Four novel Cit7GlcTs functional in flavonoid 7-O-glucoside biosynthesis are vital to flavonoid biosynthesis shunting in citrus

Abstract Citrus fruits have abundant flavonoid glycosides (FGs), an important class of natural functional and flavor components. However, there have been few reports about the modification of UDP-glycosyltransferases (UGTs) on flavonoids in citrus. Notably, in flavonoid biosynthesis, 7-O-glucosylation is the initial and essential step of glycosylation prior to the synthesis of flavanone disaccharides, the most abundant and iconic FGs in citrus fruits. Here, based on the accumulation of FGs observed at the very early fruit development stage of two pummelo varieties, we screened six novel flavonoid 7-O-glucosyltransferase genes (7GlcTs) via transcriptomic analysis and then characterized them in vitro. The results revealed that four Cg7GlcTs possess wide catalytic activities towards various flavonoid substrates, with CgUGT89AK1 exhibiting the highest catalytic efficiency. Transient overexpression of CgUGT90A31 and CgUGT89AK1 led to increases in FG synthesis in pummelo leaves. Interestingly, these two genes had conserved sequences and consistent functions across different germplasms. Moreover, CitUGT89AK1 was found to play a role in the response of citrus to Huanglongbing infection by promoting FG production. The findings improve our understanding of flavonoid 7-O-glucosylation by identifying the key genes, and may help improve the benefits of flavonoid biosynthesis for plants and humans in the future.


Introduction
Citrus fruits are extensively consumed and cultivated on a global scale [1].In 2021, the global citrus fruit production reached about 13.9 million tons, with China accounting for 42.88% of the total production (FAO, https://www.fao.org).Besides fresh consumption, citrus fruits are also processed into juice, cans, and some pharmaceutical materials [2].For example, 'Huazhou' pummelo (C.grandis var.'Tomentosa') is the source material of traditional Chinese medicine 'Huajuhong', which has been used as an antitussive and expectorant in clinic for thousands of years due to its particularly high content of f lavonoids [3].In recent years, there has been continuously increasing demand of the consumers for food products with medical and health-promoting properties, which has greatly stimulated research initiatives to explore the functional components in different foods, such as f lavonoids, carotenoids, vitamin C, minerals, and dietary fiber.Flavonoids are dominant secondary metabolites in citrus fruits [4].According to their structural characteristics, f lavonoids in plants can be generally categorized into f lavanones, f lavones, isof lavones, f lavonols, f lavanols, and anthocyanidins [5], which contribute to the formation of pigments [6] and flavors [7], and play crucial roles in defense against various biotic [8,9] and abiotic [10][11][12] stresses.Citrus f lavonoids are mostly glycosyl derivatives, which confer them with high stability, solubility, and bioactivity [13].Citrus fruits are known to contain high content of f lavonoid glycosides (FGs), particularly 'Huazhou' pummelo, whose FGs level can reach 256.48 mg/g and account for over 25% of the fruit dry weight [14].Recently, FGs have attracted increasing attention due to their diverse beneficial effects to human health.For example, naringin is the main pharmacological component of 'Huajuhong', which has various positive effect on bone regeneration, metabolic syndrome, oxidative stress response, and central nervous system diseases [15].Furthermore, FGs also play certain roles in plant defense against various diseases, such as citrus canker [16] and Huanglongbing (HLB) [17,18].'Valencia' oranges with HLB infection exhibit elevated concentrations of f lavonoids in the peel, pulp, and juice, including the FGs hesperidin and narirutin [19].Grapefruits with HLB infection significantly produce naringin [20].Therefore, it is particularly important to investigate the biosynthesis of FGs so as to better utilize citrus f lavonoids for human welfare and plant health.
Production of plant f lavonoids is an important branch of the phenylpropanoid metabolic pathway.The process is initiated with the enzymatic catalysis of the precursor phenylalanine by various enzymes, including phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate: CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI), resulting in the formation of naringenin, which serves as the intermediate precursor for f lavonoid synthesis [4].In citrus, naringenin is mostly modified by oxygenmethylase or hydroxylase to form other f lavanone aglycones (hesperetin, eriodictyol or isosakuranetin) [21].Additionally, naringenin can also be catalyzed by f lavone synthase (FNS) and f lavonol synthase (FLS) to form f lavone and f lavanol aglycones, respectively.Then, the above f lavonoid aglycones are mostly glucosylated at the 7-position by f lavonoid 7-O-glucosyltransferase (7GlcT) to form corresponding f lavonoid 7-O-glucosides, which are further glycosylated by either 1,2-rhamnosyltransferase (1,2RhaT) to yield bitter f lavonoid neohesperidosides or 1,6-rhamnosyltransferase (1,6RhaT) to generate non-bitter f lavonoid rutinosides [22] (Fig. S1).Both f lavonoid neohesperidosides and rutinosides are f lavonoid disaccharides, with f lavanone disaccharides as the most abundant and iconic FGs as the end products in citrus fruits.Previous research has documented the existence of two functional UDPglycosyltransferases, namely Cm1,2RhaT [23] and Cs1,6RhaT [7], from pummelo and sweet orange, respectively.These enzymes play vital roles in the formation of f lavonoid disaccharides, the predominant and characteristic metabolites in the f lavonoid profile of pummelo or sweet orange.Although it is clear that production of f lavonoid 7-O-glucosides is the initial step in the biosynthesis of f lavonoid disaccharides in citrus, there have been few reports on the genes governing this crucial process.
Flavonoid 7GlcT belong to the UGT family and have catalytic activity on the 7-hydroxy position of f lavonoid substrates.To date, there have been many reports on f lavonoid 7GlcT in different plants.For example, At7GlcT can take f lavonols as the substrate to produce 7-O-glucosides in Arabidopsis thaliana [24].In tea (Camellia sinensis), CsUGT75L12 specifically transports the glucose unit from UDP-glucose to the 7-hydroxy position of the f lavonoid to produce 7-O-glucoside [25].Sb7GlcT can catalyze baicalein to produce baicalein 7-O-glucoside in hairy roots in response to wounding or salicylic acid treatments in the traditional Chinese medicine Scutellaria baicalensis [26].CsUGT76F1 has been reported to catalyze f lavonoids at the 7-hydroxy position in sweet orange, with special preference to catalyze f lavonols [27].In general, most 7GlcTs currently characterized are for f lavonol or f lavone substrates, and there have been few reports on 7GlcTs involved in the f lavanone biosynthetic pathway.Recently, two CgUGTs with the functions of f lavonoid 7-O-glucosyltransferase were identified in mature pummelo fruits via mGWAS analysis [28].However, as f lavonoid accumulation usually occurs at the early stage of citrus fruit development, but tends to decline after 90 days after f lowering (DAF) [29], there may be some other 7GlcT members involved in the synthesis of citrus FGs, which may have greater contributions to the accumulation of f lavonoids in citrus fruit before 90 DAF.Thus, identification of key citrus 7GlcTs is crucial for determining the shunting of downstream FGs so as to better understand the biosynthetic patterns of FGs in citrus.
In this study, an integrated analysis of transcriptomics and metabolomics was applied to mine candidate 7GlcTs from pummelo (C.grandis).Functional analysis revealed that four out of the six Cg7GlcTs candidates were functional in glucosylation of the 7-hydroxy position to various f lavonoid substrates.Transient overexpression in pummelo leaves demonstrated the effect of CgUGT90A31 and CgUGT89AK1 to promote the accumulation of FGs.We also found that the homologous genes of CgUGT90A31 and CgUGT89AK1 in Poncirus trifoliata had a 7-O-glucosylation function as well.In addition, HLB infection upregulated the expression of CitUGT89AK1 and the accumulation of FGs in pummelo and loose skin mandarin.Overall, this study screened and characterized the key genes governing the initial step of glucosylation, which may improve our understanding of the f lavonoid biosynthetic pathway in citrus and facilitate the tuning of f lavonoid biosynthesis for plant defense and human health benefits.

FGs constitute the primary metabolites of flavonoids in citrus fruits
To investigate the accumulation pattern of FGs in citrus fruits, we analyzed the FG profiles in mature fruit pulp of five representative citrus species.The HPLC results showed that there were eight kinds of dominant f lavanone disaccharides in citrus fruits (Fig S2 ), and the accumulation of FGs in the pulp had certain species specificity.In addition, some FGs such as rhoifolin were also present in small amounts in citrus fruit pulp, which constitute stable FG profiles together with the dominant FGs in citrus.To further reveal the accumulation pattern of FGs during citrus fruit development, we selected 'Huazhou' pummelo (C.grandis) and 'Fenghuang' pummelo (C.grandis) fruits at distinct developmental stages as the experimental materials.HPLC elution profiles indicated that there were two FGs in the fruits (Fig. 1A), namely naringin (compound 1) and rhoifolin (compound 2) respectively belonging to f lavanones and f lavones, based on comparison of their retention time and ion fragmentation patterns with those of the standards (Fig. 1B).The levels of naringin and rhoifolin were the highest at the initial phase of the peel at 30 DAF or the pulp at 60 DAF, followed gradual decreases (Fig. 1C).Significantly, 7-Oglucosylation was the first step in transforming naringenin and apigenin into the final disaccharide derivatives of naringin and rhoifolin in pummelo, respectively (Fig. 1D).And Cit7GlcTs might play a key role in this process.

Identification of putative Cit7GlcTs based on the pummelo genome
To mine possible Cit7GlcTs involved in the synthesis of FGs in citrus, we firstly annotated the UGT genes from the genome of C. grandis.Subsequently, a search was conducted for conserved domains of plant secondary product glycosyltransferase (PSPG) box, yielding 144 potential UGTs and 149 transcript sequences.The predicted UGTs had lengths ranging from 214 to 679 amino acids (aa), molecular weights ranging from 22.9 to 76.9 kDa, and pI values ranging from 5.11 to 9.18 (Table S1).Then, a phylogenetic tree was constructed by alignment of the full-length amino acid sequences with those of functionally characterized UGTs from citrus and other plants (Fig. 2A).The phylogenetic tree comprised five distinct groups, including 3-O-glycosyltransferases (3GTs), 5-O-glycosyltransferases (5GTs), 7-O-glycosyltransferases (7GTs), C-glycosyltransferases (CGTs), and glycoside glycosyltransferase (GGTs).As a result, 23 possible UGT sequences were clustered in the 7GTs group, which could be preliminarily identified as candidate genes encoding citrus f lavonoid 7GlcTs.
The Cit7GlcT enzymes play a pivotal role in the initial step to catalyze the formation of FGs in citrus.We analyzed the transcription levels of 23 members from the 7GTs group in the 'Huazhou' pummelo and 'Fenghuang' pummelo fruit at different developmental stages.According to the transcription levels, candidate 7GlcTs could be divided into three types: type I genes had high expression levels throughout the development; type II  genes had low expression levels throughout the development; and type III genes only had high expression in some specific periods (Fig. 2B).Furthermore, we identified candidate 7GlcTs involved in the accumulation of f lavonoids through correlation coefficient (Pearson r) analysis between the transcription levels of 7GlcTs and the contents of FGs (sum of naringin and rhoifolin).
According to the screening criteria of correlation coefficient > 0.6 and the gene expression level of FPKM > 10 in at least one developmental stage, two (Cg7g015690 and Cg2g006310) and three genes (Cg7g015690, Cg5g012040, and Cg8g004210) were found in the peel and pulp of 'Huazhou' pummelo, respectively; in addition, three genes (Cg2g012130, Cg7g015690, Cg1g019780) in peel and pulp of 'Fenghuang' pummelo were found as candidates (Fig. 2C).Therefore, a total of six 7GlcTs were screened, including Cg2g012130 (CgUGT73CG30), Cg7g015690 (CgUGT89D30), Cg2g006310 (CgUGT90A31), Cg1g019780 (CgUGT73CG31), Cg5g012040 (CgUGT89AK1), and Cg8g004210 (CgUGT73AC12), named by the UGT Nomenclature Committee.The full-length sequences of six Cg7GlcTs were isolated from the cDNA library of 'Huazhou' pummelo pulp, and the sequencing analysis revealed that each Cg7GlcT contained a PSPG box that comprises 44 aa with glutamine (Q) as the last amino acid, which has been reported as the key residue that determines whether UGTs utilize UDPglucose as a sugar donor [30,31] (Fig. S3).

Candidate Cg7GlcTs show broad-spectrum 7-hydroxy glucosylation properties in vitro
The protein encoded by the six Cg7GlcTs was expressed as a recombinant protein fused with maltose-binding protein tags, and the recombinant proteins were approximately 100 kDa as indicated by SDS-PAGE (Fig. S4).Hence, the enzymatic activity of Cg7GlcT recombinase on three distinct types of f lavonoids (f lavanones, f lavones, and f lavonols) was determined in vitro.Glucosylated products were identified by comparing the retention time and MS/MS data with those of the authentic compounds.When the commercial standards for predicted products were not available, their identities were inferred based on the results of analogous substrates and mass spectra (Fig. S5).
The product content comparison showed that when feeding with the above nine f lavonoid substrates (Table 1), CgUGT90A31 had the most pronounced catalytic activity towards hesperetin and isosakuranetin; CgUGT73AC12 displayed the highest catalytic activity towards kaempherol; and CgUGT89AK1 exhibited the most pronounced enzymatic activity towards the remaining six substrates.These results indicated that the four Cg7GlcTs possess versatile catalytic capabilities towards various f lavonoid substrates.

Four functional Cg7GlcT proteins exhibit different substrate preference
The optimal pH and temperature for the in vitro catalytic reaction of four Cg7GlcTs were determined by using naringenin as a substrate (Fig. S7).The results showed that CgUGT89D30 exhibited the highest enzymatic activity at pH 7.0 (in 50 mM PBS buffer) and temperature of 30 • C. Nevertheless, CgUGT90A31, CgUGT89AK1, and CgUGT73AC12 displayed equivalent highest enzymatic activities at pH of 7.0 and temperature of 35 • C.
To investigate the preference of Cg7GlcT recombinants for f lavonoid substrates, their kinetic properties were determined using the Michaelis-Menten equation under optimal reaction conditions (Table 2, Fig. S8).Among the different tested f lavonoid substrates, CgUGT89AK1 exhibited the most notable catalytic efficiency towards quercetin with a Kcat/Km value of 1352.49M -1 s -1 , followed by diosmetin and eriodictyol, with Kcat/Km valued of 284.12 M -1 s -1 and 154.19 M -1 s -1 , respectively.Hesperetin had the lowest Kcat/Km values (16.64 M -1 s -1 ).For CgUGT89D30, it exhibited the greatest catalytic efficiency for the f lavone compound of apigenin, with a Kcat/Km value of 88.23 M -1 s -1 .Notably, CgUGT90A31 exhibited greater catalytic efficiency towards f lavanones, with Kcat/Km values of 52.02 M -1 s -1 for naringenin and 98.31 M -1 s -1 for isosakuranetin.CgUGT73AC12 had a comparatively higher catalytic efficiency against isosakuranetin, with a Kcat/Km value of 37.57 M -1 s -1 .Overall, CgUGT89AK1 exhibited a superior catalytic efficiency relative to other three Cg7GlcTs.

Transient overexpression of CgUGT90A31 and CgUGT89AK1 enhances FG accumulation in pummelo leaves
'Huazhou' pummelo leaves are known to have abundant FGs 14 .Hence, transient overexpression was conducted in 'Huazhou' pummelo leaves to investigate the potential role of Cg7GlcTs in the glucosylation of f lavonoids in vivo.The results demonstrated considerable upregulation of all four Cg7GlcTs in 'Huazhou' pummelo leaves (Fig. 4A).However, only those leaves treated with the bacterial solution containing CgUGT90A31-pBI121 and CgUGT89AK1-pBI121 exhibited significant increases of 1.13 and 1.16 folds in the overall content of FGs (naringin and rhoifolin), respectively (Fig. 4B).

Homologues of CgUGT90A31 and CgUGT89AK1 exhibit similar glucosylation function in other citrus species
We performed a genetic variability analysis of citrus UGT90A31 and UGT89AK1 across several germplasms from loose skin mandarins (C.reticulata 'Chachiensis'), sweet orange ('Fengjie 72-1' Navel Orange), and trifoliata orange.The results demonstrated that both two genes exhibited high degrees of conservation in sequences (Fig. S9).Subsequently, we obtained the transcriptome data from fruit pulp of the three selected cultivars.The data showed that the expression level of UGT90A31 was higher at the initial developmental stages of loose skin mandarins and sweet orange, whereas at the mature stage of trifoliata orange (Fig. 5A).On the contrary, the expression level of UGT89AK1 significantly increased at the later developmental stage of loose skin mandarins and trifoliata orange pulp.However, in sweet orange pulp, the levels of UGT89AK1 were comparatively stable throughout the development.
We further carried out sequence analysis among various species.The results revealed that PtUGT90A31 and PtUGT89AK1 exhibited the most significant sequence divergence from CgUGT90A31 and CgUGT89AK1 in trifoliata orange (Fig. S9).A total of eight f lavonoids, including four f lavanones, two f lavones, and two f lavonols, were selected as substrates to evaluate the activity of PtUGT90A31 and PtUGT89AK1.The HPLC results revealed that PtUGT90A31 and PtUGT89AK1 can catalyze naringenin and isosakuranetin, two main f lavonoid aglycones, in the formation of corresponding 7-O-glucosides in Poncirus trifoliata (Fig. 5B).Among them, PtUGT90A31 exhibited the highest catalytic activity for isosakuranetin, while PtUGT89AK1 had the strongest catalytic activity for naringenin, followed by isosakuranetin (Fig. 5C).

CitUGT89AK1 may be involved in the stress response of citrus to HLB infection
Flavonoids have been documented to play an important role in modulating plant stress response [32].Citrus HLB is the most devastating citrus disease worldwide.After infection with HLB, biological replicate 1 and biological replicate 2 of 'Shatian' pummelo showed leaf mottle, and all exhibited significant increases (averagely 2.82 folds) in the content of FGs (naringin and rhoifolin) in comparison with the control leaves, accompanied by substantial upregulation of CgUGT89AK1 (averagely 1.74 folds) and downregulation of the other three Cg7GlcTs (Fig. 6A).For loose skin mandarins, HLB infection led to uneven fruit coloring, and significantly increased the content of the major FG hesperidin in 'Shatang' mandarin fruit peel (about 4.35 folds) along with significant upregulation of CrUGT89AK1 (1.46 folds) compared with the control fruit (Fig. 6B).After being infected with HLB, 'Newhall' navel orange fruits also exhibited uneven coloring.The content of the main FG hesperidin was significantly increased, but the significantly upregulated gene was CsUGT89D30 instead of CsUGT89AK1 (Fig. 6C).
Furthermore, there was no significant change in the content of FGs in 'Hybrid' pummelo leaves after infection by citrus canker.However, a 1.5-fold increase in the transcript level of CgUGT90A31 was still observed (Fig. S10 A).Moreover, in the leaves of 'Newhall' navel oranges infected with citrus canker, no significant changes in both FG contents and gene expression levels were observed (Fig. S10 B).

Citrus flavonoid 7-O-glucosyltransferases are encoded by multiple UGT genes
As a superfamily of genes in plant f lavonoid metabolism pathways, the UGT genes are often subjected to glycosylation modification [25,33].In Lotus japonicus seeds, it was observed that the production of f lavonol glucosides is due to the collaborative effect of at least three genes in the UGT72 family [34], while in Pueraria lobata, three PlUGT recombinant enzymes could glucosylate the hydroxyl groups at the 7-position of genistein and daidzein isof lavones in vitro, with PlUGT15 demonstrating the highest catalytic efficiency [35].In this study, four Cg7GlcTs exhibited catalytic activities towards the 7-hydroxy site of f lavonoids in vitro (Fig. 3), as well as different affinities to nine f lavonoid substrates (Table 2).Furthermore, the transcriptome analysis of two pummelo fruit demonstrated that CgUGT89D30 and CgUGT89AK1 have the most pronounced expression levels at the initial development stage of peel and pulp along with the highest FG content.However, the expression levels of CgUGT90A31 and CgUGT73AC12 were reached to peak during the initial development stage of 'Huazhou' pummelo peel and pulp, respectively (Fig. 2B).Interestingly, Shen et al. used mGWAS to analyze the content of melitidin, an acylated FG, in mature pummelo fruits and identified four CgUGT genes.Functional analysis showed that CgUGT1 and CgUGT2 have catalytic activities of 7-O-glucosylation only for f lavones, while CgUGT3 and CgUGT4 have stronger 7-O-glucosylation activities for f lavanones and f lavones, the expression patterns of four CgUGTs varied across different citrus tissues [28].Therefore, it can be speculated that several 7GlcTs jointly regulate the biosynthesis of FGs in citrus, which can ensure the production of f lavonoid 7-O-glucoside derivatives across different citrus tissues and developmental stages.

CitUGT89AK1 may be a key gene for boosted FG biosynthesis in citrus fruits
Although Cit7GlcTs have been isolated in the 1990s from grapefruit (C.paradisi) [36] and lemon (C.lemon) [37] cell-free extracts, the genes encoding f lavonoid 7GlcTs were rarely reported in the last two decades.In the present study, we successfully discovered four Cg7GlcTs from pummelo.Unlike CsUGT76F1, which has been previously reported to favor catalyzing f lavonol substrates, the four Cg7GlcTs exhibit higher catalytic activity in glucosylating the 7-hydroxy position of f lavanone and f lavone substrates.However, CgUGT89AK1 showed enhanced catalytic efficiency specifically to quercetin.Among the four Cg7GlcTs, the recombinant enzyme CgUGT89AK1 exhibited notable affinity and catalytic efficiency towards f lavonoid substrates in vitro (Table 1).Transient overexpressed four Cg7GlcT genes in 'Huazhou' pummelo leaves, in which the overexpression of CgUGT90A31 and CgUGT89AK1 promoted the FG contents.While CgUGT89D30 and CgUGT73AC12 have not significantly affected the FG contents in pummelo leaves due to their weak catalytic activity towards the f lavonoid 7-hydroxy position (Fig. 4).Furthermore, we selected the homologous sequence PtUGT89AK1, a candidate with the highest sequence divergence from CgUGT89AK1, for functional validation in other citrus species.The results showed that PtUGT89AK1 could proficiently catalyze a diverse range of f lavonoid substrates (Fig. 5), implying the consistent functions of CitUGT89AK1 in various citrus species.
In addition, 'Huazhou' pummelo is a source material for the precious traditional Chinese medicine 'Huajuhong', which is characterized by particularly high accumulation of naringin that contributes to its medical value [38].Compared with the commonly cultivated 'Fenghuang' pummelo, 'Huazhou' pummelo fruits showed higher FG contents throughout the entire development period, which was even 4-fold that of 'Fenghuang' pummelo in the pulp at 60 DAF (Fig 1C).Accordingly, the expression level of CgUGT89AK1 in the 60 DAF pulp of the 'Huazhou' pummelo was 6-fold higher than that in 'Fenghuang' pummelo.However, no significant difference was observed in the peel, which is consistent with the early FG metabolism in the 'Huazhou' pummelo.Therefore, we speculate that the expression level of UGT89AK1 may be responsible for the difference in FG content among different citrus cultivars.Collectively, it can be speculated that CitUGT89AK1 may be a promising gene in citrus fruits that can be utilized to improve FG production.

CitUGT89AK1 may participate in the stress response of citrus to HLB infection by promoting flavonoid synthesis
Citrus HLB caused by the infection of 'Candidatus Liberibacter spp.' is the most devastating citrus disease worldwide [39], which can seriously affect the yield, fruit quality, and life span of citrus.There have been no effective treatments to date, but the disease can be efficiently alleviated by some special measures.It has been demonstrated that enhancing the synthesis ability of antioxidants such as f lavonoids and inhibiting the generation of reactive oxygen species is an effective method for controlling HLB [17].In a previous study, the leaves of Hamlin sweet orange and Valencia infected with HLB showed more than ten folds of increases in f lavonoid content [40].It was found that nine f lavonoids, including vitexin, cosmetin (apigenin 7-O-glucoside), and rhoifolin, exhibited considerable increases at the early stage of citrus leaves infected with HLB [18].A recent study has indicated that rutin has the potential to mitigate the detrimental effect associated with HLB [41].
In this study, we observed a considerable rise in the FG content in two tissues infected with HLB in 'Shatian' pummelo and 'Shatang' mandarin.Simultaneously, there was observable upregulation of CitUGT89AK1, suggesting that it is likely involved in the response of citrus to HLB by facilitating the synthesis of downstream FGs.In contrast, in 'Newhall' navel orange fruits infected with HLB, the expression level of CsUGT89AK1 remained unchanged, while CsUGT89D30 was significantly upregulated by 1.5 folds.Compared with other citrus germplasm infected with HLB, 'Newhall' navel orange only showed slight increases in FG content, which may be due to the unchanged expression of CsUGT89AK1.Therefore, subsequent research can focus on the binding elements related to stress response in the promoter region of CitUGT89AK1.It is worth noting that HLB invasion is rarely found in the 'Huazhou' pummelo production area, indicating the presence of a tolerant phenotype associated with the accumulation of FGs.Furthermore, after citrus canker infection of the tissues of 'Hybrid' pummelo and 'Newhall' navel orange, no significant change was observed in the FG contents, despite the upregulation of CgUGT90A31 in 'Hybrid' pummelo.This result indicate that CgUGT90A31 plays a weaker role in 7-hydroxy position glucosylation than CgUGT89AK1, though it might be involved in the response of citrus to canker infection.

Plant materials
The information on the 10 citrus accessions used in this study is listed in Table S2.For fruits at different development stages, uniform 'Huazhou' pummelo (C.grandis) and 'Fenghuang' pummelo (C.grandis) fruits without mechanical damage were harvested at 30, 60, 90, 120, 150 and 210 DAF.'Chachiensis' (C.reticulata) and trifoliata orange (P.trifoliata) fruits without mechanical damage were harvested at 60, 90, 120, 150 and 210 DAF.The fruits of other citrus germplasms were collected at maturity.Except for 30 DAF (no pulp tissue was developed at the stage, and thus the whole fruit was investigated as the peel), the fruits of the above plant materials were divided into two parts, peel (including f lavedo and albedo) and pulp (including segment membrane and juice sacs) tissues.At each stage, 15-25 fruits were harvested.In addition, for citrus leaves, thirty healthy and fully expanded adult leaves were collected.All samples were randomly divided into three biological replicates and washed with water, then promptly immersed in liquid nitrogen, and stored at −80 • C until further analysis.

Extraction, identification, and quantification of FGs
Extraction of FGs were performed as described in our previous study [22].Citrus fruit and leaf samples were ground into powder under liquid nitrogen and then lyophilized using Heto Lylab 3000 (Heto-Holten A/S, Allerød, Denmark).Around 0.1 g of powdered was sonicated in 5 mL of 70% aqueous methanol for 1 hour in an DTC-15J ultrasonic cleaning machine (Toptll Technology, Hubei, China).After centrifugation at 8000 rpm for 10 min, supernatant was extracted and filtered through a 0.22 μm micropore membrane filter for LC-MS or HPLC analysis.
FGs were determined according to Wang et al [42].Mass spectrometry data were acquired using a 1200 Series Rapid Resolution UPLC system coupled with a 1260 infinity array detector and a 6520 accurate-Mass Quadrupole Time-of-Flight (Q-TOF) MS system (Agilent Technologies, CA, USA).Separation of f lavonoids was conducted using a Shim-pack VP-ODS C18 (Shimadzu, Kyoto, Japan).Ultrapure water and acetonitrile containing 0.04% (v/v) formic acid were utilized as mobile phases A and B, respectively.Raw data were underwent processing using Agilent MassHunter Qualitative Analysis B.04.00 software.FGs were identified by comparing retention times, UV spectra, mass spectra, and ion fragments between the samples and f lavonoid standards.The identities of compounds without standards can be speculated based on their molecular weight and ion fragments.
FGs were quantified utilizing an HPLC system consisting of a 1525 Binary HPLC pump, a 2717 Autosampler and a 2998 Photodiode Array Detector (Waters, Milford, MA).Flavonoids were separated via a C18 Hypersil GOLD column (Thermo Fisher Scientific, Waltham, MA).Mobile phases A and B comprised ultrapure water and acetonitrile containing 0.15% (v/v) formic acid, respectively.The gradient elution system and instrument conditions were consistent with those described by Peng et al [43].The ultravioletvisible spectra were recorded within the range of 210 to 400 nm.Quantification of the f lavonoid 7-O-glucosides were performed using the calibration curves made with the corresponding commercial standards (Table S3).The f lavonoid disaccharide calibration curves refer to Chen et al [22].

Phylogenetic analyses and sequence alignment
The PSPG box was taken as a query according to Wu et al [44].A BLASTP search of the pummelo genome (http://citrus.hzau.edu.cn/index.php)was performed using the UGT conserved sequence.HMM file (PF00201, UGT.HMM) for the conserved domain hidden Markov model of the UGT gene family was downloaded from Pfam (http://pfam.xfam.org/).The amino acid sequences of predicted UGTs were used for phylogenetic analysis.Different protein sequences were aligned using the ClustalW program, and the phylogenetic tree was constructed using MEGA 7 software (Mega Software, State College, PA, USA).The construction of a phylogenetic tree was based on the Neighbor-Joining statistical method and was tested with 1000 bootstrap replicates.Genbank accession numbers of sequences employed in the study can be found in supplemental Table S4.
qRT-PCR was performed using a LightCycler 480 (Roche, Switzerland) with the SYBR Green system (Yeasen, Shanghai, China), as described by Lu et al [46].To verify the expression levels of 7GlcTs, each qPCR was performed with three independent biological replicates.The primers are listed in Supplemental Table S5 and are based on Citrus Pan-genome (http://citrus.hzau.edu.cn/index.php)database gene sequences [47].The actin gene was used as the reference control [48].Expression of genes was calculated by the 2 -ΔΔCt method.

Isolation, cloning, protein expression and purification of 7GlcTs
The 7GlcT genes were isolated based on the Citrus Pan-genome database.The full-length coding sequence (CDS) of 7GlcT genes was amplified using the primers described in Table S5, and then inserted into the pMal-c2x vector, followed by transfer to Escherichia coli BL21 (DE3) for expression.Transformants carrying the expression plasmid 7GlcTs were incubated to OD 600 =0.6 in Luria-Bertani (LB) medium containing 0.1 g L -1 ampicillin at 37 • C, followed by the addition of isopropyl β-D-thiogalactopyranoside to a final concentration of 0.3 mM and then cultured at 16 • C after shaking at 130 rpm for 18 h.Protein purification was performed according to Li et al [49].Subsequently, the protein was concentrated by ultrafiltration using 30 mL (50 kDa MWCO) Ultrafiltration Spin Columns (Millipore, Billerica, MA, USA).Purified proteins were evaluated by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE).

Assay of enzyme activity and kinetics
The enzyme activity assay was determined with reference to Peng et al [12].The reaction total volume was 100 μL, containing phosphate buffered saline (50 mM, pH 7.3), 1 mM UDP-glucose, 5 mM MgCl 2 , 0.2 mM f lavonoid substrates and 20 μg recombinant proteins.The reaction was incubated at 35 • C for 60 min and terminated by adding 200 μL methanol.Then, the resulting products were filtered through a 0.22 μm filter and analyzed by HPLC or LC-MS.
The optimal reaction conditions were determined by varying pH and temperature.Following the method described above, three distinct buffer solutions are employed to regulate pH levels for enzyme determination: acetic acid and sodium acetate buffer (pH 3.6-5.8),phosphate buffered saline (pH 6.0-8.0), and glycine and sodium hydroxide buffer (pH 8.6-10.6).The optimum reaction temperature was determined to be in the range of 25 • C to 60 • C in phosphate buffered saline (pH 7.3).
To determine the enzymatic kinetics for different candidate UGTs, recombinant proteins (10 μg) were added to reaction mixtures containing 50 mM phosphate buffered saline and 1 mM UDP-glucose in a final volume of 100 μL.Reactions were incubated at optimum reaction pH and temperature for 30 min, and the concentrations of tested f lavonoid substrates ranged from 0 to 400 μM.All in vitro enzyme assays were repeated at least three times.Kinetic parameters were calculated using the Michaelis-Menten nonlinear curve fitting.

Transient overexpression of Cg7GlcTs in citrus leaves
The transient overexpression assay was performed in citrus leaves as described by Hussain et al [50] with some modifications.Specifically, the CDS of Cg7GlcTs was inserted into the pBI121 vector.The Cg7GlcTs-pBI121 recombinants and the empty vector (as control) were transferred into Agrobacterium tumefaciens strain EHA105, respectively.The transformants harboring the recombinant vectors and the empty vector were initially cultured in LB Broth at 28 • C.Then, the bacterial cells were separated by centrifugation and re-suspended to an OD 600 of 0.7-0.8 in infiltration buffer (10 mmol L -1 MES, 10 mmol L -1 MgCl 2 , 200 μmol L -1 acetosyringone, pH 5.6 in equal volume).The suspensions containing the target genes and the control were injected separately into the lower epidermis of 'Huazhou' pummelo leaves on opposite sides.After five days, the treated leaves' infiltrated regions were collected for subsequent analyses.FG contents and transcription levels of Cg7GlcTs were measured as described above.The primers utilized in constructing Cg7GlcTs-pBI121 and for the qRT-PCR analysis are provided in Table S5.

Statistical analysis
Statistical analysis was performed using Microsoft Excel 2019.All data were obtained with at least three biological replicates, and error bars indicate standard errors (SE).Significant differences between the samples were analyzed with a two-tailed Student's t-test.Figures were produced with GraphPad Prism 8.0 (GraphPad SoftwareInc., San Diego, CA, USA) and OriginPro 2021 (Origin Lab Corporation, Northampton, USA).Structural formulae were drawn using ChemDraw ultra (PerkinElmer Informatics, Waltham, USA).

Figure 2 .
Figure 2. Identification of putative 7GlcTs based on the pummelo genome.(A) Phylogenetic analysis of pummelo UGTs with other plant UGTs.Only bootstrap values > 60% are displayed, and each value is visualized using a light blue circle, whose size represents the corresponding metadata value in the middle of each branch.(B) Transcript level of the 7GlcTs cluster in HZ and FH pummelo fruits at different developmental stages, red circles mark the genes screened for this article.(C) Correlation between the expression of 7GlcTs and FG contents throughout fruit development.* P < 0.05, * * P < 0.01, * * * P < 0.001.

Figure 5 .
Figure 5. Expression patterns and functional activities of UGT90A31 and UGT89AK1 in citrus.(A) Heat map of the expression patterns of UGT90A31 and UGT89AK1 genes in the pulp of three citrus germplasms during development.The color scale on the right represents the FPKM values.The results are averages of FPKM of three biological replicates.(B) HPLC chromatograms of glucosylation of naringenin and isosakuranetin catalyzed by PtUGT90A31 and PtUGT89AK1 in Nar, naringenin; Nar-7Glc, naringenin 7-O-glucoside; Iso, isosakuranetin; Iso-7Glc, isosakuranetin 7-O-glucoside.(C) F7G product content under catalysis by PtUGT90A31 and PtUGT89AK1 on three groups of f lavonoid substrates: f lavonones, f lavones, and f lavonols.F7G, f lavonoid 7-O-glucoside.Values are means ± SE (n = 3); ND, not detected.

Table 1 .
Flavonoid 7-O-glucoside products from the catalysis of Cg7GlcTs on different types of f lavonoid substrates

Table 2 .
Kinetic parameters of four Cg7GlcTs