Increased ACS Enzyme Dosage Causes Initiation of Climacteric Ethylene Production in Tomato

Abstract

Fruits of wild tomato species show different ethylene-dependent ripening characteristics, such as variations in fruit color and whether they exhibit a climacteric or nonclimacteric ripening transition. 1-Aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase (ACO) are key enzymes in the ethylene biosynthetic pathway encoded by multigene families. Gene duplication is a primary driver of plant diversification and angiosperm evolution. Here, interspecific variations in the molecular regulation of ethylene biosynthesis and perception during fruit ripening in domesticated and wild tomatoes were investigated. Results showed that the activated ACS genes were increased in number in red-ripe tomato fruits than in green-ripe tomato fruits; therefore, elevated dosage of ACS enzyme promoted ripening ethylene production. Results showed that the expression of three ACS isogenes ACS1A, ACS2, and ACS4, which are involved in autocatalytic ethylene production, was higher in red-ripe tomato fruits than in green-ripe tomato fruits. Elevated ACS enzyme dosage promoted ethylene production, which corresponded to the climacteric response of red-ripe tomato fruits. The data suggest that autoinhibitory ethylene production is common to all tomato species, while autocatalytic ethylene production is specific to red-ripe species. The essential regulators Non-ripening (NOR) and Ripening-Inhibitor (RIN) have experienced gene activation and overlapped with increasing ACS enzyme dosage. These complex levels of transcript regulation link higher ethylene production with spatiotemporal modulation of gene expression in red-ripe tomato species. Taken together, this study shows that bursts in ethylene production that accompany fruit color changes in red-ripe tomatoes are likely to be an evolutionary adaptation for seed dispersal.

Keywords: climacteric; ethylene biosynthesis; fruit ripening; gene duplication; wild tomato.

Figure 1

Comparison of fruit ripening and ethylene production among tomato species and cultivars. Changes in skin color (A) and quantification of ethylene production during fruit development (B). Scale bar = 1 cm. DAP: days after pollination. Fruits were observed from 20 to 55 DAP for each species and cultivar. Error bars in (B) represent the standard deviation (SD) of five biological replicates.

Figure 2

Quantitative RT-PCR analysis of ethylene biosynthetic genes and carotenoid biosynthetic genes. Relative quantification revealing dynamic change in gene expression of LEACS genes (A), LEACO genes (B), carotenoid biosynthetic genes (C), and the key ripening transcription factors (D). The white bars indicate the relative expression levels of genes at the immature stage. Gray bars indicate relative gene expression levels at the start of fruit ripening, and black bars correspond to relative gene expression levels at the over-ripening stage. The gene expression level of S. lycopersicum cv. Ailsa Craig at the ripening stage was selected as a reference sample. Significant differences (p < 0.05) are indicated by lowercase letters. ACS, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene; ACO, ACC oxidase genes; PSY1, Phytoene synthase 1; ZDS, z-carotene desaturase; LCYE, lycopene ε-cyclase; LCYB, lycopene β-cyclase; RIN, Ripening-Inhibitor; NOR, Non-Ripening.

Figure 3

In silico analysis of cis-regulatory elements. Regulatory elements are found at upstream sequences of ethylene biosynthetic genes (A) and carotenoid biosynthetic genes (B). Insertions and deletions at the regulatory sequences are connected by black lines. Yellow bars, ABA-responsive element (ABRE); green bars, ethylene-responsive element (ERE); blue bars, MYB protein-binding domain (MYB); red bars, MYC family protein-binding domain (MYC); gray bars, WRKY TF-binding domain (W-box). LY, S. lycopersicum; CER, S. lycopersicum var. cerasiforme; PIM, S. pimpinellifolium; PEN, S. pennellii.

Figure 4

Responsiveness of tomato species to exogenous ethylene. Triple response assay of seedlings (A,B) and gene expression in fruits exposed to exogenous ethylene (C,D). Gray bars represent controls, while black bars indicate ethylene-treated samples. Ailsa Craig, S. lycopersicum cv. Ailsa Craig; PIM, S. pimpinellifolium; PEN, S. pennellii; PER, S. peruvianum. Asterisks indicate significant differences as determined by the t-test (* p < 0.05, ** p < 0.01).

Figure 5

Analysis of DNA methylation by qAMP in upstream regions of NOR. Levels of methylation change over time. DAP: days after pollination.

Figure 6

Current understanding of genetic control of ethylene biosynthesis and ethylene-mediated coloration in tomato. Bold lines and thin lines indicate the strength in corresponding activity. Characters in black indicate highly accumulated intermediates or enzymes, and characters in gray indicate relatively lowly accumulated intermediates or enzymes. * Significant response in S. lycopersicum cv. Ailsa Craig; ※ significant response in S. pennellii; # significant response in S. peruvianum.