Regulation Of Leaf Senescence By Exogenous Melatonin And Functional Analysis Of Related Autophagy Genes In Malus

Apple is one of the most important fruits in the world, and occupies an important position in fruit production. The Northwest Loess Plateau region is the largest production base of apple in China. However, there are often limiting factors in apple production in this region because of less rain and lack of soil fertility. Long-term drought stress or nutrient starvation could induce early leaf senescence, which severely affects the nutrition of apple trees, thus affecting fruit yield and quality. Melatonin is an ancient and conserved indolamine widespread in all living organisms. Melatonin protects organisms from various damage for its highly effective activity of scavenging free radicals, and improves the adaptability to the adverse environment. Autophagy is an important degradation pathway for proteins and organelles, and plays important roles in leaf senescence, nutrient starvation and oxidative stress.Research on biological functions of melatonin in plants is still in its infancy and there are few reports on the identification and function analysis of autophagy-related genes?ATGs? in Malus. In this study, we firstly studied the regulation of exogenous melatonin on different types of leaf senescence, thus revealed the anti-senescence effect of melatonin from physiological and proteomic levels. Based on the regulation of melatonin on ATGs expressions during leaf senescence and on the autophagy occurrence under oxidative stress, we analyzed the functions of Md ATG8 i and Md ATG3 s cloned from Malus domestica, using molecular biology techniques, and revealed the important biological roles of autophagy in leaf senescence and nutrient starvation conditions, providing a theoretical basis for the biological function and stress resistance improvement of apple. The main results are documented as followings.1. Exogenous melatonin delayed dark-induced senescence of detached leaves. Using mature leaves from Malus domestica Borkh. cv. Golden Delicious, during dark-induced leaf senescence, 10 m M exogenous melatonin treatment slowed down the decrease rate of Fv/Fm and chlorophyll degradation, relatively inhibited the expression of senescence-associated genes SAG12 and PAO, as well as inhibited the accumulation of H2O2 and improved antioxidative system, especially As A-GSH recycle ability.2. Exogenous melatonin delayed drought-induced leaf senescence. Using two-year-old trees of ‘Hanfu' apple?Malus domestica Borkh.? grafted onto rootstock Malus hupehensis, when 100 ?M melatonin was regularly added to soils under long-term drought conditions, this molecule significantly reduced chlorophyll degradation and suppressed the up-regulation of SAG12 and PAO. Such treatment also alleviated the inhibition of photosynthesis and maintained better function of PS? under drought. The addition of melatonin also reduced the content of H2O2 and enhanced the activities of antioxidative enzymes and the capacity of the As A-GSH cycle.3. Exogenous melatonin delayed natural leaf senescence through its regulation of metabolic status and protein degradation in trees of Malus hupehensis Rehd. Leaves from treated plants had significantly higher photosynthetic activity, chlorophyll concentrations and levels of three photosynthetic end products?sorbitol, sucrose and starch? when compared with the control. The significant inhibition of hexose?fructose and glucose? accumulation possibly repressed the signaling of Md HXK1, which will trigger leaf senescence. The plants also exhibited better preservation of nitrogen, total soluble proteins and Rubisco protein than the control. The slower process of protein degradation might be a result of melatonin-linked inhibition on the expression of apple autophagy-related genes. At the same time, proteomic data analysis showed that melatonin down-regulated proteins that are normally up-regulated during leaf senescence. The melatonin-related delay in senescence might have occurred due to the altering of proteins involved in processes associated with senescence, such as proteins involved in degradation of macromolecules and transport. Melatonin also up-regulated some of the proteins involved in photosynthesis and redox and stress responses, and down-regulated proteins involved in DNA or RNA regulation. These information will provide a set of fundamental data about the potential roles of melatonin in plants.4. Exogenous melatonin enhanced the occurrence of autophagy induced by oxidative stress in Arabidopsis seedlings. We investigated the influence of different concentrations of melatonin?0.0, 0.5, 5.0, 10.0, or 50.0 ?M? on autophagy under methyl viologen?MV?-induced oxidative stress. Arabidopsis seedlings that were pre-treated with 10 ?M melatonin underwent relatively strong induction of autophagy. Pretreatment with 10 ?M melatonin also significantly reduced the accumulation of oxidized proteins. Those responses might have been due to the strong up-regulation of genes that involved in At ATG8-PE conjugation pathway, which enhanced the capacity for autophagy. Histochemical staining revealed that both O2-· and H2O2 were highly accumulated upon MV exposure, although the response did not differ significantly between control and melatonin-pretreated seedlings. By contrast, exogenous melatonin up-regulated the expression of two genes for H2O2-scavenging enzymes, i.e., At APX1 and At CATs. The activation of autophagy by melatonin without an alteration in ROS production may be part of a survival mechanism that is enhanced by melatonin after cellular damage. Therefore, it represents a second level of defense to remove damaged proteins when antioxidant activities are compromised.5. An autophagy-related gene Md ATG8 i was cloned from Malus domestica. Md ATG8 i located on apple chromosome 13. The full length of Md ATG8 i c DNA was 1816 bp, with an ORF 357 bp encoding a 118 amino acid protein. The Gen Bank accession number is KF438037. Md ATG8 i belonged to UBQ superfamily, and had conserved GABARAP domain as ATG8 in mammals, with predicted ATG7 binding sites. Md ATG8 i expressed in all tested tissues, including root, stem, leaf, flower, fruit and bud, but expressed most abundant in senesenced leaves. It responded to leaf senescence, nitrogen starvation and oxidative stress. The inducible expression of Md ATG8 i in yeast could complement the recovery phenotype of yeast Atg8 mutant atg8 after nitrogen starvation. The subcellular localization of Md ATG8 i was in nucleus, cell membrane and cytoplasm.6. The overexpression of Md ATG8 i transgenic Arabidopsis lines showed early bolting, promoted leaf senescence and better resistance phenotypes growing on nitrogen or sugar starvation mediums. The overexpression of Md ATG8 i transgenic Orin callus also showed better growth on low sugar mediums. The overexpression of Md ATG8 i transgenic Gala apple plants showed better resistance than wide type, growing on different nutrient starvation and oxidative stress conditions. The consistent phenotype between transgenic Arabidopsis, Orin callus and Gala plants, with resistence improvement in nutrient starvation, indicated that autophagy plays important roles in nutrient starvation stress.7. Two autophagy-related gene ATG3 homologs---Md ATG3-1 and Md ATG3-2 were also cloned from Malus domestica. Their Gen Bank accession numbers are KF438032 and KR024682. Md ATG3-1 and Md ATG3-2 shared high homology but located on apple chromosome 6 and 7, respectively. Both of them contained 927 bp in ORF and encoded 308 amino acid proteins, which had conserved Autophagy_N, Autophagy_act_C and Autophagy_Cterm domains. Md ATG3-1 and Md ATG3-2 expressed in all tested tissues, with the highest in senescenced leaves. Their expression also responded to leaf senescence, nitrogen starvation and oxidative stress. The subcellular localization of Md ATG3-1 and Md ATG3-2 were also in nucleus, cell membrane and cytoplasm. Their induced expression in yeast could complement the recovery phenotype of yeast Atg3 mutant atg3 after nitrogen starvation. Yeast two-hybrid experiments showed that both Md ATG3-1 and Md ATG3-2 could interact with Md ATG8 i in yeast.8. The promoter of Md ATG3-2 was isolated from Malus domestica, which was 1764 bp in length. Its cis-acting elements and promoter activities were analyzed as well. By using yeast one-hybrid screening system, we obtained one new gene Md ROATG3 interacting with the promoter of Md ATG3-2. Md ROATG3 was comprised of 1912 bp in full length c DNA, contained 888 bp in ORF and encoded a 295 amino acid protein. It located on apple chromosome 12. The Gen Bank accession number is XP₀08389698.1. Currently, there is no conserved known domains in this protein, belonging to unknown proteins. The expression of Md ROATG3 was relatively high in senescenced leaves and fruits, and responded to leaf senescence, nitrogen starvation and oxidative stress. Md ROATG3 was considered as a transcriptional factor for its subcellular localization in nucleus and its self-transcriptional activation in yeast. After verification using yeast one-hybrid and Ch IP-PCR, protein Md ROATG3 could bind to the 10-120 bp and 1-154 bp of 5' region in Md ATG3-2 promoter.9. The overexpression of Md ATG3-2 and Md ROATG3 transgenic Arabidopsis lines and Orin callus exhibited the same growth phenotype and resistance as Md ATG8 i transgenic plants. Meanwhile the overexpression of Md ROATG3 transgenic Gala apple plants showed similar resistance phenotype under nutrient starvation stress, which demonstrated that Md ROATG3 positively improved plant resistance against nutrient starvation through regulating autophagy gene, reinforcing the important biological roles of autophagy. In addition, the Md ROATG3 transgenic Gala apple plants also alleviated the damage caused by drought stress, and relatively increased the resistance against natural drought stress.