Transcriptome and selected metabolite analyses reveal multiple points of ethylene control during tomato fruit development

Abstract

Transcriptome profiling via cDNA microarray analysis identified 869 genes that are differentially expressed in developing tomato (Solanum lycopersicum) pericarp. Parallel phenotypic and targeted metabolite comparisons were employed to inform the expression analysis. Transcript accumulation in tomato fruit was observed to be extensively coordinated and often completely dependent on ethylene. Mutation of an ethylene receptor (Never-ripe [Nr]), which reduces ethylene sensitivity and inhibits ripening, alters the expression of 37% of these 869 genes. Nr also influences fruit morphology, seed number, ascorbate accumulation, carotenoid biosynthesis, ethylene evolution, and the expression of many genes during fruit maturation, indicating that ethylene governs multiple aspects of development both prior to and during fruit ripening in tomato. Of the 869 genes identified, 628 share homology (E-value < or = 1 x 10(-10)) with known gene products or known protein domains. Of these 628 loci, 72 share homology with previously described signal transduction or transcription factors, suggesting complex regulatory control. These results demonstrate multiple points of ethylene regulatory control during tomato fruit development and provide new insights into the molecular basis of ethylene-mediated ripening.

Figure 1.

Experimental Design. For each of the nine sequential time-point comparisons, Cy-labeled cDNAs were hybridized to six independent microarrays using a triple dye-flip design. Equivalent age fruit tissue was collected and pooled from a normal line (cv Ailsa Craig) and a line nearly isogenic and homozygous for the Nr mutation. Cy3, cyanine3 fluor; Cy5, cyanine5 fluor; B, breaker stage; B+5, 5 d after breaker; B+10, 10 d after breaker; B+15, 15 d after breaker; DAP, days after pollination; MG, mature green.

Figure 2.

Effect of the Nr Lesion on Transcriptome Dynamics. Nr⁺ has a global effect on expression in tomato pericarp and regulates thousands of genes prior to ripening. These data also indicate that many ripening-related genes are not influenced by the Nr allele in this cultivar. Two different perspectives of each transcriptome are shown: (A) and (B) show relative gene activation and gene repression, respectively. (C) is a color map depicting relative transcript accumulation in (A) and (B). DAP, days after pollination.

Figure 3.

Coordinated Expression during Fruit Development. Transcriptome profiling in wild-type fruit (left) reveals highly coordinated expression in developing pericarp. In some cases, this coordination correlates with gene function (e.g., 40 cytosolic ribosome genes, top panel). Comparison of wild-type and Nr fruit (right) shows that C₂H₄ dictates the expression of many ripening-related genes. Each trace represents a different gene, and the number of genes shown in each panel is noted in the wild-type plot; genes shown for the Nr tissue are the same genes shown for the wild-type tissue. The vertical dashed line denotes the breaker stage of fruit development. DAP, days after pollination; Ribo, cytosolic ribosome genes.

Figure 4.

Candidate Regulatory Genes for Tomato Ripening. Ten of the seventy-two candidate regulatory genes identified in this study are shown. Gray lines show pseudoreplicates in the TOM1 array for the gene shown; black lines show a running mean when pseudoreplicates exist or expression of the gene shown if no pseudoreplicates exist. Only ESTs with substantial sequence homology were considered (E-value <1 × 10⁻¹⁰), and all EST clones shown have been sequence verified. The vertical dashed line denotes the breaker stage of fruit development. 14-3-3, 14-3-3 domain; DAP, days after pollination; G-protein, transducin-like heterotrimeric G-protein; PP2A, Ser/Thr protein phosphatase 2A regulatory chain.

Figure 5.

Transcriptional Regulation of C₂H₄ Biosynthesis. Seven genes thought to encode C₂H₄ biosynthetic enzymes are expressed in a ripening-related manner, five of which are under C₂H₄ control. (A) Pathway of C₂H₄ biosynthesis in tomato. (B) Expression profiles for TS, CGS, two likely MSR homologs, SAM1, ACS2, ACS4, and ACO1. Gray lines show pseudoreplicates in the TOM1 array for the gene shown; black lines show a running mean when pseudoreplicates exist or expression of the gene shown if no pseudoreplicates exist. Only ESTs with substantial sequence homology were considered (E-value <1 × 10⁻¹⁰), and all EST clones have been sequence verified. The vertical dashed line denotes the breaker stage of fruit development. E4, ripening-related protein E4.

Figure 6.

Transcriptional Regulation of Carotenoid Biosynthesis. Six genes thought to be required for carotenoid biosynthesis are expressed in a ripening-related manner, four of which are under C₂H₄ control. (A) Pathway of carotenoid biosynthesis in tomato. (B) Expression profiles for DXS, r⁺, pds⁺, gh⁺, B⁺, and CrtR-b⁺. Gray lines show pseudoreplicates in the TOM1 array for the gene shown; black lines show a running mean when pseudoreplicates exist or expression of the gene shown if no pseudoreplicates exist. Only ESTs with substantial sequence homology were considered (E-value <1 × 10⁻¹⁰), and all EST clones have been sequence verified. The vertical dashed line denotes the breaker stage of fruit development.

Figure 7.

Carotenoid Metabolite Profiling in Tomato Fruit. Ethylene regulates carotenoid metabolism in developing tomatoes. Carotenoid metabolite profiles for wild-type (A) and Nr (B) pericarp are shown. Carotenoid content (top panels) was determined from mean HPLC peak areas (n = 8, ±se). Mean trans-lycopene and β-Car content at 57 DAP in wild-type fruit was 91.6 ± 1.076 μg/g FW and 9.3 ± 0.350 μg/g FW, respectively. Mean trans-lycopene and β-Car content at 57 DAP in Nr fruit was 9.6 ± 0.702 μg/g FW and 8.3 ± 0.526 μg/g FW, respectively. The vertical dashed line denotes the breaker stage of fruit development.

Figure 8.

Model for C₂H₄ Regulatory Control in Tomato Fruit. Time-series transcriptome analysis, morphometric analysis, and selective metabolite profiling reveal novel regulatory points in pathways associated with ripening and identify numerous candidate regulatory loci. Only a fraction of the candidate regulatory genes identified in this study are shown here. A complete list of C₂H₄-regulated genes, candidate regulatory factors, Sol Genomics Network (SGN) unigene identifiers, and corresponding expression profiles are included in Supplemental Tables 1 and 3 online.