Effect of Reflected Sunlight on Differential Expression of Anthocyanin Synthesis-Related Genes in Young Apple Fruit

Figures & data

Figure 1. Orchard-based apple reflected sunlight was conducted to increase light intensity on the fruit of apple ‘Summer Prince’ cultivar at the early developmental stage (60 DAFB). (a) Fruits were sampled for anthocyanin analysis showed the difference red-coloration levels between the three groups: reflected sunlight, bagged, and control. (b) The fruits in the control group showed the significant difference red-coloration level between the exposed and shaded sides in the same fruit. (c). For the reflected sunlight treatment, the fruit was set up with a paper wrapped-aluminum disc (28 cm diameter, V-shaped with angle of 140°) by hanging the disc on below the fruit in order to the shaded side of fruit to be exposed to sunlight (d). The same experimental layout was also applied to apple ‘Arisoo” cultivar (data not show)

Table 1. Fruit characteristics and colorimetric coordinates of two apple ‘Arisoo’ and ‘Summer Prince’ cultivars

		Fruit characteristics							Colorimetric coordinates
Cultivar	Group	Weight (g)	Vertical length (cm)	Horizontal length (cm)	Firmness (N)	SSC (^oBrix)	Acidity (%)	Starch index	L		a		b
									Exposed	Shaded	Exposed	Shaded	Exposed	Shaded
Arisoo	Reflected	104.4	54.9	61.4	98.1	11.4	0.60	5.0	46.10	48.86	15.21	13.96	16.44	17.98
	Control	137.4	59.4	68.2	91.3	10.7	0.70	5.0	38.56	64.41	13.04	−16.57	12.42	29.05
	Bagged	144.5	62.8	67.6	89.5	10.3	0.69	5.0	60.20	67.82	−10.60	−17.08	25.27	29.28
Summer Prince	Reflected	113.5	57.6	65.3	69.1	11.0	0.68	4.7	46.11	48.01	17.20	19.72	16.08	15.78
	Control	141.0	60.7	69.8	71.9	9.6	0.68	5.0	52.25	66.33	0.83	−10.99	20.89	28.81
	Bagged	105.7	58.3	66.4	70.7	8.8	0.57	4.5	64.45	65.53	−14.58	−10.88	27.00	27.56

• L scale: Light vs. dark where a low number (0–50) indicates dark and a high number (51–100) indicates light.

• a scale: Red vs. green where a positive number indicates red and a negative number indicates green.

• b scale: Yellow vs. blue where a positive number indicates yellow and a negative number indicates blue.

Table 2. Sequences of primers used in qRT-PCR

Gene	Encoded protein		Primer sequence (5ʹ-3ʹ)
PAL	Phenylalanine ammonialyase	F	TTCCTTGGCAACCCTGTCAC
		R	ATGTCGACCGCCTCAAAGG
CHS	Chalcone synthase	F	GGACTGGAACTCACTCTTC
		R	GCCGTAATCTGACAACAC
CHI	Chalcone isomerase	F	AGGATCACTAACGATCAGCTTCTCT
		R	TGGCCAAACTTTGCTTTGCTG
F3H	Flavanone 3-hydroxylase	F	CCAAGCAGTGGTGAACTC
		R	CTTCTCTCCCTCCCTCAC
DFR	DihydroXavonol 4-reductase	F	AAGCCTCATCACTGGACT
		R	ATGTGAGAAAGGCAGAGG
ANS	Anthocyanin synthase	F	GCTGGAGAAAGAAGTTGG
		R	GGAGGATGAAGGTGAGTG
UFGluT	UDP-glucose: flavonoid-3-O-glucosyltransferase	F	CAACATCCAAGGTCTCTC
		R	GTCCCATCTGAAGTAGCA
MYB10	R3R3 myb-type transcipt factor	F	TCATCTCTCTACTGCAGTGCCTA
		R	CCAGAAAGACACCTTAGACCTTCG
MYB110a	R3R3 myb-type transcipt factor	F	CTTGAGTGTGATGAGAAAAGGTGC
		R	CCTGTTTAAGCCTGCTTTGTATGG
COP1	E3 ubiquitin-protein ligase	F	CGCCAGCATTTTCCCCAATTT
		R	TCGAAATCTCGCAGCCCTTG
PIF3	Phytochrome interacting factor 3	F	TCCAAGAGAAGCAGAGCAGC
		R	TCGAAGCTTTGTCCACCTTGT

Table 3. Coloration patterns of two apple cultivars with the different light treatments

		Chroma (C)		Hue (°)		Color Index (CI)
		Exposed	Shaded	Exposed	Shaded	Exposed	Shaded
Arisoo	Reflected	22.39	22.76	47.23	52.19	1.81^b	1.81^b
	Control	18.01	33.44	43.62	110.88	2.38^a	0.62^d
	Bagged	27.40	33.89	112.76	111.55	0.77 ^c	0.59^d
Summer Prince	Reflected	23.54	25.25	41.66	38.66	1.93^a	1.88^a
	Control	20.91	30.84	87.74	110.88	1.28^b	0.70 ^c
	Bagged	30.69	29.63	118.36	111.55	0.65 ^c	0.71 ^c

Data are means ± S.E. of three replications. Different letters above the bars indicate significant (P < 0.05) differences according to the t-test.

Figure 2. Expression profiles of anthocyanin synthesis-related structural genes PAL, CHS, CHI, F3H, DFR, ANS, and UFGluT in the skin and flesh of three apple fruit groups: reflected, control, and bagged on the exposed side and shaded side of the apple ‘Arisoo’ (left panel) and ‘Summer Prince’ (right panel). All qRT-PCR reactions were normalized to an apple SGF 29 tudor-like domain-containing protein (MDP0000336547) gene. Data are means ± S.E. of seven replications

short-legendFigure 2.

Figure 3. Expression profiles of anthocyanin synthesis-related transcription factors MYB1 and MYB110a in the skin and flesh of three apple fruit groups: reflected, control, and bagged on the exposed side and shaded side of the apple ‘Arisoo’ (left panel) and ‘Summer Prince’ (right panel). All qRT-PCR reactions were normalized to an apple SGF 29 tudor-like domain-containing protein (MDP0000336547) gene. Data are means ± S.E. of seven replications

Figure 4. Expression profiles of anthocyanin synthesis-related light-responsive genes PIF3 and COP1 in the skin and flesh of three apple fruit groups: reflected, control, and bagged on the exposed side and shaded side of the apple ‘Arisoo’ (left panel) and ‘Summer Prince’ (right panel). All qRT-PCR reactions were normalized to an apple SGF 29 tudor-like domain-containing protein (MDP0000336547) gene. Data are means ± S.E. of seven replications

Table 4. Correlation analysis of anthocyanin-related gene expression levels and coloration patterns

	PAL	CHS	CHI	F3H	DFR	ANS	UFGluT	MYB10	MYB110a	COP1	PIF3
Arisoo	0.870*	0.915*	0.763	0.817*	0.906*	0.853*	0.752	0.713	0.919*	−0.530	−0.686
Summer	0.793	0.980**	0.940**	0.832*	0.879*	0.944**	0.935**	0.971**	0.932**	−0.900*	−0.881*

Level of significance: *p ≤ .05; **p ≤ .01.

**. Correlation is significant at the 0.01 level (2-tailed), *. Correlation is significant at the 0.05 level (2-tailed), -: negative correlation.

The correlation coefficient varied from 0.8–1.0 indicating extremely strong correlation; 0.6–0.8 indicating strong correlation; 0.4–0.6 indicating medium correlation; 0.2–0.4 indicating weak correlation; 0.0–0.2 indicating very weakly correlated or not correlated.