Dual role for tomato heat shock protein 21: protecting photosystem II from oxidative stress and promoting color changes during fruit maturation

Abstract

The tomato (Lycopersicon esculentum) chloroplast small heat shock protein (sHSP), HSP21, is induced by heat treatment in leaves, but also under normal growth conditions in developing fruits during the transition of chloroplasts to chromoplasts. We used transgenic tomato plants constitutively expressing HSP21 to study the role of the protein under stress conditions and during fruit maturation. Although we did not find any effect for the transgene on photosystem II (PSII) thermotolerance, our results show that the protein protects PSII from temperature-dependent oxidative stress. In addition, we found direct evidence of the protein's role in fruit reddening and the conversion of chloroplasts to chromoplasts. When plants were grown under normal growth temperature, transgenic fruits accumulated carotenoids earlier than controls. Furthermore, when detached mature green fruits were stored for 2 weeks at 2 degrees C and then transferred to room temperature, the natural accumulation of carotenoids was blocked. In a previous study, we showed that preheat treatment, which induces HSP21, allowed fruit color change at room temperature, after a cold treatment. Here, we show that mature green transgenic fruits constitutively expressing HSP21 do not require the heat treatment to maintain the ability to accumulate carotenoids after cold storage. This study demonstrates that a sHSP plays a role in plant development under normal growth conditions, in addition to its protective effect under stress conditions.

Figure 1.

Molecular Analyses (PCR, RNA Gel Blot, and Protein Gel Blot) of Transgenic Tomato Plants Transformed with 35S-HSP21 Construct. DNA, RNA, and proteins were extracted from a control nontransgenic plant and three T1 plants originating from three independent transformation events.

Figure 2.

Effect of Heat and High-Light Stresses on PSII Activity in Control and Transgenic Tomato Plants Expressing High Levels of HSP21. (A) Detached leaves from transgenic line (T2-311, black bars) and control (white bars) were exposed to 40 or 50°C for 2.5 h, and then chlorophyll fluorescence yield (variable fluorescence/maximum fluorescence [Fv/Fm]) was measured. (B) to (D) Detached leaves (from three independent T2 transgenic lines and control) were exposed to 40°C (B), 47°C (C), or 50°C (D) for 2 h and then to high white light (1200 μmol·m⁻²s⁻¹) for 2 min. Chlorophyll fluorescence yield was then measured. Black bars represent chlorophyll fluorescence yield before treatments, and gray bars represent chlorophyll fluorescence yield after stress treatments. Each value represents an average of 10 measurements taken from 10 different leaves ± se.

Figure 3.

Effect of Cold and High-Light Stresses on PSII Activity in Control and Transgenic Tomato Plants Expressing High Levels of HSP21. (A) Detached leaves from a control and transgenic lines (T2) were incubated in vials with tap water for 3 d at 4°C and then exposed for 2 min to high light (1200 μmol·m⁻² s⁻¹). Chlorophyll fluorescence yield (Fv/Fm) was then measured. (B) Detached leaves from a control and transgenic lines (T2) were incubated for 3 d at 4°C and then chlorophyll fluorescence yield was measured. (C) Detached leaves from a control and transgenic line (T2-831) were incubated for 3 d at 4°C and then exposed for 2 min to low (600 μmol·m⁻²s⁻¹) or high white light (1200 μmol·m⁻² s⁻¹). Chlorophyll fluorescence yield was then measured. HL, high light; LL, low light. Black bars represent chlorophyll fluorescence yield before treatments, and gray bars represent chlorophyll fluorescence yield after stress treatments. Each value represents an average of at least 10 measurements taken from 10 different leaves ± se.

Figure 4.

Expression of HSP21 during Tomato Fruit Development in Control and Transgenic Plants (T2-311). Developmental stages: IG, immature green; MG, mature green; T, turning; R, red ripe. The top panel shows control fruits at the four different developmental stages.

Figure 5.

Effect of HSP21 Overexpression on Tomato Fruit Reddening. (A) Flowers of the control VF36 cultivar and three independent T2 transgenic lines were labeled at anthesis, and the time for fruits to reach turning stage was monitored. Each value represents an average of at least 50 different fruits from at least five different plants (control and three independent T2 transgenic lines) ± se. (B) Carotenoids were extracted from control and transgenic (T4-311) plants at different time point after anthesis (40, 50, and 60 d) and measured with a spectrophotometer. Results are average values of five different fruits. FW, fresh weight.

Figure 6.

HSP21 Promotes Carotenoid Accumulation in Cold-Treated Tomato Fruits. (A) Expression of HSP21 in a control VF36 cultivar and transgenic T3 plant (T3-311) before and during storage at 2°C and 48 h after transferring to room temperature (25°C). Lanes 1 and 2, control (1) and transgenic (2) mature green fruits at harvest; lanes 3 and 4, control (3) and transgenic (4) fruits after 10 d at 2°C; lanes 5 and 6, control (5) and transgenic (6) fruits 48 h after transferring from cold to room temperature. (B) Evaluation of color change (from green to red) in fruits from control VF36 and two T3 transgenic lines, stored for 2 weeks at 2°C and then transferred to 25°C. Color was evaluated at the end of the cold storage (white bars), 7 (gray bars) and 9 (black bars) d after transferring to room temperature. Each value represents an average of at least 30 different fruits from at least four different plants ± se. (C) Control and transgenic fruits 7 d after the end of the 2-week cold storage. (D) HPLC analysis of carotenoids extracted from cold-treated control and transgenic (T4-311) fruits 7 d after transferal to room temperature. 1, lutein; 2, lycopene; 3, β-carotene.