| China is a major apple producing country,and the area and production of apple cultivation accounts for more than half of the world production,while in the major apple producing areas of China,soil salinization,especially secondary salinization,occurs commonly,which seriously restricts the development of apple industry in China.How to resist soil salt damage is of great importance to how to improve fruit quality.Sugar plays an important role in the physiological metabolism,growth and development of plants.Sugar is not only an energy supply substance to provide energy for plant growth,but also a key to determine the quality and commercial value of certain agricultural by-products.Carbohydrates can serve as the carbon skeleton for plant growth and development,and improve the resistance of plants to abiotic stresses.Sugar metabolism is an important link in the biological metabolic process of plants,which is the information carrier of lipid metabolism and nucleic acid metabolism and provides sufficient nutrients for cell growth and development.In a previous study,we found that apple MdCIPK13 is a salt stress-responsive CIPK（CBL-interacting protein kinase）protein kinase,which interacts with sucrose transporter protein MdSUT2.2 and phosphorylates MdSUT2.2 under salt stress to enhance its transport activity,thus promoting sucrose accumulation in apple plants and fruits.However,the upstream regulators of MdCIPK13 in response to salt stress are not known.CBLs can interact with CIPKs in response to variety stress signals.Meanwhile,studies showed that the Arabidopsis palmitoyltransferase protein（Protein Acyl-Transferase,PAT）interacts with the calcium phosphatase-like B subunit protein（CBL,Calcineurin B-like protein）and is involved in the response to salt stress,but we do not know how it regulates salt stress response.Based on the above studies,we hypothesized that palmitoyltransferase proteins may regulate the function of MdCIPK13-MdSUT2.2 through MdCBLs.A palmitoyltransferase gene,MdPAT16,was identified in apple by combining transcriptome data with fluorescence quantitative PCR data,and transgenic apple seedlings overexpressing apple MdPAT16 were obtained by genetic transformation.On this basis,the function of apple MdPAT16 was investigated and the following results were obtained:1.Relationship between palmitoyltransferase and salt tolerance and sugar accumulation in apple:The sugar content of Malus hupehensis live seedlings under moderate salt stress（1mmol/L as well as 10 mmol/L Na Cl treatment）increased significantly,indicating that salt stress was closely related to sugar accumulation.When 10μm 2-bromopalmitate（2-BP,an inhibitor of palmitoylation）was used to treat Malus hupehensis live seedlings,it was found that the plants treated with 2-bromopalmitate had significantly lower salt tolerance compared with the control treatment,indicating that palmitoylation could affect the salt tolerance of the plants.A total of 30 MdPAT family members were identified in the whole genome of apple,and one MdPATs,MdPAT16,was identified as highly expressed under salt stress by fluorescence quantitative PCR analysis of 30 members and cross-comparison with transcriptome data.We found that MdPAT16 in apple is most closely related to PAT16 in white pear（Pyrus×bretschneideri）.the full-length ORF of MdPAT16 is 1146 bp and encodes 381 amino acids,and structural domain analysis of the protein sequence of MdPAT16 revealed that the protein contains DHHC and ANK structural domains unique to the PAT family,which are typical members of the PAT family.Subcellular localization analysis showed that MdPAT16 was localized to the plasma membrane.The q RT-PCR results indicated that MdPAT16 was induced by salt stress.2.Functional characterization of MdPAT16:Five 35S::MdPAT16-GFP transgenic apple seedling lines were obtained by Agrobacterium infestation with the full-length CDS of MdPAT16 linked with a GFP tag,and the m RNA levels and protein levels of MdPAT16 were significantly up-regulated.The MdPAT16 overexpression apple lines and lines transferred into the empty vector were treated with 150 mmol/L Na Cl,respectively.The results showed that the salt resistance and soluble sugar content of MdPAT16 overexpression lines were significantly higher than that of the control.Similarly,we obtained three lines of MdPAT16-OE and MdPAT16-RNAi by Agrobacterium tumefaciens infestation and treated them with salt and examined the sugar content.The results showed that salt resistance and sugar content were significantly increased in the MdPAT16 overexpression lines,while in the MdPAT16-RNAi lines,salt resistance and sugar content were significantly lower.These results indicate that the palmitoyltransferase encoded by MdPAT16 can positively regulate salt resistance and sugar content.To further verify whether MdPAT16 encodes a palmitoyltransferase protein,we expressed MdPAT16 in the yeast palmitoyltransferase mutant akr1,and functional complementation experiments showed that MdPAT16 protein was able to complement the palmitoyltransferase deficient phenotype of akr1,while The DHHC structural domain mutant of MdPAT16,MdPAT16C244A,lost the ability to compensate for the palmitoyltransferase deficient phenotype,and the acyl-biotin exchange assay also demonstrated that MdPAT16 has self-palmitoylation activity.These results suggest that MdPAT16 is a typical palmitoyltransferase.3.MdPAT16 interacts with MdCBL1 at the protein level:To further verify how MdPAT16 functions to resist salt stress and promote sugar accumulation,we performed a series of genetic transformation and correlation analyses using apple calli and found that MdCBL1 may interact with MdPAT16.Subsequently,the interactions were verified in vivo as well as in vitro using Co-IP,Pull-Down and Bi FC assays,respectively.Meanwhile,the dual luciferase complementation assay further verified the interaction of MdPAT16 with MdCBL1.The addition of Na Cl to the interactions revealed that the external application of Na Cl could significantly increase the intensity of the two interactions.In vivo Co-IP experiments demonstrate that MdPAT16 is able to palmitoylate MdCBL1,while the results of ABE experiments show that the 3rd cysteine residue of MdCBL1 is a key site for palmitoylation to occur.The absence of MdCBL1 palmitoylation leads to the migration of the subcellular localization profile of MdCBL1 from the plasma membrane localization to the nucleus and cytoplasm.Protein degradation experiments likewise showed that MdPAT16 stabilized the protein expression of MdCBL1 through palmitoylation.4.MdPAT16 regulates plant sugar content via MdCBL1:To further analyze how MdPAT16 regulates sugar content,we obtained transgenic root lines with MdCBL1overexpression,RNAi,and mutation of the 3rd cysteine residue using Agrobacterium tumefaciens-mediated transformation.Assays of the transgenic root system indicators as well as sugar content showed that MdCBL1 was able to positively regulate the sugar content changes.A series of transient expression assays were performed on the background of MdPAT16 overexpression and found that co-injected fruits also showed the same results.Thus,MdPAT16 may ultimately regulate sugar and anthocyanin accumulation through palmitoylation of MdCBL1.Previous studies demonstrated that the MdCBL1/MdCIPK13-MdSUT2.2 pathway can induce sugar accumulation within vesicles,and given the interaction between MdCBL1 and MdCIPK13,we hypothesized that MdCIPK13 is involved in the regulation of salt stress response and sugar accumulation by MdPAT16.To test the above hypothesis,a transient expression assay was performed on apple fruits using a viral vector,and the MdCIPK13-IL60 overexpression and repression vector MdCIPK13-TRV was constructed and tested by apple fruit injection.MdCIPK13-IL60 promoted sugar accumulation in apple fruits,while MdCIPK13-TRV inhibited sugar accumulation.Furthermore,MdCIPK13-TRV inhibited sugar accumulation due to MdPAT16-IL60+MdCIPK13-TRV co-injection,suggesting that it is MdPAT16-MdCBL1-mediated sugar accumulation that requires the involvement of MdCIPK13. |
