The Phytoene synthase gene family of apple (Malus x domestica) and its role in controlling fruit carotenoid content

Abstract

Background: Carotenoid compounds play essential roles in plants such as protecting the photosynthetic apparatus and in hormone signalling. Coloured carotenoids provide yellow, orange and red colour to plant tissues, as well as offering nutritional benefit to humans and animals. The enzyme phytoene synthase (PSY) catalyses the first committed step of the carotenoid biosynthetic pathway and has been associated with control of pathway flux. We characterised four PSY genes found in the apple genome to further understand their involvement in fruit carotenoid accumulation.

Results: The apple PSY gene family, containing six members, was predicted to have three functional members, PSY1, PSY2, and PSY4, based on translation of the predicted gene sequences and/or corresponding cDNAs. However, only PSY1 and PSY2 showed activity in a complementation assay. Protein localisation experiments revealed differential localization of the PSY proteins in chloroplasts; PSY1 and PSY2 localized to the thylakoid membranes, while PSY4 localized to plastoglobuli. Transcript levels in 'Granny Smith' and 'Royal Gala' apple cultivars showed PSY2 was most highly expressed in fruit and other vegetative tissues. We tested the transient activation of the apple PSY1 and PSY2 promoters and identified potential and differential regulation by AP2/ERF transcription factors, which suggested that the PSY genes are controlled by different transcriptional mechanisms.

Conclusion: The first committed carotenoid pathway step in apple is controlled by MdPSY1 and MdPSY2, while MdPSY4 play little or no role in this respect. This has implications for apple breeding programmes where carotenoid enhancement is a target and would allow co-segregation with phenotypes to be tested during the development of new cultivars.

Fig. 1

Alignment of the four apple Phytoene synthase (PSY) compared with Arabidopsis PSY and apple squalene synthase (MdSQS1 and MdSQS2). Multiple sequence alignment was conducted using ClustalW and manually adjusted in Geneious. The predicted chloroplast cleavage site by ChloroP is indicated by a black triangle. The stop codon present in PSY3 is indicated by a circled asterisk. Boxed sequence indicate the putative active site DXXXD [18]. Highlighting indicates similarity among residues ignoring the gaps in sequence; black, 100 %, dark grey >80 % and grey, >50 %

Fig. 2

Phylogenetic tree was constructed using MEGA6 [37] from PSY and apple squalene synthase sequences retrieved from the GenBank database (except where noted): Arabidopsis, AtPSY; loquat, EjPSY1, EjPSY2A, EjPSY3; apple, MdPSY1, MdPSY2, MdPSY3, MdPSY4, MdSQS1, MdSQS2; cassava, MePSY1, MePSY2, MePSY3; rice, OsPSY1, OsPSY2, OsPSY3; sorghum, SbPSY1, SbPSY3; tomato, SlPSY1, SlPSY2, SlPSY3; maize, ZmPSY1, ZmPSY2, ZmPSY3. Evolutionary relationships were inferred using the Neighbor-joining method [85], with 1000 bootstrap re-sampling strategy. The four apple PSY sequences are indicated by diamond

Fig. 3

Functional complementation of apple PSY proteins. a. Escherichia coli cells harbouring the pACCRTE vector (which encodes CRTE, the enzyme catalyzing formation of geranyl geranyl pyrophosphate) were additionally transformed with apple PSY constructs or empty vector. Cells carrying the pAC-PHYT vector confer accumulation of phytoene [40] and were used as a positive control. HPLC chromatograms for the extracted pigments are shown. The peak representing phytoene (indicated by an arrow) was observed in cells with PSY1 and PSY2 constructs, but not with PSY4. The inset shows the absorption spectrum of the phytoene peak. b. E. coli cells harbouring pACCAR25ΔcrtB were transformed with the apple PSY constructs. Cells carrying the plasmid pAC-ZEAX [86] accumulating zeaxanthin were used as a positive control. The peak representing zeaxanthin diglucoside is shown. The inset shows the absorption spectra of the zeaxanthin diglucoside peak of pAC-ZEAX, which was similar to that from both PSY1 and PSY2 constructs with ΔcrtB

Fig. 4

Transient expression of apple PSY-YFP fusion constructs in etiolated maize leaf protoplasts. PSY1 and PSY2 were localized throughout the plastids based on the fluorescent distribution pattern. PSY4 localized to speckles, suggesting localization to plastoglobuli [18]. CHL, chlorophyll autofluorescence; Bars = 10 μm

Fig. 5

Carotenoid concentrations in fruit of apple cultivars selected based on the pigmentation of their skin and flesh. a. Ripe fruit (150 DAFB) of ‘Granny Smith’ (left) and ‘Royal Gala’ (right). b. Total carotenoid concentration as measured by HPLC in apple fruit skin (top panel) and flesh (lower panel). Fruit were harvested at different time points (days after full bloom) and separated into skin and flesh for carotenoid extraction and analysis. Error bars are standard errors from three biological replicates

Fig. 6

Gene expression profiles of PSY genes assessed in ‘Royal Gala’ and ‘Granny Smith’ apple fruit . PSY transcript levels in fruit skin and flesh picked at different time points (days after full bloom). The data were analysed using the target-reference ratios measured with LightCycler 480 software (Roche) using apple Actin and Elongation factor1-α (EF1-α) as reference genes. Data are analysed from biological replicates and presented as means ± SE (n = 4). Fisher's least significant difference (LSD) at P < 0.05 is shown

Fig. 7

PSY transcript levels in different apple tissues from ‘Royal Gala’. Data were analyzed from biological replicates as described in Fig. 6 and presented as means ± SE (n = 4). Fisher’s least significant difference (LSD) at P < 0.05 is shown

Fig. 8

Transient activation of PSY promoters by apple AP2/ERF domain (AP2D) transcription factors. a. Alignment of ‘Royal Gala’ PSY1 and PSY2 promoters conducted using ClustalW in Geneious. Highlighting (black) indicate nucleotide similarity. RAP2.2 motifs are indicated by black (PSY1) and grey (PSY2) bars. b. Ratio of fluorescent signals measured from Nicotiana benthamiana leaves co-infiltrated with Agrobacterium constructs with AP2/ERF genes and a vector with firefly luciferase (LUC) under control of PSY promoter and Renilla luciferase (REN) under the control of CaMV 35S promoter. LUC/REN signal ratios were normalised to the basal promoter activity. Bars represent means ± SE (n = 4). Fisher's least significant difference (LSD) at P < 0.05 is shown