Fengwang Ma

Delayed senescence of apple leaves by exogenous melatonin treatment: toward regulating the ascorbate–glutathione cycle

Abstract:  The objectives of this study were to test the effects of exogenous melatonin on apple (Malus domestica Borkh. cv. Golden Delicious) leaves and investigate its possible physiological role in delaying leaf senescence. Detached leaves treated with 10 mm melatonin solutions clearly showed a slowing in their process of dark‐induced senescence, as evidenced by both biochemical and molecular parameters. Melatonin delayed the normal reduction in chlorophyll content and maximum potential photosystem II efficiency (Fv/Fm). It also suppressed the transcript levels of a key chlorophyll degradation gene, pheide a oxygenase (PAO), and the senescence‐associated gene 12 (SAG12). This outcome was thought to be because of the enhanced antioxidant capabilities of melatonin. Indeed, H2O2 accumulation was inhibited by exogenous melatonin, which might have resulted from direct reactive oxygen species scavenging by melatonin and a great enhancement of ascorbate peroxidase (APX; EC 1.11.1.11), which acted on both mRNA and protein activity levels. Melatonin treatment led to the maintenance of higher contents of ascorbic acid (AsA) and glutathione (GSH) but less dehydroascorbate (DHA) and oxidized glutathione (GSSG) compared with the control, possibly through its regulation of the AsA–GSH cycle.

The mitigation effects of exogenous melatonin on salinity‐induced stress in Malus hupehensis

Abstract: As an indoleamine molecule, melatonin mediates many physiological processes in plants. We investigated its role in regulating growth, ion homeostasis, and the response to oxidative stress in Malus hupehensis Rehd. under high‐salinity conditions. Stressed plants had reduced growth and a marked decline in their net photosynthetic rates and chlorophyll contents. However, pretreatment with 0.1 μm melatonin significantly alleviated this growth inhibition and enabled plants to maintain an improved photosynthetic capacity. The addition of melatonin also lessened the amount of oxidative damage brought on by salinity, perhaps by directly scavenging H2O2 or enhancing the activities of antioxidative enzymes such as ascorbate peroxidase, catalase, and peroxidase. We also investigated whether melatonin might control the expression of ion‐channel genes under salinity. Here, MdNHX1 and MdAKT1 were greatly up‐regulated in the leaves, which possibly contributed to the maintenance of ion homeostasis and, thus, improved salinity resistance in plants exposed to exogenous melatonin.

Long‐term exogenous application of melatonin delays drought‐induced leaf senescence in apple

To examine the potential roles of melatonin in drought tolerance, we tested the effects of its long‐term exogenous application on ‘Hanfu’ apple (Malus domestica Borkh.). When 100 μm melatonin was added to soils under drought conditions, the resultant oxidative stress was eased and leaf senescence was delayed. This molecule significantly reduced chlorophyll degradation and suppressed the up‐regulation of senescence‐associated gene 12 (SAG12) and pheophorbide a oxygenase (PAO). Such treatment also alleviated the inhibition of photosynthesis brought on by drought stress. We also investigated quenching and the efficiency of Photosystem II (PSII) photochemistry under dark and light conditions and found that melatonin helped to maintain better function of PSII under drought. The addition of melatonin also controlled the burst of hydrogen peroxide, possibly through direct scavenging and by enhancing the activities of antioxidative enzymes and the capacity of the ascorbate–glutathione cycle. Thus, understanding this effect of melatonin on drought tolerance introduces new possibilities to use this compound for agricultural purposes.

Exogenous melatonin improves Malus resistance to Marssonina apple blotch

We examined whether exogenously applied melatonin could improve resistance to Marssonina apple blotch (Diplocarpon mali) by apple [Malus prunifolia (Willd.) Borkh. cv. Donghongguo]. This serious disease leads to premature defoliation in the main regions of apple production. When plants were pretreated with melatonin, resistance was increased in the leaves. We investigated the potential roles for melatonin in modulating levels of hydrogen peroxide (H2O2), as well the activities of antioxidant enzymes and pathogenesis‐related proteins during these plant–pathogen interactions. Pretreatment enabled plants to maintain intracellular H2O2 concentrations at steady‐state levels and enhance the activities of plant defence‐related enzymes, possibly improving disease resistance. Because melatonin is safe and beneficial to animals and humans, exogenous pretreatment might represent a promising cultivation strategy to protect plants against this pathogen infection.

Melatonin mediates the regulation of ABA metabolism, free-radical scavenging, and stomatal behaviour in two Malus species under drought stress

Melatonin pre-treatment significantly increases the tolerance of both drought-tolerant Malus prunifolia and drought-sensitive M. hupehensis plants. Its beneficial effects include better water conservation in leaves, less electrolyte leakage, steady chlorophyll contents, and greater photosynthetic performance under stress conditions. Melatonin selectively down-regulates MdNCED3, an abscisic acid (ABA) synthesis gene, and up-regulates its catabolic genes, MdCYP707A1 and MdCYP707A2, thereby reducing ABA contents in drought-stressed plants. Melatonin also directly scavenges H2O2 and enhances the activities of antioxidant enzymes to detoxify H2O2 indirectly. These two mechanisms work synergistically to improve the functions of stomata, i.e. causing them to re-open. Plants can effectively regulate their water balance under drought conditions by up-regulating the expression of melatonin synthesis genes MdTDC1, MdAANAT2, MdT5H4, and MdASMT1. Therefore, inducing melatonin production is an important mechanism by which plants can counteract the influence of this abiotic stressor.

Delay in leaf senescence of Malus hupehensis by long‐term melatonin application is associated with its regulation of metabolic status and protein degradation

Melatonin has an important anti‐aging role in plant physiology. We tested the effects of long‐term melatonin exposure on metabolic status and protein degradation during natural leaf senescence in trees of Malus hupehensis Rehd. The 2‐month regular supplement of 100 μm melatonin to the soil once every 6 days altered the metabolic status and delayed protein degradation. For example, 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 regulated the signaling of MdHXK1, a gene for which expression was also repressed by melatonin during senescence. The plants also exhibited better preservation of their nitrogen, total soluble protein, and Rubisco protein concentrations 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 (ATGs). Our results are the first to provide evidence for this delay in senescence based on the metabolic alteration and protein degradation.

Influence of drought stress on the cellular ultrastructure and antioxidant system in leaves of drought-tolerant and drought-sensitive apple rootstocks

We compared two apple rootstocks -Malus prunifolia and Malus hupehensis - that differ in their tolerance to this abiotic stress. The former is considered drought-tolerant, the latter, sensitive. We monitored changes in their leaf ultrastructure and responses by their antioxidant defense systems. Irrigation was withheld for 12 d from two-year-old potted plants. Compared with the control, this treatment led to considerable ultrastructural alterations in organelles. Plants of M. prunifolia maintained their structural cell integrity longer than did M. hupehensis. M. hupehensis was more vulnerable to drought than was M. prunifolia, resulting in larger increases in the levels of H(2)O(2), O(2)(-), and MDA from the former. Except for catalase (CAT) and monodehydroascorbate reductase (MDHAR), the activities of superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR), and dehydroascorbate reductase (DHAR) analyzed here were enhanced to a greater extent in M. prunifolia than in M. hupehensis in response to drought. This was also true for levels of ascorbic acid (AsA) and glutathione (GSH). Under well-watered conditions, changes in lipid peroxidation and relevant antioxidant parameters were not significantly different between the two species throughout the experimental period. These results demonstrate that, in order to minimize oxidative damage, both the activities of antioxidant enzymes and antioxidant concentrations are increased in the leaves of M. prunifolia and M. hupehensis in response to water stress. Moreover, plants of M. prunifolia exhibit higher antioxidant capacity and a stronger protective mechanism, such that their cell structural integrity is better maintained during exposure to drought.

Ascorbate Biosynthesis during Early Fruit Development Is the Main Reason for Its Accumulation in Kiwi

Background Ascorbic acid (AsA) is a unique antioxidant as well as an enzyme cofactor. Although it has multiple roles in plants, it is unclear how its accumulation is controlled at the expression level, especially in sink tissues. Kiwifruit (Actinidia) is well-known for its high ascorbate content. Our objective was to determine whether AsA accumulates in the fruits primarily through biosynthesis or because it is imported from the foliage. Methodology/Principal Findings We systematically investigated AsA levels, biosynthetic capacity, and mRNA expression of genes involved in AsA biosynthesis in kiwi (A. deliciosa cv. Qinmei). Recycling and AsA localization were also monitored during fruit development and among different tissue types. Over time, the amount of AsA, with its capacity for higher biosynthesis and lower recycling, peaked at 30 days after anthesis (DAA), and then decreased markedly up to 60 DAA before declining more slowly. Expression of key genes showed similar patterns of change, except for L-galactono-1,4-lactone dehydrogenase and L-galactose-1-phosphate phosphatase (GPP). However, GPP had good correlation with the rate of AsA accumulation. The expression of these genes could be detected in phloem of stem as well as petiole of leaf and fruit. Additionally, fruit petioles had greater ascorbate amounts, although that was the site of lowest expression by most genes. Fruit microtubule tissues also had higher AsA. However, exogenous applications of AsA to those petioles did not lead to its transport into fruits, and distribution of ascorbate was cell-specific in the fruits, with more accumulation occurring in larger cells. Conclusions These results suggest that AsA biosynthesis in kiwi during early fruit development is the main reason for its accumulation in the fruits. We also postulate here that GPP is a good candidate for regulating AsA biosynthesis whereas GDP-L-galactose-1-phosphate phosphorylase is not.

Melatonin regulates carbohydrate metabolism and defenses against Pseudomonas syringae pv. tomato DC3000 infection in Arabidopsis thaliana.

Melatonin has been reported to promote plant growth and development. Our experiments with Arabidopsis thaliana showed that exogenous applications of this molecule mediated invertase inhibitor (C/VIF)‐regulated invertase activity and enhanced sucrose metabolism. Hexoses were accumulated in response to elevated activities by cell wall invertase (CWI) and vacuolar invertase (VI). Analyses of sugar metabolism‐related genes revealed differential expression during plant development that was modulated by melatonin. In particular, C/VIF1 and C/VIF2 were strongly down‐regulated by exogenous feeding. We also found the elevated CWI activity in melatonin‐treated Arabidopsis improved the factors (cellulose, xylose, and galactose) for cell wall reinforcement and callose deposition during Pseudomonas syringae pv. tomato DC3000 infection, therefore, partially induced the pathogen resistance. However, CWI did not involve in salicylic acid (SA)‐regulated defense pathway. Taken together, this study reveals that melatonin plays an important role in invertase‐related carbohydrate metabolism, plant growth, and pathogen defense.

Melatonin regulates proteomic changes during leaf senescence in Malus hupehensis

Despite the relationship between melatonin and aging, the overall changes and regulation of proteome profiling by long‐term melatonin exposure during leaf senescence is not well understood. In this study, leaf senescence in Malus hupehensis plants was delayed when exogenous melatonin was regularly applied to the roots for 2 months compared with natural leaf senescence. Proteins of samples 0 and 50 day for both treatments were extracted and labeled with TMT regents before being examined via NanoLC‐MS/MS. The proteomics data showed that 622 and 309 proteins were altered by senescence and melatonin, respectively. Our GO analysis by Blast2GO revealed that most of the altered proteins that are involved in major metabolic processes exhibited hydrolase activity and were mainly located in the plastids. These proteins were classified into several senescence‐related functional categories, including degradation of macromolecules, redox and stress responses, transport, photosynthesis, development, and other regulatory proteins. We found that melatonin treatment led to the downregulation of proteins that are normally upregulated during senescence. The melatonin‐related delay in senescence might have occurred due to the altering of proteins involved in processes associated with senescence. And as well, there are many unknown regulatory proteins possibly being involved in the melatonins function. This study is the first to demonstrate changes at the proteome level in response to exogenous melatonin in plants. Our findings provide a set of informative and fundamental data about the role of melatonin in apple leaf senescence.