The apple palmitoyltransferase MdPAT16 influences sugar content and salt tolerance via an MdCBL1-MdCIPK13-MdSUT2.2 pathway.

Protein S-acyltransferases (PATs) are a category of eukaryotic transmembrane proteins that mediate the S-acylation of their target proteins. S-acylation, commonly known as palmitoylation, is a reversible protein modification that regulates the membrane association and function of target proteins. However, the functions and mechanisms of PATs in apple (Malus domestica) remain poorly understood. In this study, an MdPAT family member, MdPAT16, was identified and shown to have palmitoyltransferase activity. We demonstrated that this gene responds to salt stress and that its expression improves plant salt stress resistance. In addition, its overexpression significantly promotes the accumulation of soluble sugars. The same phenotypes were observed in transgenic tissue culture seedlings, transgenic roots, and Arabidopsis thaliana that ectopically expressed MdPAT16. MdPAT16 was shown to interact with MdCBL1 and stabilize MdCBL1 protein levels through palmitoylation. The N-terminal sequence of MdCBL1 contains a palmitoylation site, and its N-terminal deletion led to changes in MdCBL1 protein stability and subcellular localization. The phenotypes of MdCBL1 transgenic roots and transiently injected apple fruits were fully consistent with the sugar accumulation phenotype of MdPAT16. Mutation of the palmitoylation site interfered with this phenotype. These findings suggest that MdPAT16 palmitoylates its downstream target proteins, improving their stability. This may be a missing link in the plant salt stress response pathway and have an important impact on fruit quality.

MdCER2 conferred to wax accumulation and increased drought tolerance in plants.

Drought can activate many stress responses in plant growth and development, including the synthesis of epidermal wax and the induction of abscisic acid (ABA), and increased wax accumulation will improve plant drought resistance. Therefore, an examination of wax biosynthesis genes could help to better understand the molecular mechanism of environmental factors regulating wax biosynthesis and the wax associated stress response. Here, we identified the MdCER2 gene from the 'Gala' (Malus× domestica Borkh.) variety of domestic apple, which is a homolog of Arabidopsis AtCER2. It possesses a transferase domain and the protein localizes on the cell membrane. The MdCER2 gene was constitutively expressed in apple tissues and was induced by drought treatment. Finally, we transformed the MdCER2 gene into Arabidopsis to identify its function, and found ectopic expression of MdCER2 promoted accumulation of cuticular wax in both leaves and stems, decreased water loss and permeability in leaves, increased lateral root number, changed plant ABA sensitivity, and increased drought resistance.

MdWRKY15 improves resistance of apple to Botryosphaeria dothidea via the salicylic acid-mediated pathway by directly binding the MdICS1 promoter.

Isochorismate synthase (ICS) plays an essential role in the accumulation of salicylic acid (SA) and plant disease resistance. Diseases caused by Botryosphaeria dothidea affect apple yields. Thus, it is important to understand the role of ICS1 in disease resistance to B. dothidea in apple. In this study, SA treatment enhanced the resistance to B. dothidea. MdICS1 was induced by B. dothidea and enhanced the resistance to B. dothidea. MdICS1 promoter analysis indicated that the W-box was vital for the response to B. dothidea treatment. MdWRKY15 was found to interact with the W-box using yeast one-hybrid screening. Subsequently, the interaction was confirmed by EMSA, yeast one-hybrid, ChIP-PCR, and quantitative PCR assays. Moreover, luciferase and GUS analysis further indicated that MdICS1 was transcriptionally activated by MdWRKY15. Finally, we found the function of MdWRKY15 in the resistance to B. dothidea was partially dependent on MdICS1 from the phenotype of transgenic apples and calli. In summary, we revealed that MdWRKY15 activated the transcription of MdICS1 by directly binding to its promoter to increase the accumulation of SA and the expression of disease-related genes, thereby resulting in the enhanced resistance to B. dothidea in the SA biosynthesis pathway.

MdWRKY46-Enhanced Apple Resistance to Botryosphaeria dothidea by Activating the Expression of MdPBS3.1 in the Salicylic Acid Signaling Pathway.

Salicylic acid (SA) is closely related to disease resistance of plants. WRKY transcription factors have been linked to the growth and development of plants, especially under stress conditions. However, the regulatory mechanism of WRKY proteins involved in SA production and disease resistance in apple is not clear. In this study, MdPBS3.1 responded to Botryosphaeria dothidea and enhanced resistance to B. dothidea. Electrophoretic mobility shift assays, yeast one-hybrid assays, and chromatin immunoprecipitation and quantitative PCR demonstrated that MdWRKY46 can directly bind to a W-box motif in the promoter of MdPBS3.1. Glucuronidase transactivation and luciferase analysis further showed that MdWRKY46 can activate the expression of MdPBS3.1. Finally, B. dothidea inoculation in transgenic apple calli and fruits revealed that MdWRKY46 improved resistance to B. dothidea by the transcriptional activation of MdPBS3.1. Viral vector-based transformation assays indicated that MdWRKY46 elevates SA content and transcription of SA-related genes, including MdPR1, MdPR5, and MdNPR1 in an MdPBS3.1-dependent way. These findings provide new insights into how MdWRKY46 regulates plant resistance to B. dothidea through the SA signaling pathway.

MdHIR4 transcription and translation levels associated with disease in apple are regulated by MdWRKY31.

KEY MESSAGE: Here we describe that the regulation of MdWRKY31 on MdHIR4 in transcription and translation levels associated with disease in apple. The phytohormone salicylic acid (SA) is a main factor in apple (Malus domestica) production due to its function in disease resistance. WRKY transcription factors play a vital role in response to stress. An RNA-seq analysis was conducted with 'Royal Gala' seedlings treated with SA to identify the WRKY regulatory mechanism of disease resistance in apple. The analysis indicated that MdWRKY31 was induced. A quantitative real-time polymerase chain reaction (qPCR) analysis demonstrated that the expression of MdWRKY31 was induced by SA and flg22. Ectopic expression of MdWRKY31 in Arabidopsis and Nicotiana benthamiana increased the resistance to flg22 and Pseudomonas syringae tomato (Pst DC3000). A yeast two-hybrid screen was conducted to further analyze the function of MdWRKY31. As a result, hypersensitive-induced reaction (HIR) protein MdHIR4 interacted with MdWRKY31. Biomolecular fluorescence complementation, yeast two-hybrid, and pull-down assays demonstrated the interaction. In our previous study, MdHIR4 conferred decreased resistance to Botryosphaeria dothidea (B. dothidea). A viral vector-based transformation assay indicated that MdWRKY31 evaluated the transcription of SA-related genes, including MdPR1, MdPR5, and MdNPR1 in an MdHIR4-dependent way. A GUS analysis demonstrated that the w-box, particularly w-box2, of the MdHIR4 promoter played a major role in the responses to SA and B. dothidea. Electrophoretic mobility shift assays, yeast one-hybrid assay, and chromatin immunoprecipitation-qPCR demonstrated that MdWRKY31 directly bound to the w-box2 motif in the MdHIR4 promoter. GUS staining activity and a protein intensity analysis further showed that MdWRKY31 repressed MdHIR4 expression. Taken together, our findings reveal that MdWRKY31 regulated plant resistance to B. dothidea through the SA signaling pathway by interacting with MdHIR4.

Functional identification of apple on MdHIR4 in biotic stress.

In plants, hypersensitive-induced reaction (HIR) proteins are involved in stress responses, especially biotic stress. However, the potential molecular mechanisms of HIR-mediated biotic resistance in plants are rarely reported. We found that apple (Malus domestica) MdHIR4 was localized in the cell nucleus and membrane similar to AtHIR1 in Arabidopsis. Moreover, salicylic acid and the bacterial flagellin flg22 (a conserved, 22-amino acid motif), which are relevant to biotic stress, could induce MdHIR4 expression. Additionally, the transcription level of MdHIR4 was increased by Methyl jasmonate treatment. Ectopic expression of MdHIR4 in Arabidopsis and Nicotiana benthamiana reduced sensitivity to Methyl jasmonate and enhanced resistance to the bacterial pathogen Pst DC3000 (Pseudomonas syringae tomato DC3000). The interaction between MdHIR4 and AtJAZs proteins (AtJAZ3, AtJAZ4, and AtJAZ9) implied that MdHIR4 participated in the jasmonic acid (JA) signaling pathway. We found the expression of JA-related genes and PRs to change in transgenic plants, further demonstrating that MdHIR4 mediated biotic stress through the JA signaling pathway. Repressing the expression of MdHIR4 in apple leaves and calli increased resistance to Botryosphaeria dothidea by influencing the transcription of resistance-related genes. Our findings reveal the resistant function to biotic stress of MdHIR4 in transgenic plants, including Arabidopsis, tobacco, and apple, and identify the regulating mechanism of MdHIR4-related biotic resistance.