Transcriptome Analysis Provides a Preliminary Regulation Route of the Ethylene Signal Transduction Component, SlEIN2, during Tomato Ripening

Abstract

Ethylene is crucial in climacteric fruit ripening. The ethylene signal pathway regulates several physiological alterations such as softening, carotenoid accumulation and sugar level reduction, and production of volatile compounds. All these physiological processes are controlled by numerous genes and their expression simultaneously changes at the onset of ripening. Ethylene insensitive 2 (EIN2) is a key component for ethylene signal transduction, and its mutation causes ethylene insensitivity. In tomato, silencing SlEIN2 resulted in a non-ripening phenotype and low ethylene production. RNA sequencing of SlEIN2-silenced and wild type tomato, and differential gene expression analyses, indicated that silencing SlEIN2 caused changes in more than 4,000 genes, including those related to photosynthesis, defense, and secondary metabolism. The relative expression level of 28 genes covering ripening-associated transcription factors, ethylene biosynthesis, ethylene signal pathway, chlorophyll binding proteins, lycopene and aroma biosynthesis, and defense pathway, showed that SlEIN2 influences ripening inhibitor (RIN) in a feedback loop, thus controlling the expression of several other genes. SlEIN2 regulates many aspects of fruit ripening, and is a key factor in the ethylene signal transduction pathway. Silencing SlEIN2 ultimately results in lycopene biosynthesis inhibition, which is the reason why tomato does not turn red, and this gene also affects the expression of several defense-associated genes. Although SlEIN2-silenced and green wild type fruits are similar in appearance, their metabolism is significantly different at the molecular level.

Fig 1. Acquisition of TRV-SlEIN2 fruits.

(A). Main phases of VIGS. (B). Non-ripening phenotype of attached and dissected TRV-SlEIN2 tomato fruit compared with the two control groups. (C). qPCR analysis of SlEIN2 expression in VIGS and control fruits. The error bar indicates the standard deviation, based on three biological replicates. Asterisks indicate significant differences, according to Student’s t-test (***, P < 0.001). (D). pTRV transmission in fruits. M is the 2 kb marker, and C is the control, using the pTRV2 plasmid as template. 1, 2, and 3 represent the three biological replicates.

Fig 2. Phenotype of SlEIN2-silenced tomato three months after picking, BK+5 phase was initiated.

Fig 3. Analyses of differential expressed genes.

(A, C). Volcano diagrams of DEGs. Spots above the threshold line (Q-value = 0.05), indicate that differences are significant. Genes whose expression was less than a half than that displayed in the control group for Q-value < 0.05 are displayed in the green area, while those whose expression was more than two-fold that of the control group are displayed in the red area. Genes in the grey area were neither over- or under-expressed. (B, D). Percentage of up/down regulated SDEGs. SDEGs were screened in the green and red areas. (E) Venn diagram showing the numbers of non-overlapped and overlapped SDEGs in the four conditions tested. OU and OD separately means upregulated and downregulated TRV-OR/TRV-SlEIN2 SDEGs. GU and GD represents upregulated and downregulated TRV-GR/TRV-SlEIN2 SDEGs respectively.

Fig 4. Molecular function and pathway enrichment analysis of SDEGs.

(A). Molecular function of TRV-GM/TRV-SlEIN2 SDEGs, considering a corrected P-value < 0.05. The X axis indicates the gene number, and Y represents classification. (B). Top 3 pathway enrichment of TRV-GM/TRV-SlEIN2 SDEGs, with a Q-value < 0.05. Rich factor means the number of gene from SDEGs/all gene numbers, in a pathway. (C, D). Molecular function and top 4 pathway enrichment of TRV-OR/TRV-SlEIN2 SDEGs. All the graphic descriptions and parameters are identical to TRV-GM/TRV-SlEIN2.

Fig 5. Relative expression of 28 genes covering several aspects associated with ripening.

The error bar indicates the standard deviation, based on three biological replicates. Asterisks indicate significant differences, according to Student’s t-test (*, P<0.05, **, P<0.01, ***, P< 0.001).

Fig 6. Regulation route in SlEIN2-silenced fruits.

Red represents upregulated substances and proteins, and blue downregulated substances and proteins.