Synthesis of melatonin in mitochondria was reported in animals. However, there is no report on whether plant mitochondria also produce melatonin. Herein, we show that plant mitochondria are a major site for melatonin synthesis. In an in vitro study, isolated apple mitochondria had the capacity to generate melatonin. Subcellular localization analysis documented that an apple SNAT isoform, MzSNAT5, was localized in the mitochondria of both Arabidopsis protoplasts and apple callus cells. The kinetic analysis revealed that the recombinant MzSNAT5 protein exhibited high enzymatic activity to catalyze serotonin to N-acetylserotonin with the K and V of 55 μmol/L and 0.909 pmol/min/mg protein at 35°C, respectively; this pathway functioned over a wide range of temperatures from 5 to 75°C. In an in vivo study, MzSNAT5 was drought inducible. The transgenic Arabidopsis ectopically expressing MzSNAT5 elevated the melatonin level and, hence, enhanced drought tolerance. The mechanistic study indicated that the ectopically expressing MzSNAT5 allows plant mitochondria to increase melatonin synthesis. As a potent free radical scavenger, melatonin reduces the oxidative stress caused by the elevated reactive oxygen species which are generated under drought stress in plants. Our findings provide evidence that engineered melatonin-enriched plants exhibit enhanced oxidative tolerance.

Within the chloroplasts reactive oxygen species (ROS) are generated during photosynthesis and stressful conditions. Excessive ROS damages chloroplasts and reduces photosynthesis if not properly detoxified. In this current study, we document that chloroplasts produce melatonin, a recentlydiscovered plant antioxidant molecule. When N-acetylserotonin, a substrate for melatonin synthesis, was fed to purified chloroplasts, they produced melatonin in a dose-response manner. To further confirm this function of chloroplasts, the terminal enzyme for melatonin synthesis, N-acetylserotonin-O-methyltransferase (ASMT), was cloned from apple rootstock, Malus zumi. The in vivo fluorescence observations and Western blots confirmed MzASMT9 was localized in the chloroplasts. A study of enzyme kinetics revealed that the K m and V max of the purified recombinant MzASMT9 protein for melatonin synthesis were 500 μM and 12 pmol/min·mg protein, respectively. Arabidopsis ectopicallyexpressing MzASMT9 possessed improved melatonin level. Importantly, the MzASMT9 gene was found to be upregulated by high light intensity and salt stress. Increased melatonin due to the highlyexpressed MzASMT9 resulted in Arabidopsis lines with enhanced salt tolerance than wild type plants, as indicated by reduced ROS, lowered lipid peroxidation and enhanced photosynthesis. These findings have agricultural applications for the genetic enhancement of melatonin-enriched plants for increasing crop production under a variety of unfavorable environmental conditions.Chloroplasts are critically important plant cellular organelles with the major function of performing photosynthesis. The chlorophyll pigments in chloroplasts capture energy from photons, with this energy being used to synthesize organic molecules from carbon dioxide via the Calvin cycle. Large quantities of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated during photosynthesis, especially under the stressful conditions such as exposure to excessive light intensity, heat, salt, cold, drought and environmental pollutants 1-4 . ROS and RNS, if not properly detoxified, damages chloroplasts and reduces their photosynthetic efficiency, ultimately killing the cell. This damage is referred to as oxidative stress. During evolution, plants developed an array of mechanisms to protect themselves against oxidative stress 5 . One mechanism is the production of antioxidants; which include ascorbic acid, carotenoids, tocopherol, glutathione and polyphenols 6 . Interestingly another antioxidant, melatonin, initially thought to be exclusively an animal hormone, was identified in plants in 1995 7,8 . Since then melatonin has been detected in many different species of plants and plant products [9][10][11][12][13] . The significance of melatonin in plants is related to its potent free radical scavenging and antioxidant capacity [14][15][16][17] . In contrast to several other antioxidants, melatonin can enter every sub-cellular compartment due to its amphiphilic nature 16 . In addition, melaton...

Waterlogging, one of the notorious abiotic stressors, retards the growth of apple plants and reduces their production. Thus, it is an urgent agenda for scientists to identify the suitable remedies for this problem. In the current study, we found that melatonin significantly improved the tolerance of apple seedlings against waterlogging stress. This was indicated by the reduced chlorosis and wilting of the seedlings after melatonin applications either by leaf spray or root irrigation. The mechanisms involve in that melatonin functions to maintain aerobic respiration, preserves photosynthesis and reduces oxidative damage of the plants which are under waterlogging stress. Melatonin application also enhances the gene expression of its synthetic enzymes (MbT5H1, MbAANAT3, MbASMT9) and increases melatonin production. This is the first report of a positive feedback that exogenous melatonin application promotes the melatonin synthesis in plants. A post-transcriptional regulation apparently participated in this regulation. When exogenous melatonin meets the requirement of the plants it is found that the protein synthesis of MbASMT9 was suppressed. Taken together, the results showed that melatonin was an effective molecule to protect plant, particularly apple plant, against waterlogging stress.

Glomerella leaf spot (GLS), a fungal disease caused by Colletotrichum fructicola (C. fructicola), severely affects apple quality and yield, yet few resistance genes have been identified in apple (Malus domestica Borkh.). Here we found a transcription factor MdWRKY17 significantly induced by C. fructicola infection in the susceptible apple cultivar ‘Gala’. MdWRKY17 overexpressing transgenic ‘Gala’ plants exhibited increased susceptibility to C. fructicola, whereas MdWRKY17 RNA-interference plants showed opposite phenotypes, indicating MdWRKY17 acts as a plant susceptibility factor during C. fructicola infection. Furthermore, MdWRKY17 directly bound to the promoter of the salicylic acid (SA) degradation gene Downy Mildew Resistant 6 (MdDMR6) and promoted its expression, resulting in reduced resistance to C. fructicola. Additionally, Mitogen-activated protein kinase 3 (MdMPK3)directly interacted with and phosphorylated MdWRKY17. Importantly, predicted phosphorylation residues in MdWRKY17 by MAPK kinase 4 (MdMEK4)-MdMPK3 were critical for the activity of MdWRKY17 to regulate MdDMR6 expression. In the six susceptible germplasms, MdWRKY17 levels were significantly higher than the six tolerant germplasms after infection, which corresponded with lower SA content, confirming the critical role of MdWRKY17-mediated SA degradation in GLS tolerance. Our study reveals a rapid regulatory mechanism of MdWRKY17, which is essential for SA degradation and GLS susceptibility, paving the way to generate GLS resistant apple.

Dwarfing rootstocks enable high-density planting and are therefore highly desirable in modern apple (Malus domestica) production. M26 is a semi-dwarfing rootstock that is used worldwide, but identifying intensive dwarfing rootstock is a major goal of apple breeding programs. Herein, we show that MdWRKY9 mediates dwarfing by directly inhibiting the transcription of the brassinosteroid (BR) rate-limiting synthetase MdDWF4 and reducing BR production. We found that the transcriptional factor MdWRKY9 is highly expressed in all tested dwarfing rootstocks. Transgenic lines of M26 rootstock overexpressing MdWRKY9 exhibit further dwarfing, which resulted from the reduced BR levels and was reversed via exogenous brassinolide treatment. Both an in vivo chromatin immunoprecipitation (ChIP) analysis and an in vitro electrophoretic mobility shift assay (EMSA) indicated that MdWRKY9 binds to the promoter of MdDWF4. Furthermore, MdWRKY9 repressed MdDWF4 expression in stable transgenic apple plants as determined by quantitative PCR. In addition, RNA-interfered expression of MdWRKY9 in transiently transformed apple calli led to a significant increase of MdDWF4, suggesting MdWRKY9 plays a critical role in regulating the expression of MdDWF4. We report a novel dwarfing mechanism in perennial woody plants that involves WRKY-controlled BR production, and present a new dwarfing M26 rootstock for potential applications in apple production.