The apple C2H2-type zinc finger transcription factor MdZAT10 positively regulates JA-induced leaf senescence by interacting with MdBT2

Abstract

Jasmonic acid (JA) plays an important role in regulating leaf senescence. However, the molecular mechanisms of leaf senescence in apple (Malus domestica) remain elusive. In this study, we found that MdZAT10, a C2H2-type zinc finger transcription factor (TF) in apple, markedly accelerates leaf senescence and increases the expression of senescence-related genes. To explore how MdZAT10 promotes leaf senescence, we carried out liquid chromatography/mass spectrometry screening. We found that MdABI5 physically interacts with MdZAT10. MdABI5, an important positive regulator of leaf senescence, significantly accelerated leaf senescence in apple. MdZAT10 was found to enhance the transcriptional activity of MdABI5 for MdNYC1 and MdNYE1, thus accelerating leaf senescence. In addition, we found that MdZAT10 expression was induced by methyl jasmonate (MeJA), which accelerated JA-induced leaf senescence. We also found that the JA-responsive protein MdBT2 directly interacts with MdZAT10 and reduces its protein stability through ubiquitination and degradation, thereby delaying MdZAT10-mediated leaf senescence. Taken together, our results provide new insight into the mechanisms by which MdZAT10 positively regulates JA-induced leaf senescence in apple.

Fig. 1. MdZAT10 promotes leaf senescence.

a MdZAT10 transcript level in late-senescent (LS), early-senescent (ES), and nonsenescent (NS) leaves. b Detached leaves from 3-week-old wild-type (Col) plants and three transgenic lines (MdZAT10-L1, L2, and L3) were incubated in 3 mM MES buffer in the dark for 5 days. c The chlorophyll content and d Fv/Fm before and after dark treatment were determined. e Leaf senescence phenotype and f the total chlorophyll content of apple leaves transiently expressing empty vector (EV), overexpressing MdZAT10 or MdZAT10 antisense vector in the dark for 15 days. g, h The expression levels of MdNYC1 and MdNYE1 in apple leaves after 15 days of dark treatment. Asterisks indicate significant differences determined by t-test (^*P < 0.05, ^**P < 0.01)

Fig. 2. MdZAT10 physically interacts with MdABI5.

a A yeast two-hybrid (Y2H) assay showed that MdZAT10 interacts with MdABI5. Full-length MdZAT10 and truncated MdZAT10 sequences were cloned into the pGAD424 vector. Full-length MdABI5 was cloned into the pGBT9 vector. The empty pGAD vector was used as a negative control. b A Y2H assay showed that MdABIs (MdABI1, MdABI2, and MdABI4) and MdZAT10 interact. c A Y2H assay showed that MdABI5 interacts with MdZAT proteins (MdAZF1, MdZAT5, MdZAT6, MdZAT10, MdZAT11, MdZAT14, MdZAT16, and MdZAT18). d In vitro MdZAT10 and MdABI5 pull-down assay. The MdABI5-His protein was incubated with MdZAT10-GST and GST. Proteins pulled down with GST beads were detected using anti-GST and anti-His antibodies. e BiFC assay. The MdZAT10-cYFP and MdABI5-nYFP constructs were transiently expressed in Nicotiana benthamiana leaves, and the fluorescence signal was observed by fluorescence microscopy. Nuclei are indicated by DAPI staining. Scale bars, 10 μm

Fig. 3. MdZAT10 accelerates MdABI5-regulated leaf senescence.

a Leaf senescence phenotype and b total chlorophyll content of detached leaves from Col, MdABI5-L2, and MdZAT10-L1/MdABI5-L2 transgenic Arabidopsis floated on 3 mM MES buffer in the dark for 5 days. c Leaf senescence phenotype and d total chlorophyll content of apple leaves transiently expressing empty vector (EV), overexpressing MdABI5 alone or overexpressing MdZAT10 and MdABI5 that were floated on 3 mM MES buffer in the dark for 14 days. e, f The expression levels of MdNYC1 and MdNYE1 in wild-type (WT) calli and MdABI5-OX, MdZAT10-OX, MdZAT10-OX/MdABI5-OX transgenic calli were measured by qRT-PCR assay. g Schematic diagram of reporter and effector constructs. h, i A luciferase assay showed that transient cotransformation of MdZAT10 and MdABI5 into tobacco leaves activated the expression of MdNYC1 and MdNYE1. The empty vector (SK + LUC) served as a negative control. The LUC/REN ratio represents the ability of MdABI5 to activate MdNYC1 and MdNYE1 expression. Asterisks indicate significant differences at ^*P < 0.05, ^**P < 0.01

Fig. 4. MdZAT10 accelerates JA-induced leaf senescence.

a Transcript level of MdZAT10 in response to 100 μM MeJA. b GUS staining and c GUS activity in proMdZAT10::GUS transgenic calli with (MeJA) or without (Mock) 100 µM MeJA treatment for 12 h. d Leaf senescence phenotype and e chlorophyll content of detached leaves from 3-week-old Col and transgenic Arabidopsis MdZAT10-L1, L2, and L3 floated on 3 mM MES buffer with or without 100 μM MeJA for 3 days. f Leaf senescence phenotype and g chlorophyll content of apple leaves transiently expressing empty vector (EV), overexpressing MdZAT10 or MdZAT10 antisense vector floated on 3 mM MES buffer with or without 100 μM MeJA for 14 days. Asterisks indicate significant differences by t-test (^*P < 0.05, ^**P < 0.01)

Fig. 5. MdZAT10 physically interacts with MdBT2.

a, b A Y2H assay showed the interaction between MdZAT10 and MdBT2. Full-length and truncated MdZAT10 and MdBT2 sequences were cloned into the pGBD and pGAD vectors. c In vitro pull-down assay with MdZAT10 and MdBT2. The MdZAT10-His protein was incubated with GST-MdBT2 and GST. Proteins that were pulled down with GST beads were detected using anti-GST and anti-His antibodies. d BiFC assay. The MdZAT10-cYFP and MdBT2-nYFP constructs were transiently expressed in Nicotiana benthamiana leaves, and the fluorescence signal in the nucleus was observed via fluorescence microscopy. Nuclei are indicated by DAPI staining. Scale bars, 10 μm

Fig. 6. MdBT2 mediates MdZAT10 protein stability.

a Cell-free MdZAT10-His degradation assay. The MdZAT10-His fusion protein was incubated with total protein extracted from wild-type (WT), MdBT2-OX, and MdBT2-Anti transgenic calli for the indicated time periods. b MdBT2 ubiquitinated the MdZAT10-His protein in vivo. MdZAT10-His was immunoprecipitated using an anti-His antibody and then examined using anti-His and anti-Ubi antibodies. IP, immunoprecipitate; IB, immunoblot; Ubi, ubiquitin. c The MdZAT10-GFP protein abundance in transgenic apple calli (MdZAT10-GFP and MdZAT10-GFP/MdBT2-OX) was assessed by immunoblotting using an anti-GFP antibody

Fig. 7. MdBT2 delayed the MdZAT10-promoted leaf senescence.

a Leaf senescence phenotype and b chlorophyll content of detached leaves from wild-type (GL-3) and transgenic apple seedlings overexpressing MdBT2 (MdBT2-OE-L1, MdBT2-OE-L5) and MdBT2 antisense (MdBT2-Anti-L13, MdBT2-Anti-L23) that were floated on 3 mM MES buffer with 100 μM MeJA in the dark for 12 days. c Leaf senescence phenotype and d chlorophyll content of detached leaves from Col and transgenic Arabidopsis MdZAT10-L1, MdZAT10-L1/MdBT2-L1, and MdZAT10-L1/MdBT2-L2 that were floated on 3 mM MES buffer in the dark for 6 days. e Leaf senescence phenotype and f chlorophyll content of apple leaves transiently expressing empty vector (EV) or overexpressing MdZAT10 alone or MdZAT10 and MdBT2 were floated on 3 mM MES buffer in the dark for 13 days. Asterisks indicate significant differences at ^*P < 0.05, ^**P < 0.01

Fig. 8. A working model of the role of MdZAT10 in apple leaf senescence.

On the one hand, MdZAT10 positively regulates leaf senescence by interacting with MdABI5, enhancing the transcriptional activity of MdABI5 for MdNYC1 and MdNYE1. On the other hand, in the absence of JA, MdBT2 interacts with MdZAT10 and ubiquitinates MdZAT10 to degrade MdZAT10, thereby negatively regulating MdZAT10-promoted leaf senescence. In the present of JA, JA accelerates the degradation of MdBT2, releasing MdZAT10, which contributes to JA-induced leaf senescence.