MdWER interacts with MdERF109 and MdJAZ2 to mediate methyl jasmonate- and light-induced anthocyanin biosynthesis in apple fruit.

Anthocyanin generation in apples (Malus domestica) and the pigmentation that results from it may be caused by irradiation and through administration of methyl jasmonate (MeJA). However, their regulatory interrelationships associated with fruit coloration are not well defined. To determine whether MdERF109, a transcription factor (TF) involved in light-mediated coloration and anthocyanin biosynthesis, has synergistic effects with other proteins, we performed a yeast two-hybrid assessment and identified another TF, MdWER. MdWER was induced by MeJA treatment, and although overexpression of MdWER alone did not promote anthocyanin accumulation co-overexpression with MdERF109 resulted in significantly increase in anthocyanin biosynthesis. MdWER may form a protein complex with MdERF109 to promote anthocyanin accumulation by enhancing combinations between the proteins and their corresponding genes. In addition, MdWER, as a MeJA responsive protein, interacts with the anthocyanin repressor MdJAZ2. Transient co-expression in apple fruit and protein interaction assays allowed us to conclude that MdERF109 and MdJAZ2 interact with MdWER and take part in the production of anthocyanins upon MeJA treatment and irradiation. Our findings validate a role for the MdERF109-MdWER-MdJAZ2 module in anthocyanin biosynthesis and uncover a novel mechanism for how light and MeJA signals are coordinated anthocyanin biosynthesis in apple fruit.

The apple RING-H2 protein MdCIP8 regulates anthocyanin accumulation and hypocotyl elongation by interacting with MdCOP1.

COP1, an important RING ubiquitin ligase E3, is a molecular switch for light regulation in plant development. As an interacting protein of COP1, CIP8 contains a RING-H2 domain, but its biological function is unclear. Here, the apple MdCIP8 was identified based on its homology with AtCIP8 in Arabidopsis. MdCIP8 was constitutively expressed at different levels in various apple tissues, and the expression level of MdCIP8 was not affected by light and dark conditions. MdCIP8 reversed the short hypocotyl phenotype of the cip8 mutant under light conditions. Furthermore, the yeast two-hybrid experiment showed that MdCIP8 interacted with the RING domain of MdCOP1 through its RING-H2 domain. MdCIP8-OX/cop1-4 exhibited the phenotype of the cop1-4 mutant, indicating that CIP8 acts upstream of COP1. In addition, an apple transient injection experiment showed that MdCIP8 inhibited anthocyanin accumulation in an MdCOP1-dependent pathway. Overall, our findings reveal that CIP8 plays an inhibitory role in the light-regulation responses of plants.

Application of a Krypton-Chlorine Excilamp To Control Alicyclobacillus acidoterrestris Spores in Apple Juice and Identification of Its Sporicidal Mechanism.

The aim of this study was to investigate the sporicidal effect of a krypton-chlorine (KrCl) excilamp against Alicyclobacillus acidoterrestris spores and to compare its inactivation mechanism to that of a conventional UV lamp containing mercury (Hg). The inactivation effect of the KrCl excilamp was not significantly different from that of the Hg UV lamp for A. acidoterrestris spores in apple juice despite the 222-nm wavelength of the KrCl excilamp having a higher absorption coefficient in apple juice than the 254-nm wavelength of the Hg UV lamp; this is because KrCl excilamps have a fundamentally greater inactivation effect than Hg UV lamps, which is confirmed under ideal conditions (phosphate-buffered saline). The inactivation mechanism analysis revealed that the DNA damage induced by the KrCl excilamp was not significantly different (P > 0.05) from that induced by the Hg UV lamp, while the KrCl excilamp caused significantly higher (P < 0.05) lipid peroxidation incidence and permeability change in the inner membrane of A. acidoterrestris spores than did the Hg UV lamp. Meanwhile, the KrCl excilamp did not generate significant (P > 0.05) intracellular reactive oxygen species, indicating that the KrCl excilamp causes damage only through the direct absorption of UV light. In addition, after KrCl excilamp treatment with a dose of 2,011 mJ/cm2 to reduce A. acidoterrestris spores in apple juice by 5 logs, there were no significant (P > 0.05) changes in quality parameters such as color (L*, a*, and b*), total phenolic compounds, and DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity.IMPORTANCEAlicyclobacillus acidoterrestris spores, which have high resistance to thermal treatment and can germinate even at low pH, are very troublesome in the juice industry. UV technology, a nonthermal treatment, can be an excellent means to control heat-resistant A. acidoterrestris spores in place of thermal treatment. However, the traditionally applied UV sources are lamps that contain mercury (Hg), which is harmful to humans and the environment; thus, there is a need to apply novel UV technology without the use of Hg. In response to this issue, excilamps, an Hg-free UV source, have been actively studied. However, no studies have been conducted applying this technique to control A. acidoterrestris spores. Therefore, the results of this study, which applied a KrCl excilamp for the control of A. acidoterrestris spores and elucidated the inactivation principle, are expected to be utilized as important basic data for application to actual industry or conducting further studies.

Smartphone Application-Enabled Apple Fruit Surface Temperature Monitoring Tool for In-Field and Real-Time Sunburn Susceptibility Prediction.

Heat stress and resulting sunburn is a major abiotic stress in perineal specialty crops. For example, such stress to the maturing fruits on apple tree canopies can cause several physiological disorders that result in considerable crop losses and reduced marketability of the produce. Thus, there is a critical technological need to effectively monitor the abiotic stress under field conditions for timely actuation of remedial measures. Fruit surface temperature (FST) is one of the stress indicators that can reliably be used to predict apple fruit sunburn susceptibility. This study was therefore focused on development and in-field testing of a mobile FST monitoring tool that can be used for real-time crop stress monitoring. The tool integrates a smartphone connected thermal-Red-Green-Blue (RGB) imaging sensor and a custom developed application ('AppSense 1.0') for apple fruit sunburn prediction. This tool is configured to acquire and analyze imagery data onboard the smartphone to estimate FST. The tool also utilizes geolocation-specific weather data to estimate weather-based FST using an energy balance modeling approach. The 'AppSense 1.0' application, developed to work in the Android operating system, allows visual display, annotation and real-time sharing of the imagery, weather data and pertinent FST estimates. The developed tool was evaluated in orchard conditions during the 2019 crop production season on the Gala, Fuji, Red delicious and Honeycrisp apple cultivars. Overall, results showed no significant difference (t110 = 0.51, p = 0.6) between the mobile FST monitoring tool outputs, and ground truth FST data collected using a thermal probe which had accuracy of ±0.4 °C. Upon further refinements, such tool could aid growers in real-time apple fruit sunburn susceptibility prediction and assist in more effective actuation of apple fruit sunburn preventative measures. This tool also has the potential to be customized for in-field monitoring of the heat stressors in some of the sun-exposed perennial and annual specialty crops at produce maturation.

Systematic identification of long noncoding RNAs expressed during light-induced anthocyanin accumulation in apple fruit.

Anthocyanin pigments contribute to the red color of apple (Malus × domestica) fruit and have a major influence on their ornamental, dietary and market value. In this study, we investigated the potential role of long noncoding RNAs (lncRNAs) in anthocyanin biosynthesis. RNA-seq analysis of apple peels from the 'Red Fuji' cultivar during light-induced rapid anthocyanin accumulation revealed 5297 putative lncRNAs. Differential expression analysis further showed that lncRNAs were induced during light treatment and were involved in photosynthesis. Using the miRNA-lncRNA-mRNA network and endogenous target mimic (eTM) analysis, we predicted that two differentially expressed lncRNAs, MLNC3.2 and MLNC4.6, were potential eTMs for miRNA156a and promoted the expression of the SPL2-like and SPL33 transcription factors. Transient expression in apple fruit and stable transformation of apple callus showed that overexpression of the eTMs and SPLs promoted anthocyanin accumulation, with the opposite results in eTM and SPL-silenced fruit. Silencing or overexpressing of miR156a also affected the expression of the identified eTMs and SPLs. These results indicated that MLNC3.2 and MLNC4.6 function as eTMs for miR156a and prevent cleavage of SPL2-like and SPL33 by miR156a during light-induced anthocyanin biosynthesis. Our study provides fundamental insights into lncRNA involvement in the anthocyanin biosynthetic pathway in apple fruit.

Melatonin increases the performance of Malus hupehensis after UV-B exposure.

Melatonin, an evolutionarily conserved molecule, is implicated in numerous physiological processes in plants. To explore the potential roles of melatonin in response to UV-B radiation, we examined the influence of exogenous melatonin on Malus hupehensis Rehd. seedlings under two levels of UV-B radiation. Under UV-B stress, seedlings showed significant reduction in plant growth, biomass production, and root system development. However, 1 µM melatonin solution markedly alleviated these effects, especially at the higher dosage of UV-B radiation. The inhibitory effects of UV-B radiation on photosynthetic parameters, chlorophyll fluorescence parameters, stomatal apertures, chlorophyll levels and leaf membrane damages were also markedly alleviated with melatonin application. Melatonin treatment was also associated with higher activity and expression of genes encoding antioxidant enzymes (ascorbate peroxidase, catalase and peroxidase) and greater decline of H2O2 content in leaves exposed to UV-B. Moreover, exogenous melatonin treatment and UV-B stress increased the concentration of endogenous melatonin. The content of several phenolic compounds, including chlorogenic acid, phloridzin and quercetin-3-galactoside, also increased under UV-B stress, and these were further elevated significantly with melatonin addition. This study provides insight into the role(s) of endogenous melatonin in response to UV-B stress, and will facilitate application of exogenous melatonin in agriculture.

A feedback loop involving MdMYB108L and MdHY5 controls apple cold tolerance.

The MYB transcription factors are important for many aspects of plant stress responses. In this study, we isolated and identified an apple MYB gene, MdMYB108L, whose expression is induced by light and cold stresses. An analysis of MdMYB108L-overexpressing transgenic apple calli revealed that MdMYB108L enhances cold tolerance in apple by upregulating MdCBF3 expression. Interestingly, the expression of MdHY5, which encodes an integrator of light and cold signals, was significantly downregulated in transgenic calli. Yeast one-hybrid and electrophoretic mobility shift assays indicated that MdMYB108L positively regulates cold tolerance by binding to the MdCBF3 promoter. Additionally, MdHY5 functions upstream of MdMYB108L, and the resulting increase in MdMYB108L abundance downregulates MdHY5 transcription. The results of this study elucidate a new pathway for the regulation of apple cold tolerance via a feedback mechanism involving MdMYB108L and MdHY5.

The B-box zinc finger protein MdBBX20 integrates anthocyanin accumulation in response to ultraviolet radiation and low temperature.

Ultraviolet-B (UV-B) radiation and low temperature promote the accumulation of anthocyanins, which give apple skins their red colour. Although many transcription regulators have been characterized in the UV-B and low-temperature pathways, their interregulation and synergistic effects are not well understood. Here, a B-box transcription factor gene, MdBBX20, was characterized in apple and identified to promote anthocyanin biosynthesis under UV-B conditions in field experiments and when overexpressed in transgenic apple calli. The transcript level of MdBBX20 was significantly induced by UV-B. Specific G-box elements in the promoters of target genes were identified as interaction sites for MdBBX20. Further experimental interrogation of the UV-B signalling pathways showed that MdBBX20 could interact with MdHY5 in vitro and in vivo and that this interaction was required to significantly enhance the promoter activity of MdMYB1. MdBBX20 also responded to low temperature (14°C) with the participation of MdbHLH3, which directly bound a low temperature-response cis elements in the MdBBX20 promoter. These findings demonstrate the molecular mechanism by which MdBBX20 integrates low-temperature- and UV-B-induced anthocyanin accumulation in apple skin.

MdCOL4 Interaction Mediates Crosstalk Between UV-B and High Temperature to Control Fruit Coloration in Apple.

In many plants, anthocyanin biosynthesis is affected by environmental conditions. Ultraviolet-B (UV-B) radiation promotes anthocyanin accumulation and fruit coloration in apple skin, whereas high temperature suppresses these processes. In this study, we characterized a B-box transcription factor, MdCOL4, from 'Fuji' apple, and identified its role in anthocyanin biosynthesis by overexpressing its encoding gene in apple red callus. The expression of MdCOL4 was reduced by UV-B, but promoted by high temperature. We explored the regulatory relationship between heat shock transcription factors (HSFs) and MdCOL4, and found that MdHSF3b and MdHSF4a directly bound to the heat shock element cis-element of the MdCOL4 promoter. MdCOL4 interacted with MdHY5 to synergistically inhibit the expression of MdMYB1, and MdCOL4 directly bound to the promoters of MdANS and MdUFGT, which encode genes in the anthocyanin biosynthetic pathway, to suppress their expression. Our findings shed light on the molecular mechanism by which MdCOL4 suppresses anthocyanin accumulation in apple skin under UV-B and high temperature.

Anthocyanin synthesis and light utilisation can be enhanced by reflective mulch - Visualisation of light penetration into a tree canopy.

With the increasing use of hailnets and decrease in light availability in the ripening period of apple fruit, insufficient light exposure often causes poor colouration viz anthocyanin synthesis on certain parts of the fruit and on certain fruit within the tree. The aim of this study was to investigate the potential of improving anthocyanin synthesis, in terms of fruit colouration, the major incentive for a consumer. Therefore, the reflective ground cover Lumilys® was spread between 'Braeburn Mariri Red' apple rows under a crystalline hailnet seven weeks prior to harvest and colour measured on 240 attached apple fruit. The reflective ground cover increased the reflected light by 1.6 to 3.9 times 1.0 m above ground. The improved light utilization led to an improved peel colouration, especially on the shaded side of the apple fruit and fruit in the lower inner part of the canopy, A coloured visualization from orange (high light intensity), yellow (medium) to green (low light intensity) as a result of the individual PAR measurements every 20 cm inside the canopy showed how the reflective mulch influences the light penetration into the different parts of the tree canopy.

Biological Evaluation of Golden Delicious Apples Exposure to UV Lights in Rats.

BACKGROUND AND OBJECTIVE: Anthocyanin is responsible for the red color of apple. Ultraviolet light plays a key role in activating the genes responsible for anthocyanin biosynthesis. However, the most important concern is using UV light irradiation on fruit to increase anthocyanins level and its nutritional quality. In this study, the accumulation of anthocyanin in green apple using UV-B and UV-C was investigated and its biological influence was evaluated in rats. MATERIAL AND METHOD: Green Golden delicious apples were irradiated with doses of UV-C and UV-B light for a period of 3 h/day each for 3 days. Two Groups of rats were fed on balanced diet or balanced diet supplemented with 10% apple exposure to UV (AP-UV) for a month. RESULTS: The HPTLC and spectrophotometric determination of anthocyanin revealed that color development was significantly increased by 90% in treated apple compared to the control apples. Histological difference was observed between the 2 groups. Plasma levels of uric acid, the activity of transaminases (ALT and AST) as well as malondialdehyde (MDA) were significantly elevated in AP-UV rats. Plasma total cholesterol, triglycerides and creatinine level did not differ among the 2 groups. Liver MDA and catalase levels were eminent in AP-UV rats compared to control. Gene expression of selected inflammatory cytokines (TNF-α, IL-6 and IL-1ß) was significantly up-regulated in liver of AP-UV rats in comparison to control rats. CONCLUSION: The result revealed that there is a health-hazard linked to feeding rats on diet containing irradiated-apple with UV-B and UV-C, which represented by body weight reduction, inflammation development, liver function and oxidative stress elevation.

Transcription profiles reveal the regulatory mechanisms of spur bud changes and flower induction in response to shoot bending in apple (Malus domestica Borkh.).

KEY MESSAGE: Shoot bending, as an effective agronomic measure, has been widely used to promote flowering in 'Fuji' apple trees. Here, we examined the transcriptional responses of genes in 'Fuji' apple buds at different flowering stages under a shoot-bending treatment using RNA sequencing. A complex genetic crosstalk-regulated network, involving abscisic acid-related genes, starch metabolism and circadian rhythm-related genes, as well as stress response-related genes, was up-regulated by shoot bending, in which were contrbuted to apple flower bud formation in response to shoot-bending conditions. Flower induction plays an important role in the apple tree life cycle, but young trees produce fewer and inferior flower buds. Shoot bending, as an effective agronomic measure, has been widely used to promote flowering in 'Fuji' apple trees. However, little is known about the gene expression network patterns and molecular regulatory mechanisms caused by shoot bending during the induced flowering. Here, we examined the transcriptional responses of genes in 'Fuji' apple buds at different flowering stages under a shoot-bending treatment using RNA sequencing. A steady up-regulation of carbon metabolism-related genes led to relatively high levels of sucrose in early induced flowering stages and starch accumulation during shoot bending. Additionally, global gene expression profiling determined that cytokinin, indole-3-acetic acid, gibberellin synthesis and signalling-related genes were significantly regulated by shoot bending, contributing to cell division and differentiation, bud growth and flower induction. A complex genetic crosstalk-regulated network, involving abscisic acid-related genes, starch metabolism- and circadian rhythm-related genes, as well as stress response-related genes, was up-regulated by shoot bending. Additionally, some transcription factor family genes that were involved in sugar, abscisic acid and stress response signalling were significantly induced by shoot bending. These important flowering genes, which were mainly involved in photoperiod, age and autonomous pathways, were up-regulated by shoot bending. Thus, a complex genetic network of regulatory mechanisms involved in sugar, hormone and stress response signalling pathways may mediate the induction of apple tree flowering in response to shoot-bending conditions.

Effects of curcumin-based photodynamic treatment on the storage quality of fresh-cut apples.

Photodynamic treatment (PDT) is an innovative technology with non-thermal and environmentally sound merits, but the evaluation on the storage qualities of fresh produce was scarce. In this study, the effects of curcumin-based PDT on the quality of fresh-cut 'Fuji' apple slices during storage at 4 °C were investigated. The impacts on the survival of Escherichia coli, color and weight loss were examined under different curcumin concentrations, illumination time or incubation time. Curcumin-based photodynamic inactivation of E. coli on the surface of apple slices reached 0.95 log. Curcumin-based PDT was proven to prevent browning and weight loss. Additionally, PDT significantly reduced the activity of polyphenol oxidase and peroxidases to 48% and 51%, respectively. Moreover, there were few negative changes in total phenolic, ascorbic acid content and anti-oxidant activity of the treated apples. These results indicated that curcumin-based PDT was a viable and promising non-thermal technology to preserve the quality of fresh produce.

Analysis of apple beverages treated with high-power ultrasound: a quality function deployment approach.

BACKGROUND: The objective of this paper was to demonstrate application of quality function deployment in analysing effects of high power ultrasound on quality properties of apple juices and nectars. In order to develop a quality function deployment model, joint with instrumental analysis of treated samples, a field survey was performed to identify consumer preferences towards quality characteristics of juices/nectar. RESULTS: Based on field research, the three most important characteristics were 'taste' and 'aroma' with 28.5% of relative absolute weight importance, followed by 'odour' (16.9%). The quality function deployment model showed that the top three 'quality scores' for apple juice were treatments with amplitude 90 µm, 9 min treatment time and sample temperature 40 °C; 60 µm, 9 min, 60 °C; and 90 µm, 6 min, 40 °C. For nectars, the top three were treatments 120 µm, 9 min, 20 °C; 60 µm, 9 min, 60 °C; and A2.16 60 µm, 9 min, 20 °C. CONCLUSION: This type of quality model enables a more complex measure of large scale of different quality parameters. Its simplicity should be understood as its practical advantage and, as such, this tool can be a part of design quality when using novel preservation technologies. © 2017 Society of Chemical Industry.

Optimization of Ultrasonic-Assisted Aqueous Two-Phase Extraction of Phloridzin from Malus Micromalus Makino with Ethanol/Ammonia Sulfate System.

Application of an aqueous two-phase system (ATPS) coupled with ultrasonic technology for the extraction of phloridzin from Malus micromalus Makino was evaluated and optimized by response surface methodology (RSM). The ethanol/ammonium sulfate ATPS was selected for detailed investigation, including the phase diagram, effect of phase composition and extract conditions on the partition of phloridzin, and the recycling of ammonium sulfate. In addition, the evaluation of extraction efficiency and the identification of phloridzin were investigated. The optimal partition coefficient (6.55) and recovery (92.86%) of phloridzin were obtained in a system composed of 35% ethanol (w/w) and 16% (NH4 )2 SO4 (w/w), 51:1 liquid-to-solid ratio, and extraction temperature of 36 °C. Comparing with the traditional solvent extraction with respective 35% and 80% ethanol, ultrasonic-assisted aqueous two-phase extraction (UAATPE) strategy had significant advantages with lower ethanol consumption, less impurity of sugar and protein, and higher extracting efficiency of phloridzin. Our result indicated that UAATPE was a valuable method for the extraction and preliminary purification of phloridzin from the fruit of Malus micromalus Makino, which has great potential in the deep processing of Malus micromalus Makino industry to increase these fruits' additional value and drive the local economic development.

High efficiency stratification of apple cultivar Ligol seed dormancy by phytohormones, heat shock and pulsed radio frequency.

The aim of the study was to improve the effect of stratification of apple "Ligol" seeds by application of selected compounds, phytohormones, and physical methods For this purpose the seeds were stratified at 3°C in distilled water or in the presence of potassium nitrate (KNO3), ethephon (ET), carbon monoxide (CO), hydrogen peroxide (H2O2), a mixture of KNO3, ET, CO, H2O2, gibberellins (GA3), 6-benzylaminopurine (BAP), jasmonic acid (JA), salicylic acid (SA) and a mixture of SA, GA3, BAP, JA, nitric oxide (NO), hydrogen chloride (HCL). Arranged protocols included various durations and combinations of selected compounds and phytohormones as well as laser and red light, heat shock - 2h heat shock (45°C) and Pulsed Radio Frequency (PRF) were investigated by germination tests and the activity of selected enzymes, gas exchange and index of chlorophyll in leaves. The obtained results showed the possibility to shorten more effectively the time of the apple 'Ligol' dormancy removal by treatments of the stratified seeds at 3°C with different biological and physical methods Selected compounds and phytohormones acted collectively as a regulatory complex controlling the course of release from dormancy. Physical methods (PRF and heat shock) additionally contributed to dormancy breakage. Duration of phytohormones or compounds impacts during stratification should be prolonged to minimum 7days to assure more balanced conditions of the regulatory complex for the acceleration of dormancy a removal. The most beneficial results were obtained after seed stratification for 7days on filter paper moistened in KNO3+Etephon+CO+H2O2 at 3°C, and then on filter paper moistened in phytohormones (GA3+BAP+JA) till the end of seed germination (3°C). The application of this protocol could be a very useful tool in a shortening the apple breeding cycle since the period of removing dormancy was reduced by 38days in comparison to stratified in water. PRF has also the additive role in breaking dormancy of apple 'Ligol' seed. Positive effects of compounds and phytohormones applied during stratification remarkably accelerated the growth of developed from them seedlings. Further research is needed to optimize stratification methods with appropriate contents and concentrations of compounds and phytohormones combined with PRF exposure.

Heterogeneous Photochemistry of Agrochemicals at the Leaf Surface: A Case Study of Plant Activator Acibenzolar-S-methyl.

The photoreactivity of plant activator benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH), commonly named acibenzolar-S-methyl, was studied on the surfaces of glass, paraffinic wax films, and apple leaves. Experiments were carried out in a solar simulator using pure and formulated BTH (BION). Surface photoproducts were identified using liquid chromatography coupled with electrospray ionization and high-resolution Orbitrap mass spectrometry, while volatile photoproducts were characterized using an online thermal desorption system coupled to a gas chromatography-mass spectrometry (GC-MS) system. Pure BTH degraded quickly on wax surfaces with a half-life of 5.0 ± 0.5 h, whereas photolysis of formulated BTH was 7 times slower (t1/2 = 36 ± 14 h). On the other hand, formulated BTH was found to photolyze quickly on detached apple leaves with a half-life of 2.8 h ± 0.4 h. This drastic difference in photoreactivity was attributed to the nature and spreading of the BTH deposit, as influenced by the surfactant and surface characteristics. Abiotic stress of irradiated apple leaf was also shown to produce OH radicals which might contribute to the enhanced photodegradability. Eight surface photoproducts were identified, whereas GC-MS analyses revealed the formation of gaseous dimethyl disulfide and methanethiol. The yield of dimethyl disulfide ranged between 1.5% and 12%, and a significant fraction of dimethyl disulfide produced was found to be absorbed by the leaf. This is the first study to report on the formation of volatile chemicals and OH radicals during agrochemical photolysis on plant surfaces. The developed experimental approach can provide valuable insights into the heterogeneous photoreactivity of sprayed agrochemicals and could help improve dissipation models.

Chloroplastic biosynthesis of melatonin and its involvement in protection of plants from salt stress.

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 recently-discovered 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 Km and Vmax of the purified recombinant MzASMT9 protein for melatonin synthesis were 500 µM and 12 pmol/min·mg protein, respectively. Arabidopsis ectopically-expressing 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 highly-expressed 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.

A robust impact assessment that informs actionable climate change adaptation: future sunburn browning risk in apple.

Climate change impact assessments are predominantly undertaken for the purpose of informing future adaptation decisions. Often, the complexity of the methodology hinders the actionable outcomes. The approach used here illustrates the importance of considering uncertainty in future climate projections, at the same time providing robust and simple to interpret information for decision-makers. By quantifying current and future exposure of Royal Gala apple to damaging temperature extremes across ten important pome fruit-growing locations in Australia, differences in impact to ripening fruit are highlighted, with, by the end of the twenty-first century, some locations maintaining no sunburn browning risk, while others potentially experiencing the risk for the majority of the January ripening period. Installation of over-tree netting can reduce the impact of sunburn browning. The benefits from employing this management option varied across the ten study locations. The two approaches explored to assist decision-makers assess this information (a) using sunburn browning risk analogues and (b) through identifying hypothetical sunburn browning risk thresholds, resulted in varying recommendations for introducing over-tree netting. These recommendations were location and future time period dependent with some sites showing no benefit for sunburn protection from nets even by the end of the twenty-first century and others already deriving benefits from employing this adaptation option. Potential best and worst cases of sunburn browning risk and its potential reduction through introduction of over-tree nets were explored. The range of results presented highlights the importance of addressing uncertainty in climate projections that result from different global climate models and possible future emission pathways.