Melatonin is a potent naturally occurring reactive oxygen species (ROS) and reactive nitrogen species (RNS) scavenger in plants. Melatonin protects plants from oxidative stress and, therefore, it improves their tolerance against a variety of environmental abiotic stressors. N-acetylserotonin-O-methyltransferase (ASMT) is a specific enzyme required for melatonin synthesis. In this report, an ASMT gene was cloned from apple rootstock (Malus zumi Mats) and designated as MzASMT1 (KJ123721). The MzASMT1 expression was induced by drought stress in apple leaves. The upregulation of MzASMT1 in the apple leaf positively relates to melatonin production over a 24-hr dark/light cycle. Purified MzASMT1 protein expressed in E. coli converted its substrates to melatonin with an activity of approximately 5.5 pmol/min/mg protein. The transient transformation in tobacco identified that MzASMT1 is located in cytoplasm of the cell. When MzASMT1 gene driven by 35S promoter was transferred to Arabidopsis, melatonin levels in transgenic Arabidopsis plants were 2-4 times higher than those in the wild type. The transgenic Arabidopsis plants had significantly lower intrinsic ROS than the wild type and therefore these plants exhibited greater tolerance to drought stress than that of wild type. This is, at least partially, attributed to the elevated melatonin levels resulting from the overexpression of MzASMT1. The results elucidated the important role that membrane-located melatonin synthase plays in drought tolerance. These findings have significant implications in agriculture.

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.

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.

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