Promoters of tps10 and tps23 are dissimilar among maize lines

The promoters of tps10 and tps23 of several maize lines were sequenced and analyzed for putative regulatory sequences. Promoter sequences were obtained by PCR or with the help of the Institute of Genomic Diversity from Cornell University. The maize lines selected for sequence analysis differed in (E)-ß-caryophyllene production. The lines Delprim, NC358, Du101, and F476 possess an active TPS23 enzyme and produced herbivore-induced

(E)-ß-69

caryophyllene. In the lines B73, CML247, OH7B, and MS71, on the other hand, transcript of tps23 could not be detected (Degen et al. 2004; Köllner et al. 2008). Sequence analysis of

~800 bp revealed only minor nucleotide exchanges among the tps23 promoter of all lines (Fig.3.15).

-766 -697 Du101 CAAAGGTTGT AT-GCACAAA AGGCTGCTTC TTTGTGGTGT AGCAGCTTTG TTT-GGTCGA TTTGGCTTTG NC358 CAAAGGTTGT AT-GCACAAA AGGCTGCTTC TTTGTGGTGT AGCAGCTTTG TTT-GGTCGA TTTGGCTTTG F476 CAAAGGTTGT ATTGCACNAA AGGCTGCTTC TTTCTAGCGT TGCGGCTTTG TTTTGGTCGA TTTGGCTTTG OH7B CAAAGGTTGT AT-GCACAAA NGGCTGCTTC TTTCTAGCGT TGCGGCTTTG TTTTGGTCGA TTTGGCTTTG Delprim CAAAGGTTGT AT-GCACAAA AGGCTGCTTC TTTCTAGCGT TGCGGCTTTG TTTTGGTCGA TTTGGCTTTG B73 CAAAGGTTGT AT-GCACAAA AGGCTGCTTC TTTCTAGCGT TGCGGCTTTG TTTTGGTCGA TTTGGCTTTG MS71 CAAAGGTTGT AT-GCACAAA NGGCTGCTTC TTTCTAGCGT TGCGGCTTTG TTTTGGTCGA TTTGGCTTTG CML247 CAAAGGTTGT AT-GCACAAA AGGCTGCTTC TTTCTAGCGT TGCGGCTTTG TTTTGGTCGA TTTGGCTTTG -696 -627 Du101 CCGTAATGAT GTGGTCTTTA ATCATAACCA TATATCGTCA ATTATACAGG TGATCTTTAG AGATACATAT NC358 CCGTAATGAT GTGGTCTTTA ATCATAACCA TATATCGTCA ATTATACACG TGATCTTTAG AGATACATAT F476 TCGTAATGAT GTGGTGTTTA ATCATAACCA TATACCGTCA ACTATACATG TGATCTTTAG AGGTACATAT OH7B TCGTAATGAT GTGGTGTTTA ATCATAACCA TATACCGTCA ACTATACATG TGATCTTTAG AGGTACATAT Delprim TCGTAATGAT GTGGTGTTTA ATCATAACCA TATACCGTCA ACTATACATG TGATCTTTAG AGGTACATAT B73 TCGTAATGAT GTGGTGTTTA ATCATAACCA TATACCGTCA ACTATACATG TGATCTTTAG AGGTACATAT MS71 TCGTAATGAT GTGGTGTTTA ATCATAACCA TATACCGTCA ATTATACATG TGATCTTTAG AGGTACATAT CML247 TCGTAATGAT GTGGTGTTTA ATCATAACCA TATACCGTCA ACTATACATG TGATCTTTAG AGGTACATAT -626 -557 Du101 TGGTTCAGAT TTTGGGGACT CCTGCAGAAG GAAGAAACAT AGCAGCAAAT CCTAGTCGTG TATCATACTT NC358 TGGTTCAGAT TTTGGGGACT CCTGCAGAAG GAAGAAACAT AGCAGCAAAT CCTAGTCGTG TATCATACTT F476 TG-TTCACAT TTTAGAGACT CCTGCAGATG GAAGAAACAC AACAGCAAAT CTTAGTCGTG TATCAGACTT OH7B TG-TTCACAT TTTAGAGACT CCTGCAGATG GAAGAAACAC AACAGCAAAT CTTAGTCGTG TATCAGACTT Delprim TG-TTCACAT TTTAGAGACT CCTGCAGATG GAAGAAACAC AACAGCAAAT CTTAGTCGTG TATCAGACTT B73 TG-TTCACAT TTTAGAGACT CCTGCAGATG GAAGAAACAC AACAGCAAAT CTTAGTCGTG TATCAGACTT MS71 TG-TTCAGAT TTTAGAGACT CCTGCAGAAG GAAGAAACAC AACAGCAAAT CTTAGTCGTG TATCAGACTT CML247 TG-TTCACAT TTTAGAGACT CCTGCAGATG GAAGAAACAC AACAGCAAAT CTTAGTCGTG TATCAGACTT

-556 -487 Du101 TGGAGGTGAT GGCCATGGAG TTCTTCGCGA GTCATGGGTG GCGGTCTAAT GCAAGGATCG ATGTGGTTTA NC358 TGGAGGTGAT GGCCATGGAG TTCTTCGCGA GTCACGGGTG GCGGTCTAAT GCAAGGATCG ATGTGGTTTA F476 TGGAGGTTAT GGCCATGGAG TTCTTCACGA GTCACGGGCG GCGGTCTAAT GCAAGGATCG ATGCCGTTTA OH7B TGGAGGTTAT GGCCATGGAG TTCTTCACGA GTCACGGGTG GCGGTCTAAT GCAAGGATCG ATGCCGTTTA Delprim TGGAGGTTAT GGCCATGGAG TTCTTCACGA GTCACGGGTG GCGGTCTAAT GCAAGGATCG ATGCCGTTTA B73 TGGAGGTTAT GGCCATGGAG TTCTTCACGA GTCACGGGTG GCGGTCTAAT GCAAGGATCG ATGCCGTTTA MS71 TGGAGGTTAT GGCCATGGAG TTCTTCACGA GTCACGGGTG GCGGTCTAAT GCAAGGATCG ATGACGTTTA CML247 TGGAGGTTAT GGCCATGGAG TTCTTCACGA GTCACGGGTG GCGGTCTAAT GCAAGGATCG ATGYCGTTTA

-486 -417 Du101 GTGTTTATGG TCTTCTATTC CATATCATGT TTTAAGTTGA AGTTTGTTTG ATCCACAAAT TCTTGGGATG NC358 GTGTTTATGG TCTTCTATTC CATATCATGT TTTAAGTTGA AGTTTGTTTG ATCCACAAAT TTTTGGGATG F476 GTGTTTATGA TCTTTTATTC CATATCATGT TTTAAGTTGA AGTTTGTTTG ATCCATGAAT TCCTGGGATG OH7B GTGTTTATGA TCTTTTATTC CATATCATGT TTTAAGTTGA AGTTTGTTTG ATCCATGAAT TCCTGGGATG Delprim GTGTTTATGA TCTTTTATTC CATATCATGT TTTAAGTTGA AGTTTGTTTG ATCCATGAAT TCCTGGGATG B73 GTGTTTATGA TCTTTTATTC CATATCATGT TTTAAGTTGA AGTTTGTTTG ATCCATGAAT TCCTGGGATG MS71 GTGTTTATGA TCTTTTATTC CATATCATGT TTTAAGTTGA AGTTTGTTTG ATCCATAAAT TCCTGGGATG CML247 GTGTTTATGA TCTTTTATTC CATATCATGT TTTAAGTTGA AGTTTGTTTG ATCCATGAAT TCCTGGGATG -416 -347 Du101 ATGTAATATT TGAGGTGTAT GGTTGTATGC ATGAAAATGC AAAAGTTCGG AACGATATTT CCATTTCAAA NC358 ATGTAATATT TGAGGTGTAT GGTTGTATGC ATGAAAATGC AAAAGTTCGG AACGATATTT CTATTTCAAA F476 ATGTAACATT TGAGGTGTAT GGTTGTATGC ATGAAAATGC AAAAGTCCGG AACGATATTT CCATTTCAAA OH7B ATGTAACATT TGAGGTGTAT GGTTGTATGC ATGAAAATGC AAAAGTCCGG AACGATATTT CCATTTCAAA Delprim ATGTAACATT TGAGGTGTAT GGTTGTATGC ATGAAAATGC AAAAGTCCGG AACGATATTT CCATTTCAAA B73 ATGTAACATT TGAGGTGTAT GGTTGTATGC ATGAAAATGC AAAAGTCCGG AACGATATTT CCATTTCAAA MS71 ATGTGACATT TGAGGTGTAT GGTTGTATTC ATGAAAATGC AAAAGTCCGG AACGATATTT CCATTTCCAA CML247 ATGTAACATT TGAGGTGTAT GGTTGTATGC ATGAAAATGC AAAAGTCCGG AACGATATTT CCATTTCAAA

70

-346 -277 Du101 AAAAGTCTAC TGGACGGCCA AACCAACATT GTTGAGTGGA CGACCTACGA TATTATTAAC AATTCCATGT NC358 AAAAGTCTAC TGGACGGCCA AACCAACATT GTTGAGTGGA CGACTTACGA TATTATTAAC AATTCCACGT F476 AAAAGTCTAC TGGACGGCCA AACCAACATT GTTGAGTGGA CGACCTACGA TATTATTAAC AATTCCATGT OH7B AAAAGTCTAC TGGACGGCCA AACCAACATT GTTGAGTGGA CGACCTACGA TATTATTAAC AATTCCATGT Delprim AAAAGTCTAC TGGACGGCCA AACCAACATT GTTGAGTGGA CGACCTACGA TATTATTAAC AATTCCATGT B73 AAAAGTCTAC TGGACGGCCA AACCAACATT GTTGAGTGGA CGACCTACGA TATTATTAAC AATTCCATGT MS71 AAAAGTCTAC TGGACGGCCA AACCAACATT GTTGAGTGGA CGACCTACGA TATTATTAAC AATTCCATGT CML247 AAAAGTCTAC TGGACGGCCA AACCAACATT GTTGAGTGGA CGACCTACGA TATTATTAAC AATTCCATGT -276 -207 Du101 GTTGCACATG CCTGTCGGCA GATCTATATT TAAAAAAAA- -CGAGTGGAC TACGACGCTC AGATCCTTCC NC358 GTTGCACATG CCTGTCGGCA GATCTATATT TAAAAAAAAA ACTAGTGGAC TACGACGCTC AGATCCTTCC F476 GTTGCACATG CCTGTCGGCA GATCTATATT TAAAAAAAAA -CGAGTGGAC TACGACGCTC AGATCCTTCC OH7B GTTGCACATG CCTGTCGGCA GATCTATATT TAAAAAAAAA -CGAGTGGAC TACGACGCTC AGATCCTTCC Delprim GTTGCACATG CCTGTCGGCA GATCTATATT TAAAAAAAAA -CGAGTGGAC TACGACGCTC AGATCCTTCC B73 GTTGCACATG CCTGTCGGCA GATCTATATT TAAAAAAAAA -CGAGTGGAC TACGACGCTC AGATCCTTCC MS71 GTTGCACATG CCTGTCGGCA GATCTATATT TAAAAAAAA- -CGAGTGGAC TACGACGCTC AGATCCTTCC CML247 GTTGCACATG CCTGTCGGCA GATCTATATT TAAAAAAAAA –CGAGTGGAC TACGACGCTC AGATCCTTCC -206 -137 Du101 AGGAAAAAAA A-CAATATAA TGCAAATTGA GTACGTACGT GCCCACGTGC ATCACGAGTA CCACTCCACG NC358 AGGAAAAAAA AACAATATAA TGCAAATTGA GTACGTACGT GCCCACGTGC ATCACGAGTA CCACTCCACG F476 AGGAAAAAAA A-CAATATAA TGCAAATTGA GTACGTACGT GCCCACGTGC ATCACGAGTA CCACTCCACG OH7B AGGAAAAAAA A-CAATATAA TGCAAATTGA GTACGTACGT GCCCACGTGC ATCACGAGTA CCACTCCACG Delprim AGGAAAAAAA A-CAATATAA TGCAAATTGA GTACGTACGT GCCCACGTGC ATCACGAGTA CCACTCCACG B73 AGGAAAAAAA A-CAATATAA TGCAAATTGA GAACGTACGT GCCCACGTGC ATCACGAGTA CCACTCCACG MS71 AGGAAAAAAA AACAATATAA TGCAAATTGA GTACGTACGT GCCCACGTGC ATCACGAGTA CCACTCCACG CML247 AGGAAAAAAA A-CAATATAA TGCAAATTGA GTACGTACGT GYCCACGTGC ATCACGAGTA CCACTCCACG -136 -67 Du101 CTGGCTACCT ATATTAAGAA CACCATAGGA TCCAGGTGCT TGCCAGTGCA TGCATTGTAT ATCTGCACTG NC358 CTGGCTACCT ATATTAAGAA CACCATAGGA TCCAGGTGCT TGCCAGTGCA TGCATTGTAT ATCTGCTCTG F476 CTGGCTACCT ATATTAAGAA CACCATAGGA TCCAGGTGCT TGCCAGTGCA TGCATTGTAT ATCTGCACTG OH7B CTGGCTACCT ATATTAAGAA CACCATAGGA TCCAGGTGCT TGCCAGTGCA TGCATTGTAT ATCTGCACTG Delprim CTGGCTACCT ATATTAAGAA CACCATAGGA TCCAGGTGCT TGCCAGTGCA TGCATTGTAT ATCTGCACTG B73 CTGGCTACCT ATATTAAGAA CACCATAGGA TCCAGGTGCT TGCCAGTGCA TGCATTGTAT ATCTGCACTG MS71 CTGGCTACCT ATATTAAGAA CACCATAGGA TCCAGGTGCT TGCCAGTGCA TGCATTGTAT ATCTGCGCTG CML247 CTGGCTACCT ATATTAAGAA CACCATAGGA TCCAGGTGCT TGCCAGTGCA TGCATTGTAT ATCTGCACTG -66 -1 Du101 CTACGCCGGT GGGCACGTAC GTGCAACAGG CAGCAGGAGA GTAGCTAGCT AGTATACTAT ACTTTCATG NC358 CTACGCCGGT GGGCACGTAC GTGCAACAGG CAGCAGGAGA GTAGCTAGCT AGTATACTAT ACTTTCATG F476 CTACGCCGGT GGGCACGTAC GTGCAACAGG CAGCAGGAGA GTAGCTAGCT AGTATACTAT ACTTTCATG OH7B CTACGCCGGT GGGCACGTAC GTGCAACAGG CAGCAGGAGA GTAGCTAGCT AGTATACTAT ACTTTCATG Delprim CTACGCCGGT GGGCACGTAC GTGCAACAGG CAGCAGGAGA GTAGCTAGCT AGTATACTAT ACTTTCATG B73 CTACGCCGGT GGGCACGTAC GTGCAACAGG CAGCAGGAGA GTAGCTAGCT AGTATACTAT ACTTTCATG MS71 CTACGCCGGT GGGCACGTAC GTGCAACAGG CAGCAGGAGA GTAGCTAGCT AGTATACTAT ACTTTCATG CML247 CTACGCCGGT GGGCACGTAC GTGCAACAGG CAGCAGGAGA GTAGCTAGCT AGTATACTAT ACTTTCATG

Fig.3.15: Sequence comparison of the promoter of tps23 from (E)-ß-caryophyllene producing and non-producing maize lines. Start codon is underlined. Nucleotide differences are highlighted in red.

Nucleotide changes were present between the promoters of caryophyllene-producing and non-producing maize lines, as well as within both groups. Therefore, predictions regarding the ability to transcribe tps23 cannot be made from the promoter nucleotide sequences.

Only the lines B73, OH7B, MS71, Delprim, NC358, and CML 247 could be used for the comparison of the tps10 promoter. Sequences from NC358, CML247, and OH7B were obtained from the HAPMAP2 database from Cornell University because PCR on genomic

71

DNA was not successful (data provided by Jeff Glaubitz and Edward Buckler).

Unfortunately, data from that database did not cover promoter fragments long enough for useful comparisons (Fig.3.16).

-1491 -1422 B73 GGCAAAGAGC TCCTTTACTA AATTTTTTAT TTTACCGAGG GTTTTTTTTT TGCTTAGCAC TCGGCAAAGA OH7B GGCAAAGAGC TCCTTTACCA AATGTTTTAT TTTACCGAGG GTTTTTTT-- -GCTTAGCAC TCGGCAAAGA MS71 CCG CCCCCCAAAA Delprim GGCAAAGAGC TCCTTTACCA AATGTTTTAT TTTACTGAGG GTTTTTTT-- -GCTTAGCAC TCGGCAAAGA NC358 CML247

-1421 -1352 B73 GTTTATTTAC CGAGTGTCCG AAAAAAACAC TCGGCAAAAC ATTTGGCACT CGGCAAAGAG CCGAAATCCG OH7B GTTTATTTGC CGAGTGTCCG AAAAAAACAC TCGGCAAAAC ATTTGGCACT CGGCAAAGAG CCGAAATCCG MS71 GTTT--TTAC CGAGTGTCCG AAAAAAACAC TCGGCAAAAA AATTCGT--- --GTGAAGTT TTCAAATTTA Delprim GTTTATTTGC CGAGTGTTCG AAAAAAACAC TCGGCAAAAA GC--- ---CAAAATC CGGTAATGCG NC358 CML247 -1351 -1282 B73 GTAATGTTTA GTATTAATAT TAACATTTAG GAATCTTCGT ATTGAATATT TTAGCTCCCG CAAGATCTCA OH7B GTAATGTTTA GTATTAATAT TAACATTTAG GAATCTTCGT ATTGAATATT TTAGCTCCC- -AAGATCTCA MS71 GTAATGTTTA GTATTAATAT TAACATTTAG GAATCTTCGT ATTGAATATT TTAGCTCCCG CAAGATCTCA Delprim GTAATGTTTA GTATTAATAT TAACATTTAG GAATCTTCGT ATTGAATATT TTAGCTCCCG CAAGATCTCA NC358 CML247 -1281 -1212 B73 AATGAAAACT ATTTTAACTA TAGAGTTTTA GATCCATCTT ATCAAAGCAC TACATTTTTT GACATAAGTA OH7B AATGATAACT ATTTTAACTA TAGAGTTTTA GATCGATCTT ATCAAAGCAC TACATTTTTT GACATAAGTA MS71 AATGAAAACT ATTTTAACTA TAGAGTTTTA GATCGATCTT ATCAA-GCAC TACATTTTTT GACATAAGTA Delprim AATGAAAACT ATTTTAACTA TAGAGTTTTA GATCGATCTT ATCAAAGCAC TACATTTTTT GACATAAGTA NC358 TTT GACATAAGTA CML247 TCTT ATCAAAGCAC TATATTTTTT GACATAAGTA

-1211 -1142 B73 TGTCTCATCC AAGATAATTT TAAATTTTGA GTATCAAAAT CTGGTTAATT TCATAATTAA TATTTAGGAC OH7B TGTCTCATTC AAGATAATTT TAAATTTTGA GTATGAAAAT CTGATTAATT TCATAATTAA TATTTAGGAC MS71 TGTCTCATCC GAGATAATTT TAAATTTTGA ATATCAAAAT CTGATTAATT TCATAATTAA TATTTAGGAC Delprim TGTCTCATCC AAGATAATTT TAAATTTTGA GTATCAAAAT CTGATTAATT TCATAATTAA TATTTAGGAC NC358 TGTCTCATTC AAGATAATTT TAAATTTTGA GTATGAAAAT CTGATTAATT TCATAATTAA TATTTAGGAC ML247 TGTCTCATTC AAGATAATTT TAAATTTTGA GTATGAAAAT CTGATTAATT TCATAATTAA TATTTAGGAC

-1141 -1072 B73 TAAATGATTT CAGATGAAAA GCTTACCAAT TATAATGCGA TAGATCACGT TGAATACCAC AAACTTAATA OH7B TAAATGATTT CAGATGAAAA GCTTACCAAT TATAATGCGA TAAATCACGT TGAATACCAC AACCTTAATA MS71 TAAATGATTT CAGATGAAAA GCTTATCAAT TATAATGCGA TAGATCACGT TGAATACCAC AAACTTAATA Delprim TAAATGATTT CAGATGAATA GCTTACCAAT -ATAATGCGA TAGATCACGT TGAATACCAC AACCTTAATA NC358 TAAATGATTT CAGATGAAAA GCTTACCAAT TATAATGCGA TAAATCACGT TGAATACCAC AACCTTAATA CML247 TAAATGATTT CAGATGAAAA GCTTACCAAT TATAATGCGA TAGATCACGT TGAATACCAC AACCTTAATA

-1071 -1002 B73 TACACCGCGT TAGCATCTGG GCATTAACCG ATCAGACCAT TTGAAGGTGG TGGCCAGGCT GCCTCTAAAA OH7B TACACCGCGT TAGCATCTGG GCATTAACCG ATCAGACCAT TTGAAGGTGG TGGCCAGGCT GCCTCTAAAA MS71 TACACCGCGT TAGCATCTGG GCATTAACCG ATCAGACCAT TTGAAGGTGG TGGCCAGGCT GCCTCTAAAA Delprim TACACCGCGT TAGCATCTGG GCATTAACCG ATCAGACCAT TTGAAGGTGG TGGCCAGGCT GCCTCTAAAA NC358 TACACCGCGT TAGCATCTGG GCATTAACCG ATCAGACCAT TTGAAGGTGG TGGCCAGGCT GCCTCTAAAA CML247 TACACCGCGT TAGCATCTGG GCATTAACCG ATCAGACCAT TTGAAGGTGG TGGCCAGGCT GCCTCTAAAA -1001 -932 B73 ATGAGTTTTG TAATAGTAGA TTAGTAGCGT AAGGAACCCT AAACCATATA TATATATATA TATATATATA OH7B ATGAGTTTTG TAATAGTAGA TTAGTAGCGT AAGGAACCCT AAACCATATA TATATATATA TA--- MS71 ATGAGTTTTG TAATAGTAGA TTAGTAGCGT AAGGAACCCT AAACCATATA TATATATATA TATATATATA Delprim ATGAGTTTTG TAATAGTAGA TTAGTAGCGT AAGG--- ----CGTTTG TATATATAAA --- NC358 ATGAGTTTTG TAATAGTAGA TTAGTAGCGT AAGGAACCCT AAACCATATA TATATATATA TATATATATA CML247 ATGAGTTTTG TAATAGTAGA TTAGTAGCGT AAGGAACCCT AAACCATATA TATATA---- --- -931 -862

72

B73 TATATATATA TATATATATA TATATATATA TATAGTGAAC AAATTAGTAT GGAATAGAAA AAAAATCAAG OH7B --- --- --- ----GTGAAC AAATTAGTAT GGAATAGAAA AAAA-TCAAG MS71 TATATATATA TATATATATA TATATATATA TATAGTGAAC AAATTAGTAT GGAATAGAAA AAAAATCAAG Delprim --- --- --- ----GTGAAC AAATTAGTAT GGAATAGAAA AAAAATCAAG NC358 TATATATATA TATATATATA TATATATATA TATAGTGAAC AAATTAGTAT GGAATAGAAA AAAAATCAAG CML247 --- --- --- ----GTGAAC AAATTAGTAT GGAATAGAAA AAAAATCAAG

-861 -792 B73 ACAAAACGAT CAATATAGAT CACGTTGAAT ACCACAACCT TAATTAATAT ACACCGCGTT AGCATCTGGG OH7B ACAAAACGAT CAATATAGAT CACGTTGAAT ACCACAACCT TAATTAATAT ACACCCTGTT AGCATCTGGG MS71 ACAAAACGAT CAATATAGAT CACGTTGAAT ACCACAACCT TAATTAATAT ACACCGCGTT AGCATCTGGG Delprim ACAAAACGAT CAA--- --- --- --- --- --- NC358 ACAAAACGAT CA-TATAGAT CACGTTGAAT ACCACAACCT TAATTAATAT ACACCCTGTT AGCATCTGCG CML247 ACAAAACGAT CAATATAGAT CACGTTGAAT ACCACAACCT TAATTAATAT ACACCCTGTT AGCATCTGCG

-791 -722 B73 CATTAACCGA TCAGACCATT TGAGGAAGGT GGCCAGGCTG CCTCTAAAAA TGAGTTTTGT AATAGTAGAT OH7B CATTAACCGA TCAGACCATT TGAGGACGGT AGCC--- --TCTAAAAA TGAGTTTTGT AATAGTAGAT MS71 CATTAACCGA TCAGACCATT TGAGGAAGGT GGCCAGGCTG CCTCTAAAAA TGAGTTTTGT AATAGTAGAT Delprim --- -CATACC--- --- --- --- --- --- NC358 CATTAACCGA TCAGACCATT TGAGGACGGT AGCC--- --TCTAAAAA TGAGTTTTGT AATAGTAGAT CML247 CATTAACCGA TCAGACCATT TGAGGACGGT AGCC--- --TCTAAAAA TGAGTTTTGT AATAGTAGAT

-721 -652 B73 TAGTAGCGTA AGGCGTTTGT GTAAATTAAT TAGTTAAA-- ---TA TGAAGTAGAT TCCTAAGAGA OH7B TAGTAGCGTA AGGCGTTTGT GTAAATTAAT TAGTTAAA-- ---TA TGAAGTAAAT TCCTAAGAGA MS71 TAGTAGCGTA AGGCGTTTGT GTAAATTAAT TAGTTAAAAT TAGTTAAATA TGAAGTAAAT TCCTAAGAGA Delprim --- ---T GTAAATTAAT TAGTTAGA-- ---TA TGAAGTAAAT TCCTAAGATA NC358 TAGTAGCGTA AGGCGTTTGT GTAAATTAAT TAGTTAAA-- ---TA TGAAGTAGAT TCCTAAGAGA CML247 TAGTAGCGTA AGGCGTTTGT GTAAATTAAT TAGTTAAA-- ---TA TGAAGTAAAT TCCTAAGAGA

-651 -582 B73 TTCTAGGGCA CAACTGT--C TCTCTGTAGC AAAAACTAGT TTAATCAGTA GGCATGATGT CATATCCCAA OH7B TTCTAGGGCA CAACTGT--C TCTCTGTAGC AAAAACTAGT TTAATCAGTA GGCATGATGT CATATCCCAA MS71 TTCTAGGGCA CAACTGT--C TCTCTGTAGC AAAAACTAGT TTAATCAGTA GGCATGATGT CATATCCCAA Delprim TTCTAGGGCA CAACTGTGTC TCTCTCTAGC AAAAACTAGT TTAATCAGTA GGCATGATGT CATATCCCGA NC358 TTCTAGGGCA CAACTGT--C TCTCTGTAGC AAAAACTAGT TTAATCAGTA GGCATGATGT CATATCCCAA CML247 TTCTAGGGCA CAACTGT--C TCTCTGTAGC AAAAACTAGT TTAATCAGTA GGCATGATGT CATATCCCAA

-581 -512 B73 A-TGGTTGTT CATTGCATAA -TGTGACTCC TAGAACCAAC TAGCTTGAGC CTAAAGAAGC TCACACACA- OH7B AATGGTTGTT CATTGCATAA ATGTGACTCC TATAACCAAC TAGCTTGAGC CTAAAGAAGC TCACACACA- MS71 AATGGTTGTT CATTGCATAA -TGTGACTCC TAGAACCAAC TAGCTTGAGC CTAAAGAAGC TCACACACA- Delprim A-TGGTTGTT CATTGCATAA -TGTGACTCC TAGAACCAAC TAGCTTGAGC CTAAAGAAGC TCACACACAC NC358 AATGGTTGTT CATTGCATAA -TGTGACTCC TATAACCAAC TAGCTTGAGC CTAAAGAAGC TCACACACA- CML247 AATGGTTGTT CATTGCATAA ATGTGACTCC TATAACCAAC TAGCTTGAGC CTAAAGA-GC TCAC-CNCA- -511 -442 B73 -GCAATCTAG CTCAATTAGT TGTACACATG TGAGCATACA TCACGTGCAA TACAATGGCA GTTCTGATTT OH7B -GCAATCTAG CTCAATTAGT TGTACACATG TGAGCATACA TC-CGTGCAA TACAATGGCA GTTCTGATTT MS71 -GCAATCTAG CTCAATTAGT TGTACACATG TGAGCATACA TCACGTGCAA TACAATGGCA GTTCTGATTT Delprim AGCAATCTAG CTCAATTAGT TGTACA--TG TGAGCATACA TCACGTGCAA TACAATGGCA GTTCTGATTT NC358 -GCAATCTAG CTCAATTAGT TGTACACATG TGAGCATACA TC-CGTGCAA TACAATGGCA GTTCTGATTT CML247 -NCAATCTAG CTCAATTAGT TGTACACATG TGAGCATACA TC-CGTGCAA TACAATGGCA GTTCTGATTT

-441 -372 B73 AATTTGCAAC TCCATTTTGT CTCACAGCTA GGATAAATAC TGGTGCCAGC CCATGCATCC TTATTGTTCT OH7B AATTTGCAAC TCCATTTTGT CTCA--GATA GGATAAATAC TGGTGCCAGC CCATGCATCC TTATTGTTCT MS71 AATTTGCAAC TCCATTTTGT CTCA--GCTA GGATAAATAC TGGTGCCAGC CCATGCATCC TTATTGTTCT Delprim AATTTGCAAC TCCATTTTGT CTCA--GCTA GGATAAATAC TGGTGCCAGC CCATGCATCC TTATTGTTCT NC358 AATTTGCAAC TCCATTTTGT CTCACAGCTA GGATAAATAC TGGTGCCAGC CCATGCATCC TTATTGTTCT CML247 AATTTGCAAC TCCATTTTGT CTCACAGCTA GGATAAATAC TGGTGCCAGC CCATGCATCC TTATTGTTCT

-371 -302 B73 TCAGTGCGAA ACCAACCATA TATATACGGA GCGGAGCTTG TGGCGGCAAT TCTCCCGTCT TGGACGGCCC OH7B TCAGTGCGAA ACCAACCATA TATATACGGA GCGGAGCTTG TGGCGGCAAT TCTCCCGTCT TGGACGGCCC MS71 TCAGTGCGAA A---ACCAT- ----TAC--- --- --- --- --- Delprim TCAGTGCGAA A----CCATT ACCAGACTTG T--GAGCTTG TGGCGGCAAT TCTCCCGTCT TGGACGGCCC NC358 TCAGTGCGAA ACCAACCATA TATATACGGA GCGGAGCTTG TGGCGGCAAT TCTCCCGTCT TGGACGGCCC CML247 TCAGTGCGAA ACCAACCATA TATATACGGA GCGGAGCTTG TGGCGGCAAT TCTCCCGTCT TGGACGGCCC

73

-301 -232 B73 AACGTTTTGC ACGCAGACGT AATAGTTGAG AGGAAGTACC AGACTTGTGT ACATCAGCCG TCTCTATCTT OH7B AACGTTTTGC ACGCAGAAGT AATAGTTGAG AGGAAGTACC AGACTTGTGT ACATCAGCCG TCTCTATCTT MS71 --- ---CAGACGT AATAGTTGAG AGGAAGTACC AGACTTGTGT ACATCAGCCG TCTCTATCTT Delprim AACGTTTTGC ACGCAGACGT AATAGTTGAG AGGAAGTACC AGACTTGTGT ACATCAGCCG TCTCTATCTT NC358 AACGTTTTGC ACGCAGACGT AATAGTTGAG AGGAAGTACC AGACTTGTGT ACATCAGCCG TCTCTATCTT CML247 AACGTTTTGC ACGCAGAAGT AATAGTTGAG AGGAAGTACC AGACTTGTGT ACATCAGCCG TCTCTATCTT

-231 -162 B73 CTGCATGCAT CATGCATCAT TATTT-GACT GCTGGTCTCT GCTGCTTCAT TGTGCACAGC GCCCTATAAG OH7B CTGCATGCAT CATGCATCAT TATTT-GACT GCTGGTCTCT GCTGCTTCAT TGTGCACAGC GCCCTATAAG MS71 CTGCATGCAT CATGCATCAC TATTTTGACT GCTGGTCTCT GCTGCTTCAT TGTGCACAGC GCCCTATAAG Delprim CTGCATGCAT CATGCATCAC TATTTTGACT GCTGGTCTCT GCTGCTTCAT TGTGCACAGC GCCCTATAAG NC358 CTGCATGCAT CATGCATCAT TATTT-GACT GCTGGTCTCT GCTGCTTCAT TGTGCACAGC GCCCTATAAG CML247 CTGCATGCAT CATGCATCAC TATTTTGACT GCTGGTCTCT GCTGCTTCAT TGTGCACAGC GCCCTATAAG

-161 -92 B73 TACCTGGCGC AATGCAACAC ACTGAAACAA TCAGAAGCTG TAGACGTACT ACCAAAGATC GATCTCAAAA OH7B TACCTGGCGC AATGCAACAC ACTGAAACAA TCAGAAGCTG TAGACGTACT ACCAAAGATC GATCTCAAAA MS71 TACCTGGCGC AATGCAACAC ACTGAAACAA TCAGAAGCTG TAGACGTACT ACCAAAGATC GATCTCAAAA Delprim TACCTGGCGC AATGCAACAC ACTGAAACAA TCAGAAGCTG TAGACGTACT ACCAAAGATC GATCTCAAAA NC358 TACCTGGCGC AATGCAACAC ACTGAAACAA TCAGAAGCTG TAGACGTACT ACCAAAGATC GATCTCAAAA CML247 TACCTGGCGC AATSCAACAC ACTGAAACAA TCAGAAGCTG TAGGCGTACT ACCAAAGATC GATCTCAAAA -91 -22

B73 CAAA-GGGTA ATA----CCA TTACCAGCTA GAAGATGTGC AGG--GGCTG CTGCAATTTG CTCCGTCAAC OH7B CAAAAGGGTA ATA----CCA TTACCAGCTA GAAGATGTCC CTG

MS71 CAAA-AGGTA ATA----CCA TTACCAGCTA GAAGATGTCC CTGACGACGC CTGCAGTTTG CTCCGTCAAC Delprim CAAAAGGGTA ATATATACCA TTACCAGCTA GAAGATGTCC CTGACGACGC CTGCAGTTTG CTCCGTCAAC NC358 CAAAAGGGTA ATA----CCA TTACCAGCTA GAAGATGTCC CTGACGACGC CTGCAGTTTG CTCCGTCAAC CML247 CAAATCGGAA GAG---CG T--CGTG

-21 -1

B73 GATGTGCAGG GGCTGCTGCA AATG OH7B

MS71 GATGTGCAGG GGCTGCTGCA AATG

Delprim -ATGTGCAGG GGCTGCTGCA AATG NC358 GATGTGCAGG GGCTGCTGCA AATG CML247

Fig.3.16: Sequence comparison of the promoter fragment of tps10 in different maize lines. Start codon is underlined. Differences in the sequences are marked in red.

Interestingly, far more nucleotide changes could be observed in the tps10 promoter than in the promoter of tps23. While line MS71 and CML247 are not able to produce TPS10-specific sesquiterpenes, all other lines have an active TPS10 enzyme. Looking at the raw promoter sequence, an essential difference between TPS10-producing and non-producing cannot be predicted. In a next step, both promoter fragments from line Delprim were analyzed for regulatory sequences. Database analysis was conducted using either PLACE (plant cis-acting regulatory DNA elements) or PlantCARE (cis-acting regulatory element). 530 potential boxes were revealed in the promoter of tps23 and 379 in the promoter of tps10. An overview of all identified regulatory elements is attached in the appendix (7.8, 7.9). Several core promoter elements were identified in both promoters as well as stress- and defense-related

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cis-acting elements. Truncation experiments were conducted to isolate essential regulatory elements involved in the herbivore-induced responses (3.4.3).

3.4.2 Analysis of the promoters of tps10 and tps23 for binding activity to