Physiological and Transcriptomic Analysis of Citrus Fruit Segment Drying under Facility-Forcing Cultivation

Abstract

Facility-forcing cultivation could effectively improve the quality of ‘Beni Madonna’ citrus (Citrus nankao × C. amakusa) and advance the ripening period. However, segment drying (KS) before harvest caused fruit quality deterioration and commodity value loss. In this research, we investigated the physiological and molecular characteristics involved in citrus segment drying under facility-forcing cultivation. The juice yield, sugar, acid, vitamin C, and lignin contents in KS fruits were significantly decreased, and the contents of pectin and cellulose were significantly increased. The relative contents of abscisic acid and abscisic acid glucosyl ester in KS fruits were significantly decreased. A total of 1215 differentially expressed genes (DEGs) were screened by transcriptome sequencing. DEGs were significantly enriched in water metabolism, sugar metabolism, transportation, cell wall and phenylpropanoid biosynthesis, and plant hormone signal transduction. The decrease in water absorption and sugar synthesis, the increase of pectin and cellulose synthesis, and the decrease in ABA accumulation may be the main reasons that cause citrus fruit segments to dry under facility-forcing cultivation.

1. Introduction

Facility-forcing cultivation could promote crop ripening by using greenhouses, mulching film, hot fans, air conditioners, and other facilities. Facility-forcing cultivation alters the external environmental factors, including temperature, light, humidity, and atmosphere, of crop growth, thereby breaking crop dormancy, encouraging early growth, and modifying the phenology period to accelerate fruit maturation. Facility-forcing cultivation is widely used in horticultural crops such as flowers, vegetables, and fruit trees; grapes, jujubes, citrus, strawberries, peaches, bayberries, cherries, and blueberries have all been successfully grown by facility-forcing cultivation [1,2,3].

Citrus fruit is one of the most important fruit crops worldwide. In China, the citrus ripening period is relatively integrated, with most citrus ripening from November to December [4]. A large quantity of ripe citrus fruits causes significant pressure on storage and marketing. To prolong the supply period of fresh fruit, facility cultivation has been widely applied in Zhejiang, China. Facility-forcing cultivation could advance the maturity of citrus effectually, and improve fruit quality, which increases economic benefits for farmers. However, in recent years, segment drying was found in citrus fruits under facility-forcing cultivation in the maturity stage. The occurrence rate of segment drying was as high as 20%. When fruit segment drying occurred, the juice sacs atrophied, the sugar and acid contents dramatically decreased, and the fruits had little commercial value [5].

As a common physiological disorder of citrus, fruit segment drying often occurs both in the maturity stage and post-harvest storage [5], such as guanxi pomelo (Citrus maxima) [6], navel orange (Citrus sinensis) [7], Shatangju (Citrus reticulata) [8,9], and Huyou (Citrus changshanensis) [10]. When segment drying occurred, the fruit weight and water content decreased significantly, the juice sacs were granulated or atrophied, the sugar and acid content decreased, and the edible value was lost [5]. Citrus fruit segment drying is a complex physiological process involving cell development, nutrient metabolism, water absorption, mineral element balance, and plant hormone metabolism [11]. Early studies suggested that nutrient loss of fruit pulp caused by excessive senescence or secondary growth of fruit peel caused juice vesicle granulation and collapse [12]. Recent studies have shown that the metabolic disorder of the pulp cell wall is the primary cause of citrus segment drying [8,11]. For example, studies on navel orange showed that the abnormal metabolism of the fruit cell wall components caused by low temperatures, below 0 °C in winter, led to a large accumulation of pectin and lignin, which was the main reason for segment drying [7]. Studies on pomelo showed that excessive accumulation of cell wall components and hormone metabolism induced juice granulation and segment drying [10]. During the storage process of sweet oranges, the vesicle gradually granulated from the stem to the stylar region, the sugar and acid content decreased significantly, and the pectin, cellulose, and lignin accumulated in large quantities [8]. An excessive accumulation of abscisic acid led to an increased expression of CgMYB58, activating lignin synthesis, which was the main cause of granulation [13].

Plants undergo numerous physiological reactions during growth, development, and abiotic and biotic stress responses. The analysis of physiological indexes can effectively define changes in the plant life cycle. Transcriptome sequencing analysis could qualitatively and quantitatively analyze mRNA during plant growth and development, and abiotic and biotic stress responses, screen differentially expressed genes, and perform functional analysis of genes. Combined physiological and transcriptomic analysis could investigate plant physiological characteristics at both the molecular and physiological levels, revealing transcriptional regulation mechanisms. For example, the integrated physiochemical, hormonal, and transcriptomic analysis revealed the underlying mechanisms for granulation in Huyou fruits [10]. Physiological and transcriptomic analysis shows the potential mechanism of Morinda officinalis response to freezing stress [14]. Physiological and transcriptomic analysis revealed the molecular mechanism of Pinus koraiensis responses to light [15].

‘Beni Madonna’ is a new hybrid citrus of Citrus nankao and C. amakusa with a gorgeous peel, delicious taste, thin segment membrane, soft pulp, and rich aromas, which is favored by consumers and has more significant economic benefits [4,16]. Segment drying has affected the application and technology promotion of facility-forcing cultivation of hybrid citrus. A better understanding of segment drying may guide the prevention of this disorder. In this study, a physiological, including juice yield, water content, total soluble solid content, total acid content, Vc content, soluble sugar contents, citric acid content, propectin content, lignin relative content, and cellulose relative content, and transcriptomic analysis was performed to explore the formation mechanism of segment drying in ‘Beni Madonna’ fruit under facility-forcing cultivation.

2. Materials and Methods

2.1. Materials

The experiment was performed in Chenghu orchard (28.66 N 121.25 E) in Huangyan District, Zhejiang province, China. Huangyan District is the main citrus-producing area in Zhejiang Province, China. Currently, over 3600 hectares of citrus are grown in Huangyan District, with about 300 hectares growing in facilities. There is a wide variety of citrus, including Bendizao, Zaoju, Manju, Zhuju, Beni Madonna, Kanpei, Himekoharu tangor, and Cocktail grapefruit, which are important economic crops in the Huangyan District. The area of the experimental greenhouse is 1440 m², the length is 40 m, the width is 36 m, the span is 8 m, the ridge height is 5 m, and the shoulder height is 3 m. The greenhouse is insulated with a double layer of plastic film. An air-source heat pump system (Sunrain, Lianyungang, China) was used to control the temperature in the greenhouse. The experimental orchard soil is acidic red and yellow soil with a pH value of 5.69 with an organic matter content of 3.14%. The available nitrogen is 77 mg kg⁻¹, the available phosphorus is 160 mg kg⁻¹, the available potassium is 149 mg kg⁻¹, the exchangeable calcium is 242 mg kg⁻¹, and the available magnesium is 67 mg kg⁻¹. A 6-year-old ‘Beni Madonna’ hybrid citrus grafted on trifoliate orange (Poncirus trifoliata) and cultivated in forcing facilities was used for the experimental materials. The segment drying (KS) and normal (CK) fruits were harvested on 15 September 2022. Each treatment contains 3 biological replicates and each replicate contains 10 fruits. The fruits were frozen in liquid nitrogen immediately and stored at −80 °C for later use.

2.2. Determination of Fruit Physiological

The juice yield was measured by weighing after extracting the juice using a juicer. Fruit water content was measured by drying and weighing. The total soluble solid content was measured by using a PAL-1 saccharometer (ATAGO, Tokyo, Japan). The total acid content was determined by using a PLA-Easy ACID1 (ATAGO, Tokyo, Japan). The content of vitamin C was determined by the titration of 2, 6-dichlorophenol indophenol [17]. The contents of sucrose, glucose, fructose, and citric acid were determined by high-performance liquid chromatography [18]. The contents of pectin, cellulose, and lignin were detected after freeze-drying juice sacs and using an assay kit (Beijing Boxbio Science & Technology Co., Ltd., Beijing, China) according to the manufacturer’s instructions, and three biological experimental repeats were performed.

2.3. Plant Hormone Assay

The juice sacs of KS and CK citrus fruits were frozen with liquid nitrogen and ground into powder. About 50 mg samples were weighed (3 biological replicates were performed for each sample) and transferred into the centrifuge tubes. A total of 1 mL of extraction solution (methanol–water–formic acid = 15:4:1, volume ratio), and 10 μL internal standard mixed solution were added into the tubes. The mixture was swirled for 10 min, centrifuged for 5 min (12.000 r min⁻¹, 4 °C), the supernatant was transferred into clean plastic microtubules, evaporated and dried with nitrogen, dissolved in 100 μL 80% methanol (volume ratio), filtered through a 0.22 μm membrane filter, and an LC-MS/MS analysis was performed. The analysis was performed by Wuhan Metville Biotechnology Co., Ltd. (Wuhan, China). The different accumulation hormones were screened using the online OmicShare tools (http://www.omicshare.com/tools, accessed on 6 June 2024) through OPLS-DA analysis according to rules of fold change ≥ 2 or fold change ≤ 0.5, a variable important for the projection ≥ 1, and p ≤ 0.05.

2.4. RNA-Seq and Fluorescence Quantitative Real-Time PCR Analysis

The total RNA was extracted from juice sacs of KS and CK citrus fruits by using a plant total RNA extraction kit (Takara, Dalian, China). Each treatment contained three biological replicates and each biological replicate was a mixture of juice sacs from 3 fruits. The RNA degradation and contamination were monitored by 1% agarose gel electrophoresis. The RNA purity was checked using the NanoPhotometer^® spectrophotometer (IMPLEN, Westlake Village, CA, USA). The sequencing Library was constructed with NEBNext^®UltraTM RNA Library Prep Kit (NEB, Ipswich, USA). The sequencing was completed by Matwell Biotechnology Co., Ltd. (Wuhan, China). The clean reads were mapped to the citrus genome (http://citrus.hzau.edu.cn/download.php, accessed on 28 December 2022). The detailed RNA-Seq data analysis process was performed according to previous research [19]. Nine differentially expressed genes were randomly selected to verify the transcriptome data by fluorescent quantitative PCR (qRT-PCR). β-actin was used as the internal reference gene [20]. The qRT-PCR primers were designed with Primer 5.0 and the sequence information are listed in Supplementary Table S1. The fluorescence quantitative PCR methods were followed from previous research [19,21].

2.5. Data Analysis

All data are shown as mean ± standard error (SE). GraphPad Prism 8 software was used for plotting. The heatmaps were drawn using TBtools-II (v2.096) [22]. The tests for normality and homogeneity of variances of physiological and gene expression data were analyzed using SPSS 13.0. The significant differences were analyzed using SPSS 13.0 with the t-test (LSD) at the 0.05 level.

3. Results

3.1. Effect of Segment Drying on Citrus Fruit Quality

The juice sacs of CK fruits were full and bright orange, while the juice sacs of KS fruits were atrophied and dark (Figure 1). The juice yield, contents of total soluble solids, total acid, and vitamin C in KS fruits were 40.52%, 7.6%, 0.29 mg (100 mL)⁻¹, and 15.2 mg (100 mL)⁻¹, respectively, which decreased by 37%, 33%, 29%, and 30% compared with CK. The contents of sucrose, glucose, fructose, and citric acid in KS fruits were 26.0 mg g⁻¹, 22.9 mg g⁻¹, 25.8 mg g⁻¹, and 6.5 mg g⁻¹, respectively, which decreased by 36%, 20%, 15%, and 51% compared with CK. The contents of pectin and cellulose in KS fruits were 126 mg g⁻¹ and 7.7%, respectively, which increased by 14% and 135% compared with CK (Figure 2). Lignin content in KS fruits was 38%, which decreased by 57% compared with CK.

3.2. Effect of Segment Drying on Hormone Content of Citrus Fruit

HPLC-MS/MS was used to analyze the hormone contents between KS and CK citrus fruits. A total of 66 phytohormones, including abscisic acid, auxin, cytokinin, ethylene, gibberellin, jasmonic acid, salicylic acid, and strigolactone, were detected between KS and CK citrus fruits (Supplementary Table S2). OPLS-DA analysis indicated that the fruit hormone content of these two groups could be divided into two categories (Figure 3A). The relative contents of Abscisic acid (ABA) and ABA-glucosyl ester (ABA-Ge) in KS fruits were 73.82 and 468.54, respectively, which were significantly lower than those in CK fruits (Figure 3B).

3.3. Transcriptome Analysis on Segment Drying of Citrus Fruit

Transcriptome sequencing was performed on KS fruit and CK fruit using RNA-seq. A total of 43~50 M clean reads were obtained from six libraries, and the percentage of Q30 bases in each sample was greater than 90%. More than 91% of reads were mapped to the reference genome (Supplementary Table S3). The gene expression of KS and CK was obviously divided into two groups by principal component analysis (Figure 4A). A total of 1215 differentially expressed genes (DEGs) were screened by transcriptome sequencing. Compared with CK, 740 genes were up-regulated and 475 genes were down-regulated in KS fruit (Figure 4B, Supplementary Table S4). GO analysis showed that DEGs were mainly enriched in the cell wall macromolecule metabolic process, cellular polysaccharide metabolic process, hemicellulose metabolic process, phenylpropanoid metabolic process, response to salicylic acid, secondary metabolic process, xyloglucan metabolic process, disaccharide transmembrane transporter activity, glucosyltransferase activity, oligosaccharide transmembrane transporter activity, polysaccharide binding, transferase activity, transferring hexosyl groups, water channel activity, water transmembrane transporter activity, and xyloglucan: xyloglucosyl transferase activity (Figure 5).

3.4. Heat Map Analysis of Important Metabolic Pathway Genes in Segment Drying Fruits

Aquaporins are membrane channels that facilitate the transport of water and small neutral molecules, which are involved in water absorption and transport in plants. The juice yield of KS fruit was significantly lower than that of normal fruit, so the expression of the aquaporin gene was analyzed. In this study, nine genes encoding aquaporin were differently expressed, of which seven genes, including NIP1-2, PIP1-2, PIP1-5, PIP2-1, PIP2-2, PIP2-2L, and TIP2-1, were down-regulated in KS fruit (Figure 6A). Due to the significant reduction of soluble solid content in KS fruits, the analysis of gene expression of glucose and acid metabolism showed that 20 differentially expressed genes were significantly enriched in the metabolic pathways of starch and sucrose, of which 4 genes were up-regulated and 16 genes were down-regulated (Figure 6B). The expression levels of SUS and SPS, the key genes of sucrose synthesis, were significantly lower in KS fruit than in control. At the same time, the analysis of the transporter genes in the process of sugar accumulation in fruits showed that seven genes encoding SWEET protein were differentially expressed, among which the expression levels of SWEET7, SWEET12, SWEET14, SWEET15, and SWEET16 in the degradation of KS fruits were significantly lower than those of the control (Figure 6B). Pectin is an important component of the cell wall, and 15 genes related to pectin metabolism are differentially expressed, among which 3 genes are up-regulated in KS fruit and 12 genes are down-regulated in KS fruit (Figure 6C). Lignin is an important branch of phenylpropyl metabolism, and 32 genes related to phenylpropyl metabolism are differentially expressed, of which 10 genes are up-regulated in KS fruits and 21 genes are down-regulated in KS fruits (Figure 6D). Cellulose and hemicellulose are also important components of the cell wall. A total of 30 genes related to the metabolism of cellulose and hemicellulose are differentially expressed, of which 5 genes are up-regulated and 25 genes are down-regulated in KS fruits (Figure 6E). Plant hormone signaling plays a key regulatory role in the growth and development of citrus fruits. A total of 50 genes related to hormone signaling were found to be differentially expressed between KS and CK fruits, with 23 up-regulated and 27 down-regulated in KS fruits (Figure 6F).

3.5. qRT-PCR Analysis of Different Expressed Genes in Segment-Drying Fruits

To verify the reliability of transcriptome data, nine differentially expressed genes were randomly selected for expression analysis by fluorescent quantitative PCR. The results showed that the results of transcriptome showed a similar trend to those of fluorescent quantitative PCR (Figure 7), indicating that the transcriptome data were reliable.

4. Discussion

Segment drying is a common physiological disease of citrus fruits, which usually occurs during maturation and storage. Its typical symptoms are the granulation or atrophy of juice, reduced water content, sugar and acid content of pulp, and partial or complete loss of edible ability [8]. The formation of segment-drying symptoms in citrus fruits involves a large number of biological processes, including sucrose and organic acid metabolism [23], pectin synthesis and structural changes [24,25], lignin synthesis [26,27], and hormone synthesis and signal transduction [7,10]. In this research, the contents of soluble sugar and citric acid in KS ‘Beni Madonna’ fruits were significantly lower than those in CK fruits and the contents of pectin and cellulose were significantly lower than those in CK fruits, which was consistent with previous research results in sugar tangerine [28], pomelo [10], hybrid citrus [27] and navel orange [7]. The lignin content of ‘Beni Madonna’ fruit in segment drying was significantly lower than that of normal fruits, which is contrary to previous studies, indicating that the changes in the cell wall components of ‘Beni Madonna’ fruit in cultivation were different from those of other citrus varieties. The fruit ripening period of the ‘Beni Madonna’ was in mid-September, when the summer temperature was high, so it is speculated that the segment drying of the fruit might have been caused by the high temperature. Transcriptome sequencing was further used to analyze the gene expression of the KS and CK fruits of the cultivated ‘Beni Madonna’. A total of 1215 genes were found to be differentially expressed, including 475 genes expressed in strips and 740 genes expressed down-regulated. The differentially expressed genes were mainly concentrated in many biological processes and molecular functions, such as phenylpropanoid biosynthesis, plant hormone signaling, glucose metabolism and transport, cell wall macromolecular substance metabolism, and aquaporin, which indicated that these metabolic reactions of KS fruit had significantly changed. Previous research also showed that activation of the phenylpropanoid pathway caused the collapsed vesicles during segment drying in Citrus sinensis [26], Huyou [10], and Shatangju [28].

The most typical symptom of segment drying is the decrease in fruit water content. In navel oranges, it was found that the water content of segment-drying fruit decreased by 11% [7]. In this study, there was no significant difference in absolute water content between the segment drying and the normal fruit, but the juice yield of the KS ‘Beni Madonna’ decreased by 37% indicating that the mobile water in juice sacs may be fixed by other substances. Aquaporins are a kind of membrane intrinsic proteins widely present in the intimal system of plant membranes and vacuoles, which are involved in water balance and stress response [29,30]. The expression of the gene encoding aquaporin was significantly down-regulated in the late storage stage of orange. Overexpression of CsNIP5 increased the accumulation of osmotic regulators in citrus fruits and enhanced the resistance of citrus to water loss [31]. In this study, nine genes encoding aquaporins were differentially expressed between segment drying and normal fruits, and seven genes were significantly down-regulated in KS fruits. The blocking of water transport into juices during fruit ripening may be an important reason for the formation of segment drying in fruits of ‘Beni Madonna’ cultivated in facilities. At the same time, high temperatures at maturity will destroy plant membrane structure, resulting in the leakage of cell fluid [32], which may also lead to the atrophy of juice sacs.

The contents of total soluble solid and soluble sugar in segment drying fruits were significantly lower than normal fruits. In this study, compared with the control, the total soluble solid content of KS fruits was reduced by 33%, and the contents of sucrose, fructose, and glucose were also significantly reduced. Enrichment analysis also found that 20 differentially expressed genes were enriched in starch and sugar metabolism pathways, and 16 of them were down-regulated. SUS and SPS play a crucial regulatory role in the sucrose synthesis of citrus fruits [32,33]. In this study, the expression levels of SUS6 and SPS4 in the KS fruits of ‘Beni Madonna’ were significantly lower than those of CK fruits, indicating that the obstruction of sucrose synthesis in KS fruits may be an important reason for the low content of soluble solids. The SWEET gene encodes a class of higher plant sugar transporter, which has the transport activity of glucose, fructose, and sucrose and is involved in the accumulation of sugar during the development of fruits [34]. Studies on sweet oranges found that the expression level of SWEET15 in high-sugar varieties was significantly higher than that in low-sugar varieties [35]. In this study, seven genes encoding the SWEET protein were differentially expressed between KS and CK fruit, among which six genes were significantly down-regulated in KS fruit, and SWEET15 had the highest FPKM value. This suggests that the low expression of SWEET15 may be the main reason for the low sugar content and tasteless flavor of the fruit.

Pectin, cellulose, hemicellulose, and lignin were the main components of the citrus juice sac wall. Numerous genes related to pectin synthesis and degradation were differently expressed in segment-drying Huyou fruit [10]. In this study, the pectin content in KS fruit was significantly higher than in CK. The increased pectin content can stabilize the mobile water in the juice sacs [24], which might cause a decrease in juice yield. A total of 19 genes related to pectin metabolism, including the genes encoding polygalacturonase, galacturonosyltransferase, pectin acetylesterase, and pectate lyase pectinesterase inhibitor were differentially expressed between KS and CK fruit. Cellulose and hemicellulose were important components of cell wall morphogenesis. It was found that the up-regulated expression of 11 genes encoding cellulose synthase in segment-drying pomelo resulted in higher cellulose content than normal fruits [10]. In this study, 30 genes related to the metabolism of cellulose and hemicellulose are differentially expressed, including the genes encoding cellulose synthase, xyloglucosyl transferase, xyloglucan O-acetyltransferase, endoglucanase, and galacturonosyltransferas. The differential expression of these genes may be an important reason for the abnormal accumulation of cellular cellulose in citrus juice sacs leading to fruit segment drying. The massive synthesis of pectin and cellulose in KS juice sacs resulting in a decrease in sugar and acid content. A large number of studies have shown that lignin accumulation caused by phenylpropanoid metabolism disorder is the key factor of citrus segment-drying granulation. CAD and POD were key enzymes in the synthesis and polymerization of the lignin monomer. PAL is a key enzyme and rate-limiting enzyme in the lignin metabolic pathway and initiates the phenylpropane metabolic pathway [36]. Overexpression of PAL in Arabidopsis Thaliana significantly increased the lignin content in transgenic plants [37]. The inhibition of 4CL activity resulted in a decrease in lignin content [38]. Studies on navel orange, Dayagan, and pomelo showed that excess lignin accumulates in the juice sacs, resulting in segment drying, and lignin synthesis-related genes were highly expressed in segment-drying citrus fruits [7,10,26,27]. In our research, the lignin content in KS ‘Beni Madonna’ juice secs was significantly lower than that of CK. RNA-seq results showed that two PAL, one 4CL, four CAD, and six PODs were down-regulated in KS fruits, indicating that lignin synthesis was inhibited in ‘Beni Madonna’ KS fruits. These results showed that the lignin metabolism in segment drying of ‘Beni Madonna’ fruit was different from previous research. Lignin is an important component of the cell wall and plays an important role in enhancing the mechanical strength of cells [36]. Reducing the accumulation of lignin in the KS and inferior fruits of cultivated ‘Beni Madonna’ may block cell wall synthesis and reduce cell mechanical strength, which will lead to the atrophy and KS of juice sacs.

Plant hormones play a crucial regulatory role in citrus fruit development and maturation [39]. Several studies showed that the occurrence of segment drying in citrus was related to changes in phytohormones. In this study, 51 genes of plant hormone signaling pathways were differentially expressed between KS and CK fruits. This suggests that the disturbance of hormone signaling may be closely related to the segment drying of ‘Beni Madonna’ fruit under facility-forcing cultivation. ABA is a key hormone regulating the development and ripening of non-respiratory jump fruits such as citrus [35], and mutations in ABA synthesis and signal transduction genes can lead to inhibition of citrus fruit ripening [40]. The ABA content of granulated segment-drying fruits, such as navel orange and pomelo, was significantly higher than that of normal fruits [7,10], and studies on pomelo also found that exogenous ABA treatment accelerated the granulation of juice sacs [13]. In this study, the ABA content of KS fruits was significantly lower than that of CK fruits, indicating that ABA had opposite regulatory effects on the formation of two types of segment drying. The decrease in ABA content in ‘Beni Madonna’ fruit may be caused by the catabolism and inactivation of ABA induced by high temperatures in the facility, and similar reports have also been reported on cherries [41].

5. Conclusions

The ‘Beni Madonna’ citrus fruits under facility-forcing cultivation showed symptoms of segment drying. The juice yield and the contents of sucrose, glucose, fructose, citric acid, and vitamin C in KS fruit were significantly lower than those in CK fruits. The pectin and cellulose contents in KS fruits were significantly higher than those in CK fruits, but the lignin content was significantly reduced. The ABA and ABA-Ge contents in KS fruits were also significantly lower than those in CK fruits. Numerous genes related to cell wall synthesis, water metabolism, sugar metabolism, and plant hormone signaling were differentially expressed between KS and CK fruits. The disturbance of these substances’ synthesis resulted in the segment drying of ‘Beni Madonna’ citrus fruits, which might be caused by improper environmental factors under facility-forcing cultivation. More mechanisms of the complex regulatory network in the process of segment drying under facility-forcing cultivation remain to be explored in future studies.