Changes in transcriptional profiles are associated with early fruit tissue specialization in tomato

Abstract

The cell expansion phase contributes in determining the major characteristics of a fleshy fruit and represents two-thirds of the total fruit development in tomato (Solanum lycopersicum). So far, it has received very little attention. To evaluate the interest of a genomic scale approach, we performed an initial sequencing of approximately 1,200 cell expansion stage-related sequence tags from tomato fruit at 8, 12, and 15 d post anthesis. Interestingly, up to approximately 35% of the expressed sequence tags showed no homology with available tomato expressed sequence tags and up to approximately 21% with any known gene. Microarrays spotted with expansion phase-related cDNAs and other fruit cDNAs involved in various developmental processes were used (1) to profile gene expression in developing fruit and other plant organs and (2) to compare two growing fruit tissues engaged mostly in cell division (exocarp) or in cell expansion (locular tissue surrounding the seeds). Reverse transcription-polymerase chain reaction analysis was further used to confirm microarray results and to specify expression profiles of selected genes (24) in various tissues from expanding fruit. The wide range of genes expressed in the exocarp is consistent with a protective function and with a high metabolic activity of this tissue. In addition, our data show that the expansion of locular cells is concomitant with the expression of genes controlling water flow, organic acid synthesis, sugar storage, and photosynthesis and suggest that hormones (auxin and gibberellin) regulate this process. The data presented provide a basis for tissue-specific analyses of gene function in growing tomato fruit.

Figure 1.

Design of microarray experiment. A, Comparison of transcript abundance in developing tomato fruit, root, leaf, and seed from cherry tomato (cv WVa106). RNA was extracted from each organ sample (two RNA samples per organ), reverse transcribed, Cy labeled (Cy3 and Cy5 dye-swap for each RNA sample), and hybridized to tomato cDNA arrays for comparison with 15-DPA fruit sample, used as common reference (for each comparison, four slides, i.e. eight subarrays analyzed). B, Comparison of transcript abundance in exocarp and locular tissue from expanding tomato fruit (cv Ferum). Cytological observations and comparison of growth curves indicate that the developmental stage of 22-DPA Ferum fruit is equivalent to that of 15-DPA WVa106 fruit (data not shown). Exocarp and locular tissue samples were obtained by dissecting and pooling fruit tissues from 150 Ferum fruit harvested at 22 DPA. RNA was extracted from pooled tissue samples (two RNA samples per tissue), reverse transcribed, Cy labeled (Cy3 and Cy5 dye-swap for each RNA sample), and hybridized to tomato cDNA arrays (four slides, i.e. eight subarrays analyzed).

Figure 2.

RT-PCR analyses of developing fruit confirm microarray data. The expression of 14 genes preferentially expressed in 8-DPA fruit, in 15-DPA fruit, or at both stages of fruit development, according to the microarray data, was assayed in developing fruit by semiquantitative RT-PCR using gene-specific primers. Total RNA was isolated from fruits harvested at different developmental stages (anthesis [A], 3, 6, 8, 15, 20, and 25 DPA; mature green [MG], orange [Or], and red ripe [RR]) and from seed (S), root (R), young leaf (YL), and mature leaf (ML). LeActin (U60480) was used as a control. C−, PCR-negative control without added DNA; C+, PCR-positive control using purified cDNA insert.

Figure 3.

Hierarchical clustering of 607 tomato genes expressed in developing fruit. A, Each gene is represented by a single row of colored boxes. The five columns represent the different organs: fruit at 8 DPA (8), fruit at 15 DPA (15), leaf (L), root (R), and seed (S). Induction (or repression) ranges from pale to saturated red (or green). B, Four clusters showing distinctive expression profiles of genes preferentially expressed in fruit and root (Groups Ia and Ib) and in fruit and leaf (Group IIa and IIb) are presented. N indicates the number of transcripts in each group.

Figure 4.

Structure of tomato fruit during early development. A, Fresh section from 22-DPA Ferum fruit. B, Tissue sections from ovary at anthesis (A) and from 6-, 12-, and 25-DPA fruit were cut from the equatorial region of the fruit (cv Ferum). Numbers 1 to 5 indicate fruit regions where samples from 12- and 25-DPA fruits were taken. P, Pericarp; Sep, septum; E, exocarp; M, mesocarp; En, endocarp; L, locular tissue; S, seed; C, columella; V, vascular bundles.

Figure 5.

RT-PCR analyses confirm differential gene expression in exocarp and locular tissue. The expression of 21 genes preferentially expressed in fruit exocarp or locular tissue according to the microarray data was assayed by semiquantitative RT-PCR in different tissues dissected from 22-DPA fruit (cv Ferum) using gene-specific primers. E, Exocarp; M, mesocarp; C, columella; L, locular tissue; F, fruit without seeds; S, seed. A, LeActin (U60480) was used as control. For each gene and tissue, the relative abundance of mRNA was normalized toward that of tomato actin in the corresponding tissue. The results are presented as percentage of the highest relative expression for the considered gene. B, Transcripts showing preferential expression in the exocarp. C, Transcripts showing preferential expression in the locular tissue.

Figure 6.

Schematic representation of the major mechanisms and regulations in early developing fruit tissues. A, Exocarp; B, locular tissue.