Differential expression and internal feedback regulation of 1-aminocyclopropane-1-carboxylate synthase, 1-aminocyclopropane-1-carboxylate oxidase, and ethylene receptor genes in tomato fruit during development and ripening

Abstract

We investigated the feedback regulation of ethylene biosynthesis in tomato (Lycopersicon esculentum) fruit with respect to the transition from system 1 to system 2 ethylene production. The abundance of LE-ACS2, LE-ACS4, and NR mRNAs increased in the ripening fruit concomitant with a burst in ethylene production. These increases in mRNAs with ripening were prevented to a large extent by treatment with 1-methylcyclopropene (MCP), an ethylene action inhibitor. Transcripts for the LE-ACS6 gene, which accumulated in preclimacteric fruit but not in untreated ripening fruit, did accumulate in ripening fruit treated with MCP. Treatment of young fruit with propylene prevented the accumulation of transcripts for this gene. LE-ACS1A, LE-ACS3, and TAE1 genes were expressed constitutively in the fruit throughout development and ripening irrespective of whether the fruit was treated with MCP or propylene. The transcripts for LE-ACO1 and LE-ACO4 genes already existed in preclimacteric fruit and increased greatly when ripening commenced. These increases in LE-ACO mRNA with ripening were also prevented by treatment with MCP. The results suggest that in tomato fruit the preclimacteric system 1 ethylene is possibly mediated via constitutively expressed LE-ACS1A and LE-ACS3 and negatively feedback-regulated LE-ACS6 genes with preexisting LE-ACO1 and LE-ACO4 mRNAs. At the onset of the climacteric stage, it shifts to system 2 ethylene, with a large accumulation of LE-ACS2, LE-ACS4, LE-ACO1, and LE-ACO4 mRNAs as a result of a positive feedback regulation. This transition from system 1 to system 2 ethylene production might be related to the accumulated level of NR mRNA.

Figure 1

Comparison of the deduced amino acid sequences among the four tomato ACC oxidase proteins (LE-ACO1, LE-ACO2 [accession no. Y00478], LE-ACO3 [accession no. Z54199], and LE-ACO4). The asterisks indicate sequence identity. Highly conserved regions for ACC oxidase are boxed, and the nine shaded amino acid residues are conserved in all members of the Fe(II) ascorbate family of dioxygenases (Lasserre et al., 1996).

Figure 2

Changes in the rate of ethylene production in tomato fruit during development and ripening and the effect of MCP. Fruit were harvested at six stages: immature green (IM), mature green (MG), turning (T), pink (P), red (R), and full ripe (FR), based on the observations described in Methods. Fruit harvested at the turning and pink stages were treated with 10 to 20 nL L⁻¹ MCP for 6 h and then ripened at 22°C. The ripening stages of MCP-treated fruit corresponding to the control fruit were determined as described in Methods. Vertical bars are the se of three replications; missing error bars are smaller than the symbols.

Figure 3

Agarose/ethidium bromide gel image of RT-PCR products amplified using specific primers for LE-ACS1A and LE-ACS1B. Each primer was designed to amplify the corresponding region in LE-ACS1A and LE-ACS1B but with two different nucleotides at the 3′ ends either upstream or downstream set to avoid cross-amplification. The LE-ACS1A primers were used for the reaction of lanes 2, 3, 4, and 9, and the LE-ACS1B primers were used for lanes 5 to 8. Templates used for RT-PCR were the combined single-strand cDNAs prepared from preclimacteric and ripening fruits in a ratio of 1:1 (lanes 2 and 6), the genomic DNA extracted from tomato leaves (lanes 3 and 7), and the plasmid inserted with the LE-ACS1A (lanes 4 and 5) or LE-ACS1B (lanes 8 and 9) fragment. Lane 1 shows a 100-bp DNA ladder as a size marker.

Figure 4

Expression of LE-ACS, LE-ACO, and ethylene receptor genes in tomato fruit during development and ripening and effect of MCP. mRNAs were prepared from the fruit immediately after the determination of ethylene levels as shown in Figure 2. Lane 1, Control fruit at the immature stage; lane 2, control fruit at the mature green stage; lane 3, control fruit at the turning stage; lane 4, control fruit at the pink stage; lane 5, control fruit at the red stage; lane 6, control fruit at the full-ripe stage; lane 7, turning-stage fruit 2 d after MCP treatment; lane 8, turning-stage fruit 4 d after MCP treatment; lane 9, turning-stage fruit 6 d after MCP treatment; lane 10, pink-stage fruit 2 d after MCP treatment; and lane 11, pink-stage fruit 4 d after MCP treatment. Each lane contained 3 μg of mRNA. Actin was used as an internal control to normalize the amount of mRNA loaded.

Figure 5

Effect of propylene on the accumulation of mRNAs corresponding to LE-ACS and ethylene receptor gene families and the LE-ACO1 gene in immature green fruit. mRNAs were isolated from the same fruit sample shown in Table III. Lane 1, Control fruit at harvest; lane 2, control fruit 2 d after harvest; lane 3, control fruit 4 d after harvest; lane 4, propylene-treated fruit for 2 d; lane 5, propylene-treated fruit for 4 d. Each lane contained 3 μg of mRNA. Actin was used as an internal control to normalize the amounts of mRNAs loaded.

Figure 6

Changes in the accumulation of mRNAs corresponding to LE-ACS and ethylene receptor gene families and the LE-ACO1 gene in fruit with different rates of ethylene production from the mature green stage to the turning stage. Lane 1, MG1 fruit (0.18 nL g⁻¹ h⁻¹ ethylene production); lane 2, MG2 fruit (0.36 nL g⁻¹ h⁻¹ ethylene production); lane 3, MG3 fruit (0.96 nL g⁻¹ h⁻¹ ethylene production); and lane 4, turning fruit (1.46 nL g⁻¹ h⁻¹ ethylene production). Each lane contained 3 μg of mRNA. Actin was used as an internal control to normalize the amounts of mRNAs loaded.