Figure 1.
Localization of putative cis-acting elements in SlPPC2 promoter sequence.
Sequence was analyzed using PLACE and PlantCARE databases.
Figure 2.
GUS activity in tomato (cv. «Ferum») stably transformed with SlPPC2 promoter:GUS transgenes.
(A) Representative images of GUS activity in seedlings, leaf, flower and 6, 14 and 21 days after anthesis (daa) tomato fruit with the 35S:GUS and SlPPC2 promoter:GUS constructs. (B) Representative images of GUS activity in «Ferum» fruits at the various stages of fruit development (daa) as indicated.
Figure 3.
Transient reporter-gene expression analysis of the SlPPC2 promoter in developing tomato fruit («WVa 106» cherry).
Fruit slices at the indicated stages of development from 6 to 30 days after anthesis (daa) were transformed by biolistic with a 35S:GUS plasmid co-delivered with a promoter:LUC fusion plasmid (promoter:LUC construct pPPC2pro1:LUC) that included the nucleotides –1528 to +439 of SlPPC2 (with respect to the transcription start site). The pGr (plasmid alone) and pLUC (promoterless LUC construct) plasmids were used as negative controls to transform 8 daa fruit slices. Data were normalized using the 35S:GUS construct as internal standard and are expressed as % of maximum activity (8 daa fruit). The mean values and SE of 6 to 12 independent transformations are shown. Insert represents the RT-PCR analysis of SlPPC2 expression during wild-type tomato fruit development.
Figure 4.
Transient reporter-gene expression analysis of SlPPC2 promoter deletions in 8 daa tomato fruit.
Slices from 8 daa fruit («WVa 106» cherry) were transformed by biolistic with a 35S:GUS plasmid co-delivered with SlPPC2 promoter:LUC fusion plasmids (pPPC2pro1-4:LUC construct; sizes in nucleotides from the transcription start indicated; grey box indicates leader intron).The pLUC plasmid (promoterless LUC construct) was used as a negative control. Data were normalized using the 35S:GUS construct as internal standard and are expressed as % of maximum activity (pPPC2pro1:LUC construct). The mean values and SE of 6 to 12 independent transformations are shown.
Figure 5.
Transient reporter-gene expression analysis of the SlPPC2 promoter deleted from its leader intron in developing tomato fruit and in leaf.
(A) Details of the constructs. (B) Young leaf discs and slices of tomato fruit («WVa 106» cherry) at the indicated stages of development were transformed by biolistic with the –1528 to +439 construct (pPPC2pro1:LUC, in black) or with the –1528 to +195 construct (pPPC2proΔ:LUC, in grey) as indicated in Figure 1. Data are expressed as % of the pPPC2pro1:LUC activity at 8 daa. The mean values and SE of 10 independent transformations are shown.
Figure 6.
Influence of ethylene (ACC) on SlPPC2 promoter activity.
Fruit slices from 8 daa fruit («WVa 106» cherry) were transformed by biolistic with a 35S:GUS plasmid co-delivered with SlPPC2 promoter:LUC fusion pPPC2pro1:LUC plasmid and incubated for 20 h on CPW4 medium supplemented with 1-aminocyclopropane-1-carboxylic acid (ACC, 20 µM and 200 µM), silver thiosulfate (AgTS), or ACC (20 µM) plus AgTS or NaTS. Control was CPW4 medium. Data were normalized using the 35S:GUS construct as internal standard and are expressed as % of the control. The mean values and SE of 12 independent transformations are shown. * indicates a significant statistical difference using a Student’s t-test (*: P<0.05; **: P<0.001).
Table 1.
Malic and citric acid contents of 8 daa tomato fruit slices.
Figure 7.
Influence of sugars on SlPPC2 promoter activity.
Fruit slices from 8 daa fruit («WVa 106» cherry) were transformed by biolistic with a 35S:GUS plasmid co-delivered with SlPPC2 promoter:LUC fusion pPPC2pro1:LUC plasmid and incubated for 20 h on CPW4 medium supplemented with sucrose concentrations ranging from 5 mM to 100 mM as indicated, 3-O methylglucose (3-OMG, 50 mM), 2-deoxyglucose (2-dG, 50 mM), glucose (50 mM) or fructose (50 mM). Control was CPW4 medium supplemented with mannitol (50 mM). Data were normalized using the 35S:GUS construct as internal standard and are expressed as % of the control. The mean values and SE of 12 independent transformations are shown. * indicates a significant statistical difference using a Student’s t-test (*: P<0.05; **: P<0.001).