The regulation of 1-aminocyclopropane-1-carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato

Abstract

1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is one of the key regulatory enzymes involved in the synthesis of the hormone ethylene and is encoded by a multigene family containing at least eight members in tomato (Lycopersicon esculentum). Increased ethylene production accompanies ripening in tomato, and this coincides with a change in the regulation of ethylene synthesis from auto-inhibitory to autostimulatory. The signaling pathways that operate to bring about this transition from so-called system-1 to system-2 ethylene production are unknown, and we have begun to address these by investigating the regulation of ACS expression during ripening. Transcripts corresponding to four ACS genes, LEACS1A, LEACS2, LEACS4, and LEACS6, were detected in tomato fruit, and expression analysis using the ripening inhibitor (rin) mutant in combination with ethylene treatments and the Never-ripe (Nr) mutant has demonstrated that each is regulated in a unique way. A proposed model suggests that system-1 ethylene is regulated by the expression of LEACS1A and LEACS6. In fruit a transition period occurs in which the RIN gene plays a pivotal role leading to increased expression of LEACS1A and induction of LEACS4. System-2 ethylene synthesis is subsequently initiated and maintained by ethylene-dependent induction of LEACS2.

Figure 1

ACS gene expression in wild-type cv Ailsa Craig and rin fruit. Twenty-five micrograms of total RNA from cv Ailsa Craig mature green (M), breaker (B), breaker +3 (3), and breaker +7 (7) and rin fruit harvested at 36, 42, 48, 54, and 60 DPA was hybridized to radiolabeled RNA probes specific for tomato ACS genes and RPA analysis was performed as described in “Materials and Methods.” Results are shown only for the genes that showed a positive hybridization signal. Exposure times to x-ray film were as follows: LEACS1A, 7 d; LEACS2, 20 h; LEACS4, 20 h; and LEACS6, 16 d.

Figure 2

ACS gene expression in response to ethylene. Mature green wild-type and rin tomato fruit (37 DPA) were treated with 10 μL L⁻¹ ethylene as described in “Materials and Methods” and harvested at time zero (0), 1 h (1), 2 h (2), 4 h (4), 12 h (12), and 24 h (24) after treatment. Twenty-five micrograms of total RNA was hybridized with radiolabeled RNA probes specific for LEACS1A, LEACS2, LEACS4, and LEACS6 and subjected to RPA analysis as described in the “Materials and Methods.” Exposure times to x-ray film were as follows: LEACS1A, 20 d; LEACS2, 4 d; LEACS4, 4 d; LEACS6, 14 d (cv Ailsa Craig); LEACS1A, 4 d; LEACS2, 3 d; LEACS4, 4 d; and LEACS6, 2 d (rin).

Figure 3

LEACS4 expression in 60-DPA rin fruit treated with ethylene. Sixty-DPA rin fruit were held in air (−) or in 10 μL L ⁻¹ ethylene (+) for a further 4 d. Twenty-five micrograms of total RNA was hybridized with a radiolabeled RNA probe specific for LEACS4 and subjected to RPA analysis as described in the “Materials and Methods.” RNA gel-blot analysis of E4 expression is included as a positive control. Exposure times to x-ray film were as follows: LEACS4, 4 d; and E4, 16 h.

Figure 4

ACS expression in tomato seedlings. The expression of eight ACS genes was examined in 10-d-old light-grown tomato seedlings grown in air (−) or treated with 10 μL L ⁻¹ ethylene (+) for 12 h. Protection assays were carried out according to the “Materials and Methods” using 50 μg of total RNA per assay. RNA gel-blot analysis of E4 expression is included as a positive control. Exposure times to x-ray film were as follows: LEACS1A, 7 d; LEACS1B, 5d; LEACS6, 3 d; and E4, 16 h.

Figure 5

ACS gene expression in Nr fruit. Total RNA was extracted from cv Pearson mature green (M), breaker (B), breaker +3 d (3), and breaker +7 d (7) and an isogenic line of the Nr mutant at mature green (M), breaker (B), breaker +3 d (3), breaker +7 d (7), and breaker +10 (10). The RNA was hybridized with radiolabeled RNA probes specific for LEACS1A, LEACS2, LEACS4, and LEACS6 and subjected to RPA analysis as described in the “Materials and Methods.” Exposure times to x-ray film were as follows: LEACS1A, 7 d; LEACS2, 20 h; LEACS4, 20 h; and LEACS6, 14 d.

Figure 6

Model proposing the regulation of ACS gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato. The symbols −ve (negative) and +ve (positive) refer to the action of ethylene on signaling pathways resulting in repression (−ve) or stimulation (+ve) of ACS gene expression.