RNA interference silencing of chalcone synthase, the first step in the flavonoid biosynthesis pathway, leads to parthenocarpic tomato fruits

Abstract

Parthenocarpy, the formation of seedless fruits in the absence of functional fertilization, is a desirable trait for several important crop plants, including tomato (Solanum lycopersicum). Seedless fruits can be of great value for consumers, the processing industry, and breeding companies. In this article, we propose a novel strategy to obtain parthenocarpic tomatoes by down-regulation of the flavonoid biosynthesis pathway using RNA interference (RNAi)-mediated suppression of chalcone synthase (CHS), the first gene in the flavonoid pathway. In CHS RNAi plants, total flavonoid levels, transcript levels of both Chs1 and Chs2, as well as CHS enzyme activity were reduced by up to a few percent of the corresponding wild-type values. Surprisingly, all strong Chs-silenced tomato lines developed parthenocarpic fruits. Although a relation between flavonoids and parthenocarpic fruit development has never been described, it is well known that flavonoids are essential for pollen development and pollen tube growth and, hence, play an essential role in plant reproduction. The observed parthenocarpic fruit development appeared to be pollination dependent, and Chs RNAi fruits displayed impaired pollen tube growth. Our results lead to novel insight in the mechanisms underlying parthenocarpic fruit development. The potential of this technology for applications in plant breeding and biotechnology will be discussed.

Figure 1.

Schematic overview of the flavonoid biosynthesis pathway in plants. The pathway normally active in tomato fruit peel, leading to flavonol production, is indicated by solid arrows. Abbreviations: STS, stilbene synthase; CHI, chalcone isomerase; F3H, flavanone hydroxylase; FNS, flavone synthase; IFS, isoflavone synthase; FLS, flavonol synthase; F3′H, flavonoid-3′-hydroxylase; F3′5′H, flavonoid-3′,5′-hydroxylase; DFR, dihydroflavonol-4-reductase; ANS, anthocyanidin synthase.

Figure 2.

Schematic drawing of the tomato Chs RNAi construct. Transgene expression was under control of the CaMV double 35S promoter (Pd35S) and terminated by Tnos. An inverted repeat was generated by cloning a sense Chs1 cDNA fragment (801 bp) followed by the full-length cDNA sequence encoding tomato Chs1 in anti-sense orientation.

Figure 3.

Comparison of flavonoid levels between wild-type and Chs RNAi tomato. A, Total flavonoid levels in leaf extracts of different CHS RNAi transgenic lines. B, HPLC chromatograms obtained from nonhydrolyzed fruit peel extracts of wild-type (top) and Chs RNAi plants (bottom). In the control plant, the major compounds found are NAR-chalcone (NC) and the flavonol rutin. C, Percentage of flavonoids in the fruit peel of Chs RNAi plants (lines 34, 39, 44, and 24) relative to the fruit peel of control tomatoes. Mean control values (milligram per kilogram fresh weight): NAR-chalcone, 212.5, sd 66.5; quercetin derivatives (rutin + rutin apioside), 80.7, sd 11.0. [See online article for color version of this figure.]

Figure 4.

Quantitative real-time PCR analysis. Steady-state mRNA levels of tomato Chs1 and Chs2 relative to the housekeeping gene L33 (encoding tomato ribosomal protein L33) were measured in fruit peel extracts of RNAi (34, 39, 44, and 24) and control lines. Values represent the average of three biological replicates, each with three technical replicates. Expression levels in control were set to 100%.

Figure 5.

CHS enzyme activity. A, Autoradiography scan of extraction of CHS assays developed on cellulose plates in chloroform:acetic acid:water. Left, CHS activity of tomato fruit peel in different Chs RNAi and control lines. Right, CHS activity in fruits obtained after (reciprocal) crossings between wild type and Chs RNAi. B, Densitometric scans of profiles of selected assays. The peak representing the CHS reaction product NAR is indicated.

Figure 6.

Tomato fruit weight in grams (black bars) and fruit size at equatorial cross section in millimeters (gray bars). Values represent mean values ± SEM. Control (wild type), n = 10; line 34, n = 4; line 44, n = 15; line 24 and 39, each n = 14.

Figure 7.

Overview of different phenotypes found in Chs RNAi tomatoes compared to wild type. A, Typical ripe wild-type tomato fruits are shiny and orange red, in contrast to dull, smaller, and more reddish Chs RNAi fruits (B, C, and D; line 24, 34, and 39, respectively). Fruits derived from flowers that were pollinated with wild-type pollen (arrow) grew to normal size and obtained their shininess (D). Transgenic line 44 yielded extreme small fruits and line 34 “fruit caves” when compared to wild type (E). In parthenocarpic Chs RNAi fruits, seed development was disturbed (G) or totally absent (H and I), whereas wild-type fruits had a normal seed set (F).

Figure 8.

Electron microscopy photograph of epidermal cells of red ripe tomato fruits. A and C, Surface view; B and D, cross section. Wild-type (A and B) fruits contain conical-shaped cells on the epidermal surface, whereas in Chs RNAi (C and D) fruits the epidermal cell layer is disturbed (absence of conical shapes and empty cells).

Figure 9.

Histochemical staining of wild-type and Chs RNAi (line 24) pollen tube growth in carpels 2 d after pollination. Fertilized carpels were stained with aniline blue to specifically stain callose present in growing pollen tubes. A, E, and I, Wild-type carpels crossed with wild-type pollen; B, F, and J, wild-type carpels × Chs RNAi pollen; C, G, and K, Chs RNAi × wild type; D, H, and L, Chs RNAi self-crossings. A to D, Pollen at the stigma. Note in D, callose in the pollen tubes is still visible at the stigma (arrow), indicating inhibited growth. E to H, Proliferation of pollen tube growth in the middle of the style, except for H, which is only one-quarter of the way down the style from the stigma. No pollen tubes were visible in the middle of the styles from Chs RNAi selfed plants. I to L, Pollen tube growth at the base of the style, except K, which grew only nine-tenths of the way down the style. In K, the tips of the pollen tubes are swollen (arrow). Pollen tubes are not visible at the base of the style in K (not shown) or L. All micrographs are the same magnification. [See online article for color version of this figure.]