Manipulation of ZDS in tomato exposes carotenoid- and ABA-specific effects on fruit development and ripening

Abstract

Spontaneous mutations in fruit-specific carotenoid biosynthetic genes of tomato (Solanum lycopersicum) have led to improved understanding of ripening-associated carotenogenesis. Here, we confirm that ZDS is encoded by a single gene in tomato transcriptionally regulated by ripening transcription factors RIN, NOR and ethylene. Manipulation of ZDS was achieved through transgenic repression and heterologous over-expression in tomato. CaMV 35S-driven RNAi repression inhibited carotenoid biosynthesis in all aerial tissues examined resulting in elevated levels of ζ-carotene isomers and upstream carotenoids, while downstream all trans-lycopene and subsequent photoprotective carotenes and xanthophylls were diminished. Consequently, immature fruit displayed photo-bleaching consistent with reduced levels of the photoprotective carotenes and developmental phenotypes related to a reduction in the carotenoid-derived phytohormone abscisic acid (ABA). ZDS-repressed ripe fruit was devoid of the characteristic red carotenoid, all trans-lycopene and displayed brilliant yellow pigmentation due to elevated 9,9' di-cis-ζ-carotene. Over-expression of the Arabidopsis thaliana ZDS (AtZDS) gene bypassed endogenous co-suppression and revealed ZDS as an additional bottleneck in ripening-associated carotenogenesis of tomato. Quantitation of carotenoids in addition to multiple ripening parameters in ZDS-altered lines and ABA-deficient fruit-specific carotenoid mutants was used to separate phenotypic consequences of ABA from other effects of ZDS manipulation and reveal a unique and dynamic ζ-carotene isomer profile in ripe fruit.

Keywords: Solanum lycopersicum; abscisic acid; carotenoid biosynthesis; fruit development; fruit ripening; zeta-carotene desaturase.

Figure 1

Tomato ZDS and CRTISO gene expression. (a) mRNA levels in aerial tissues in wild‐type (AC++). 20DPA, 20 days post‐anthesis; MG, mature green; Br, breaker; 3DPB and 7DPB, 3 and 7 days post‐breaker. (b) mRNA levels during wild‐type (AC++) fruit development. Fruit stages analysed are 7, 17, 27 DPA, MG (38 DPA), Br‐1day (40 DPA), Br (41 DPA), 1, 5, 10 and 15 DPB. (c) ZDS mRNA levels in ripening mutants compared to wild‐type (AC++). rin, ripening inhibitor; nor, non‐ripening; Nr, Never ripe; Gr, Green ripe. Each stage, tissue and genotype analysed were represented by 3 biological replicates done in triplicate.

Figure 2

Phenotypic characterization of tomato fruit from ZDS‐RNAi lines. (a) Tissue‐specific repression of ZDS mRNA levels in ZDS‐RNAi lines relative to wild‐type (AC++) (n = 3 and performed in triplicate). (b) Visually apparent alteration of chlorophyll and carotenoid content during fruit development in ZDS‐RNAi lines compared to wild‐type (AC++). MG, mature green; BR, breaker; RR, red ripe. Photographs are not to scale and are relevant only to fruit pigmentation.

Figure 3

Effectiveness of the AtZDS over‐expression transgene in tomato. (a) Quantitative RT‐PCR comparing tomato and Arabidopsis ζ‐carotene desaturase (SlZDS and AtZDS, respectively) transcript levels in AtZDS over‐expression lines relative to the wild‐type (AC++). Ripe fruits are 7 days post‐breaker (7DPB) (n = 3 and performed in triplicate). (b) Visual phenotypes of chromoplast rich ripe fruit (7DPB) from AtZDS over‐expression lines.

Figure 4

Differential accumulation of ζ‐carotene isomers in ZDS‐RNAi and AtZDS.OE ripe fruit (7DPB). (a) Carotenoid content (µg/g FW) of 7 ζ‐carotene isomers found in ZDS‐RNAi ripe fruit compared to Ailsa Craig wild‐type (AC++) (n ≥ 3). (b) Carotenoid content (µg/g FW) of 4 ζ‐carotene isomers most commonly found in wild‐type (AC++) ripe fruit (7DPB) compared to AtZDS.OE 7DPB fruit (n ≥ 5). The legend in A is the same for B. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 5

Decreased synthesis of photoprotective carotenoids and ABA in ZDS‐repressed tomato fruit. (a) Changes in accumulation of photoprotective carotenoids, lutein; β‐carotene; neoxanthin; and violaxanthin, in ZDS‐RNAi lines and not compared to wild‐type (AC++) controls through early fruit development. 1 cm, 7–10 days post‐anthesis (DPA); 15 DPA; and 25 DPA (n ≥ 3). (b) Decreased ABA synthesis in ZDS‐RNAi lines and not compared to wild‐type (AC++) through early stages of fruit development (n ≥ 3). (c) Elevated FtsZ mRNA levels in ZDS‐RNAi lines, ZDS.2 and ZDS.7, and not relative to wild‐type (AC++) in 3 early stages of fruit development (n = 3 and performed in triplicate). (d) Altered chlorophyll a and chlorophyll b content of ZDS‐RNAi lines and not compared to wild‐type (AC++) during early stages of fruit development (n ≥ 3). Inset picture of photobleached ZDS‐RNAi 25 DPA fruit compared to not and AC++ 25 DPA fruit. Scale bar = 1 cm. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6

Manipulation of tomato ZDS alters fruit quality and development. (a) Primary metabolite profiling of AC++ control and ZDS‐RNAi fruit. Intensity of fold change to wild‐type (AC++) is visualized by indicating colour (log₂FC > 2.0 shows red, log₂FC > −2.0 shows blue). Fruit stages analysed were mature green (MG) and red ripe (RR) (n = 6). For metabolite guidelines check list and overview of metabolite list see Table S3 and S4, respectively. (b) Brix^o content in 7 DPB fruit pericarp and locule of ZDS‐RNAi lines, ZDS.2 and ZDS.7, compared to other carotenoid mutants, r/r and tangerine (t/t), and wild‐type (AC++) (n ≥ 5; *, P < 0.05). (c) Decrease in fruit weight of ZDS‐RNAi lines, ZDS.2, ZDS.5 and ZDS.7, compared to wild‐type (AC++). (d) Fruit weight of AtZDS.OE lines, AtZDS.OE.1A, 7 and 9 compared to wild‐type (AC++). (e) Number of days to reach the initiation of ripening in ABA‐deficient ZDS‐RNAi lines, ZDS.2 and ZDS.7, and not compared to wild‐type (AC++) (DPA, days post‐anthesis). (f) Number of days post‐anthesis (DPA) to reach the initiation of ripening (breaker) in AtZDS.OE lines compared to wild‐type (AC++) (n ≥ 3; *, P < 0.05; **, P < 0.01).

Figure 7

Changes in ethylene production during tomato fruit ripening as a consequence of ZDS manipulation. (a) Ethylene content (ng/g/hr) in breaker, 3DPB and 7DPB fruit of ZDS‐RNAi lines, ZDS.2, ZDS.5 and ZDS.7, compared to wild‐type (AC++) (n ≥ 3; *, P < 0.05). (b) Ethylene content (ng/g/hr) in 3DPB and 5DPB fruit of AtZDS.OE lines, AtZDS.OE.1A, AtZDS.OE.7 and AtZDS.OE.9, compared to wild‐type (AC++) (n > 5).