Study On The Function And Mechanism Of Apple Tyrosine Decarboxylase (MdTyDC) In Drought And Salt Tolerance

Drought alone causes more annual loss in crop yield when compared with other stresses.In the past decade,global losses in crop production due to drought totaled?$30 billion.Drought stress affects the growth and development which reduced the crop yield and quality.Salt stress caused osmotic stress,ionic stress and oxidative stress which slowed the plant growth and reduced the plant yield.Dopamine mediates numerous physiological processes in plants,and response to many biotic stresses.Tyrosine decarboxylase(Ty DC,EC 4.1.1.28)plays a fundamental role in many secondary metabolite reactions in higher plants,but little was known in the resistance in plant biotic and abiotic stresses,especially in apple during salt and drought stresses.Transgenic apple materials were obtained of MdTyDC and MdbHLH93 to verify their function,the results as list:1.The MdTyDC was cloned from‘Golden delicious'and the transgenic apple plants were got by using the genetic transformation technology of GL-3.The long-term moderate drought was performed using the transgenic apple lines after transplanted to the pots.Results showed that overexpression of MdTyDC increased the dopamine content.By detecting the development parameters during drought and results showed that,overexpression of MdTyDC(OE)in apple showed better growth performance,higher photosynthetic capacity and higher capacity for photochemical reactions(Fv/Fm value)than wild type lines(WT).Under long-term moderate drought stress,OE lines showed higher WUE,increased ABA content,decreased stomatal aperture.During drought stress,increases in amino acids(such as proline,phenylalanine and lysine)were detected in OE lines.Meanwhile,OE lines showed higher antioxidant activity,lower accumulation of ROS.These results indicate that MdTyDC confers long-term moderate drought tolerance by increasing the dopamine content,improving photosynthetic capacity,WUE,antioxidant activity and changing the contents of amino acids(such as proline accumulation).2.Overexpression of MdTyDC enhances salt tolerance.Here,apple plants and apple calli‘Orin'were utilized to study the function of MdTyDC under salt conditions.Overexpression of MdTyDC(OE)significantly increased dopamine content in overexpression MdTyDC lines,while,RNAi lines showed lower dopamine content when compared with WT lines.The OE apple lines exhibited increased growth performance and higher photosynthetic capacity and contained more photosynthetic pigments than those of in the wild-type lines(WT)under salt conditions.The OE lines had lower H₂O₂ and O₂.^-according to DAB and NBT-staining compared to the WT lines.Meanwhile,OE lines showed higher antioxidant activities.Overexpressing MdTyDC maintained lower Na⁺content and higher K⁺content in leaf and root when compared with WT lines.In addition,OE lines showed increased expression levels of ion homeostasis associated genes.Furthermore,overexpressing MdTyDC enhanced proline content,increased fresh weight,and reduced malondialdehyde(MDA)content in apple calli;however,RNA interference lines showed higher MDA content and lower fresh weight and proline content.Our findings demonstrate that overexpressing MdTyDC induced higher dopamine levels and increased scavenging of reactive oxygen species and the Na⁺/K⁺balance;thus,enhancing salt tolerance.3.Transcription factor MdbHLH93 can positively regulate drought resistance by binding to the promoter of MdTyDC.Analysis of promoter sequence of MdTyDC showed that there are three E-box cis-acting elements,which are the binding sites of b HLH transcript.We used part of the promoter which content E-box cis-acting elements as bites and through yeast one-hybrid screening to find the potential transcript factor.Results showed that,MdbHLH93 was found to interact with the E-box in the promoter of MdTyDC.Then,Yeast one-hybrid,EMSA and Luciferase assay were used to identify that MdbHLH93 directly bind to the E-box element on the promoter of MdTyDC.4.Overexpression of MdbHLH93 or MdTyDC enhanced drought tolerance.Agrobacterium rhizogenes infection was used to get the MdTyDC and MdbHLH93 OE and RNAi lines.MdTyDC and MdbHLH93-OE and RNAi lines were used in the short-term drought experiment.Results showed that,overexpression of MdTyDC or MdbHLH93enhanced the drought tolerance by maintained higher root parameters(total length,surface area,et al.)and TTC activity,with a higher Pn and Fv/Fm overground parts.In contrast,MdTyDC and MdbHLH93 RNAi lines showed weak root development and lower plant growth paraments overground,which results a weak drought resistance when compared with WT.Based on the MdTyDC-OE lines,we use agrobacterium rhizogenes of MdbHLH93-OE injected the MdTyDC-OE lines.Results showed that co-expression of MdbHLH93 and MdTyDC showed higher drought tolerance by improving the root development and maintained higher Pn and Fv/Fm values.Dopamine content detection showed that,MdbHLH93-OE/MdTyDC-OE lines was the highest among all transgenic lines(?1.5-fold)and WT(?4-fold),with a higher MdTyDC expression levels when compared with WT lines.In addition,co-expression of MdbHLH93/MdTyDC lines showed lower ROS but higher antioxidant activity than WT.Amino acids were changed during drought stress,but some osmotic substances,such as proline were higher in the MdTyDC-OE and MdbHLH93-OE/MdTyDC-OE lines when compared with WT.Hormone content during transgenic and WT lines showed that MdbHLH93-OE/MdTyDC-OE lines and MdTyDC-OE lines with higher IAA contents under short-term drought stress.MdbHLH93-OE/MdTyDC-OE lines and MdTyDC-OE lines had higher ABA and Me JA content when compared with WT under short-term drought stress,thus,enhanced the drought resistance of transgenic apple plants.