Expression Differences of Pigment Structural Genes and Transcription Factors Explain Flesh Coloration in Three Contrasting Kiwifruit Cultivars

Abstract

Fruits of kiwifruit cultivars (Actinidia chinensis and A. deliciosa) generally have green or yellow flesh when ripe. A small number of genotypes have red flesh but this coloration is usually restricted to the inner pericarp. Three kiwifruit cultivars having red ('Hongyang'), or yellow ('Jinnong-2'), or green ('Hayward') flesh were investigated for their color characteristics and pigment contents during development and ripening. The results show the yellow of the 'Jinnong-2' fruit is due to the combined effects of chlorophyll degradation and of beta-carotene accumulation. The red inner pericarps of 'Hongyang' fruit are due to anthocyanin accumulation. Expression differences of the pathway genes in the inner pericarps of the three different kiwifruits suggest that stay-green (SGR) controls the degradation of chlorophylls, while lycopene beta-cyclase (LCY-β) controls the biosynthesis of beta-carotene. The abundance of anthocyanin in the inner pericarps of the 'Hongyang' fruit is the results of high expressions of UDP flavonoid glycosyltransferases (UFGT). At the same time, expressions of anthocyanin transcription factors show that AcMYBF110 expression parallels changes in anthocyanin concentration, so seems to be a key R2R3 MYB, regulating anthocyanin biosynthesis. Further, transient color assays reveal that AcMYBF110 can autonomously induce anthocyanin accumulation in Nicotiana tabacum leaves by activating the transcription of dihydroflavonol 4-reductase (NtDFR), anthocyanidin synthase (NtANS) and NtUFGT. For basic helix-loop-helix proteins (bHLHs) and WD-repeat proteins (WD40s), expression differences show these may depend on AcMYBF110 forming a MYB-bHLH-WD40 complex to regulate anthocyanin biosynthesis, instead of it having a direct involvement.

Keywords: color diversity; expression differences; kiwifruit; structural genes; transcription factors.

FIGURE 1

Changes of color in three kiwifruit cultivars. (A–C) Bisected fruits of ‘Hongyang’ (HY), ‘Jinnong-2’ (JN) and ‘Hayward’ (HWD) at nine developmental stages. The red star indicates a fruit harvested at 145 DAP (days after pollination). (D) Changes of hue angle (h°) in the outer and inner pericarps during development and ripening. OP, outer pericarp; IP, inner pericarp; black arrows mark stages were selected for following analyses. (E) PCA analyses of hue angles of all samples.

FIGURE 2

Chlorophyll and carotenoid analyses in ‘Hongyang’ (HY, red), ‘Jinnong-2’ (JN, yellow) and ‘Hayward’ (HWD, green). (A–D) Concentrations of chlorophyll b, chlorophyll a, lutein and beta-carotene in the outer and (E–H) inner pericarps of the three kiwifruit cultivars during development. Stages S1–S6, represent 45, 85, 125, 145, 155, 170/190 DAP (days after pollination), respectively. Results represent means ± SE of three replicates.

FIGURE 3

Changes of anthocyanin content of ‘Hongyang’ kiwifruits. (A) Outer pericarps. (B) Inner pericarps. Results represent means ± SE of three replicates.

FIGURE 4

Expression profiles of chlorophyll pathway genes in the inner pericarps of ‘Hongyang’ (HY, red), ‘Jinnong-2’ (JN, yellow) and ‘Hayward’ (HWD, green). (A) Biosynthesis genes, (B) degradation genes. Error bars are SE for three replicates. CAO, Chlorophyll a oxygenase; CBR, Chlorophyll b reductase; GLUTR, Glutamyl tRNA reductase; PAO, Pheophorbide a oxygenase; PPH, Pheophytin pheophorbide hydrolase; RBCS, Small subunit of ribulose-1,5-bisphosphate Carboxylase; SGR, Stay-green.

FIGURE 5

Expression profiles of carotenoid pathway genes in the inner pericarps of ‘Hongyang’ (HY, red), ‘Jinnong-2’ (JN, yellow) and ‘Hayward’ (HWD, green). (A) Biosynthesis genes and (B) degradation genes. Error bars are SE for three replicates. PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, zeta-carotene desaturase; CRTISO, carotene isomerase; LCY-β, lycopene beta-cyclase; LCY-𝜀, lycopene epsilon-cyclase; CCD, carotenoid cleavage dioxygenease; NCED, 9-cis-epoxycarotenoid dioxygenase.

FIGURE 6

Expression profiles of anthocyanin pathway genes in the inner pericarps of three kiwifruit cultivars ‘Hongyang’ (HY, red), ‘Jinnong-2’ (JN, yellow) and ‘Hayward’ (HWD, green). Error bars are SE for three replicates. CHI, Chalcone isomerase; CHS, Chalcone synthase; F3H, Flavonoid 3-hydroxylase; F3’H, Flavonoid 3’-hydroxylase; DFR, Dihydroflavonol 4-reductase; ANS, Anthocyanidin synthase; UFGT, UDP flavonoid glycosyltransferases.

FIGURE 7

Expression profiles of anthocyanin transcription factors in the inner pericarps of ‘Hongyang’ (HY, red), ‘Jinnong-2’ (JN, yellow) and ‘Hayward’ (HWD, green). (A) MYBs, (B) bHLHs and (C) WD40s. Error bars are SE for three replicates.

FIGURE 8

Analyses of AcMYBF110. (A) Amino acid sequence alignment of AcMYBF110 and other plant anthocyanin-promoting MYBs. The R2 and R3 MYB motifs are indicated. The bHLH motif indicates residues needed for the interaction with the bHLH partner, box A and box B are well-conserved in anthocyanin-promoting MYBs. (B) Anthocyanin contents and expression profiles of AcMYBF110 in various tissues of ‘Hongyang’ and fruit cultivars. Le, leaves; St, stems; Pe, petals; Ov, ovary; YF, young fruits (10 days after pollination); QH, ‘Qihong’; DH, ‘Dohong’; ZG, Actinidia arguta var. purpurea; XX, ‘Xuxiang’; CX, ‘Cuixiang’; JK, Jinkui’. Data represent means ± SE of three replicates. (C) Subcellular localization of AcMYBF110 in Nicotiana benthamiana leaves. Scale bars: 50 μm. (D) Transient assays demonstrate the function of AcMYBF110 as a regulator of anthocyanin biosynthesis. (i) Assay leaves of AcMYBF110 exhibited anthocyanin accumulation. (ii) HPLC analysis of leaves injected with empty vector (CK) and 35S:AcMYBF110-GFP (OE). (iii,iv) Expression analysis of AcMYBF110 and key anthocyanin biosynthesis genes in CK and OE leaves. Data were analyzed with t-test. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001.