Overexpression of Solanum habrochaites microRNA319d (sha-miR319d) confers chilling and heat stress tolerance in tomato (S. lycopersicum)

Abstract

Background: MicroRNA319 (miR319) acts as an essential regulator of gene expression during plant development and under stress conditions. Although the role of miR319a in regulating leaf development has been well studied in tomato (Solanum lycopersicum), the function of the recently discovered wild tomato Solanum habrochaites miRNA319d (sha-miR319d) remains poorly understood. In this study, we overexpressed sha-miR319d in cultivated tomato 'Micro-Tom' to further investigate its role in tomato temperature stress responses.

Results: Under chilling or heat stress, sha-miR319d-overexpressing plants showed enhanced stress tolerance, including lower relative electrolyte leakage (REL), malondialdehyde (MDA) concentration, O₂^- generation and H₂O₂ concentration and higher chlorophyll contents and Fv/Fm values than wild-type (WT) plants. Overexpression of sha-miR319d enhanced the activities of superoxide dismutase (SOD) and catalase (CAT), with possible correlation with elevated expression levels of the genes FeSOD, CuZnSOD and CAT. Moreover, different expression levels of key genes involved in chilling (MYB83 and CBF1), heat (HsfA1a, HsfA1b and Hsp90), and reactive oxygen species (ROS) (ZAT12 and ZAT10) signaling in transgenic plants and WT were determined, suggesting a role for sha-miR319d in regulating tomato temperature stress via chilling, heat and ROS signaling. Silencing GAMYB-like1 increased tomato chilling tolerance as well as the expression levels of CBF1, CuZnSOD, CAT, APX1, APX2, ZAT12 and ZAT10. Additionally, overexpression of sha-miR319d in tomato caused plant leaf crinkling and reduced height.

Conclusions: Overexpression of sha-miR319d confers chilling and heat stress tolerance in tomato. Sha-miR319d regulates tomato chilling tolerance, possibly by inhibiting expression of GAMYB-like1 and further alters chilling, heat and ROS signal transduction. Our research provides insight for further study of the role of sha-miR319d in tomato growth and stress regulation and lays a foundation for the genetic improvement of tomato.

Keywords: Chilling stress; Heat stress; Solanum habrochaites; Tomato; miR319.

Fig. 1

Identification of sha-miR319d overexpression transgenic plants. a Genomic PCR analysis of sha-MIR319d in WT and transgenic plants. b Expression levels of sha-miR319d in WT and T2 generations of transgenic plants. The reference gene for sha-miR319d was U6snRNA. Each value was the mean of three biological repeats ± the standard deviation (SD). c Leaf morphogenesis of WT and transgenic lines (T1 generations). d, e The plant height of 30-day-old WT and transgenic lines (T2 generations). Asterisks indicate significant differences between WT and transgenic plants. *, P < 0.05; **, P < 0.01, Student’s t test

Fig. 2

The effect of overexpression of sha-miR319d on plant growth under control and temperature-stress conditions. a Phenotypes of WT and transgenic lines (T2 generations) under control (25 °C) conditions. b Phenotypes of WT and transgenic lines (T2 generations) under chilling (4 °C) conditions for 5 d. c Phenotypes of WT and transgenic lines (T2 generations) under heat (40 °C) conditions for 5 d. d, e Effects of chilling stress on REL (D) and the MDA content (f) in WT and transgenic lines (T2 generations). f, g Effects of heat stress on REL (F) and MDA (g). The data in (d-g) are the means of three biological repeats ± the standard deviation (SD). Asterisks indicate significant differences between transgenic lines and WT. *, P < 0.05; **, P < 0.01, Student’s t test

Fig. 3

The effect of overexpression of sha-miR319d on the chlorophyll content and Fv/Fm. a Chlorophyll content of WT and transgenic plants under control or chilling (4 °C) treatment for 5 d. b Chlorophyll content of WT and transgenic plants under control or heat (40 °C) treatment for 5 d. C Fv/Fm of WT and transgenic plants under chilling (4 °C) treatment. d Fv/Fm of WT and transgenic plants under heat (40 °C) treatment. T2 generations of transgenic plants as well as the WT were used for measurements of Fv/Fm and the chlorophyll content. The data in (a) and (b) are the mean of three biological repeats ± the standard deviation (SD). Asterisks indicate significant differences between transgenic lines and WT. *, P < 0.05; **, P < 0.01, Student’s t test. The data in (c) and (d) are the means ± the standard deviation (SD) obtained from 24 seedlings, and letters above indicate the statistical significance of differences (P < 0.05) analyzed by variance (ANOVA) using SPSS 18.0

Fig. 4

The effect of overexpression of sha-miR319d on ROS accumulation. NBT staining for O₂⁻ in the leaves of WT and transgenic plants under control conditions and after 1 d of chilling (4 °C) or heat (40 °C) treatment. b DAB staining for H₂O₂ in the leaves of WT and transgenic plants under control conditions and after 1 d of chilling (4 °C) or heat (40 °C) treatment. c Quantitative analysis of O₂⁻ in the leaves of WT and transgenic plants under control conditions and after 1 d of chilling (4 °C) or heat (40 °C) treatment. d Quantitative analysis of H₂O₂ in the leaves of WT and transgenic plants under control conditions and after 1 d of chilling (4 °C) or heat (40 °C) treatment. T2 generations of transgenic plants as well as WT were used for O₂⁻ and H₂O₂ histochemical staining and quantitative analysis. Each value is the mean of three biological repeats ± the standard deviation (SD). Asterisks indicate significant differences between transgenic lines and WT. *, P < 0.05; **, P < 0.01, Student’s t test. The bar in (a) and (b) indicates 1 cm

Fig. 5

The effect of overexpression of sha-miR319d on the activities of antioxidant enzymes. a SOD activities of WT and transgenic plants under control, chilling stress (4 °C) and heat stress (40 °C) conditions. b CAT activities of WT and transgenic plants under control, chilling stress (4 °C) and heat stress (40 °C) conditions. c APX activities of WT and transgenic plants under control, chilling stress (4 °C) and heat stress (40 °C) conditions. T2 generations of transgenic plants as well as WT were used. Each value is the mean of three biological repeats ± the standard deviation (SD). Asterisks indicate significant differences between transgenic lines and WT. *, P < 0.05; **, P < 0.01, Student’s t test.

Fig. 6

Relative expression levels of key genes involved in chilling, heat and ROS signaling. qPCR analysis of CBF1 (a), MYB83 (b), HsfA1a (c), HsfA1b (d), Hsp90 (e), CuZnSOD (f), FeSOD (g), CAT (h), APX1 (i), APX2 (j), ZAT12 (k) and ZAT10 (l) in sha-miR319d-overexpressing lines and WT. T2 generations of transgenic plants as well as WT were treated with chilling or heat for 0 (control), 4 and 8 h. The reference gene was Actin. Each value is the mean of three biological repeats ± the standard deviation (SD). Asterisks indicate significant differences between WT and transgenic plants. *, P < 0.05; **, P < 0.01, Student’s t test.

Fig. 7

The effect of GAMYB-like1-silencing on tomato seedlings under chilling stress. a Relative expression levels of GAMYB-like1 in WT, Ve and VGAMYB-like1 plants. b Phenotypes of WT, Ve and VGAMYB-like1 lines under control (25 °C) conditions. c Phenotypes of WT, Ve and VGAMYB-like1 lines under chilling (4 °C) conditions for 4 h; d Phenotypes of WT, Ve and VGAMYB-like1 lines under chilling (4 °C) conditions for 8 h. e and f Effects of chilling stress on REL (e) and MDA (f) in WT, Ve and VGAMYB-like1 lines. g Relative expression levels of key genes involved in chilling and ROS signaling. The data are means of three biological repeats ± the standard deviation (SD). Asterisks indicate significant differences between gene-silencing lines and WT. *, P < 0.05; **, P < 0.01, Student’s test

Fig. 8

A possible regulatory model of sha-miR319d in tomato temperature stress. Arrows indicate positive regulation, and T bars indicate negative regulation