in this assay and found that d1 to d6 , 
  and f3 all significantly ( p < 0.05 ) advanced the timing of bud 
  outgrowth in wild - type , with the advancement ranging from 1 day ( d1 ) to 3 days 
  ( d3 ) ( fig . this earlier bud 
  outgrowth was equivalent to that observed in the atccd7-null mutant 
  max3 - 9 ( fig . 5 ) , and 
  indicates an inhibition of atccd7 and/or possibly atccd8 in this tissue . the 
  effect of the inhibitors in this assay only partially mirrored the activities 
  in the e. coli assay , with compounds f1 and f2 having a relatively 
  small activity in both the bud outgrowth assay 
  ( fig . 5 ) and the e. 
  coli atccd7 assay ( fig . 
  4 ) . however , in the case of f3 there was disagreement because it 
  was inactive in the e. coli assay for atccd7 , but it was active in 
  stimulating bud outgrowth . one possibility in this case is that f3 stimulated 
  branching by inhibiting atccd8 ( not tested in vitro or in e. 
  coli ) rather than atccd7 . stimulation of shoot branching in whole arabidopsis 
  plants  inhibitors were also applied to arabidopsis whole 
  plants grown under sterile conditions in agar . 6b ) and those 
  treated with d2 , d4 , d5 , and d6 ( fig . 6c shows d6-treated plants ) exhibited a bushy appearance 
  compared with the untreated wild - type controls 
  ( fig . this bushy 
  appearance was due to the increased number of side branches from the rosette 
  nodes , with max3 - 9 plants exhibiting 3 to 4 side branches compared 
  with a mean of 0.25 for wild type . inhibitor treated plants were intermediate 
  ( mean of 2 branches ) and so partially mimicked max3 - 9 
  ( fig . figure 6.branching phenotypes of arabidopsis plants grown in agar media 
  for 45 days supplemented with inhibitor d6 . images are shown : ( a ) 
  col-0 ( wt ) without inhibitor ; ( b ) max3 - 9 mutant without 
  inhibitor ; ( c ) col-0 with 100 m d6 . , 
  n = 6 to 12 . branching phenotypes of arabidopsis plants grown in agar media 
  for 45 days supplemented with inhibitor d6 . images are shown : ( a ) 
  col-0 ( wt ) without inhibitor ; ( b ) max3 - 9 mutant without 
  inhibitor ; ( c ) col-0 with 100 m d6 . f1 and f2 were toxic to whole plants at 100 m ( data not 
  shown ) . 5 ) , but in whole plants 
  d3 had a negative effect on arabidopsis growth when added to the agar 
  media , and a toxicity effect was suggested by the observation that roots grew 
  across the agar surface rather than penetrating . this general growth effect 
  may have masked any possible stimulatory effects of d3 on side branching . we have designed and tested a new class of inhibitor of the carotenoid 
  cleavage dioxygenase family that is based on a structural mimic of the 
  substrate that positions an iron - chelating hydroxamic acid group within the 
  active site . positioning was achieved by varying the distance between the 
  hydroxamic acid and an aromatic ring so that it matched the distance within 
  the carotenoid substrate between the proximal cyclic end - group and the 
  cleavage site . crystal structure of aco , a cyanobacterial ccd , indicates that 
  cleavage position is likely to be determined by the distance between the 
  fe(ii ) catalytic center and the opening of the long non - polar tunnel that 
  allows access to carotenoid substrates 
  ( 11 ) . this idea is supported 
  by the observation that for nosccd ( from nostoc sp . pcc 7120 ) 
  cleavage of the monocyclic -carotene occurs at the 7,8 
  position where the proximal terminus is linear , but at the 9,10 position when 
  the proximal terminus has a more compact cyclic end group 
  ( 48 ) ; indeed it was suggested 
  that the cyclic end group may be arrested at the entrance of the tunnel 
  ( 48 ) . we predicted from this crystal structure , and our model for the cleavage 
  mechanism ( fig . 3 ) , that 
  aryl - c1n , aryl - c2n and aryl - c3n compounds 
  would be selective for 7,8 , 9,10 and 11,12 cleavage reactions , respectively ; 
  we tested these classes against enzymes with 9,10 , 11,12 , and 15,15 
  specificities . certain aryl - c1n compounds ( f1 , f2 ) were effective 
  inhibitors of 9,10 but not 11,12 or 15,15 cleavages . the 
  aryl - c2n compounds were potent inhibitors of 9,10 enzymes , but also 
  had a moderate 11,12 inhibition activity . the aryl - c3n compounds 
  were much less potent against 9,10 enzymes , and although this group contained 
  the best 11,12 inhibitor ( d8 ) , they all still maintained a somewhat greater 
  selectivity toward the 9,10 cleavage . in comparison , a further analogue , 
  abaminesg , was reported to be more active against the 11,12 cleavage than the 
  9,10 cleavage ; at 100 m it inhibited atnced3 by 78% and atccd1 
  by 20% ( 32 ) . none of the 
  compounds tested inhibited the 15,15 enzyme , presumably because the 
  spacing was too small . thus we conclude that the strategy of varying the 
  positioning of the hydroxamic acid group was only moderately successful , since 
  some overlap existed between the classes . nevertheless , individual compounds 
  were identified with very high specificity to the 9,10 cleavage in vitro , 
  e.g. ic50 for f1 was 2.0 m but no inhibition of 
  lenced1 was detected . the inhibitors also exhibited different patterns of activity in e. 
  coli against the three different enzymes with 9,10 cleavage activity . for 
  example , f1 and f2 had high inhibitory activity against leccd1a and mmbco2 but 
  were relatively ineffective against atccd7 . such differences are not 
  surprising since mmbco2 shares only 17 - 23% amino acid identity with the two 
  plant 9,10 enzymes ( leccd1a and atccd7 ) , which are themselves highly 
  divergent , with only 19% identity to each other . this indicates that the 
  variants of the hydroxamic acid inhibitors are able to distinguish between 
  enzymes that have similar activities but highly divergent primary 
  structure . the e. coli system proved useful in measuring the efficacy of the 
  inhibitors in vivo . for example , the e. coli assays showed 
  f1 and f2 were poor inhibitors against atccd7 and this was confirmed in the 
  arabidopsis bud outgrowth assay 
  ( fig . 5 ) , which measures atccd7 
  and/or atccd8 activity . however , although d5 and d6 were poor in the e. 
  coli assays they showed the largest effect on whole arabidopsis 
  plants , giving the greatest number of side branches . also d1 and d3 appeared 
  to be good inhibitors of the 9,10 enzymes in vitro 
  ( table 1 ) , in e. coli 
  ( fig . 5 ) , but 
  d3 had negative effects on growth , which confounded the branching assay in 
  whole plants , whereas plants treated with d1 grew normally and without an 
  increase in branching . d1 and d3 both contained a more polar hydroxyl group on 
  the aryl ring , therefore it is possible that these compounds are more actively 
  transported or metabolized in the plant . overall , the different activities observed in different assays suggest that 
  factors such as uptake , metabolism , and effects on non - target processes may 
  play a role in determining the suitability and effectiveness of the inhibitors 
  in planta . our results underline the importance of performing 
  secondary screens in the biological systems where the compounds are to be 
  used . here we have been able to demonstrate that d2 , d4 , d5 , and d6 appear to 
  inhibit ccds in all the assays tested , including in planta , without 
  negative unintended effects on whole plants . these compounds represent useful 
  chemical genetic agents to explore the function of ccds in plants , animals , 
  and micro - organisms . using the inhibitors described here , it will now be possible to inhibit the 
  ccd(s ) involved in branching in a wide range of plant species and then look 
  for changes in carotenoids and apocarotenoids ; this could provide a powerful 
  approach for the identification of the precursors of strigolactone , the 
  identity of other active strigolactone - related compounds , and to the further 
  elucidation of the biosynthetic pathway . the inhibitors could also be used to 
  probe for functional variation in the role of strigolactone between species . branching - promoting chemicals may have applications in horticulture where 
  compact plant architecture is often highly desirable , e.g. in orchard 
  crops ( 50 ) . other biological systems where genetic manipulations are not practical 
  include the production of saffron 
  ( 9 ) and bixin 
  ( 49 ) in crocus and 
  bixa plants respectively , where the in vivo substrates of 
  the ccds involved are not clear , and also in the study of the functions of 
  mycorradicin and strigolactone in plant interactions with mycorrhyza 
  ( 51 ) and parasitic weeds 
  ( 15 ) , respectively . finally , 
  there may be pharmaceutical applications for inhibitors of bco2 in humans 
  because products from 9,10 carotenoid cleavage have been implicated in dna 
  damage and carcinogenesis ( 52 , 
  53 ) .
members of the carotenoid cleavage dioxygenase family catalyze the 
  oxidative cleavage of carotenoids at various chain positions , leading to the 
  formation of a wide range of apocarotenoid signaling molecules . to explore the 
  functions of this diverse enzyme family , 
 we have used a chemical genetic 
  approach to design selective inhibitors for different classes of carotenoid 
  cleavage dioxygenase . 
 a set of 18 arylalkyl - hydroxamic acids was synthesized 
  in which the distance between an iron - chelating hydroxamic acid and an 
  aromatic ring was varied ; these compounds were screened as inhibitors of four 
  different enzyme classes , either in vitro or in vivo . 
 potent 
  inhibitors were found that selectively inhibited enzymes that cleave 
  carotenoids at the 9,10 position ; 50% inhibition was achieved at submicromolar 
  concentrations . 
 application of certain inhibitors at 100 m to 
  arabidopsis node explants or whole plants led to increased shoot 
  branching , consistent with inhibition of 9,10-cleavage .