Overexpression of tomato SlNAC1 transcription factor alters fruit pigmentation and softening

Abstract

Background: Fruit maturation and ripening are genetically regulated processes that involve a complex interplay of plant hormones, growth regulators and multiple biological and environmental factors. Tomato (Solanum lycopersicum) has been used as a model of biological and genetic studies on the regulation of specific ripening pathways, including ethylene, carotenoid and cell wall metabolism. This model has also been used to investigate the functions of upstream signalling and transcriptional regulators. Thus far, many ripening-associated transcription factors that influence fruit development and ripening have been reported. NAC transcription factors are plant specific and play important roles in many stages of plant growth and development, such as lateral root formation, secondary cell wall synthesis, and embryo, floral organ, vegetative organ and fruit development.

Results: Tissue-specific analysis by quantitative real-time PCR showed that SlNAC1 was highly accumulated in immature green fruits; the expression of SlNAC1 increased with fruit ripening till to the highest level at 7 d after the breaker stage. The overexpression of SlNAC1 resulted in reduced carotenoids by altering carotenoid pathway flux and decreasing ethylene synthesis mediated mainly by the reduced expression of ethylene biosynthetic genes of system-2, thus led to yellow or orange mature fruits. The results of yeast one-hybrid experiment demonstrated that SlNAC1 can interact with the regulatory regions of genes related lycopene and ethylene synthesis. These results also indicated that SlNAC1 inhibited fruit ripening by affecting ethylene synthesis and carotenoid accumulation in SlNAC1 overexpression lines. In addition, the overexpression of SlNAC1 reduced the firmness of the fruits and the thickness of the pericarp and produced more abscisic acid, resulting in the early softening of fruits. Hence, in SlNAC1 overexpression lines, both ethylene-dependent and abscisic acid-dependent pathways are regulated by SlNAC1 in fruit ripening regulatory network.

Conclusions: SlNAC1 had a broad influence on tomato fruit ripening and regulated SlNAC1 overexpression tomato fruit ripening through both ethylene-dependent and abscisic acid-dependent pathways. Thus, this study provided new insights into the current model of tomato fruit ripening regulatory network.

Figure 1

qRT-PCR analysis of SlNAC1 expression and phenotypes of OE and WT fruits. (A) Transcripts of SlNAC1 accumulated in different tissues. Rt, root; St, stem; L, leaf; F, flower; Sp, sepal; Gf, green fruit; Sd, seed. The pericarp tissues of the green fruits were used. (B) The relative mRNA level of SlNAC1 as fruit ripened. The pericarp tissues of fruits at different stages were used to perform the experiment. (C) qRT-PCR expression analysis of SlNAC1 in OE lines and WT. Total RNA from the pericarp tissues of fruits at B7 stage was subjected to quantitative RT-PCR analysis. (D) Phenotypes of OE and WT fruits along with the developmental stages. IM, immature green; MG, mature green; Br, breaker; B3, 3 d after breaker; B7, 7 d after breaker; B15, 15 d after breaker; R, ripe. Data are the means ± SD of three independent experiments. The WT expression data are normalised to 1.

Figure 2

Expression of four other NAC transcription factors in OE and WT fruits. The pericarp tissues of fruits in different stages were used to perform the experiment. MG, mature green; Br, breaker; B3, 3 d after breaker; B7, 7 d after breaker. Data are the means ± SD of three independent experiments.

Figure 3

Carotenoids contents and expression of carotenoid biosynthesis genes in OE and WT fruits. (A) Total carotenoid content in OE and WT fruits at B20. (B) Contents of lutein, β-carotene and lycopene in OE and WT fruits at B20. (C) to (F) Expression analysis of genes related to carotenoid synthesis. The relative mRNA levels of SlPSY1 (C), SlLCYb (D), SlLCYe (E) and SlCYCB (F) at indicated developmental stages were shown. MG, mature green; Br, breaker; B3, 3 d after breaker; B7, 7 d after breaker. Data are the means ± SD of three independent experiments. The asterisks indicate statistically significant differences between OE and WT fruits (*P < 0.05, **P < 0.01).

Figure 4

Ethylene emission and expression of ethylene synthesis genes in OE and WT fruits. (A) Ethylene production of OE and WT fruits was detected at the indicated stage. (B) to (D) qRT-PCR analysis of genes related to ethylene synthesis. The expression of SlACS2 (B), SlACS4 (C) and SlACO1 (D) were detected between OE and WT fruits. (E) Changes in the phenotypes of OE-8 fruits after these fruits were treated with ethephon. MG, mature green; Br, breaker; B3, 3 d after breaker; B7, 7 d after breaker. Data are the means ± SD of three independent experiments. The asterisks indicate statistically significant differences between OE and WT fruits (*P < 0.05, **P < 0.01).

Figure 5

Yeast one-hybrid assay between SlNAC1 and SlPSY1 , SlACS2 and SlACO1 promoters. (A) Structure of SlNAC1. The five subdomains (A to E) comprising the NAC domain and the C-terminal are shown. We selected the region from the 65^th amino acid to the 149^th amino acid containing the DNA binding domain (DBD) to construct the pGADT7 AD-SlNAC1 recombinant plastid. (B) Yeast one-hybrid assay results. SD/-Ura, SD medium without Ura; SD/-Leu, SD medium without Leu; SD/-Leu/AbA, SD medium without Leu but containing Aureobasidin A. The p53-AbAi control vector and the pAbAi vector were used as positive and negative controls, respectively.

Figure 6

Fruit firmness and pericarp thickness of OE and WT fruits. (A) Fruit firmness of OE and WT fruits was evaluated at the indicated stage. Br, breaker; B3, 3 d after breaker; B5, 5 d after breaker; B7, 7 d after breaker; B15, 15 d after breaker. (B) Cross-sections of OE-8 and WT fruits at B15. OE fruits showed thinner pericarp (white line indicated) compared with WT fruits. (C) The statistics of pericarp thickness between OE and WT fruits in the breaker stage. Data are the means ± SD of at least 10 individual fruits. The asterisks indicate statistically significant differences between OE and WT fruits (*P < 0.05, **P < 0.01).

Figure 7

ABA contents, expression of genes related to ABA synthesis and cell-wall metabolism and NDGA treatment. (A) ABA contents between OE-8 and WT fruits. (B) Expression of SlNCED1. (C) Expression of SlNCED2. (D) to (G) qRT-PCR analysis of genes related to cell-wall metabolism. The expression of SlPG (D), SlExp1 (E), SlCel1 (F) and SlWiv1 (G) were detected between OE and WT fruits. (H) The ratio of firmness of mock fruits to NDGA treated fruits. IM, immature green; McG, mature green; Br, breaker; B2, 2 d after breaker; B3, 3 d after breaker; B7, 7 d after breaker; B15, 15 d after breaker. Data are the means ± SD of three independent experiments. The asterisks indicate statistically significant differences between OE and WT fruits (*P < 0.05, **P < 0.01).