3.1 ATG8 gene in Arabidopsis and wheat
The length of coding sequence of TaATG8 gene is 360bp. The comparative analysis about amino acid sequence of TaATG8 and AtATG8 shows that they are in high similarity of 84.03% (Fig.1A). The T-DNA of atg8 locates in the sixth intron at 1322bp (Fig.1B). RT-PCR showed that AtATG8 is abundantly expressed in Col-0, but no expression of AtATG8 were detected in atg8 (Fig.1C), the result indicated that the atg8 in our study is loss-of-function mutant. The T-DNA insertion mutant of atg8 provides a good reference value for studying the function of TaATG8.
3.2 TaATG8 rescues autophagy defect in atg8
Previous studies have shown that darkness induce leaf senescence, and there are autophagosome in senescent leaves. Monodansylcadaverine (MDC) is a specific reagent used to label autophagosome. Autophagosome present as light blue dots in 488 nm exciting light when labelled by MDC (Klionsky et al., 2012). Four-week-old Arabidopsis were incubated with 1 mM MDC, the results showed that untreated Col-0 (Fig.2A) and 35S::TaATG8 (Fig.2D) leaves have no accumulation of autophagosome; When treated with dark, a small number of autophagosome accumulated in the Col-0 (Fig.2B) and atg8 35S::TaATG8 (Fig.2E) leaves, there was no accumulation of autophagosome in atg8 leaves (Fig.2C). However, there are a lot of autophagosome in TaATG8 overexpression leaves (Fig.2F). The result indicated that TaATG8 restored AtATG8’s function in the leaf senescence process when treated by dark, TaATG8 is involved in the formation of autophagy, and it shares high conserved function with that of AtATG8.
3.3 TaATG8 play roles in germination rate under some abiotic stresses
Fatal damage would happen to wheat if it encounter stress during germination. In order to study the role of TaATG8 gene on seed germination under abiotic stress, the germination rate of Arabidopsis seeds on the MS and some abiotic stresses were analyzed (Fig.3). As the result shows that there was no significant difference about germination rate among the four different genotypes of Arabidopsis on MS medium, MS medium lacking of phosphorus and nitrogen deficiency medium; However, the germination rate of atg8 was lower than that of Col-0, atg8 35S::TaATG8 and 35S::TaATG8 on the medium lacking sucrose, and on the medium containing 120 mM NaCl, 0.5 M ABA and 10 mM H2O2, The result indicated that TaATG8 gene may be related to Arabidopsis’ resistance to sucrose deficiency, high salt, drought, ABA and oxidative stress, but it is insensitive to the stress of phosphorus and nitrogen deficiency.
3.4 TaATG8 play positive roles in some abiotic stresses in Arabidopsis seedling
Because seedling stage is a sensitive period to abiotic stress, it is necessary to study the effect of TaATG8 gene on the growth and development of Arabidopsis seedlings under stresses. We treated the seedlings under some familiar stresses, such as osmotic stress, nutritional stress and oxidative stress. The result showed that there was no obvious difference among Col-0, atg8, atg8 35S::TaATG8 and 35S::TaATG8 seedlings on normal MS culture medium (Fig.4). When treated with 200 mM Mannitol, 120 mM NaCl, nitrogen deficiency, sucrose deficiency, phosphorus deficiency, 0.5 µM ABA and 10 mM H2O2, the growing status of all the seedlings behaved different degree of inhibition (Fig4, A), and the root length of atg8 was shorter than that of Col-0, atg8 35S::TaATG8 and 35S::TaATG8 (Fig4, B). The results indicated that TaATG8 plays a very important role in Arabidopsis seedlings resisting against some abiotic stresses.
3.5 TaATG8 enhanced tolerance to drought, but not salt stress in adult stage
In the adult stage, the familiar abiotic stress of wheat is mainly drought and salt stress. Does TaATG8 gene respond to adversity in the adult stage? To answer this question, we treated 4-week-old Arabidopsis with drought and salt stress to test whether TaATG8 gene is effective. As the result revealed in the drought stress, atg8 showed wilting dry, Col-0 and atg8 35S::TaATG8 showed a little wilting with only very few leaves showed dry, but 35S::TaATG8 growed better (Fig.5A). After rewatered for 24 h (Fig.5B), all of the atg8 died, most of Col-0 and atg8 35S::TaATG8 revived, 35S::TaATG8 restore best. The result demonstrated that TaATG8 gene plays an important role in response to drought stress in adult stage.
However, four genotype Arabidopsis showed no obvious phenotypic differences after a month of 170 mM NaCl treatment (Fig.5C), indicating that TaATG8 gene maybe has no obvious role in salt stress in adult stage.
3.6 TaATG8 enhanced tolerance to phosphorus deficiency stress in adult stage
In the process of plant’s growth and development, nutrition deficiency often happens, such as the lacking of phosphate fertilizer. As we observed, the semidiameter of atg8 is smaller at the early stage of the phosphorus deficiency treatment (Fig.6A). At late stage, atg8 senesced earlier than Col-0, atg8 35S::TaATG8 and 35S::TaATG8, and it is easy to get flattened because its main stem is very slim (Fig.6B).This indicated that TaATG8 gene participated in the vegetative growth of plant, it is maybe related to the uptake and utilization of phosphorus of plant.
3.7 TaATG8 enhanced resistance of Arabidopsis to powdery mildew through HR
Powdery mildew fungi are biotrophic pathogens that infect numerous plant species, including important crops such as wheat, as well as the model plant Arabidopsis thaliana (Zhou et al., 2014; Liu et al., 2005). As powdery mildew is one of the most devastating diseases of wheat worldwide and severely reduces wheat yields, it is very important to study the molecular mechanism that resist against powdery mildew. To analysis whether TaATG8 participate in the response to powdery mildew in plant, 4-week-old Arabidopsis were infected with the powdery mildew Golovinomyces cichoracearum UCSC1. Trypan blue staining were carried out to monitor the production of conidiophores in the infected leaves. As our result showed that, a large number of fungal hyphae were produced in atg8 mutant leaves while no fungal hyphae were produced in Col-0, atg8 35S::TaATG8 and 35S::TaATG8 (Fig.7A). This indicated that TaATG8 gene play a positive regulation role in the process of Arabidopsis defending against powdery mildew.
Hypersensitive Reaction (HR) refers to the rapid death of a small number of cells around the infection site when the pathogen infects the plant, thus limiting the spread of the pathogen, inhibit further pathogen proliferation in some cases (Dow et al., 2000, Lerena et al., 2010). Previous studies indicated that autophagy participates in HR of pathogen infection (Xiong et al., 2007; Lenz et al., 2011). As the marker gene of autophagy, does TaATG8 gene regulate the infection of powdery mildew through HR? To answer the question, Trypan blue staining were carried out to monitor the cell death in the infected leaves. As our result showed that, few dead mesophyll in the leaves of atg8 mutant, but there are more dead mesophyll in Col-0, atg8 35S::TaATG8 and 35S::TaATG8 (Fig.7A). The result indicated that TaATG8 gene induced cell death against powdery mildew through HR.
3.8 Hydrogen peroxide acts as a metabolic product and signaling molecule in the process of TaATG8 resisting to powdery mildew
Hydrogen peroxide is an important metabolic product and signaling molecule in the process of plant resisting against outside invasion biology (Zhou et al., 2012). To test the accumulation of hydrogen peroxide in Arabidopsis when inoculated with powdery mildew, the infected leaves were stained with DAB. The result showed that there almost no accumulation of hydrogen peroxide in the leaves of atg8 that inoculated with powdery mildew for 48 h, however, there are a lot of hydrogen peroxide can be seen on the leaves of Col-0, atg8 35S::TaATG8 and 35S::TaATG8. The result indicated that TaATG8 gene is related to the burst of H2O2 caused by the infection of powdery mildew, and H2O2 may has close connection to HR. The hydrogen peroxide signaling molecule and HR may function together to confer resistance of TaATG8 to powdery mildew.