Identification of the citrus GRF gene family and its expression in fruit peel thickening mediated by gibberellin

doi:10.21203/rs.3.rs-5297278/v1

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Identification of the citrus GRF gene family and its expression in fruit peel thickening mediated by gibberellin

https://doi.org/10.21203/rs.3.rs-5297278/v1

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Background

Growth-regulating factors (GRFs) play a crucial role in plant growth and development, particularly in cell division and expansion. Citrus fruit cracking, a prevalent issue, adversely impacts both yield and fruit quality. Gibberellins (GAs) are known to ameliorate citrus fruit cracking by inducing thicker peel formation, which is attributed to cell division and expansion. However, the mechanistic link between gibberellins and citrus peel thickening, and whether this process is mediated by GRF genes regulation, has not been definitively established.

Results

In this study, 8 CsGRFs (Citrus sinensis), 11 CcGRFs (Citrus clementina), and 8 CgGRFs (Citrus grandis) were identified from the citrus genome which divided into six clusters, with the genes of the same cluster sharing similar gene structures. Cis-elements analysis revealed that the promoter regions of GRF genes contained numerous hormone-responsive elements. Tissue expression profiles showed that CsGRF genes had higher expression levels in young tissues, including early fruit tissues, one-year-leaf, ovules, and root tips. RNA-seq and qPCR analyses revealed that the expression levels of CsGRF3, 4, 7, and 8 were significantly regulated in response to GA₃ treatment. Notably, CsGRF8 was the most significantly induced by GA₃ and highly expressed in the early stages of peel development. These findings indicate gibberellins may exert regulatory effects on peel development through the induction of CsGRF genes.

Conclusion

This study systematically analyzed the characteristics of the citrus GRF gene family, as well as the changes in citrus peel thickness and the expression patterns of CsGRF genes under gibberellin treatment. These findings provide valuable insights for advancing research on the role of CsGRF genes in regulating citrus peel development, which could help reduce the occurrence of fruit cracking.

GRF

Citrus

Gibberellins

Peel thickness

Fruit cracking

The thickness of the peel limits the quality of citrus fruits, where excessively thin peels can precipitate pre-harvest cracking and compromise post-harvest storage, whereas overly thick peels are often associated with rough disorder, both conditions significantly contributing to an overall decline in fruit quality [1, 2]. In lemons, it has been observed that the peel layer (including the cuticle, epidermis, and hypodermis) of cracked fruits is noticeably thinner compared to normal fruits [3]. In murcott, the period of frequent natural fruit cracking correlated with a significant thinning of the peel development [4]. Moreover, a specific disorder characterized by a pronounced thick peel has been documented in Citrus unshiu, presenting with a constellation of undesirable traits including rough fruit, non-degrading pulp, lower soluble solids content, and a bland flavor [5]. Therefore, unraveling the molecular mechanisms that regulate peel thickness is of paramount importance for advancing citrus fruit quality.

Gibberellins, a class of plant growth regulators, are integral to a multitude of plant developmental processes including leaf expansion [6], stem elongation [7], and root growth [8]. Despite this, studies focusing on their impact during fruit development, and specifically on peel thickness modulation, are comparatively scant. Notably, gibberellin application has been shown to enhance peel thickness in Citrus unshiu, which is associated with the formation of rough fruit [2]. Early maturing ponkan varieties exhibit an increase in peel thickness in response to gibberellin treatment, which correlates with a decreased incidence of fruit cracking, without compromising fruit quality [9]. In tomatoes, silencing the SlPRE2 gene upregulates GA biosynthesis and catabolism genes, resulting in a significant reduction in peel cell size and overall peel thickness [10]. Conversely, interfering with the expression of the SlDELLA protein, a key repressor in the GA signaling pathway, increases the length of peel cells, particularly in the mesocarp, where cell length nearly doubles [11]. While the influence of gibberellin on peel cell development and thickness has been established, the molecular mechanisms underlying these processes warrant further investigation.

GRFs are plant-specific transcription factors that have been identified across many species, including Arabidopsis thaliana [12], Zea mays [13], Solanum lycopersicum [14], Glycine max [15], Oryza sativa [16], Nicotiana tabacum [17], Arachis hypogaea [18], Brassica napus [19]. GRF genes are extensively studied for their roles in leaf morphology [20, 21], grain size regulation [22, 23], stress responses [24], and hormone responses [25, 26]. GRF genes were first discovered in rice, where they play a crucial role in regulating gibberellin-induced stem elongation and leaf development [6, 27]. Overexpression of the OsGRF7 gene upregulates the GA signaling negative regulators SLR1 and SLR1L, ultimately leading to altered leaf morphology and reduced plant height [28]. In maize, overexpression of the ZmGRF10 gene results in a reduced rate of cell division, leading to smaller leaf size and shorter plant height [29]. In Arabidopsis, mutants of GRF1, 2, and 3 result in an enlargement of the meristematic region in the root tips, whereas overexpression of the AtGRF2 and AtGRF3 exhibit the opposite phenotype [30]. Several studies have established that miR396 can directly target and regulate GRFs [31–33]. In tomatoes, the disruption of miR396 leads to a significant upregulation of SlGRF1/2/3/4/5, resulting in the enlargement and thickening of fruit peel cells, and further observations revealed that overexpression of SlGRF2/3/4/5 also caused an increase in the thickness of the fruit pericarp, particularly with SlGRF4 [14, 34].

The N-terminal region of GRF proteins contains two highly conserved domains: the QLQ (Gln, Leu, Gln) and WRC (Trp, Arg, Cys) domains [12, 31]. The QLQ domain interacts with the SNH domain (SYT N-terminal homology domain) of GIF (GRF-interacting factor) proteins, thereby facilitating its transcriptional activation function [27, 35]. The WRC domain contains two distinctive features, namely a putative nuclear localization signal and a zinc-finger motif that consists of the conserved spacing of three Cys and one His residue [12, 27]. Unlike the highly conserved N-terminal region, the C-terminal region of GRF proteins, characterized by less conserved motifs such as TQL (Thr, Gln, Leu) and FFD (Phe, Phe, Asp), exhibits significant variability in length and amino acid composition and have a role in transactivation [13, 29, 36]. These unconservative structural features in C-terminal implying that GRF genes may possess diverse biological functions.

Fruit cracking presents a considerable challenge in the citrus industry, adversely affecting both yield and fruit quality. Understanding whether GRF genes regulate citrus peel thickness in response to gibberellin could help mitigate fruit cracking. This study performed a comprehensive genome-wide identification of the GRF gene family in Citrus sinensis, Citrus clementina, and Citrus grandis. It analyzed their evolutionary relationships, gene structures, cis-elements, chromosomal locations, duplication events, and expression patterns across various citrus tissues. Additionally, the study investigated the effects of different concentrations of gibberellins on peel thickness and CsGRFs transcription levels. The aim is to identify CsGRF genes affecting citrus peel thickness, providing a foundation for improving citrus quality.

Genome-wide identification of GRF gene family in C. sinensis, C. clementina and C. grandis

To comprehensively identify potential GRF genes in the citrus genomes of C. sinensis, C. clementina, and C. grandis, we utilized the HMM model based on the conserved QLQ and WRC domains. This screening identified 8 GRF genes in C. sinensis, 11 in C. clementina, and 8 in C. grandis. We conducted the basic characteristics for these GRFs, encompassing aspects such as chromosome localization, number of introns, protein length, isoelectric point, molecular weight, and subcellular localization (Table S1).

In CsGRFs, CcGRFs, and CgGRFs, intron numbers ranged 1–3, 1–3, and 2–3, respectively. Protein lengths spanned 232 aa (CsGRF5)-600 aa (CsGRF2), 232 aa (CcGRF3)-600 aa (CcGRF10), and 312 aa (CgGRF6)-600 aa (CgGRF1), respectively. Isoelectric points (pI) ranged 5.48 (CsGRF4)-9.75 (CsGRF5), 5.83 (CcGRF4)-9.09 (CcGRF8), and 5.98 (CgGRF5)-9.03 (CgGRF3), respectively. Molecular weights (Mw) ranged 25.26 KDa (CsGRF5)-65.05KDa (CsGRF2), 25.40 KDa (CcGRF3)-65.17 KDa (CcGRF10), and 34.98 KDa (CgGRF6)-65.05 KDa (CgGRF1), respectively. All predicted subcellular localizations indicated nuclear localization.

Phylogenetic analysis of GRF gene family between C. sinensis, C. clementina, C. grandis, and A. thaliana

Phylogenetic analysis based on protein sequence data from C. sinensis, C. clementina, C. grandis, and A. thaliana clustered the 36 GRFs from these four species into six groups (Ⅰ-Ⅵ) (Fig. 1). Group Ⅲ contained the most members (9), followed by Groups IV and V (7 each), Groups Ⅰ and Ⅱ (5 each), and Group Ⅵ, which had the fewest members (3) and lacked CgGRF members. Interestingly, group Ⅴ included only GRFs from the Citrus genus, suggesting a potential genus-specific uniqueness.

Gene structures, conserved motifs, and conserved domains analysis of GRF gene family in C. sinensis, C. clementina, and C. grandis

To explore evolutionary relationships and gene function, we analyzed the gene structures and conserved motifs in CsGRFs, CcGRFs, and CgGRFs. The gene structure analysis revealed that GRFs contained 2–4 exons (Fig. 2). The WRC and QLQ domains were consistently located at the N-terminus of the GRF proteins. Through motif analysis, 10 conserved motifs were identified, with motif 1 and motif 2 present in all GRF proteins, indicating that this structural domain was highly conserved within the GRF gene family. Group Ⅳ had the fewest motifs, containing only motifs 1, 2, and 3, whereas Group Ⅴ had the most, with 7–8 motifs. Similar motif patterns were observed within the same group, suggesting analogous gene functions, while variations in motif order and number across different groups might contribute to functional diversities.

Cis -elements in the promoters of GRF gene family in C. sinensis, C. clementina, and C. grandis

Cis-elements in the promoter region, crucial for gene regulation, were identified in the GRF genes of C. sinensis, C. clementina, and C. grandis. We screened for hormone-responsive and stress-responsive cis-elements, including auxin-responsive elements (TGA-element, AuxRE, TGA-box, AuxRR-core), gibberellin-responsive elements (TATC-box, P-box, GARE-motif), MeJA-responsive elements (TGACG-motif, CGTCA-motif), abscisic acid-responsive elements (ABRE), ethylene-responsive elements, salicylic acid-responsive elements (TCA-element); defense and stress-responsive elements (TC-rich repeats), wound-responsive elements (WUN-motif), MYB binding sites involved in drought-inducibility (MBS), low temperature-responsive elements (LTR), and anaerobic induction elements (ARE), totaling 16 types of cis-elements (Fig. 3).

MeJA-responsive elements (TGACG-motif or CGTCA-motif) were the most abundant hormone-responsive elements in the promoter regions of 27 GRF genes, with 20 GRF gene promoters containing this element, particularly abundant in Group V. Additionally, abscisic acid-responsive (ABRE), auxin-responsive (TGA-element, AuxRE, TGA-box, or AuxRR-core), and gibberellin-responsive (TATC-box, P-box, or GARE-motif) elements were identified in 16, 13, and 13 gene promoters, respectively. In stress responses, anaerobic induction (ARE) elements were found in 21 gene promoters, while drought-inducibility (MBS) and low temperature-responsive (LTR) elements were found in 12 and 11 gene promoters, respectively. These findings suggest that the GRF gene family plays significant roles in various hormonal and stress responses.

Duplication events of GRF gene family

To further investigate the evolutionary mechanisms of GRF gene family, we analyzed the chromosomal localization and tandem/segmental duplication events of CsGRFs, CcGRFs, and CgGRFs. This study showed that CsGRFs, CcGRFs, and CgGRFs were distributed on chromosomes chr1, chr3, chr5, and chr6 simultaneously (Fig. S1).

No tandem duplication events were found in CsGRFs, CcGRFs, and CgGRFs. C. sinensis and C. clementina contained a pair of segmental duplicated genes (CsGRF4/6, CcGRF2/6, respectively), while C. grandis did not contain any (Fig. 4A). These results imply that segmental duplication predominantly facilitate the expansion of GRF genes.

Comparative syntenic maps constructed among C. sinensis, C. clementina, C. grandis, and A. thaliana revealed 12, 8, and 8 syntenic pairs between CsGRFs/CcGRFs, CsGRFs/CgGRFs and CcGRFs/CgGRFs, respectively. 9, 10, and 7 syntenic pairs between CsGRFs/CcGRFs/CgGRFs and AtGRFs, respectively (Fig. 4B, Table S2). Notably, CgGRF4 contained 6 collinear gene pairs, while CsGRF4/6 and CcGRF2/4 each contained 5 collinear gene pairs. This suggests that these GRF genes may play a crucial role in the evolution of GRF gene family.

Influence of gibberellins on peel thickness

To evaluate the influence of exogenous gibberellin (GA₃) on peel thickness, different GA₃ concentrations (25, 50, and 75 mg/L) were applied to ‘Zaomi’ (C. reticulata Blanco) during the early fruit expansion stage, and peel thickness was measured in the late expansion stage and maturity stage, respectively. The results showed that GA₃ application was associated with an enhancement in peel roughness and an expansion in fruit size, as well as fruit coloring (Fig. S2-S4). After treatment with 25/50/75 mg/L of GA₃, the peel thickness increased from 1.76 mm to 2.03, 2.25, 2.25 mm in the late expansion stage, and from 2.28 mm to 2.64, 2.59, 2.57 mm in the maturity stage, respectively (Fig. 5). The results indicate that different concentrations of GA₃ promote an increase in peel thickness.

Tissue expression profiles of CsGRF genes

The expression profiles of CsGRF genes were analyzed across different citrus tissues, including different developmental stages of leaves, ovules, and roots. The expression level of CsGRF genes were higher in ovules and young leaves, while lower in roots and mature leaves. This indicates that CsGRF genes exhibits spatiotemporally expression specificity pattern, which higher expression in ovules suggest that its potential role in fruit development (Fig. 6A).

To further explore the role of CsGRF genes in fruit development, we analyzed the expression levels of CsGRF genes in different development stages of the epicarp, albedo, juice sacs, and segment membrane in C. sinensis fruits (Fig. 6B). CsGRF3 and CsGRF7 showed high expression levels in all tissues, while CsGRF1 and CsGRF6 were barely expressed across all tissue. CsGRF2, 4, 5, and 8 were highly expressed at the early stages of different fruit tissues, with their expression gradually decreasing during fruit maturation. Interestingly, CsGRF2 and CsGRF8 exhibited higher expression levels in the epicarp during the early stages of development, suggesting a potential involvement in the progression of peel development.

RNA-seq and qPCR analyses of CsGRF genes expression under GA₃ treatment

To explore whether CsGRFs respond to the regulation of GA₃, RNA-seq analyses of peel samples were performed by different concentrations of GA₃ treatment to ‘Zaomi’ (Fig. 6C). Results demonstrated that the expression levels of CsGRF3, 4, 6, and 8 significantly increased post-GA₃ treatment, particularly CsGRF8; whereas CsGRF7 expression decreased. CsGRF1, 2 and 5 expression levels were almost undetectable. These findings indicated CsGRF genes were regulated by GA₃, which suggests that GA₃ may regulate fruit peel development by affecting the expression of GRFs.

To further analyze the RNA-seq results, qPCR analysis was performed to assess the expression levels of CsGRF genes in ‘Zaomi’ under different concentrations of GA₃ treatment (Fig. 7). After treatment with GA₃, the expression levels of CsGRF3, 4, and 8 were significantly upregulated, especially CsGRF8. Compared with the control, CsGRF8 showed increments of 459%, 314%, and 186% under 25, 50, and 75 mg/L GA₃ treatment, respectively. In contrast, the expression of CsGRF1, 2, and 7 were significantly downregulated, with CsGRF7 showing the most considerable decrease, recording reductions of 40%, 34%, and 78% at corresponding GA₃ treatments. Overall, the expression of CsGRF3, 4, 7, and 8 was notably responsive to GA₃ modulation, consistent with the RNA-seq results. The expression profiles of tissue- specificity and GA₃ treatment indicate that GRF8 is highly probable to play a crucial role in fruit peel development.

Predicting the interaction network of CsGRFs protein

GRF-GIF often act as partner proteins to form plant-specific transcription complexes that influence organogenesis through involvement in cellular mechanisms [20, 37, 38]. CsGRF3, 4, 7, and 8 were significantly regulated by gibberellins, thus their homologous protein interaction networks in Arabidopsis were predicted using the STRING online tool (Fig. 8). The results indicate that CsGRF3, 4, 7, and 8 homologs in Arabidopsis are predicted to interact with GIF1, 2, and 3. Such predicted interactions reinforce the proposed role of the GRF-GIF complexes in the regulatory mechanisms of cellular development.

Fruit cracking severely impacts citrus yield and quality, with peel thickness being a critical factor in fruit cracking susceptibility. Previous studies have indicated that cell size and number play an important role in determining peel thickness [10, 39, 40]. The GRF gene family, unique to plants, has been widely reported to regulate cell size and number, thus contributing to the control of organ size in plants [32, 41, 42]. However, it remains unclear whether citrus GRF genes are induced by gibberellin and participate in the formation of fruit peel thickness. In this study, phylogenetic analysis categorized the 36 GRF members from CsGRFs, CcGRFs, CgGRFs, and AtGRFs into six distinct groups. Notably, Group V was exclusively composed of GRFs from citrus, hinting at a possible unique evolutionary pathway for this group within the citrus clade. The presence of both motif 1 and motif 2 in all citrus GRFs (Fig. 2) underscores the strong evolutionary conservation characterizing the GRF gene family. Exon-intron structure and motif distribution analyses were consistent with phylogenetic analysis, revealing high structural similarity among GRF genes within the same group but lower similarity between different groups.

Gene duplication provides the raw genetic material for the evolution of novel functions [43]. It is established that the GRF gene family has undergone two notable duplication events: an initial whole-genome triplication event in the shared ancestor of all eudicots, followed by species-specific whole-genome duplications within plants [44]. Citrus, as a core eudicot within the Rutaceae [45], appears to retain a high degree of conservation with limited gene duplication events—often presenting as a single pair or even without duplications. This suggests that GRF genes have been preserved throughout the evolutionary trajectory of the citrus lineage. Combining phylogenetic and syntenic analyses based on gene expression can better predict the functions of GRF genes in different species. Previous reports indicated that AtGRF1, 2, and 3 increase leaf size through cell expansion [12]. In this study, CsGRF2 was syntenic with AtGRF1 and AtGRF2, while CsGRF7 and CsGRF8 were syntenic with AtGRF2 and AtGRF3, respectively, emphasizing the potential functions of CsGRF2, 7, and 8 in organ formation similar to Arabidopsis by regulating cell expansion.

The structure of citrus fruit comprises the epicarp, albedo, juice sacs and segment membrane, with their formation being closely linked to early differentiation. Citrus peel cells begin dividing before flowering, and the peel thickens most rapidly during flowering [46]. The GRF gene family is known for its involvement in cell division and expansion particularly in young plant tissues, with their expression typically elevated in the early phases of tissue development and subsequently declining as maturation ensues [47–49]. Consistent with previous findings, CsGRF genes also exhibited a specific expression pattern at cell division phase, indicating that they played a major role in the early stages of plant growth and development (Fig. 6A, B). The significant expression of CsGRF2 and CsGRF8 within the developing ovule and epicarp coincides with the phase of sharp peel thickening in citrus, indicative of their potential contribution to the development of peel thickness.

Gibberellins are critical hormones in plants, implicated in a wide range of life processes, particularly influencing organ size by modulating both cell division and expansion [50–53], similar to the function of GRF genes. The application of exogenous GA₃ can significantly alter the GRF gene expression levels [6, 17, 54]. In tomatoes, overexpression of SlGRF2/3/4/5 leads to different degrees of peel thickening, particularly overexpression of SlGRF4 increasing it by nearly 50% [34]. In this study, different concentrations of GA₃ increased the peel thickness of ‘Zaomi’ and caused changes in the transcriptional levels of most CsGRF genes. This suggests an intrinsic association between GRF genes, fruit peel thickness and gibberellins, wherein GRF genes potentially play an active role in sustaining or enhancing cellular division and expansion via feedback mechanisms, contributing to organ size regulation. Interestingly, compared to other tissues, CsGRF8 not only exhibits higher expression levels in the fruit peel but also shows the most significant induction by GA₃. Meanwhile, CsGRF8 exhibits the closest evolutionary relationship with SlGRF4 (Table S3), implying that CsGRF8 is highly likely to play a similar role in the development of citrus fruit peel thickness. In other words, it seems reasonable to propose that gibberellin promotes peel thickening by inducing the expression of CsGRF8.

In this study, 8 CsGRFs, 11 CcGRFs, and 8 CgGRFs were identified in the citrus genome. The characteristics, phylogenetic relationships, conserved motifs, gene structures, cis-elements, chromosomal distribution, and gene duplication of the 27 citrus GRF genes were characterized. Additionally, we investigated the changes in citrus fruit peel thickness following GA₃ treatment and analyzed the expression patterns and transcription levels of the 8 CsGRF genes in various citrus tissues and under gibberellin treatment. In fruit tissues, CsGRF2 and CsGRF8 exhibited significantly higher expression levels in the epicarp during the early development stages. Further analysis revealed that CsGRF8 still maintained higher expression levels during the fruit expansion stage and displayed the most significant induction in response to GA₃. This provides a new direction for further research into how GRF genes are associated with GA-mediated fruit peel thickness development.

Identification and physicochemical property analysis of GRF gene family

The protein sequences and genome annotations for C. sinensis, C. clementina, and C. grandis were retrieved from the citrus genome database (http://citrus.hzau.edu.cn). The Hidden Markov Model (HMM) files for the WRC (PF08879) and QLQ (PF08880) domains were obtained from the Pfam database (http://pfam.xfam.org/) [55]. Using the HMMER tool in a Linux system, candidate proteins containing both the WRC and QLQ domains were screened to identify CsGRFs, CcGRFs, and CgGRFs (E-value ≤ 1e^-5). Then, the presumed GRF protein sequences were further identified by means of the identification of the QLQ and WRC domains via the SMART (http://smart.embl-heidelberg.de/) and InterPro (https://www.ebi.ac.uk/interpro/search/text/null) databases.

Characterization of the CsGRFs, CcGRFs, and CgGRFs proteins, including amino acid length, isoelectric point, and molecular weight information, was analyzed through the Expasy Protparam tool (https://web.expasy.org/protparam/). Gene structural analyses were conducted using the Gene Structure View tool within TBtools [56] (https://github.com/CJ-Chen/TBtools). Subcellular localization prediction was performed using the online tool Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/). Furthermore, CsGRFs, CcGRFs, and CgGRFs were renamed based on their chromosome location. The corresponding blast gene mappings for Citrus sinensis v2.0, Citrus sinensis v1.0, Citrus clementina v1.0, and Citrus sinensis v3.0 were provided in table (Table S4).

Phylogenetic analysis of the GRF proteins

All GRF protein sequences were aligned using ClustalW within MEGA11 software [57]. Subsequently, an unrooted phylogenetic tree was constructed based on these alignments employing the neighbor-joining method, with 1000 bootstrap replications.

Gene structures, conserved motifs, and conserved domains analysis

The conserved motifs encompassed within the GRF protein sequences were identified through the MEME program (http://meme-suite.org/tools/meme). All parameter settings are default, except for the number of motifs set to 10. Conserved domain predictions for GRF proteins were conducted using the Batch CD-Search tool available at the NCBI database (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi). In instances where the QLQ and WRC domains within GRF proteins were not readily identifiable, the InterPro database (https://www.ebi.ac.uk/interpro/search/text/null) was employed as a secondary resource for confirmation. Finally, the gene structures of CsGRFs, CcGRFs, and CgGRFs were visualized with the Gene Structure View tool within TBtools.

Analysis of cis-elements in promoters

The upstream 2000 bp sequence of CsGRFs, CcGRFs, and CgGRFs in the citrus genome was extracted. The online tool PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was utilized for the prediction of cis-regulatory elements within the promoters of GRF genes [58], allowing for the determination of their types, quantities, and associated functions. Visualization of these elements was subsequently achieved using the Basic Biosequence View tool within TBtools.

Chromosomal location and gene duplication analysis

Using the genomic data of C. sinensis, C. clementina, and C. grandis, the chromosomal positions of the GRF genes were mapped using the Gene Location Visualize from GTF/GFF tool within TBtools.

The One StepMCScanX Super Fast tool within TBtools analyzes GRF genes duplication events (E-value ≤ 1e^-3), and the results were visualized through the Advanced Circos and Dual systeny plot for MCScanX tool within Tbtools.

Plant materials and gibberellins treatments

To investigate the effect of GA₃ on the peel thickness development of citrus, the citrus variety ‘Zaomi’ used in this study was sourced from Luxi County, Jishou City, Hunan Province, China. GA₃ at different concentrations (0, 25, 50, and 75 mg/L), with 0.05% Tween-80 included as a surfactant, was applied to the entire tree using a backpack sprayer. GA₃ treatment was applied three times during the early fruit expansion stage (70, 80, and 90 DAF, days after full bloom), with five trees for each treatment. Fruits were harvested during the late expansion stage (110 DAF) and maturation stage (190 DAF). From each treatment group, five fruits were selected for sampling, and placed in a cooler with ice packs for transportation on the same day to the laboratory, followed by grounding into powder using liquid nitrogen, and stored at -80℃ for further analyses.

Measurement of fruit shape indices and peel thickness

Randomly measure the maximum width of over 100 fruits treated with GA₃, and sample based on the fluctuations in the maximum width of the fruits. Measure the maximum width and height of the collected ‘Zaomi’ samples using a vernier scale, and determine the weight of each fruit using an electronic balance. Peel thickness is measured as follows: each fruit was bisected horizontally at its point of maximum width, then peel thickness was gauged at two diametrically opposed sites on the cut surface using a stereo fluorescence microscope. The mean value of these two measurements was recorded as the peel thickness for that particular fruit, with each treatment group containing at least 20 fruits.

Tissue expression profiles and RNA-seq analysis

Genome-wide expression data for leaves, root, and ovules tissue of C. sinensis were obtained from the citrus genome database (http://citrus.hzau.edu.cn/), and detailed data is provided in the table (Table S5). Furthermore, by utilizing the RNA-seq data of C. sinensis from Feng [59], the FPKM values for the 8 CsGRFs across different tissues (epicarp (EP), albedo (AL), juice sac (JS), and segment membrane (SM)) and developmental stages (50, 80, 120, 155, 180, and 220 DAF). Create a heatmap for these data using the HeatMap tool within TBtools.

Total RNA from the peel of ‘Zaomi’, collected at the late fruit expansion stage (110 DAF), was isolated using the CTAB extraction protocol. The enrichment of mRNA was subsequently achieved with the aid of magnetic oligo (dT) beads. Transcriptome libraries for sequencing were constructed for four sample groups: control peel (ZM_CK), peel treated with 25 mg/L GA₃ (ZM_GA25), peel treated with 50 mg/L GA₃ (ZM_GA50), and peel treated with 75 mg/L GA₃ (ZM_GA75). These RNA-seq libraries were sequenced on the Illumina HiSeq platform, and reads were generated in a 150-bp paired-end format. The reads were quality-filtered using the fastp toolkit that involved the removal of adapter sequences and low-quality reads containing ploy-N [60]. The quality-filtered reads were mapped to the reference cDNA sequences of Citrus clementine from the citrus genome annotation project (Citrus clementina v1.0) database using the default parameters of HISAT2 [61]. To estimate gene expression levels, fragments per kilobase per million mapped reads (FPKM) of each gene were calculated [62]. Since the sequenced genome was Citrus clementina v1.0, the CsGRFs were blasted against the Citrus clementina v1.0 genome.

Analysis of gene expression by RT-qPCR

Total RNA from ‘Zaomi’ peel was extracted using a plant RNA extraction kit (SteadyPure, ACCURATE BIOLOGY, Changsha, China). Reverse transcription was performed with a reverse transcription kit (Evo M-MLV, ACCURATE BIOLOGY, Changsha, China). Citrus Actin (Csactin) was used as the reference gene for normalization in the qPCR analysis. qPCR was performed on the CFX96^™ real-time system (Bio-Rad Laboratories). The relative quantification of the amplified products was calculated using the 2^–ΔΔCT method [63]. Statistical significance of the qPCR data was assessed using the SPSS 26, based on three independent biological replicates. The primers used for amplification of CsGRFs and the Csactin was detailed in the supplementary table (Table S6).

The protein association network predictions

The web tool STRING 12.0 (http://string-db.org) was used to predict the functional interaction network models of homologs of CsGRF proteins in Arabidopsis thaliana, and the results were visualized using Cytoscape software.

GRFs

Growth-regulating factors

GAs

Gibberellins

Citrus sinensis

Citrus clementina

Citrus grandis

DAF

days after full bloom.

Acknowledgements

Not applicable.

Author contributions

YWC and JFY designed the research. JFY and XPL modified the manuscript. XL, BY, MP, YSZ, TC, JWL, FFL, and JFY performed the experiments and analyzed the data. BY provided experimental materials. XL wrote the original manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (31872044 and 32472666), the Natural Science Foundation of Hunan Province (2024JJ6240), and the earmarked fund for China Agriculture Research System (CARS-26).

Availability of data and materials

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Gene expression profile data can be obtained at supplementary information. Fruit tissue expression profile data is available on the NCBI Gene Expression Omnibus (GEO) under the accession number GSE125726 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE125726).

Ethics approval and consent to participate

The experimental research on plants performed in this study complies with institutional, national and international guidelines.

Clinical trial number

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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Identification of the citrus GRF gene family and its expression in fruit peel thickening mediated by gibberellin

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Abstract

Background

Results

Conclusion

Figures

Background

Results

Duplication events of GRF gene family

Influence of gibberellins on peel thickness

Predicting the interaction network of CsGRFs protein

Discussion

Conclusions

Methods

Identification and physicochemical property analysis of GRF gene family

Phylogenetic analysis of the GRF proteins

Gene structures, conserved motifs, and conserved domains analysis

Chromosomal location and gene duplication analysis

Plant materials and gibberellins treatments

Measurement of fruit shape indices and peel thickness

Tissue expression profiles and RNA-seq analysis

Analysis of gene expression by RT-qPCR

The protein association network predictions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1