Abstract

   Exogenous melatonin (MT) is widely used in fruit preservation, and can
   increase the storage time and delay the quality deterioration. Firstly,
   it was found that 150 μM MT was the optimal concentration to treat
   ‘Xinli No.7’ under storage at 4 °C for 60 days. MT could significantly
   improve oxidase activity and inhibit the reduction of physiological
   indexes, including pulp hardness, weight loss, titratable acid and
   soluble solid content. MT could also reduce ethylene release and limit
   the reduction of fruit aroma. The average content of fruit aroma
   substance increased by 43.53%. A relevant RNA-Seq database was built to
   further explore the regulation mechanism of MT. A total of 2,761
   differentially expressed genes (DEGs) were identified. DEGs were
   enriched in 64 functional groups and 191 Kyoto Encyclopedia of Genes
   and Genomes (KEGG) pathways. DEGs were mainly enriched in
   alpha-linolenic acid metabolism, fatty acid metabolism and plant
   hormone signal transduction pathway. The gene pycom09g05270 belonging
   to long chain acyl-CoA synthetase family and participating in fatty
   acid metabolism pathway was identified, and its expression level was
   consistent with fragments per kilobase per million mapped reads (FPKM)
   values, implying that pycom09g05270 might play a vital role in
   maintaining quality during the storage process.

   Keywords: Pear, Melatonin, Aroma

Introduction

   Pear (Pyrus) is one of the three largest deciduous fruit trees in the
   world, and fruit aroma is one of the important indexes to evaluate the
   quality of pear fruit and determine consumers’ preference. Occidental
   pear varieties have richer fruit aromas as compared to Oriental
   varieties. The fruit aroma and storage are affected by some
   irreversible physiological and biochemical reactions occurring inside
   the fruit along with fruit ripening and development ([38]Jia et al.,
   2018). On the other hand, the pear fruit aroma would become weak under
   low temperature storage, which leads to a decrease in the edible
   quality and commercial value.

   Exogenous melatonin (MT) (N-acetyl-5-methoxytryptamine) can limit the
   decrease in fruit aroma during storage and prolong the storage time. MT
   is a kind of indole small molecule substance which is widely spread in
   animals and plants ([39]Jang, Zu & Center, 2015; [40]Wang, Yang & Li,
   2016; [41]Jemima, Bhattacharjee & Singhal, 2011), and was discovered in
   the pineal gland of cattle. Initial research held that the substance
   only existed in animals and humans. Until 1995, [42]Hattori, Migitaka &
   Iigo (1995) found that MT was also present in some plants, such as
   wheat and corn. Afterwards, MT was discovered in roots, leaves and
   flowers of plants ([43]Hattori, Migitaka & Iigo, 1995).

   Studies have shown that MT can scavenge reactive oxide species (ROS)
   ([44]Lei, Wang & Tang, 2013; [45]Pieri, Moroni & Marra, 1995), regulate
   fruit maturity and senescence ([46]Gao, Zhang & Chai, 2016; [47]Xin, Si
   & Kou, 2017; [48]Hu, Li & Rao, 2018) and improve stress resistance
   ([49]Lei et al., 2010; [50]Jia et al., 2019). In addition, MT has also
   been proven to have a better preservation effect on fruits and
   vegetables. In peach, MT treatment could reduce decay rate, delay
   senescence and maintain total soluble solids and ascorbic acid content
   during storage ([51]Gao, Zhang & Chai, 2016). A total of 0.2 mM MT
   could delay ripening, maintain and improve quality of mangoes via
   inhibiting hydrolytic enzymes and enhancing the antioxidant system
   ([52]Awad & Al-Qurashi, 2021). In the apple ripening process, the
   malondialdehyde (MDA) content was negatively correlated with the MT
   content. This may be due to the fact that MT can remove the ROS
   produced by the respiratory jump to maintain the redox balance in the
   cell ([53]Lei, Wang & Tang, 2013). Additionally, MT could regulate ROS
   signaling followed by activation of the calcineurin B-like
   1-interacting protein kinases 23 (CIPK23) pathway to regulate the
   expression of the potassium channel protein gene, which then promotes
   K^+ absorption ([54]Li et al., 2016). MT could inhibit ethylene
   biosynthesis to alleviate O[3] stress in grape leaves ([55]Liu et al.,
   2021).

   The ability of MT to scavenge radicals is higher than that of vitamin
   E, ascorbic acid and glutathione, and it is an efficient redox balance
   agent ([56]Pieri, Moroni & Marra, 1995). In cucumber, MT could inhibit
   the respiration rate and reduce ethylene release, MDA content and
   active oxygen content during storage ([57]Xin, Si & Kou, 2017). The
   carrot suspension cell line treated with MT could better maintain the
   function and integrity of the cell membrane under low temperature
   stress ([58]Jia et al., 2019). A total of 100 μmol·L^−1 exogenous MT
   treatment could alleviate the decrease in chlorophyll, ascorbic acid
   (Vc), titratable acid and soluble protein contents, inhibit membrane
   lipid peroxidation and maintain membrane function and integrity of
   cucumber after harvest ([59]Xin, Si & Kou, 2017). In cabbage tissues
   stored at low temperature after harvest, MT could increase the activity
   of superoxide dismutase (SOD) and peroxidase (POD), reduce MDA
   accumulation, delay chlorophyll degradation and maintain soluble sugar
   and soluble protein contents in stem and leaf tissues ([60]Jia et al.,
   2019). In plum fruits, suitable concentration of MT treatment could
   keep the contents of fruit soluble solids and titratable acid stable
   and ensure the fruit flavor and quality ([61]Feng et al., 2020). MT
   could inhibit phenolic compound metabolism and improve antioxidant
   capacity to delay the surface discoloration of fresh-cut Chinese water
   chestnuts and prolong the shelf life ([62]Xu et al., 2022).

   MT can induce ethylene synthesis and signal transduction, inhibit the
   accumulation of free radicals and membrane lipid peroxidation and
   promote fruit maturity and delay fruit senescence. However, research on
   the effect of MT on pear fruits are few and mainly focus on the
   quality. MT can participate in antioxidant and antibacterial process in
   ‘Huangguan’ pear during storage. [63]Liu (2019) studied the effects of
   100 µmol·L^−1 MT on the softening and senescence of three Western pear
   cultivars after low temperature storage and further analyzed aroma
   components change of ‘Korla pear’ and ‘Abate’. However, the suitable
   concentration of MT treatment in the process of low temperature storage
   was not explicably stated.

   Until now, the molecular mechanism of MT in regulating pear fruit aroma
   is still unclear. In this study, six MT concentrations were used to
   treat ‘Xinli No.7’ fruits before storage at low temperature. The pulp
   hardness, weight loss rate, soluble solid content, titratable acid,
   MDA, POD, SOD, ethylene release rate, fruit aroma content and aroma
   components were measured at different storage days. Treating the fruits
   of ‘Xinli No.7’ with 150 µmol·L^−1 MT can significantly maintain the
   physiology quality of pear and delay aroma decrease during low
   temperature storage. Gene expression types and abundance of MT-treated
   pear fruits during low temperature storage were further analyzed using
   digital expression profiling technology. Hence, the key genes involved
   in regulating aroma production under low temperature were identified
   and the molecular mechanism of MT in the regulation of fruit aroma was
   explored.

Materials and Methods

Experimental materials

   ‘Xinli No.7’ is one of the filial generations of ‘Korla pear’ ×
   ‘ZaoSu’, which belongs to P. sinkiangensis and is a new early-maturing
   pear variety. Additionally, the scientific name is P. sinkiangensis
   Xinli No.7.

   Xinli No.7 fruits were collected from Tianping Lake Experimental
   Demonstration Base of Shandong Institute of Pomology. The pear fruits
   with uniform maturity, uniform size and without mechanical damage were
   selected and divided into six groups. Six concentrations (0, 50, 100,
   150, 200 and 250 µmol·L^−1) of MT solution were set to treat six pear
   groups. The pears were immersed in MT solution for 30 min, and then
   dried in natural conditions and stored at 4 °C. Afterwards, the
   physiological indicators were determined at 20, 40, 60 and 80 days
   after storage. Additionally, the MT was purchased from Solarbio
   (Cas:73-31-4) and dissolved with dimethyl sulfoxide (DMSO).

Analysis of physiological indexes

   The fruits treated with different MT concentrations and stored at 4 °C
   were taken out every 20 days to measure physiological data and enzyme
   activity.

   Pulp hardness was measured using a GY-4 hardness tester. The skin was
   peeled off the carcass of the fruit, and the hardness of the sunny side
   and the dark side of each fruit was measured.

   The weight loss rate of pear under treatment with MT and storage at 4
   °C were calculated using the following formula: weight loss rate (%) =
   (initial weight − post storage weight)/initial weight × 100. Firstly,
   the weight of MT-treated pear before storage was recorded, and the same
   pears were taken after storage for 20, 40, 60 and 80 days to weigh and
   record the weight. Then, the weight loss rate was calculated.

   The soluble solid contents of pear were measured using ATAGO-PAL-Q type
   digital refractometer. The detection mirror was cleaned with distilled
   water, then adjusted to zero for calibration, and the detection mirror
   was dried with lens wiping paper. Squeezed juice was dropped on the
   detection mirror of the refraction instrument for measurement, and the
   value on the digital display device was read. Each treatment was
   repeated three times.

   The titratable acid content of fruits was determined using the sodium
   hydroxide titration method. Firstly, 5 g pulp was weighed and grinded
   into homogenate in a mortar, and transferred into 50 ml conical bottle.
   The solution was placed in a water bathed for 30 min at 80 °C, and then
   cooled to room temperature. Afterwards, the solution was centrifuged at
   10,000 g for 10 min, and the precipitation was removed. The supernatant
   was added into a 50 ml volumetric flask and then distilled water was
   added into it until it reached 50 ml. The supernatant solution was
   blended, and 10 ml supernatant solution was taken for determination of
   acid content. Total of ~3–5 drops of phenolphthalein indicator were
   added into the 10 ml supernatant solution, and 0.10 mol·L^−1 NaOH
   standard liquid was added until the solution turned red and did not
   fade for 30 s. Each treatment was repeated three times. The titratable
   acid content of pear under treatment with MT and storage at 4 °C was
   calculated using the following formula: titratable acid content (%) =
   (C × V[1] × k × V[T])/m × V[S]. C, V[1], k, V[T], m and Vs represent
   concentration of NaOH standard solution (mol·L^−1), the volume of NaOH
   standard liquid (ml), organic acid conversion factor, constant volume
   after extraction (ml), sample quality (g) and volume of sample solution
   used for titration (ml), respectively.

Analysis of enzyme activity

   Firstly, 2 g pulp was weighed and put into a precooled mortar. A total
   of 5 ml pre-cooled 100 mmol· L^−1 phosphoric acid buffer with pH 7.0
   was added into the mortar, and then, a little of 1%
   polyvinylpyrrolidone (PVP) and a small amount of quartz sand were
   added. The homogenate was ground in an ice bath and centrifuged at
   10,000 rpm and 4 °C for 20 min. The supernatant was the crude enzyme
   extract for determination of malondialdehyde content and antioxidant
   enzyme activity.

   MDA content was measured using the thiobarbituric acid (TBA) method.
   The solution of 0.6%TBA (2 ml) was added to the supernatant (2 ml) and
   the water was used as references, respectively. The solution was mixed