Phytochrome-Dependent Temperature Perception Modulates Isoprenoid Metabolism

Abstract

Changes in environmental temperature influence many aspects of plant metabolism; however, the underlying regulatory mechanisms remain poorly understood. In addition to their role in light perception, phytochromes (PHYs) have been recently recognized as temperature sensors affecting plant growth. In particular, in Arabidopsis (Arabidopsis thaliana), high temperature reversibly inactivates PHYB, reducing photomorphogenesis-dependent responses. Here, we show the role of phytochrome-dependent temperature perception in modulating the accumulation of isoprenoid-derived compounds in tomato (Solanum lycopersicum) leaves and fruits. The growth of tomato plants under contrasting temperature regimes revealed that high temperatures resulted in coordinated up-regulation of chlorophyll catabolic genes, impairment of chloroplast biogenesis, and reduction of carotenoid synthesis in leaves in a PHYB1B2-dependent manner. Furthermore, by assessing a triple phyAB1B2 mutant and fruit-specific PHYA- or PHYB2-silenced plants, we demonstrated that biosynthesis of the major tomato fruit carotenoid, lycopene, is sensitive to fruit-localized PHY-dependent temperature perception. The collected data provide compelling evidence concerning the impact of PHY-mediated temperature perception on plastid metabolism in both leaves and fruit, specifically on the accumulation of isoprenoid-derived compounds.

Figure 1.

PHYB1/B2 are involved in temperature perception impacting leaf chlorophyll metabolism and fluorescence parameters in tomato. A, Side view of 50-d-old tomato MM plants and phyB1, phyB2, and phyB1B2 knockout mutants grown under AT (day/night 24°C/18°C) and HT (day/night 30°C/24°C) conditions. B, Quantification of total Chl in the seventh fully expanded leaf from 85-d-old plants. Each bar represents the mean ± se. DW, Dry weight. C, PSII maximum efficiency (Fv′/Fm′), PSII operating efficiency (Fq′/Fm′), and maximum quantum efficiency of PSII (Fv/Fm) measured in the sixth fully expanded leaf from 85-d-old plants. n = at least five biological replicates. Each bar represents the mean ± se. In B and C, different letters indicate statistically significant differences according to Fisher’s multiple comparison test (P < 0.05). Asterisk indicates statistically significant differences by two-tailed Student’s t test (P < 0.05) between MM and phyB1B2 under the same environmental conditions. D, HT/AT relative expression ratios of GLK1, GGDR, CHLG, POR1, POR2, and POR3 mRNA abundance in MM and phyB1B2 mutant leaf samples from 85-d-old plants. n = at least three biological replicates. Each bar represents the mean ± se. Asterisks (*P < 0.05 and **P < 0.01) indicate statistically significant differences by two-tailed Student’s t test between AT and HT within the same genotype. Gene abbreviations are defined in the text.

Figure 2.

HT affects plastid biogenesis and development in leaves in a PHYB1/B2-dependent manner. A, Visualization of representative leaves from 21-d-old tomato MM and phyB1B2 knockout mutants after 2 weeks under AT (day/night 24°C/18°C) and HT (30°C/24°C) conditions. Red arrows indicate chlorotic leaves (MM at HT, phyB1B2 at AT, and phyB1B2 at HT). B, Quantification of total Chl in leaves cultivated under AT (blue background) and HT (yellow background) conditions. n = at least three biological replicates. Each bar represents the mean ± se. FW, Fresh weight. C, Plastid density per mesophyll cell. Values represent chloroplast quantification of ±70 cells. Each bar represents the mean ± se. In B and C, different letters indicate statistically significant differences according to Fisher’s multiple comparison test (P < 0.05). D, Representative transmission electron microscopy images of chloroplasts from MM and phyB1B2 leaves grown under AT and HT conditions. G indicates grana and DT indicates dilated thylakoids.

Figure 3.

HT enhances chlorophyll degradation in leaves in a PHYB1/B2-dependent manner. A, Schematic model of the Chl degradation pathway. Enzymes and metabolites not defined in the text are denoted as follows: Chlide a, chlorophyllide a; Pheide a, pheophorbide a; Pheo a, pheophytin a; RCC, red chlorophyll catabolite. The enzymes highlighted in red are those shown to be regulated by temperature in a PHYB1/B2-dependent manner according to B. B, Relative mRNA levels of Chl-degrading enzyme-encoding genes in MM and phyB1B2 mutant leaf samples from 85-d-old plants grown under AT (24°C/18°C; blue background) and HT (30°C/24°C; yellow background) conditions. Expression levels are relative to MM – AT conditions. n = at least three biological replicates. Each bar represents the mean ± se. Different letters indicate statistically significant differences according to Fisher’s multiple comparison test (P < 0.05).

Figure 4.

PHYB1/B2-dependent temperature perception transcriptionally regulates leaf carotenogenesis. A, Total carotenoid levels expressed in µg g⁻¹ dry weight (DW). n = at least five biological replicates. B, Relative mRNA levels of GGPS1 gene in MM and phyB1B2 mutant leaf samples from 85-d-old plants grown under AT (24°C/18°C; blue background) and HT (30°C/24°C; yellow background) conditions. Expression levels are relative to MM – AT conditions. n = at least three biological replicates. Each bar represents the mean ± se. Different letters indicate statistically significant differences according to Fisher’s multiple comparison test (P < 0.05).

Figure 5.

PHYA/B1/B2-dependent temperature perception transcriptionally regulates fruit carotenogenesis. A, Total carotenoid (phytoene, phytofluene, lycopene, lutein, and β-carotene) levels quantified from ripe fruits of MM and phyA, phyB1, phyB2, phyB1B2, and phyAB1B2 mutant plants grown under AT (day/night 24°C/18°C; blue fill) and HT (day/night 30°C/24°C; yellow fill) conditions. B, Center, Schematic model of the lycopene biosynthetic pathway. The dotted line represents more than one enzymatic step. Left, Levels of lycopene, phytoene, and phytofluene in ripe fruits. AT, blue background; HT, yellow background. Each bar represents the mean ± se. Different letters indicate statistically significant differences according to Fisher’s multiple comparison test (P < 0.05). DW, Dry weight. Right, Relative mRNA levels of carotenoid biosynthetic genes GGPS2, PSY1, and PDS in fruits at mature green (MG) and breaker (BK) stages harvested from plants grown under AT (blue) and HT (yellow) conditions. Transcript levels are expressed relative to MM MG – AT conditions. Asterisks (*P < 0.05 and **P < 0.01) indicate differences in the ANOVA in a multiple comparison test within the same fruit stage. GGDP, Geranylgeranyl diphosphate. C, Relative mRNA levels of the PIF1a carotenogenesis regulator gene in fruits at mature green and breaker stages harvested from plants grown under AT (blue) and HT (yellow) conditions. Transcript levels are expressed relative to MM MG – AT conditions. Asterisks (**P < 0.01) indicate differences in the ANOVA in a multiple comparison test within the same fruit stage.

Figure 6.

Fruit-localized PHYA and PHYB2 are involved in temperature perception impacting lycopene synthesis and master fruit-ripening regulators. A, Lycopene levels quantified in ripe fruits from MT control genotype and fruit-specific PHYA (PHYA^RNAi) and PHYB2 (PHYB2^RNAi) knockdown transgenic lines grown under AT (24°C/18°C) and HT (30°C/24°C) conditions. Lycopene levels were quantified and expressed relative to MT fruits under AT conditions, and values are means of at least three biological replicates from two independent lines for each genotype. Each bar represents the mean ± se. B and C, Relative mRNA levels of PSY1 (B) and master fruit-ripening regulator genes (C) in MT, PHYA^RNAi, and PHYB2^RNAi breaker fruit samples harvested under AT and HT conditions. Expression levels are relative to MT – AT conditions. n = at least three biological replicates. Each bar represents the mean ± se. Different letters indicate statistically significant differences according to Fisher’s multiple comparison test (P < 0.05).

Figure 7.

Effect of PHY-mediated temperature perception on tomato metabolism regulation. The rise of AT shifts the balance to the inactive Pr form, which promotes the Chl degradation pathway in source leaves through the transcriptional up-regulation of Chl catabolic enzyme-associated genes. Additionally, reduced levels of Pfr impair carotenoid accumulation in both leaves and ripe fruits, through the transcriptional down-regulation of carotenoid biosynthetic and master ripening regulator genes.