Developmental and Molecular Biological Dissection of a Heat Tolerant Tomato Mutant HT7

概要

Tomato (Solanum lycopersicum) is an undeniable importance of vegetable crop in term of the merits of fresh and industrial products. It is also well known as an incredible source of rich nutrient components such as vitamin C, β-carotene and lycopene that positively impact on human health (Bergougnoux 2014). However, the yield and quality of tomato are adversely affected as a consequence of various biotic and abiotic stresses. High temperature is one of the major global abiotic stresses, which causes the multifarious negative effects on plant morphology, physiology, biochemistry and molecular pathways at all vegetative and reproductive stages, causing the reduce of fruit set and bad quality (Hasanuzzaman et al. 2013, Bita and Gerats 2013).

　We isolated heat-tolerant (HT) mutants providing improved fruit production under long- term ambient high temperature among EMS tomato mutant populations, created by National BioResource Project. In this project, we focused on the HT7 produced normal fruits with seeds in heat stress (HS) to understand the developmental and molecular dissection of HT mutant lines. Mutant and WT were evaluated in two fixed cultivated conditions, HS (35°/25°C) or control (CO) (25°C), 16 h/8 h light/dark, 60.0 μmol m-2 s-1, and opened greenhouse in summer 2018.

　Mutant HT7 expressed several unique traits under long-term exposing to elevated temperature: HT7 kept the narrow plant canopy with producing fewer lateral shoots; HT7 had around 30-40 flowers per plant; rate of fruit setting (6 %) was five times higher than WT, resulting the fruit yield of HT7 was two times higher than WT; HT7 consistently produced normal fruits in HS, while WT could not get fruit set for every cultivation. Typically, HT7 fruits contained seeds that could germinate, while 100 % of WT fruits did not have seeds. In the opened greenhouse in summer 2018, 100 % of HT7 and WT fruits produced fruits without seeds when bud formation and anthesis were during the highest peak of summer (more than 42 °C at noon).

　In HS, mutant HT7 remained the flower structure was more suitable for self-pollination. HT7 had less than 20 % of abnormal flowers, while WT was more than 40 %; HT7 produced pollens within pollen sacs, which their walls opened for releasing pollens, while 40 % WT flowers did not produce pollens and pollen sac walls did not open well at anthesis. In long term HS, HT7 produced two times higher viable pollens and total pollens than WT; HT7 and WT pollens germinated very low in the pollen germination medium, at 7 % and 6 %, while these rates were more than 80 % in CO; HT7 and WT pollens did not elongate pollen tubes well. In short term HS, HT7 produced two times higher in viable pollens than WT in -1, -3, -5 days before anthesis, however, the pollen germinate rate was not significant differences. In the greenhouse, HT7 and WT flowers had more than 85 % of pollens died due to hot temperature.

　To adapt with the elevated temperature, tomatoes have established various heat responded reactions. The 2-weeks-old seedlings of HT7 and WT died after 12 h constantly treated at 42°C; the stomata diameters of HT7 were the biggest in HS and smallest in GRH, while those in WT were the biggest in CO and smallest in GRH. The stomata density on leaves of HT7 was smaller than in WT in three conditions; HT7 released lower at 10 days and higher at 60 days in HS in total ionic leakage than WT; Fruits of HT7 and WT released similar amount of hydrogen peroxide. The amount of Fructose, Glucose and Sucrose in HT7 fruits were lower than in WT.

　Transcription factors, heat shock proteins and other related to heat tolerance play important roles in thermotolerance. In leaves, HT7 expressed SIHsfA1a, SIHsfA1d genes lower than WT, in contrasted, HT7 expressed SIHsfA1b1 and SIHsfA1b3 higher than WT; The relative gene expression levels of HT7 in SIHsp21 and SIHsp01 were three to four times higher than WT. However, in the anther cone, the expression of SIHsfA1a, SIHsfA1b1 and SIHsp101 were not significant differences between HT7 and WT. In the greenhouse, the expression levels of ABCDE model genes were different, HT7 expressed lower in SlAGL6 and SlNAM2 genes, while higher in SlLePI, SlMC and SlTM6 than in WT. Regarding to leaf senesce genes, RbcL, SENU3 and DREB2A expressed highest at 3 hours in HS. HT7 expressed RbcL and DREB2A lower than WT at 7 days in HS.

　Detecting the heat tolerant responsible gene(s) in mutant HT7 is one important aim. The whole genome sequencing by NGS was carried out on the bulked DNA extracting from the leaves of F2 population based on the fruit setting number. Total reads 327,921,773 (WT – like F2), 326,421,481 (HT7 – like F2), 151,100,978 (WT), 158,203,158 (HT7) were called. Sanger sequencing and restriction enzyme digestion were performed in different mutative positions, however, the strongly responsible gene(s) were not detected yet.

　In conclusion, mutant HT7 had more normal flowers compatibly for self-pollination, narrow plant canopy, higher viable pollens, higher fruit setting rate, higher fruit number per plant resulting higher fruit yield. Particularly, HT7 produced normal fruits containing seeds and stayed green in HS. Therefore, HT7 can be a prominent breeding material for enhancing heat tolerance.