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Gene expression and metabolism in tomato fruit surface tissues - PubMed

Gene expression and metabolism in tomato fruit surface tissues

Shira Mintz-Oron et al. Plant Physiol. 2008 Jun.

Abstract

The cuticle, covering the surface of all primary plant organs, plays important roles in plant development and protection against the biotic and abiotic environment. In contrast to vegetative organs, very little molecular information has been obtained regarding the surfaces of reproductive organs such as fleshy fruit. To broaden our knowledge related to fruit surface, comparative transcriptome and metabolome analyses were carried out on peel and flesh tissues during tomato (Solanum lycopersicum) fruit development. Out of 574 peel-associated transcripts, 17% were classified as putatively belonging to metabolic pathways generating cuticular components, such as wax, cutin, and phenylpropanoids. Orthologs of the Arabidopsis (Arabidopsis thaliana) SHINE2 and MIXTA-LIKE regulatory factors, activating cutin and wax biosynthesis and fruit epidermal cell differentiation, respectively, were also predominantly expressed in the peel. Ultra-performance liquid chromatography coupled to a quadrupole time-of-flight mass spectrometer and gas chromatography-mass spectrometry using a flame ionization detector identified 100 metabolites that are enriched in the peel tissue during development. These included flavonoids, glycoalkaloids, and amyrin-type pentacyclic triterpenoids as well as polar metabolites associated with cuticle and cell wall metabolism and protection against photooxidative stress. Combined results at both transcript and metabolite levels revealed that the formation of cuticular lipids precedes phenylpropanoid and flavonoid biosynthesis. Expression patterns of reporter genes driven by the upstream region of the wax-associated SlCER6 gene indicated progressive activity of this wax biosynthetic gene in both fruit exocarp and endocarp. Peel-associated genes identified in our study, together with comparative analysis of genes enriched in surface tissues of various other plant species, establish a springboard for future investigations of plant surface biology.

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Figures

Figure 1.
Figure 1.

The surface and peel of tomato during consecutive stages of fruit development. A to H, Electron micrographs of Alisa Craig tomato fruit surfaces. At the IG developmental stage, the tomato fruit surface is covered by a relatively dense mixture of type VI and type I trichomes (A–D). Young (E) and mature (F) fruit do not exhibit major differences in the shape of their conical epidermal pavement cells. At the Re stage, the tomato fruit cuticle is relatively thick and surrounds the entire epidermal cells (G and H). I, Light microscopy images of fruit peel cross sections at the five developmental stages examined in the study revealed the presence of several collenchyma and parenchyma layers beneath the epidermis. Cuticle lipids were stained by Sudan IV. ep, Epidermis; tsc, trichome support cells; t-I, type I trichome; t-VI, type VI trichome.

Figure 2.
Figure 2.

Gene expression and metabolic profiles in tomato peel and flesh tissues during fruit development. A, Principal component analysis of transcriptome assays carried out using the tomato GeneChip, with samples from peel (left) and flesh (right) tissues. B, Principal component analysis of metabolic profiles obtained by UPLC-QTOF-MS analysis, with samples from peel (left) and flesh (right) tissues. Transcriptome and metabolome analyses were carried out on the same set of samples (n = 3 for every sample type [stage × tissue]).

Figure 3.
Figure 3.

RT-PCR expression analyses of selected transcripts confirmed their up-regulation in tomato fruit peel as detected by the microarray analysis. A, Tomato TC176832, a putative AtCER1 ortholog. B, Tomato AW928465, a putative AtSHN2 ortholog. C, SlTHM27 (TC174616), a putative AtMYB4 ortholog. D, Tomato TC189627, a putative snapdragon (A. majus) MIXTA-LIKE2 ortholog. Besides SlSHN2 expression at the Br, Or, and Re stages, significantly higher expression levels were detected in the peel for all tested transcripts at all other stages (Student's t test; n = 3; P < 0.05).

Figure 4.
Figure 4.

Functional categories representation of the peel-associated transcripts detected by the array analysis. The 574 transcripts that showed a 2-fold or higher expression in the peel versus flesh (in at least one tested stage of tomato fruit development) are represented. In most cases, the putative functional categories were assigned according to sequence homologies with previously studied genes from other species. The distribution of categories is given in percentage of the total 574 transcripts.

Figure 5.
Figure 5.

Five selected clusters of gene expression profiles in tomato fruit peel (Pe) and flesh (Fl) tissues along the five tested developmental stages. A, Clusters 5, 6, and 13 represent genes with higher expression at early stages of fruit peel development. B, Cluster 16 represents genes with increased expression at the Br stage of peel development and a lower expression at later stages. C, Cluster 9 includes genes with steadily increasing expression up to the Or and Re developmental stages in both peel and flesh tissues. In parentheses next to each cluster name is the number of transcripts (t) that constitute it. Black lines represent the cluster average profile. The clustering method is described in “Materials and Methods.” [See online article for color version of this figure.]

Figure 6.
Figure 6.

Expression of SlCER6 and SlCHS1 as detected by a reporter gene assay and RT-PCR. A, Reporter GUS expression driven by the upstream region of peel-associated SlCER6 leads to staining of the exocarp (Ex) and endocarp (En) tissues in fruit slice at the Or stage. B, Reporter GUS exocarp expression driven by the upstream region of the peel-associated SlCHS1 gene in fruit slice at the Br stage. A light microscopy section image, magnifying the region of the stained exocarp, reveals discernible GUS reporter staining in the epidermis cells and one or two additional cell layers beneath them (image in the open rectangle). C, RT-PCR expression analysis of tomato SlCER6 (TC172551), a putative AtCER6 (CUT1) ortholog, and TC170658, a putative AtCHS1 ortholog, during the five tested fruit developmental stages. Significantly higher expression levels of both transcripts were detected in the peel compared with the flesh at all tested developmental stages (Student's t test; n = 3; P < 0.05).

Figure 7.
Figure 7.

Forty-five peel-associated metabolites detected by UPLC-QTOF-MS analysis. On the left side is a plot representing the five developmental stages (IG, MG, Br, Or, and Re numbered as 1–5) in the peel, and on the right side is a plot representing the profile in the flesh. Metabolite numbers (e.g. Met. 1) refer to the metabolite peak numbers in Table IV. Blue, Alkaloids; red, Val metabolism; black, phenylpropanoids/flavonoids; green, amine derivatives.

Figure 8.
Figure 8.

A proposed scheme for glycoalkaloid metabolism in tomato peel during fruit development, starting from α-tomatine. The glycoalkaloids formed in early fruit development are metabolized into the glycoalkaloids detected at later stages of tomato fruit maturation (i.e. Or and Re). The accumulation profile of each metabolite (peel left and flesh right) is presented next to it (in the profile, IG, MG, Br, Or, and Re are numbered as 1–5). [See online article for color version of this figure.]

Figure 9.
Figure 9.

Accumulation of selected peel-associated polar metabolites detected by GC-MS analysis of tomato flesh and peel-derivatized extracts during tomato fruit development. Samples were similar to the ones used for the array and other types of metabolic analysis (n = 3 for each sample). See Supplemental Table S7 for a full list of GC-MS-detected polar metabolites that were found to be abundant in the peel tissue.

Figure 10.
Figure 10.

Composition of compounds identified by GC-MS-FID analysis during the five tested developmental stages of isolated fruit cuticles. A, Total wax amounts. B, Total cutin amounts. C, Amounts of aliphatic wax compounds. D, Amounts of triterpenoid wax amounts. E, Amounts of cutin monomers. Detected cutin constituents include 16-hydroxyhexadecanoic acid, hydroxyhexadecane-1,16-dioc acid, and various dihydroxyhexadecanoic acid isomers. Values are given as averages (n = 3) and

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