Inﬂuence of Citrus Rootstocks on Scion Growth, Hormone Levels, and Metabolites Proﬁle of ‘Shatangu’ Mandarin ( Citrus reticulata Blanco)

: Dwarﬁng rootstocks are a valuable genetic resource for managing high-density plantations. The selection of the appropriate scion/rootstock combination is key to improving crop performance and sustainable production in a particular environment and speciﬁc training systems. ‘Shatangju’ mandarin scion cultivar grafted onto ‘Flying Dragon’ rootstock tends to be dwarﬁng and develops short stature plants. To obtain insight into potential mechanisms underlying rootstock-induced dwarﬁng effects, we conducted a rootstock trial to examine the inﬂuence of 11 different rootstocks based on their growth vigor, antioxidants, and hormonal levels of the scion cultivar. The phenotypic observations revealed that size reduction in the ‘Flying Dragon’ rootstock is due to lower node number, shorter internodal length, and a reduced trunk diameter of the scion compared with more vigorous rootstocks. Antioxidant analysis showed that ‘Shatangju’ mandarin grafted onto ’Flying Dragon’ and ‘Trifoliate Orange’ rootstock had signiﬁcantly lower peroxidase (POD) activity than other tested rootstocks. The hormonal analysis indicated that there were markedly lower amounts of abscisic acid (ABA) in ‘Shatangju’ mandarin grafted with ‘Flying Dragon’ rootstock. In addition, trees grafted with ‘Sour Pummelo’ and ‘Flying Dragon’ depicted minimum amounts of gibberellins (GA 24 ). Moreover, several metabolites associated with organic acids, ﬂavonoids, amino acids, and alkaloids responded differently in plants grafted with ‘Flying Dragon’ (dwarﬁng) and ‘Shatang Mandarin’ (vigorous) rootstocks. This study concluded that ‘Flying Dragon’ rootstock with a strong dwarﬁng effect has been proposed to improve high-density cultivation methods. These ﬁndings will provide useful insights for future research associated with rootstock-mediated dwarﬁng mechanisms of citrus rootstocks.


Introduction
Citrus fruits are widely grown worldwide in tropical and subtropical regions [1,2]. China has taken a prominent position in the citrus industry worldwide, with about 41 million tons of production spread over 2.7 million hectares of cultivation area [3]. These 'Shatangju' citrus grafted onto 11 different rootstocks, aiming to provide detailed information about dwarfing features that could help them to choose rootstocks.

Plant Materials and Culture Conditions
The experiment was conducted in Guangzhou, China (Latitude 23 •  The plants were drip irrigated and exposed to conventional practices. The experiment was carried out in Randomized Complete Block Design (RCBD), and three biological replicates were made (every replicate containing two plants). A total of 66 plants were used in this study.

Measurement of Plant Growth Parameters
From March 2019, measurements were recorded concerning scion growth, which included plant height (cm), the diameter of rootstock (mm), the diameter of the scion (mm), apex shoot length (cm), average internodal length (cm), and node number and number of branches for each graft combination. (1) The plant height was measured with a measuring tape and expressed in cm. (2) Crown width was measured with a measuring tape in two directions (east-west and north-south), respectively. (3) The diameter of the trunk was measured with a Digital Vernier Caliper at three different points: the scion stem (3 cm above the graft union) and rootstock stem (3 cm below the graft union). (4) The number of nodes was calculated by counting the number of nodes on shoots above the graft union. (5) The internodal length was calculated by dividing the length of the scion by the total number of nodes. (6) The number of mature branches was calculated by the counting method on the trunk that can produce shoots.

Determination of Soluble Sugars Contents
The concentration of soluble sugar was analyzed using the anthrone method, as explained earlier by Shi et al. [35]. Then, 0.1 g of ground samples were combined with 2 mL of ethanol 80% (v/v) at 80 • C for 30 min. Subsequently, 100 µL of extracts were added to 2 mL of anthrone, and the mixture was heated for 10 min. At 630 nm, the absorbance was measured, and the concentration was determined by deploying a calibration curve with sucrose standard as a point of reference in the calculation.

Determination of Soluble Protein Contents
The content of soluble protein was estimated using the procedure of Coomassie brilliant blue G-250 as followed in the study of Yang et al. [36].

Determination of Enzymatic Antioxidant Activities
A total of 0.5 g of leaf tissue was mixed in 5 mL of extraction buffer (phosphate buffer, pH 7.5, containing 0.1 mM EDTA and 4% polyvinylpolypyrrolidone). The mixture was centrifuged at 12,000× g for 20 min, and to determine antioxidant enzymes, the supernatant was used [37]. The enzymatic antioxidant activity such as peroxidase (POD) and superoxide dismutase (SOD) was determined according to the methods described by Chen and Wang [38].

Determination of Endogenous Hormones Analysis
The endogenous hormones such as auxins, cytokinins, abscisic acid, 1-amino-cyclopropane-1-carboxyic acid (the ethylene precursor), salicylic acid, jasmonic acid, and gibberellins from the leaf samples were extracted using ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UPLC/ESI-MS/MS) according to the method explained by [39]. The hormone quantification was estimated using a standard curve method and expressed as ng g −1 fresh weight.

Statistical Analysis
All data were subjected to analysis of variances by using SPSS 18.0 Statistics (SPSS Inc., Chicago, IL, USA) for correlation analysis and analysis of variance (ANOVA) to perform the statistical analysis. The differences among treatment means were evaluated by least significant difference (LSD) multiple comparison tests at p ≤ 0.05, and different lowercase letters were used to represent significant differences among treatments. The experimental result correlation map was produced by Origin 2019, Tbtools, and Hiplot web pages, and the picture-related layout was completed in Adobe illustrator 2020 and In Design 2020.

Effect of Rootstocks on Scion Growth
Rootstocks influenced the growth vigor of grafted citrus trees ( Table 1). The morphological traits, including plant height (cm), crown width (cm), number of nodes, average internodal length (cm), the diameter of rootstock (mm), and the diameter of the scion (mm), were found to be significantly unlike. 'Shatangju' scion cultivar grafted onto 'Flying Dragon' rootstock had the lowest plant height (75.67 cm) and weakest growth vigor ( Table 2). Stronger growth vigor and maximum plant height were obtained for 'Shatangju' scion cultivar grafted onto 'Shatang Mandarin' (181.86 cm) and 'Sour Orange' (142.77 cm) rootstocks. Trees grown onto 'Flying Dragon' rootstocks produced a shorter internodal length (4.62 cm), while the 'Shatang Mandarin' rootstock resulted in the most extended internodal length (24.23 cm). Moreover, lower values of crown width were recorded with 'Red Limonia', 'Trifoliate Orange', and 'Flying Dragon' rootstocks, especially compared to other selected rootstocks. Trees grafted onto 'Shatang Mandarin' rootstocks produced longer values of trunk diameter of the scion, trunk diameter of rootstock, and apex shoot length, whereas the 'Trifoliate Orange' and 'Flying Dragon' rootstocks resulted in the smallest values of all these morphological traits (2.13 cm).

Effect of Rootstocks on Soluble Sugar and Soluble Protein Contents
The data regarding the soluble sugar and protein contents of 'Shatangju' mandarin scion grafted onto different rootstocks showed dissimilarly ( Figure 1). The soluble sugar contents of 'Shatangju' mandarin scion grafted onto 'Red Tangerine' rootstock was significantly lower compared with other tested rootstocks. In contrast, higher soluble sugar contents were found in plants grafted with 'Trifoliate Orange' rootstock ( Figure 1A). Further, we found that there were no significant differences between 'Shatang Mandarin', 'Goutou Orange', 'Red Limonia', and 'Flying Dragon' rootstocks. In terms of soluble protein contents, the 'Citrange' rootstock was markedly higher, followed by the 'Flying Dragon' rootstock, whereas 'Xiangcheng Orange' rootstocks displayed low values of soluble protein contents ( Figure 1B).

Effect of Rootstocks on Enzymatic Antioxidant Activities
In this study, rootstock behaved differentially regarding enzymatic antioxidant activities in the leaves of the 'Shatangju' scion cultivar ( Figure 2). The leaf SOD activity of the 'Shatangju' scion cultivar grafted with the 'Trifoliate Orange' rootstock was significantly lower than that of the other rootstocks. In contrast, maximum SOD values were recorded with the 'Sour Orange' rootstock ( Figure 2A). Regarding POD activity, the result relative to 'Rough Lemon', 'Sour Orange', and 'Citrange' rootstocks showed noticeably higher values than other tested rootstocks ( Figure 2B). In contrast, minimum values of POD activity were obtained with 'Trifoliate Orange' and 'Flying Dragon' rootstocks.

Endogenous Hormone Levels
'Shatangju' citrus scion cultivar grafted with a range of size-controlling rootstocks varied in leaf auxin contents ( Figure 3). The contents of TRA were higher for 'Shatangju' scion grafted onto 'Flying Dragon' and 'Trifoliate Orange' rootstocks, whereas minimum TRA levels were recorded for plants grafted onto 'Shatang Mandarin' and 'Goutou Orange' rootstocks ( Figure 3A). Regarding TRP and MEIAA, the result relative to the 'Trifoliate Orange' rootstock was significantly high, whereas other rootstocks displayed low values ( Figure 3B,F). Similarly, the IAA-Trp contents showed the highest with 'Flying Dragon' and 'Rough Lemon' rootstocks, whereas 'Goutou Orange' rootstock produced lower IAA-Trp contents ( Figure 3C). The contents of IAN were higher in plants grafted with 'Sour Orange' and 'Sour Pummelo' rootstocks than other tested rootstocks ( Figure 3D).

Effect of Rootstocks on Enzymatic Antioxidant Activities
In this study, rootstock behaved differentially regarding enzymatic antioxidant activities in the leaves of the 'Shatangju' scion cultivar ( Figure 2). The leaf SOD activity of the 'Shatangju' scion cultivar grafted with the 'Trifoliate Orange' rootstock was significantly lower than that of the other rootstocks. In contrast, maximum SOD values were recorded with the 'Sour Orange' rootstock ( Figure 2A). Regarding POD activity, the result relative to 'Rough Lemon', 'Sour Orange', and 'Citrange' rootstocks showed noticeably higher values than other tested rootstocks ( Figure 2B). In contrast, minimum values of POD activity were obtained with 'Trifoliate Orange' and 'Flying Dragon' rootstocks.

Endogenous Hormone Levels
'Shatangju' citrus scion cultivar grafted with a range of size-controlling rootstocks varied in leaf auxin contents ( Figure 3). The contents of TRA were higher for 'Shatangju' scion grafted onto 'Flying Dragon' and 'Trifoliate Orange' rootstocks, whereas minimum TRA levels were recorded for plants grafted onto 'Shatang Mandarin' and 'Goutou Orange' rootstocks ( Figure 3A). Regarding TRP and MEIAA, the result relative to the 'Trifoliate Orange' rootstock was significantly high, whereas other rootstocks displayed low values ( Figure 3B,F). Similarly, the IAA-Trp contents showed the highest with 'Flying Dragon' and 'Rough Lemon' rootstocks, whereas 'Goutou Orange' rootstock produced lower IAA-Trp contents ( Figure 3C). The contents of IAN were higher in plants grafted with 'Sour Orange' and 'Sour Pummelo' rootstocks than other tested rootstocks ( Figure  3D). Different rootstocks significantly affected the endogenous cytokinin levels in grafted 'Shatangju' citrus plants ( Figure 4). Dihydrozeatin (DZ) levels were relatively high in the 'Shatangju' scion cultivar grafted onto 'Xiangcheng Orange', 'Red Limonia', and 'Flying Dragon' rootstocks, whereas low DZ levels were obtained with 'Rough Lemon', 'Citrange', and 'Goutou Orange' rootstocks ( Figure 4A). The contents of dihydrozeatin-O-glucoside riboside (DHZROG) were pointedly higher in plants grafted with 'Shatang Mandarin' rootstocks, whereas minimum DHZROG levels were recorded for plants grafted onto 'Trifoliate Orange' rootstock ( Figure 4B). Similar Kinetin riboside (KR) levels were detected, being lowest in 'Citrange' and 'Trifoliate Orange' rootstocks, whereas 'Sour Orange', 'Goutou Orange', and 'Rough Lemon' rootstocks displayed higher values of KR ( Figure 4C). For the isopentenyl adenine riboside (IPR) levels, the result corresponding to 'Goutou Orange', 'Sour Orange', 'Red Tangerine', 'Trifoliate Orange' and 'Flying Dragon' rootstock was markedly higher than in the other treatments ( Figure 4D). In terms Abscisic acid contents (ABA) were significantly higher with plants grafted with 'Shatang Mandarin' rootstock, whereas minimum values of ABA were recorded with 'Flying Dragon' rootstock ( Figure 5A). Regarding ABA glucose ester (ABA-GE) contents, the result corresponding to the 'Trifoliate Orange' rootstock was higher than other rootstocks. In addition, the contents of ABA-GE were not detected in the plants grafted with 'Shatang Mandarin', 'Rough Lemon', and 'Flying Dragon' rootstocks ( Figure 5B). The contents of gibberellin (GA 24 ) were significantly higher for the 'Shatangju' scion cultivar grafted onto the 'Goutou Orange' rootstock, followed by 'Sour Orange', 'Red Tangerine', and 'Shatang Mandarin' rootstocks. In contrast, 'Flying Dragon' and 'Sour Pummelo' rootstocks displayed lower values of GA 24 ( Figure 5C). Moreover, the opposite trends were observed for GA 9 contents, whereas the 'Flying Dragon' rootstock showed a significantly higher level of GA 9 , and lower levels were recorded with 'Shatang Mandarin' rootstocks ( Figure 5D). Furthermore, the contents of 1-aminocyclopropanecarboxylic acid (ACC) were significantly higher with the 'Shatang Mandarin' rootstock followed by the 'Sour Pummelo' rootstock; ACC contents were the lowest with 'Trifoliate Orange' and 'Flying Dragon' rootstocks ( Figure 5E). In addition, there were no significant differences in the content of remaining rootstocks. 5-deoxystrigol (5DS) concentrations were lowest with 'Shatang Mandarin' and 'Sour Pummelo' rootstocks compared with the other tested rootstocks ( Figure 5F). of cis-zeatin-O-glucoside riboside (cZROG) levels, the 'Citrange' rootstock was markedly lower than other rootstock treatments, whereas high cZROG levels were obtained with 'Shatang Mandarin', 'Sour Pummelo', 'Red Tangerine' and 'Flying Dragon' rootstocks ( Figure 4F). Abscisic acid contents (ABA) were significantly higher with plants grafted with 'Shatang Mandarin' rootstock, whereas minimum values of ABA were recorded with 'Flying Dragon' rootstock ( Figure 5A). Regarding ABA glucose ester (ABA-GE) contents, the result corresponding to the 'Trifoliate Orange' rootstock was higher than other rootstocks. In addition, the contents of ABA-GE were not detected in the plants grafted with 'Shatang Mandarin', 'Rough Lemon', and 'Flying Dragon' rootstocks ( Figure 5B). The contents of gibberellin (GA24) were significantly higher for the 'Shatangju' scion cultivar grafted onto the 'Goutou Orange' rootstock, followed by 'Sour Orange', 'Red Tangerine', and 'Shatang Mandarin' rootstocks. In contrast, 'Flying Dragon' and 'Sour Pummelo' rootstocks displayed lower values of GA24 ( Figure 5C). Moreover, the opposite trends were observed for GA9 contents, whereas the 'Flying Dragon' rootstock showed a significantly higher level of GA9, and lower levels were recorded with 'Shatang Mandarin' rootstocks ( Figure 5D). Furthermore, the contents of 1-aminocyclopropanecarboxylic acid (ACC) were significantly higher with the 'Shatang Mandarin' rootstock followed by the 'Sour Pummelo' rootstock; ACC contents were the lowest with 'Trifoliate Orange' and 'Flying Dragon' rootstocks ( Figure 5E). In addition, there were no significant differences in the content of remaining rootstocks. 5-deoxystrigol (5DS) concentrations were lowest with 'Shatang Mandarin' and 'Sour Pummelo' rootstocks compared with the other tested rootstocks ( Figure 5F).

Expression, Correlation, and PCA Analysis of Differential Metabolites
Custer analysis was performed to understand the distinct expression pattern of identified metabolites between these two groups ( Figure 7A). To reveal the relationship between various metabolite classes, a correlation analysis was performed based on the accumulation pattern of identified metabolites ( Figure 7B), showing they are closely related to each other, might have similar chemical structures, or might take part in metabolic pathways. To examine the natural variations of metabolic traits among different types of cultivars, the principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) analysis were performed and successfully separated all the varieties. PCA analysis showed significant differences with PC1 (54.89%) and PCA2 (13.27%) ( Figure 7C). OPLS-DA model was performed to identify the metabolites responsible for the separation between the samples, where the T-score was (55%) and the orthogonal T-score (11.4%), indicating that both samples had significant spectral separation, which further indicated that metabolic differences between these samples were statistically significant ( Figure 7D).

Metabolite Profiles of Dwarfing and Vigorous Rootstocks
The different metabolic profiles in the leaves of two grafting combinations ['Shatangju'/ 'Flying Dragon' (dwarfing) and 'Shatangju'/'Shatang Mandarin' (vigorous)] were analyzed ( Figure 8). Moreover, 38 metabolites significantly changed between dwarfing and vigorous graft combinations ( Figure S1). These differential metabolites mainly include organic acids, amino acids, and their derivatives, flavonoids, nucleotides and their derivatives, alkaloids, phenolic acids, and lipids types. Various organic acid metabolites, including 6-aminocaproic acid, 4-acetylaminobutyric acid, and methyl anthranilate, were found significantly downregulated in the plants grafted with 'Flying Dragon' rootstocks. In contrast, other organic metabolites were significantly up-regulated in the leaves of 'Flying Dragon' rootstocks ( Figure 8A). Among the flavonoid metabolites, while Eriodictyol-7-O-(6 -acetyl)glucoside, 5,6,3 ,4 -tetrahydroxy-3,7-dimethoxyflavone-6-O-glucoside, kaempferol-3-O-rhamnoside, and kaempferol-7-O-rhamnoside were significantly up-regulated in the leaves of dwarfing 'Flying Dragon' rootstock compared with 'Shatang Mandarin' (Figure 8B). Among the amino acids and their derivatives, several metabolites including, L-Valine, L-Isoleucine, L-Norleucine, L-Histidine, L-Phenylalanine, L-Tryptophan, N -Formylkynurenine, and L-Saccharopine amino acid were significantly down-regulated in the leaves of plants grafted with 'Flying Dragon' rootstock, while other amino acids presented an up-regulated pattern in 'Shatang Mandarin' rootstock ( Figure 8C). Regarding the metabolites of nucleotides and their derivatives, the expression of Guanine and 6-methylmercaptopurine was significantly down-regulated in the leaves of plants grafted with 'Flying Dragon' rootstock. In contrast, the rest of the metabolites were noticeably up-regulated in the leaves of 'Shatang Mandarin' rootstock ( Figure 8D). In the case of alkaloid metabolites, Citpressine I, O-Phosphoryl-ethanolamine, and Feruloylcholine was significantly up-regulated in the leaves of plants grafted with 'Flying Dragon' rootstock ( Figure 8E). Regarding phenolic acids, lignans, and coumarins, the expression of Bis(p-Coumaroyl)tartaric acid and Rutaretin were significantly up-regulated in the leaves of plants grafted with 'Flying Dragon' rootstock compared with 'Shatang Mandarin' rootstock ( Figure 8F,I). Furthermore, the expression of Hinokitiol, N-(beta-D-glucosyl)nicotinate, and 12, 13 DHOME; (9Z)-12, 13-dihydroxy-9-enoic acid were significantly down-regulated in the leaves of 'Flying Dragon' rootstock ( Figure 8G). In particular, rootstocks had a significant impact on the leaf metabolite content of organic acids, flavonoids, amino acids and derivatives, alkaloids, nucleotides, and derivatives, etc. These findings showed a great difference in leaf metabolites profile between 'Flying Dragon' and 'Shatang Mandarin' grafted plants.  analysis (PLS-DA) analysis were performed and successfully separated all the varieties. PCA analysis showed significant differences with PC1 (54.89%) and PCA2 (13.27%) (Figure 7C). OPLS-DA model was performed to identify the metabolites responsible for the separation between the samples, where the T-score was (55%) and the orthogonal T-score (11.4%), indicating that both samples had significant spectral separation, which further indicated that metabolic differences between these samples were statistically significant ( Figure 7D).

Discussion
Citrus is a valuable fruit, and higher productivity is essential for growers and the economy [40]. Dwarfism is one of the most valuable traits in fruit production for dense cultivation to obtain a maximum harvest index and effective orchard management [8,41]. Rootstocks significantly influence the morphological features of grafted plants, among which the reduction of scion vigor is one of the fascinating phenomena [7,11]. Earlier research has shown that plants grafted on taller rootstocks displayed increased primary shoot lengths and scion trunk diameter [42]. On the other hand, dwarfing rootstocks or interstock modify shoot architecture by reducing the values of morphological traits such as primary shoot length, node number, sylleptic shoot, and intermodal length [25,43,44]. In the present experiment, we noticed that the 'Shatangju' scion cultivar grafted onto the 'Flying Dragon' rootstock encouraged the small stature trees. However, trees grafted with other rootstocks like 'Shatang Mandarin', 'Goutou Orange', and 'Sour Orange' increased plant height, crown width, scion trunk diameter, apex shoot length, internodal length, and the entire plant growth. Compared with other rootstocks, plants grafted on dwarfing rootstocks displayed lower values of morphological traits with higher returns [1], which is consistent with our findings. Similarly, Nasir et al. [45] examined the influence of 'Kinnow Mandarin' grafted on three different rootstocks. They reported that plants grown on more vigorous rootstock ('Rough Lemon') increased their growth concerning plant height, scion trunk diameter, leaf areas, and internodal length, while plants grafted on 'Carrizo Citrange' showed to be a dwarfing rootstock.
Soluble sugar, protein, and enzyme activities (i.e., SOD and POD) widely exist in plants, which protect plants from harmful damage and affect the metabolism and distribution of hormones in plants [46]. Zhao et al. [19] reported the effect of various dwarfing interstocks on the morphological behavior and biochemical and physical parameters of apple plants, indicating that the plant height of various dwarf interstocks was negatively correlated with the soluble sugar content. In addition, more significant dwarfing effects are related to increased leaf enzymatic activities; subsequently, these activities have a negative impact on the growth of the scion part, which leads to tree dwarfing. Our present study established the relationship between soluble sugar, protein, enzyme activity, and rootstock vigor of grafted scion parts. There was a little difference in soluble sugar contents among different scion/rootstock combinations, indicating that different rootstocks had little effect on soluble sugar in the leaves of 'Shatangju' mandarin. Moreover, the highest soluble sugar content was observed with trees grafted onto 'Trifoliate Orange' rootstock. Previous studies have also found a substantial increase in soluble sugar content in the scion buds of the 'Auksis' scion cultivar grafted with dwarfing (B.36) rootstock. In contrast, no significant differences were observed in glucose accumulation between the semi-dwarfing, dwarfing, and superdwarfing graft combinations. The differences in soluble sugar accumulation in buds produced by these rootstocks may be related to the different periods required for fruit ripening among 'Ligol' and 'Auksis' cultivars [28]. Additionally, the soluble protein content of trees grafted with 'Citrange' and 'Flying Dragon' rootstocks was significantly higher than that of other graft combinations, which could effectively enhance the antioxidant capacity of the 'Shatangju' scion to a certain extent.
Plants produce different scavenging enzymes, i.e., SOD and POD, to overcome the negative effects of ROS [46]. The enzyme activities showed significantly different behavior in various rootstocks when grafted with the 'Shatangju' scion cultivar. In this current study, we found that the SOD activity of trees grafted with 'Sour Orange' rootstock was noticeably higher. In contrast, the lowest SOD activity was obtained with trees grafted with 'Trifoliate Orange' rootstock. In addition, POD is engaged in numerous physiological processes in plants, such as peroxide scavenging, cell wall synthesis, lignification, and IAA metabolism [47]. The POD activity in the current study was found to be minimum in trees grafted with 'Flying Dragon' and 'Trifoliate Orange' rootstocks, followed by 'Red Limonia' and 'Shatang Mandarin' rootstocks. Moreover, the POD and SOD activities of 'Shatang Mandarin' rootstocks were higher than 'Flying Dragon' rootstocks. The higher activities of defense antioxidant enzymes in the vigorous graft combination reflected that their scavenging capability for ROS was stronger and thereby accelerated wound healing, allowing plants to recover regular growth earlier [48]. On the other hand, the 'Flying Dragon' presents disadvantages such as low tolerance to drought and graft incompatibility with some scion varieties [49].
Hormone synthesis and transport have been demonstrated in previous studies to restrict tree growth [39]. Auxin and cytokinin encourage tree growth and the development of axillary buds. Gibberellins promote internodal elongation, whereas ABA increases tree aging [50]. Else et al. [51] reported that the dwarfing effect of M.9 rootstock is linked with the reduced capability of IAA transport, along with greater export of ABA than more vigorous rootstocks (MM.106). Reduced IAA transport to the roots influences biomass production and activities of cytokinin and gibberellin [52]. This mechanism limits axillary bud sprouting and internode elongation, resulting in shorter internode length, decreased growth vigor, and dwarfing of the scion [53,54]. Another study reported that [55], the dwarfing of the tree may be due to the blocked IAA transport, which inhibits the synthesis of CTK in the roots, resulting in the weakened growth of the aerial parts. Gibberellins are crucial for plant growth and a controlling factor of plant architecture. GA-related dwarfing can be divided into two categories: a responsive mutant that is linked with GA signaling and a dwarf mutant that is associated with the GA anabolic pathway. The synthetic dwarf mutant is produced by a GA deficiency due to abnormalities in GA synthetase or other GA metabolic enzymes. Our present study found that the trees grafted with 'Flying Dragon' rootstock showed a significantly higher level of GA 9 , and lower levels were recorded with trees grafted onto 'Shatang Mandarin' rootstocks.
Strigolactones represent the most recently described group of plant hormones involved in many aspects of plant growth regulation [56]. It is mainly synthesized in the root system and then transported to other parts. 5-Deoxystrigol (5-DS) is ubiquitous in plants, the first product in the strigolactone synthesis pathway, and the rest are derivatives of 5-DS. SLs are closely related to plant growth and development. Moreover, IAA, ABA, GA, CTK, ETH, and other hormones work together to regulate plant shape, branching, and root system.
Anatomical structure, material transport, photosynthesis, plant hormones, and other factors are closely related to dwarfing rootstocks; however, limited studies have shown the relationships between the degree of dwarfing and metabolomics. Earlier studies have shown that small-molecule organic acids can affect plant growth and development [57]. Compared with vigorous ('Shatangju'/'Shatang Mandarin') graft combination, most of the organic acids in the dwarf ('Shatangju'/'Flying Dragon') graft combination was upregulated, which is consistent with previous research results. Flavonoids may regulate the polar transport of auxin, and phytohormones can regulate secondary metabolites in a concentration-dependent manner [58]. The differential metabolites in flavonoids are mainly down-regulated in trees grafted with dwarfing ('Flying Dragon') rootstock than that of trees grafted with vigorous rootstock. Changes in the external environment will disrupt plants' primary and secondary metabolic profiles, so hormonal changes assume the corresponding regulatory role in adapting plants to environmental changes [59]. In this trial, differential metabolites were mainly enriched in the biosynthetic pathways of secondary metabolites, which is consistent with previous studies and further shows that hormones have an important effect on plant dwarfing.

Conclusions
In the present study, we found that citrus rootstocks noticeably affected the morphological traits of grafted 'Shatangju' plants. 'Flying Dragon' rootstock significantly reduced the plant height of the scion cultivar. Leaf POD activity of trees grafted with 'Flying Dragon' rootstock was significantly lower than other graft combinations. Moreover, a low concentration of ABA in 'Shatang Mandarin' was recorded when grafted on the 'Flying Dragon' rootstock. The differential expression of leaf metabolites may be involved in the reduction of scion growth by citrus rootstocks. Overall, these findings indicate that the 'Flying Dragon' rootstock may be the best option in the high-density plantation of citrus fruits under net house conditions. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/horticulturae8070608/s1, Figure S1: KEGG enrichment analysis (A) and volcano plot (B).