Promoter-reporter gene constructs
The constructs pRJM77, pRJM209, pRJM307, pRJM293, pCGN23, pRJM199, pRJM106, pRJM160 and pCGN09 have been previously described [11, 12].
Deletions from the 5′ terminus of the POSY element of the INO promoter were produced using oligonucleotide mutagenesis  on pRJM209  to generate eight promoter fragments. Primers P55′IIDEL1, P55′IIDEL2, P55′IIDEL3, P55′IIDEL4, POS45′DEL1, POS45′DEL2, POS45′DEL3 and POS45′DEL4 (primer sequences used in this study are listed in Additional file 2) were used to generate promoter fragments that were subsequently cloned into the HindIII/NcoI sites of pRJM137 (P-INO::GUS with P-INO replaced in these clonings; ) to create pLAW138, pLAW139, pLAW140, pLAW141, pLAW64, pLAW65, pLAW66 and pLAW67, respectively.
Deletions from the 3′ terminus of the POSY element of the INO promoter were produced by PCR amplification of promoter fragments using pRJM137  as a template with the forward primer 5′POS8 with the reverse primers POS43′DEL1, POS43′DEL2, POS43′DEL3, POS43′DEL4 and POS43′DEL5. These fragments were cloned into HindII/BamHI sites with the 35S minimal promoter and GUS coding region into pMON999 to create pLAW01, pLAW02, pLAW03, pLAW04 and pLAW05, respectively.
The POS9BC element of the INO promoter was amplified from pLMK134  using the primers NEWPOS8for and NEWPOS8rev and cloned into pLITMUS28 (New England Biolabs) to create pLAW117. The tetramer (4XPOS9BC) was created by repeatedly ligating the BglII/BamHI fragment of pLAW117 into BamHI digested pLAW117 to create pLAW120 as previously described for the POS9 element . The 4XPOS9BC element was combined with the 35S minimal promoter from pLAW105 and the GUS coding region from pCGN43 and inserted into pMON999  as a PstI/StuI fragment adjacent to the nopaline synthase polyadenylation region.
PCR was used for site-specific mutagenesis of the POS9 sequence of the INO promoter using pRJM209  as a template. Mutations in the (GA)9 sequence of the POS9A regions were created using the primers GA9-5′ and GA9-3′. Mutations in a conserved sequence in the POS9B and POS9C regions were created using the primers POS9Bfor and POS9Brev, and POS9TOPfor and POS9BOTTOMrev, respectively. These PCR products were ligated into the HindIII/NcoI sites of pRJM209 to create pKLP133, pKLP132 and pKLP134, respectively. To create mutations in the conserved sequence of POS9B and POS9C, the POS9TOPfor and POS9BOTTOMrev primers were used with POS9B mutated pKLP132 template.
Deletion of the POS9 sequence from the INO promoter was produced by oligonucleotide mediated mutagenesis  on pRJM77 with the POS9DEL primer to create pLAW156. The HindIII/NcoI fragment of pLAW156 was cloned into pRJM77 to create pLAW158.
A sequence to act as a spacer between the POSX and POSY regions, in place of POS9 in the INO promoter was produced by PCR using the primers BglIIspacer5′ and BglIIspacer3′ on the ROC3 gene of Arabidopsis using pCG23 as a template  and inserted into pCR4-TOPO (Invitrogen) to make pKLP114. The BglII fragment of pKLP114 was then transferred to pLAW158 and pKLP4 to create pKLP116 and pKLP115, respectively.
The POS6 element of the INO promoter was amplified from pLAW158 using the primers POS6Bgl and POS6BamHI and cloned into the BglII and BamHI sites of pBluescript KS- which had been modified to have a BglII site between the EcoRI and BamHI sites to create pKLP57. The BamHI/SacI fragment of pRJM293 (including the 35SMP::GUS combination, ) was cloned to pKLP57 to create pKLP60, and the BglII/SacI fragment of pKLP60 was recloned to pRJM293 to create pKLP61. The tetramer of the POS6 element were produced by repeatedly cloning the BglII/BamHI fragment of pKLP57 into the BglII site of pKLP61 to create pKLP63.
The 35S enhancer sequence  was amplified using the primers 35S-5′Hind and 35S-3′SalHind and cloned into the HindIII site of pUC118 to create pKLP2. The HindIII fragment of pKLP2 was then cloned to the HindIII sites in pRJM77, pLAW158, pRJM209, pRJM307, pCGN23, pRJM199, pRJM106, pRJM160 and pCGN09 ( and see above) to create pKLP7, pKLP4, pKLP83, pKLP10, pKLP6, pKLP8, pKLP5, pKLP9 and pKLP26, respectively. The correct orientation of the 35S enhancer in these clones was confirmed by digestion at an asymmetric MspI site.
A fragment of the B. oleracea INO (BoINO) gene was amplified from B. oleracea using primers based on the Arabidopsis sequence (B-INO1, B-INO2 and B-INO3). The sequence of the BoINO fragment was used to design primers (B-INO4 and B-INO5) that were used to identify a bacterial artificial chromosome (BAC) clone containing this gene from a B. oleracea library (a gift of Carlos Quiros, UC Davis). A BglII/HindIII fragment of this BAC that included all of the 5′-flanking region of the BoINO gene was subcloned into these same sites in pLITMUS28 to create pRB9 (the sequence of the utilized part of the insert of this clone was deposited in GenBank [GenBank:JX682714]). The entire promoter 5′-flanking region (from immediately flanking gene to start codon) was amplified with primers PB-INO1 and PB-INO2, adding HindIII and BamHI sites, respectively, and inserted at these sites into pBluescript KS+ to form pRB26. This 5′-flanking fragment was combined with the AtINO cDNA into pMON999 to form pLAW181 (P-BoINO::AtINO). The 5′-flanking fragment was also combined with an INO cDNA from which the stop codon had been removed and the eGFP coding region  to create pRB29 (P-BoINO::AtINO:GFP).
All promoter constructs were shuttled as NotI fragments into the pMLBART  binary vector and pMLBART derivatives were transferred to either Agrobacterium strain ASE  or GV3101::pMP90 .