Manipulating the Light Systemic Signal HY5 Greatly Improve Fruit Quality in Tomato

Abstract

Fruit ripening in tomato fruits comprises dramatic metabolic changes that are regulated by environmental factors. Light not only drives photosynthesis but also acts as a critical signal regulating plant growth, development, and the quality of produce. However, it is unclear how plants sense light signals in the environment to regulate fruit quality. It is demonstrated that the accumulation of Long Hypocotyl 5 (HY5) protein peaks at the breaker stage of fruit maturity, independent of fruit bagging. Genetic manipulation of HY5 reveals that its knockout delays carotenoid synthesis and sucrose conversion, while its overexpression promotes fruit ripening. Molecular and biochemical analyses show that HY5 directly activates the transcript of the key carotenoid synthesis genes, such as Phytoene Synthase 1 (PSY1) and Phytoene Desaturase (PDS), as well as the sucrose metabolism genes, including Lycopersicum Invertase (LIN5, LIN6), Vacuolar Invertase (VI) and Sucrose Synthase (SS1, SS7). Importantly, grafting experiments reveal that HY5 acts as a systemic signal, translocating from leaves to fruits to promote ripening. Furthermore, nightly lighting with red or blue LED greatly improves fruit quality. In summary, the results establish that HY5 as a mobile protein that mediates the systemic light regulation of fruit ripening, offering practical applications for improving fruit quality.

Keywords: Carotenoids; Fruit ripening; Invertase; Light signaling; Solanum Lycopersicum; Transcriptional regulation.

Figure 1

The role of light in carotenoid and sugar contents during tomato fruit ripening. A) WT fruits at 35, 40, 43, 47, and 55 DPA, respectively (Bar = 2 cm). B) Contents of lycopene and β‐carotene in WT fruit at 35, 40, 43, 47, and 55 DPA. C) Sugar (including sucrose, fructose, and glucose) contents in WT fruits at different stages during tomato ripening. D,E) qPCR analysis of PSY1 and BAM1 in WT fruits at 35, 40, 43, 47, and 55 DPA. F) Diurnal changes in the accumulation of HY5 protein in WT fruits at 35, 40, 43, 47, and 55 DPA. G) Representative 25, 35, 40, 43, 47, and 55 DPA fruits from light and bagging treatments (Bar = 2 cm). H) Contents of lycopene and β‐carotene in WT fruits with light and bagging treatments at 55 DPA. I) Contents of soluble sugars, including sucrose, fructose, and glucose, in WT fruits under light and bagging treatments at 55 DPA. J) The activity of CWINV in WT fruits under light and bagging treatments at 55 DPA. K) The relative mRNA levels of PSY1 and PDS in WT fruits with light and bagging treatments at 43 DPA. L) Changes in the accumulation of HY5 protein with light and bagging treatment during WT fruit ripening. The data in B‐E and H‐K are presented as the mean values ± SD (n = 3). Different letters and asterisks indicate significant differences according to Tukey and Student's t‐test (P < 0.05). SD, standard deviation. DPA, days post anthesis; WT, wild type; CWINV, cell wall invertase; qPCR, quantitative PCR.

Figure 2

HY5 is involved in light‐regulated tomato fruit ripening. A) Fruits collected from hy5, WT, and OE‐HY5 at 35, 40, 43, 47, and 55 DPA (Bar = 2 cm). B,C) Contents of lycopene and β‐carotene in the hy5, WT, and OE‐HY5 fruits at 55 DPA. D) Contents of soluble sugars, including sucrose, fructose, and glucose, in the hy5, WT, and OE‐HY5 fruits at 55 DPA. E) The activity of CWINV enzyme in the hy5, WT, and OE‐HY5 fruits at 55 DPA. F) qPCR analysis of the carotenoid biosynthetic genes (PSY1 and PDS) in the hy5, WT, and OE‐HY5 fruits at 43 DPA. G) qPCR analysis of VI, SS1, and SS7 in the hy5, WT, and OE‐HY5 fruits at 43 DPA. H) qPCR analysis of LIN5 and LIN6 in the hy5, WT, and OE‐HY5 fruits at 43 DPA. Data are presented as the means of three replicates ± SD; (n = 3). Different letters indicate significant differences (P < 0.05, Tukey's test). WT, wild type; CWINV, cell wall invertase; SD, standard deviation.

Figure 3

HY5 directly binds to the promoters of PSY1, PDS, VI, SS1, SS7, LIN5, and LIN6 to participate in carotenoid accumulation and sugar metabolism during tomato fruit ripening. A) Y1H assay of HY5 binding to the promoters of PSY1, PDS, VI, SS1, SS7, LIN5 and LIN6. B) EMSA assays showing the direct binding of HY5‐His fusion protein to HY5 cis‐acting elements on the promoters of PSY1, PDS, VI, SS1, SS7, LIN5, and LIN6 in vitro. +, presence of corresponding proteins and probes. ‐, absence of the corresponding proteins and probes. C) Dual‐luciferase assays for the regulatory effect of HY5 on the expression of PSY1, PDS, VI, SS1, SS7, LIN5, and LIN6. The ratio of LUC/REN of the empty vector was used as the control, and its activity was taken as one. Data are presented as the means of three replicates ± SD; n = 4. The asterisks indicate significant differences according to Student's t‐test (P < 0.05). qPCR, quantitative PCR; Y1H, yeast one‐hybrid; EMSA, electromobility shift assay.

Figure 4

HY5 acts as a systemic signal moving from leaves to fruits. A) Diagram of the grafting experiment, hy5 and OE‐HY5 plants were used as plant rootstocks and hy5 plants were used as fruit scion. B) Immunoblot analysis of the protein abundance of HY5 with anti‐HY5 and anti‐HA antibodies in the fruits from hy5/hy5 and hy5/OE‐HY5 plants at 35 DPA. C) Fruits collected from hy5/hy5 and hy5/OE‐HY5 at 35, 40, 43, 47, 55 DPA (Bar = 1 cm). D) Contents of lycopene and β‐carotene in hy5/hy5 and hy5/OE‐HY5 fruits at 55 DPA. E) Contents of sugars, including sucrose, fructose, and glucose, in hy5/hy5 and hy5/OE‐HY5 fruits at 55 DPA. F) The activity of CWINV enzyme in hy5/hy5 and hy5/OE‐HY5 fruits at 55 DPA. G–I) qPCR analysis of carotenoid biosynthetic genes (PSY1 and PDS) and sugar metabolism genes (VI, SS1, SS7, LIN5 and LIN6) in hy5/hy5 and hy5/OE‐HY5 fruits at 43 DPA. Data are presented as the means of three replicates ± SD; n = 3. The asterisks indicate a significant difference according to Student's t‐test (P < 0.05). qPCR, quantitative PCR.

Figure 5

The effect of nighttime supplemental dim red and blue light on tomato fruit quality. A) Immunoblot analysis of HY5 protein abundance from WT leaves and fruits in light (L) and dark (D), respectively. B) Immunoblot analysis of HY5 protein abundance from WT leaves in dark (D), red light (RL), and blue light (BL), respectively. C) Immunoblot analysis of HY5 protein abundance from WT fruits in dark (D), red light (RL), and blue light (BL) at 35, 40, and 43 DPA. D) Fruits collected from WT and hy5 plants under dark, dim red light, and blue light treatments during fruit ripening (Bar = 1 cm). E) Contents of lycopene and β‐carotene in WT and hy5 fruits at 55 DPA with dark, dim red light, and blue light treatments. F) Contents of sugars, including sucrose, fructose, and glucose, in WT and hy5 fruits at 55 DPA with dark, dim red light, and blue light treatments. G) The activity of CWINV enzyme in WT and hy5 fruits at 55 DPA with different light treatments. H–J) qPCR analysis of carotenoid biosynthetic genes (PSY1 and PDS) and sugar metabolism genes (VI, SS1, SS7, LIN5, and LIN6) in WT and hy5 fruits at 55 DPA with different light treatments at night. K) A proposed model that HY5 in the leaves was activated by light and transmitted into the fruits. The mobile and local HY5 in fruits increased the accumulation of carotenoid and sugar by transcriptional activating PSY1, PDS, VI, SS6, SS7, LIN5, and LIN6, respectively. Arrows indicated mobile signal and activation. Data are presented as the means of three replicates ± SD; n = 3. Different letters indicate a significant difference according to Tukey's test (P < 0.05). CWINV, cell wall invertase; qPCR, quantitative PCR.