The WUSCHEL-related homeobox transcription factor CsWOX3 negatively regulates fruit spine morphogenesis in cucumber (Cucumis sativus L.)

Abstract Cucumber (Cucumis sativus L.) is a widely cultivated crop with rich germplasm resources, holding significant nutritional value. It also serves as an important model for studying epidermal cell fate and sex determination. Cucumbers are covered with multicellular and unbranched trichomes, including a specific type called spines found on the surface of the fruit. The presence and density of these fruit spines determine the visual quality of cucumber fruits. However, the key regulatory genes and mechanisms underlying cucumber fruit spine development remain poorly understood. In this study, we identified a WUSCHEL-related homeobox (WOX) family gene CsWOX3, which functioned as a typical transcriptional repressor and played a negative role in fruit spine development. Spatial–temporal expression analysis revealed that CsWOX3 exhibited a relatively high expression level in the cucumber female floral organs, particularly in the fruit exocarp. Knockout of CsWOX3 using CRISPR/Cas9 resulted in a significant 2-to-3-fold increase in the diameter of fruit spines base, while overexpression led to a 17% decrease in the diameter compared to the wild-type. A SQUAMOSA PROMOTER BINDING PROTEIN-LIKE transcription factor CsSPL15 could directly bind and activate the expression of CsWOX3, thereby suppressing the expression of downstream auxin-related genes, such as CsARF18. Additionally, the RING-finger type E3 ubiquitin ligase CsMIEL1-like interacted with the HD domain of CsWOX3, which might result in the ubiquitination and subsequent alteration in protein stability of CsWOX3. Collectively, our study uncovered a WOX transcription factor CsWOX3 and elucidated its expression pattern and biological function. This discovery enhances our comprehension of the molecular mechanism governing cucumber fruit spine morphogenesis.


Introduction
Plant trichomes are hair-like epidermal structures that widely cover the surface of most vascular plants and are formed exceptionally through the polar growth of plant epidermal cells [1][2][3][4][5].Trichomes come in diverse morphologies and structures, categorized as either unicellular and multicellular, or non-glandular and glandular based on their secretory capacity [5][6][7].Functionally, plant trichomes act as a natural mechanical barrier, protecting plants from external damage [5,8].In addition to this primary function, they also play various other important roles.For example, the cotton fibers' trichomes found on the seed epidermis hold significant economic value.Glandular trichomes in tomatoes can produce terpene secretory substances used in the production of insecticides.Another noteworthy example is Artemisinin, a terpenoid ester secreted by Artemisia annua glandular trichomes, renowned for its medicinal properties [7,[9][10][11][12][13].
Cucumber (Cucumis sativus L.), a vegetable crop belonging to the Cucurbitaceae family, is extensively cultivated worldwide [2,4,14,15].Unlike the branched unicellular trichomes in the model plant Arabidopsis thaliana, cucumber trichomes are non-branched and multicellular, and widely found in different parts of the cucumber plant, including leaves, stems, f lowers, tendrils, fruits, etc [5,[16][17][18][19].Among them, the trichomes distributed on the surface of cucumber fruits are also known as fruit spines, and which presence and density are significant factors in determining the visual quality of the fruits, making it an important characteristic for commercial purposes [2,5,[18][19][20][21].The common fruit spines, consisting of a spherical or conical base and a sharp stalk, sometimes develop on the tubercules, which are known as the fruit warty trait and hold great commercial value for cucumber fruits [18,19,21].
Several regulatory genes and endogenous hormone signals have been identified to play pivotal roles in the development of cucumber fruit spines.Dong et al. [5] observed that the trichomes on the surface of cucumber cotyledons seemed to share a time-course developmental process with fruit spines, and their process unfolded into five stages: initiation (I), first division (II), glandular trichomes head transition/non-glandular trichomes tip head formation (III), glandular head formation/nonglandular trichomes elongation (IV), and glandular trichomes active metabolism/formation of non-glandular trichomes multicellular base (V).Notably, A HD-ZIP IV transcription factor CsTril/CsGL3 plays a critical role in the fate determination and initial development of cucumber trichomes (stage I), and their mutants exhibit a complete absence of trichome on various parts of the plant, including stems, leaves, tendrils, fruits, etc [5,17,20,22].Another critical transcription factor, CsGL1/CsMict/CsTBH, is a member of the HD-Zip I family and plays a role in trichome development at stage III [2,17,20,[23][24][25][26]. It functions as a downstream factor of CsTril/CsGL3, and the loss of its bio-function results in the formation of stunted trichomes.Recent studies have brought to light several novel regulatory factors involved in the development of cucumber fruit spines.Among them, the cucumber hard spines gene CsTs, a C-type lectin receptorlike tyrosine protein kinase, plays a crucial role in tender fruit spine formation and interacts with the auxin signaling factor CsVTI11 to inf luence cell polarity and spine development [27,28].CsNS, a member of the AUX1/LAX family, is specifically expressed in the fruit peel and spines and regulates the density of fruit spines through the auxin responsive Aux/IAA family genes independently of the CsTu [29,30].Furthermore, Guo et al. [31] have demonstrated that the cucumber APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) CsTOE3 directly interacts with CsGL1 and CsTTG1 to control the development of type II nonglandular fruit trichomes partly through a specific ethylenerelated pathway.CsSBS1, a C2H2 zinc finger transcription factor, positively regulated the development of cucumber fruit spines base, and could also form a trimer complex with CsTTG1 and CsGL1 to regulate the size of cucumber fruit spines base through the ethylene signal pathway [26].Although several regulatory genes have been reported in various development stages, the molecular mechanisms underlying cucumber fruit spine development remain incompletely understood.Therefore, it is essential to explore more novel regulatory factors to enhance our comprehension of the intricate molecular network in the future.
To further investigate key genes in fruit spine development, we identified CsWOX3, a member of the WUSCHEL-related homeobox (WOX) transcription factor in cucumber.CsWOX3 is a homolog of OsWOX3B, known for its involvement in governing the initiation and elongation of rice trichomes [32][33][34][35][36]. CsWOX3 acted as a transcriptional repressor and negatively regulated the morphogenesis of cucumber fruit spines.Additionally, we identified the CsSPL15-CsWOX3-IAA/AUX transcriptional regulatory module and confirmed the physical interaction between CsWOX3 and the RINGfinger type E3 ubiquitin ligase CsMIEL1-like during cucumber spine development.In conclusion, our study uncovers CsWOX3 as a novel transcriptional regulator that negatively impacts the morphogenesis of fruit spines in cucumber, which occurs through the auxin-and ubiquitin-mediated pathway.This study contributes to enriching the molecular mechanism of cucumber trichome development.

CsWOX3 functions as a typical WOX family transcriptional repressor
OsWOX3B, a WUSCHEL-related homeobox (WOX) transcription factor, is crucial for controlling rice development, and its mutation results in reduced or complete absence of trichomes on leaves and seed glumes [32][33][34][35][36]. Through phylogenetic analysis in cucumber, we identified CsWOX3 (Csa6G301060) as the homolog of OsWOX3B (Fig. 1A).CsWOX3 belonged to the WUS sub-family, as evidenced by the conserved HOMEODOMAIN (HD) domain and WUS domain (Fig. 1B-C).Then we further characterized CsWOX3 as a member of the WOX family transcription factors by analysing its subcellular localization, transcriptional activation activity and interaction with downstream DNA-binding cis-elements (Fig. 2).We observed that CsWOX3 localized to the nucleus after fusion with GFP protein and specifically bound to the G-box like DNA cis-element, which contained the conserved core sequence TCACGTGA (Fig. 2A-B).Additionally, when CsWOX3 was fused with VP16 protein, it significantly inhibited the transcriptional activation activity of VP16, particularly through its C-terminal WUS domain (Fig. 2C-D).Collectively, these findings indicate that CsWOX3, the homolog of OsWOX3B, functions as a typical WOX family transcriptional repressor.

Relatively high expression of CsWOX3 within female floral ovary epidermis
Next, we investigated the spatial-temporal expression pattern of CsWOX3.Wild-type cucumbers 3661 were grown in pots until f lowering and fruiting.We collected samples from various aboveground tissues and analysed the relative transcript level of CsWOX3.Our analysis revealed that CsWOX3 was expressed in multiple cucumber tissues, including the stem, leaf, and tendrils.It is worth noting that its expression was relatively high in the female f loral organs, with higher levels in the ovary but the corolla (Fig. 3A).Interestingly, the expression level was more pronounced in young female f loral organs compared to those in later f lowering stages.Further validation experiments confirmed that CsWOX3 exhibited high expression in the exocarp without fruit spines (PE), while its expression in the fruit spines (FRS) and f lesh (FE) was relatively low (Fig. 3B).Furthermore, mRNA in situ hybridization assay showed strong expression signals in the epidermis of the cucumber female f loral ovary, whereas the expression signals in fruit spines and f lesh were comparatively weak (Fig. 3C-D).

CsWOX3 negatively regulates the morphogenesis of cucumber fruit spines
To determine the biological function of CsWOX3, we initially silenced its transcription using the TRSV-VIGS system (Fig. 3E-I; Fig. S1A, see online supplementary material).A significant increase of over three times was observed in fruit spines base within TRSV::Cswox3 silenced lines, while no significant differences were noted on the surface of leaves and petioles (or stems).This suggests that CsWOX3 plays a central role in the development of cucumber fruit spines.Furthermore, we generated two CsWOX3 knockout lines, Cswox3 CRISPR 1 and Cswox3 CRISPR 3, using CRISPR/Cas9 gene editing technology (Fig. S1B-C, see online supplementary material).In these lines, all types of mutations resulted in frameshift and a premature termination of CsWOX3 translation (Fig. 4A).Notably, on the ovary surface of Cswox3 CRISPR lines, the size of fruit spines base was significantly larger compared to the 6101-4 wildtype plants, particularly in the Cswox3 CRISPR 1 line (Fig. 4B).Subsequently, we generated CsWOX3 overexpression lines, CsWOX3 DOE 1 and CsWOX3 DOE 4, which exhibited stable GFP f luorescence and ectopically increased CsWOX3 transcript level (Fig. 4F; Fig. S1D-E).CsWOX3 DOE lines exhibited a slight decrease Figure 1.Phylogenetic relationship, gene structure, and protein domains of CsWOX3.A Phylogenetic analysis using maximum likelihood between all cucumber WOX proteins and selected WOX proteins from other species.CsWOX3 (marked in red) exhibits a homologous relationship with OsWOX3B.They also belong to the WUS subclass of the WOX family together with AtWOX3 in Arabidopsis, OsWOX3 in rice, ZmWOX3A/B in maize, and SlWOX3A/B in tomato, etc. B Gene structure diagram of CsWOX3.CsWOX3 is located on Chromosome 6, with a gene length of 1366 bp and a CDS length of 582 bp, including two exons and one intron.C Protein sequence and domain analysis of CsWOX3.The N-terminus of CsWOX3 features a conserved HOMEODOMAIN (HD) domain (marked in green), while the C-terminus region includes a WUS-box protein motif with the sequence 'TLRTLELFPV' (marked in yellow).Notably, there is an absence of a typical acidic region or EAR motif.Furthermore, the red triangles highlight the predicted ubiquitination modification sites. in the size of fruit spines base compared to wild-type plant (Fig. 4C).
Next, we employed scanning electron microscope (SEM) and quantitative statistics to assess the effects of CsWOX3 knockout and overexpression on the average diameter of the fruit spines base (Fig. 4D-E).The results showed a significant 2-to-3fold increase in the average diameter of fruit spines base when CsWOX3 was knocked out, while the average diameter in the CsWOX3 DOE lines decreased by 17% compared to the 3661 wildtype plants (Fig. 4G-H).These results demonstrate that CsWOX3 plays a negative role in the morphogenesis of cucumber fruit spines.Decreased CsWOX3 expression leads to an increase in the diameter of fruit spines base, while increased expression has the opposite effect.

CsWOX3 orchestrates the expression of genes involved in auxin signaling and protein ubiquitination
To further elucidate the gene regulatory networks associated with CsWOX3 in the control of fruit spine morphogenesis in cucumber, we performed transcriptome profiling on both the 3661 line and CsWOX3 overexpression lines.A total of 309 differentially expressed genes (DEGs) were identified, including 224 upregulated genes and 85 down-regulated genes (Table S2, see online supplementary material).Subsequently, we performed Gene Ontology (GO) analysis on the DEGs to understand their functional classification in biological processes.We found that 245 genes were enriched.In terms of biological processes, the enrichment was mainly observed in the regulation of transcription and DNAtemplated processes, auxin-activated signaling pathway, and cell wall modification (Fig. 5A).In terms of cell components, the enrichment was mainly observed in the integral component of membrane, nucleus, membrane and plasma membrane, with several ubiquitin ligases being enriched (Fig. 5B).Additionally, the enrichment was mainly observed in metal ion binding, heme binding, sequence-specific DNA binding and iron ion binding within molecular function (Fig. S2, see online supplementary material).
We conducted a heatmap analysis on the transcription levels of DEGs involved in auxin-activated signaling pathway and the enriched ubiquitin ligases (Fig. 5C-D).Our results revealed that all the auxin-related genes (Csa1G231530, Csa2G010920, Csa2G011420, Csa2G200420, Csa3G431430, Csa3G646010, Csa6G 384060, Csa7G378520, and Csa7G378530) showed up-regulated expression (Fig. 5C).Subsequently, endogenous IAA levels were quantified within the transgenic lines of CsWOX3 (Fig. 5E).The overexpression lines of CsWOX3 displayed elevated auxin concentrations, while the knockout lines demonstrated reduced levels of auxin.Furthermore, among the selected genes encoding ubiquitin ligases containing F-box domains, Csa4G280500, Csa5G021300, Csa5G600900, Csa6G425740, Csa6G487650, and Csa7G056500 exhibited up-regulated expression, while Csa3G389860 and Csa3G389870 were down-regulated (Fig. 5D).In conclusion, these findings suggest that the overexpression of CsWOX3 leads to transcriptional changes in a set of related genes.CsWOX3 is likely involved in the morphogenesis of fruit spines through the auxin-related pathway and ubiquitination modification pathway in cucumber.

The conserved SPL-WOX-IAA/AUX module controls the development of cucumber fruit spines
Three homologous SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SBP, known as SPL) transcription factors, ZmSPL10/14/26,   were identified in maize, and mutations in these proteins led to the complete absence of trichomes [37,38].These proteins could bind and activate the expression of ZmWOX3A, the homolog of OsWOX3B, to orchestrate the expression of downstream auxinrelated genes.This transcriptional module played a critical role in determining the fate of epidermal hair precursor cells on maize leaves.Similarly, the homolog of OsWOX3B, CsWOX3, was also involved in the development of cucumber fruit spines through auxin-related pathways.We hypothesized that the SPL-WOX-IAA/AUX transcription module may also be conserved in cucumber.To test this hypothesis, we conducted a phylogenetic analysis of all 15 cucumber SPL family proteins.The results revealed that CsSPL15 (Csa6G109120), CsSPL11 (Csa6G517960), CsSPL9 (Csa1G074980), and CsSPL5 (Csa4G631590) were closely related to ZmSPL10/14/26 and belonged to the same branch (Fig. 6A; Fig. S3A, see online supplementary material).Subsequent dual-luciferase reporter assays demonstrated that only CsSPL15 could bind and activate the expression of promCsWOX3::LUC (Fig. 6B).We identified five SBP-box DNA cis-elements (containing the conserved core sequence GTAC) that could potentially be bound by CsSPL15 within the CsWOX3 promoter.A yeast onehybrid LacZi assay showed that CsSPL15 indeed bound to the SBP-box (TTTGTACTT) located 1823 bp upstream of the CsWOX3 promoter (Fig. 6C).Furthermore, we analysed the promoter regions of various genes involved in the auxin-related pathway, including YUCCA family genes, AUX/LAX family genes, PIN family genes, ARF genes, and other related genes.We specifically focused on the genes containing more G-box like or TAAT-box DNA ciselements and conducted a dual-luciferase reporter assay for verification (Fig. 6D).The results revealed that CsWOX3 could suppress the expression of CsARF18 (Csa6G445210) and CsYUC10 (Csa2G302220), while promoting the expression of CsIAA4 (Csa2G011420).However, CsWOX3 had no effect on CsPIN2-like (Csa4G064100) and CsD6PK (Csa1G175730).Subsequent yeast onehybrid LacZi assay results confirmed that CsWOX3 specifically binds to the G-box like DNA cis-elements located within the 1000 bp upstream region of CsARF18's promoter (Fig. 6E).
To evaluate the functional conservation of this transcriptional regulatory module in cucumber, we conducted a preliminary assessment of CsSPL15's biological function using the TRSV-VIGS system (Fig. 6F-I).Our findings revealed an enlargement in the diameter of fruit spines base within the two silenced lines.The diameters in all silenced lines increased more than twice compared to TRSV2::00 lines, suggesting a negative role for CsSPL15 (Fig. 6J).Furthermore, we observed a reduced expression of CsWOX3 within CsSPL15 silenced lines, providing additional evidence for the activation of CsWOX3 transcription by CsSPL15 (Fig. 6K).Moreover, the transcriptional level of CsARF18 was found to decrease in CsWOX3 overexpression lines and increase in CsWOX3 knockout lines, indicating a regulatory relationship between CsWOX3 and CsARF18 (Fig. 6L).Taken together, we conclude that the SPL-WOX-IAA/AUX transcription module, present in monocotyledonous plants such as maize and rice, is also conserved during the development of cucumber fruit spines.

CsWOX3 interacts with a RING-finger E3 ubiquitin ligase CsMIEL1-like
We previously showed that CsWOX3 functions as a transcriptional repressor and may control the cucumber fruit spine development through a ubiquitination modification pathway (Fig. 5).To comprehensively investigate the molecular mechanisms underlying CsWOX3's role in negatively regulating fruit spine morphogenesis in cucumber, we performed a yeast two-hybrid library screening.Our aim was to identify potential interaction partners that collaborate with CsWOX3 in this process.Through this screening, we identified four RING-finger type E3 ubiquitin ligases and revealed that only the E3 ubiquitin ligase CsMIEL1-like (Csa7G394010) could interact with CsWOX3 (Fig. 7A; Table S3, see online supplementary material).This interaction was further confirmed through firef ly luciferase complementation imaging assay and bimolecular f luorescence complementation assay (Fig. 7B-C).Additionally, we analysed the interaction between different truncated CsWOX3 protein domains and CsMIEL1-like.The results indicated that CsMIEL1-like could interact with the N-terminal HD domain of CsWOX3 (Fig. 7D).Consistent with this, five ubiquitination modification sites within the CsWOX3 HD domain were predicted using the BDM-PUB website (http://bdmpub.biocuckoo.org/prediction.php)(Fig. 1C).
CsMIEL1-like was a C 3 H 2 C 3 type RING-finger E3 ubiquitin ligase, and primarily located in the nucleus and membrane (Fig. S3B-C).CsMIEL1-like exhibited high expression levels in cucumber male f lowers and f lesh, while displaying an opposite expression trend to CsWOX3 within female f loral organs (Fig. 7E).Notably, the transcriptional expression of CsMIEL1-like appears to be positively correlated with the expression of CsWOX3 (Fig. 7F).An increase in CsWOX3 expression led to a corresponding increase in CsMIEL1-like expression, while knockout of CsWOX3 resulted in decreased expression of CsMIEL1-like.These findings suggested that there may be a crosstalk between CsWOX3 and CsMIEL1like.Collectively, our results suggest that CsWOX3 physically interacts with the RING-finger type E3 ubiquitin ligase CsMIEL1like through the N-terminal HD domain.
In this study, we identified a transcription factor CsWOX3 in cucumber, which was a homolog of OsWOX3B and belonged to the WUS sub-family of the WOX family (Fig. 1).CsWOX3 contained an N-terminal HD domain and a C-terminal WUS domain but lacked the typical acidic region, EAR motif, and nuclear localization sequence (NLS).Nonetheless, CsWOX3 was localized in the nucleus and exhibited strong transcriptional inhibitory activity (Fig. 2).After all, the WUS domain and the EAR motif possess transcriptional inhibition effect, while the acidic region may have a transcriptional activation effect [40][41][42][43][44]. Furthermore, our study confirmed that CsWOX3 binds to the G-box like DNA cis-element within the promoter of downstream genes, rather than the TAATbox or TGAA-box cis-element bound by other WUS sub-family proteins (e.g., AtWUS) (Fig. 2B) [45][46][47].
Subsequently, the expression pattern of CsWOX3 was investigated in this study (Fig. 3).It was found that CsWOX3 was highly expressed in the younger female f loral organs, particularly in the exocarp of the ovary.This expression pattern is similar to that of OsWOX3B in rice and ZmWOX3A in maize.In rice, OsWOX3B exhibited higher expression in young tissues, while in maize, ZmWOX3A was localized in the early leaf micro-trichomes and their precursor cells [32][33][34][35][36][37].Given that WOX family proteins are well-known early regulators critical for plant growth and development, we hypothesize that CsWOX3 may similarly control the growth and development of cucumber ovary epidermal cells [40,[48][49][50][51]. Further investigation of CsWOX3 expression pattern using β-glucuronidase staining analysis or fused GFP protein tags is warranted in future studies.
We conducted a comprehensive investigation on the biological function of CsWOX3, which negatively inf luenced the morphogenesis of fruit spines in cucumber (Figs 3 and 4; Fig. S1, see online supplementary material).Although the size of fruit spines base in both the Cswox3 CRISPR lines was significantly larger than that in the 6101-4 wild-type plants, the mutation of CsWOX3 did not affect the fate of fruit spines in fruit cucumber genotype 6101-4 plants, which naturally aged and fell off during the later developmental stage.Interestingly, CsWOX3 seem to affect the density of epidermal trichomes upon cucumber leaves (Fig. S1F-G, see online supplementary material).Knockout of CsWOX3 decreases leaf trichomes density, whereas overexpression increased which density.The role of CsWOX3 in controlling leaf trichomes mirrors that of SlWOX3B in tomatoes, yet this function is not applicable to cucumber fruit spines [52].Overall, CsWOX3 serves as a negative regulator of the development of fruit spines in cucumber.However, in Arabidopsis thaliana, previous studies have not reported the role of AtWOX3 in trichomes development, unlike rice and maize.AtWOX3/AtPRS not only controlled f loral organ development but also redundantly inf luenced leaf development with AtWOX1 and AtWOX5 [50,[53][54][55].Although these WOX3 proteins share a homologous phylogenetic relationship, their biological functions vary among different species due to gene subfunctionalization.Moreover, WOX proteins exhibit not only redundancy and complementarity in their biological functions but also the differentiation and specific expression of their promoters.These factors contribute to a diverse range of biological functions [41,51,56].Therefore, it is essential to further investigate the functions and molecular mechanisms of WOX Overexpression of CsWOX3 in cucumber resulted in transcriptional changes in a set of related genes.Gene Ontology (GO) analysis of these differentially expressed genes (DEGs) revealed enrichment of auxin-activated signaling pathway and ubiquitin ligases containing the F-box domain, suggesting that CsWOX3 may regulate the morphogenesis of cucumber fruit spines through the auxin signaling pathway and ubiquitin-mediated proteolysis (Fig. 5; Fig. S2, see online supplementary material).Additionally, differences in endogenous IAA content were confirmed within CsWOX3 transgenic lines, and an E3 ubiquitin ligase interacting with CsWOX3 was identified.
Plant auxin is a critical endogenous hormone involved in organ morphogenesis, including the development of plant trichomes [57,58].For instance, SlARF3 was involved in the formation of epidermal cells and trichomes, while auxin transportation mediated by GhPINs played a vital role in the initiation and development of cotton fibers [59,60].In our study, we confirmed the conservation of the SPL-WOX-IAA/AUX transcription module in cucumber (Fig. 6).CsSPL15 was found to bind and activate the expression of CsWOX3, thereby suppressing the expression of downstream auxin-related genes such as CsARF18.Although we initially investigated the biological function of CsSPL15 using TRSV-VIGS system, a more in-depth examination is warranted to elucidate the molecular mechanisms of this regulatory module, and the potential miRNA156-SPL-WOX-IAA/AUX transcription cascade should also be investigated.
As a member of the early auxin response gene family in plants, Aux/IAA proteins, a short-lived nuclear protein that can be degraded through the 26S proteasome pathway, can bind to the auxin response factor ARF proteins through the Cterminal protein domains [61].The ARF protein family is a typical transcription factor family, with class A ARFs potentially acting as transcription activators, while class B and C ARFs are generally considered transcription inhibitors [62].Within the canonical auxin signaling pathway, once the concentration of free auxin in plants reaches a certain threshold range, it triggers the formation of protein co-receptors involving TIR1/AFB and Aux/IAA proteins.This interaction leads to the degradation of Aux/IAA proteins via the SCF-type E3 ubiquitinprotein ligase pathway.Consequently, more ARF proteins are released, ultimately inf luencing the transcriptional expression of downstream genes [63,64].Our study reveals a novel auxin regulatory pathway mediated by CsWOX3.Through overexpression of CsWOX3, auxin synthesis is enhanced via a specific pathway, resulting in the establishment of a high-threshold auxin environment within plants.The release of ARF proteins, such as CsARF18, might be triggered, with CsWOX3 playing a key regulatory role in maintaining the optimal concentration of ARF proteins to effectively regulate plant growth and development.Additionally, CsWOX3, functioning as a regulator, is tightly controlled by the RING-finger type E3 ubiquitin ligase CsMIEL1-like.While our study provides insights into the interaction between CsWOX3 and CsARF18, further research is warranted to fully elucidate the intricate molecular regulatory mechanisms involving CsWOX3, CsARF18, auxin, and cucumber fruit spines.
Protein ubiquitination is a critical post-translational modification in plants, and the E3 ubiquitin ligases involved in this process can be categorized into different subfamilies, such as RING-finger ligases and cullin-RING ligases (CRLs) [65][66][67].In this study, we successfully identified a RING-finger type E3 ubiquitin ligase CsMIEL1-like as a CsWOX3 interaction partner (Fig. 7).CsMIEL1like may ubiquitinate CsWOX3 by interacting with the HD domain of CsWOX3.Interestingly, we observed that CsMIEL1-like exhibited a contrasting expression pattern to CsWOX3 within the cucumber female f loral organs, suggesting a crosstalk between CsWOX3 and CsMIEL1-like.Based on these findings, we hypothesize that CsMIEL1-like may interact with CsWOX3 to maintain its gradient distribution, which is essential for a precise control of fruit spine morphogenesis.Further research is needed to determine if there is a negative feedback loop or dosage effect within this regulatory mechanism.
Numerous crucial genes have been identified in cucumber spine development, yet limited evidence exists linking CsWOX3 to these genes, despite their analogous phenotypes.In our study, we have discovered a novel regulating pathway where CsWOX3 negatively controls the morphogenesis of fruit spines in cucumber.Although we have made initial progress in understanding the potential mechanism, further extensive research is still required to gain a thorough understanding of the regulatory mechanism of CsWOX3.Such in-depth insights into this process could significantly advance our knowledge of cucumber trichomes development and establish a robust theoretical foundation for cucumber breeding.

Conclusion
CsWOX3, a member of the WUS subclass of the WOX family, functioned as a transcriptional repressor in cucumber.CsWOX3 exhibited a relatively high expression level in the cucumber ovary and played a negative role in the morphogenesis of fruit spines in cucumber.On one hand, CsSPL15 directly bound to and activated the expression of CsWOX3, thereby controlling the expression of downstream auxin-related genes.On the other, the RINGfinger type E3 ubiquitin ligase CsMIEL1-like might ubiquitinate CsWOX3 through interacting with the HD domain of CsWOX3.In sum, CsWOX3 negatively regulated the morphogenesis of cucumber fruit spines via both the auxin-related and ubiquitination pathways.

Plant materials
The inbred cucumber lines in this experiment include North China cucumber genotype 3661 (Xintai-Mici) and fruit cucumber genotype 6101-4, which were mainly used for gene expression determination and genetic transformation.Within the fruit cucumber genotype variety 6101-4, non-glandular fruit spines (type III or IV fruit trichomes) are moderately distributed on the surface of young fruit (ovary), with smaller-sized spines base that will fall off during the later commodity period.In contrast, North China cucumber genotype variety 3661 (Xintai-Mici) has dense and large-sized spines base non-glandular fruit spines (type II fruit trichomes) that remain attached to the fruit (ovary) surface without falling off.
Cucumber seeds were soaked in 55 • C warm water and germinated at 28 • C for 3 days, and then the germinated seeds were cultured in 28 • C/16 h light and 18 • C/8 h dark environment.Nicotiana benthamiana was used for subcellular localization and protein expression in this experiment.The seeds were sown for about 2 weeks and then transplanted and cultured.The whole growth process was maintained under conditions of 25 • C, with a photoperiod of 16 hours light and 8 hours dark.Subsequent experiments were carried out when the plants had developed 5 to 8 leaves.

Protein sequence alignment and phylogenetic analysis
To obtain the protein sequence information of the target proteins, we accessed data from the Cucurbit Genomics database (http:// cucurbitgenomics.org/organism/2)and NCBI (https://www.ncbi.nlm.nih.gov/).Subsequently, MEGA X software was used for protein phylogenetic analysis, and Jalview software was used for protein sequence alignment.

Subcellular localization assay
CsWOX3 or CsMIEL1-like CDS (without the termination codon) was cloned and recombined into pSUPER1300 plasmid between SmaI and SpeI sites, and the fusion plasmid was transformed into Agrobacterium tumefaciens strain GV3101.Then the Agrobacterium was resuspended with infection buffer (10 mM MES, 20 mM AS, 10 mM MgCl 2 , pH 5.6).The resulting solution was used to transiently infect the leaves of Nicotiana benthamiana.Following a 12-hour period in darkness, the plants were returned to a normal environment.After 60 hours from the injection of the Agrobacterium solution, GFP f luorescence was observed with a Nikon A1 confocal microscope (Japan) under 488 nm excitation light.
The subcellular localization of CsWOX3 in cucumber protoplasts followed our previous method.After seed sterilization, the peeled cucumber seeds were germinated in 1/2 MS medium and cultivated under weak light conditions for 7 ∼ 10 days.Cucumber cotyledons or young leaves were cut into filaments and then placed in an enzyme solution (cellulase R10 and macerozyme R10; Yakult Honsha, Tokyo, Japan) at room temperature.The cucumber tissue was gently shaken at room temperature for 4 to 6 hours, filtered through a 70 μm cell strainer to remove impurities, leaving only protoplasts, and then the fusion plasmid was transfected into cucumber protoplasts using the PEG4000 stress method.Finally, the protoplasts were placed under Nikon A1 confocal microscope (Japan) to observe GFP f luorescence at 20 h after transfection.

Verification of transcriptional activation activity of CsWOX3
Various CsWOX3 domain truncations were integrated into the pGBKT7 vector between EcoRI and SalI sites.The fusion plasmids were transformed into yeast strain AH109 (or Y2H Gold) using PEG/LiAc transformation method, and its transcriptional activation activities were verified in SD/−Trp, SD/−Trp-His and SD/−Trp-His-Ade deficient medium.Subsequently, the protein truncations were fused with VP16 and verified by yeast experiments again, or the GAL4 upstream activation sequence was serially recombined into the pGreenII-0800 vector and verified by GAL4-UAS system in N. benthamiana [74].

Total RNA extraction, cDNA reverse transcription and quantitative reverse transcription PCR
In order to detect the expression of each gene in different cucumber tissues, stems, young leaves, tendrils, male f lowers, female f lower bud (1.0 cm) and f lowering female f lowers (7.0 cm) were collected.Total RNA was extracted with Huayueyang Quick RNA isolation Kit (Beijing, China), and reverse transcribed into cDNA with Tiangen FastKing RT Kit (Beijing, China).Finally, quantitative reverse transcription PCR was performed with the Cowin Biotech Ultra SYBR Mixture (Low ROX) kit (Jiangsu, China).Experiments were performed with three biological replicates and three technical replicates.The data was analysed using the 2 -ΔΔCT method, and Cucumber CsTubulin served as the internal control.The whole process was performed under RNase-free environmental conditions.

mRNA in situ hybridization assay
A specific 194 bp sequence within the CsWOX3 mRNA was identified as a hybridization probe.The high-quality hybridization probe was obtained using Roche DIG RNA Labeling kit (SP6/T7) for transcription in vitro.Samples were collected from the ovaries of cucumber female f loral organs on the f lowering day.The assay was carried out according to the steps of sample fixation, sectioning and hybridization.The whole process was performed under RNase-free environmental conditions [4,5].

Virus-induced gene silencing assay
Transient gene silencing was achieved using the tobacco ringspot virus-based (TRSV)-VIGS system via the Agrobacterium-mediated vacuum infiltration.The specific fragment of CsWOX3 or CsSPL15 was identified by NCBI and recombined into pTRSV2 vector (SnaBI).Cucumber line 6101-4 seeds were used in this experiment.After 2 ∼ 3 days of germination under sterile conditions, the seedings were mixed with the Agrobacterium solution (EH105 strain), vacuumed at −900 kPa for 10 min, and co-cultured at 25 • C for 3 days before planting.Finally, the seedlings were grown under the conditions of 20 • C with a 16 h light/8 h dark cycle.Before phenotypic identification, qRT-PCR detection was performed on individual plants, and only plants with a silencing efficiency exceeding 60% were selected for further experiments [5,68,69].

Cucumber transformation
For our gene editing experiments, we used the PKSE402-GFP vector for CRISPR/Cas9 editing.The CsWOX3 overexpression vector, driven by a modified dual-35S promoter, was created based on the pBI121-GFP vector backbone between NheI and ApaI restriction enzymes.These recombinant plasmids were subsequently introduced into Agrobacterium strain EHA105.We employed cucumber lines 6101-4 and 3661 for the transformation process.
The cucumber seeds without coat were sterilized under sterile conditions and then germinated in SGM medium (MS solid medium, 2 mg/L 6-BA).After 40 hours, the excised cotyledons were infected with the Agrobacterium solution, resuspended in liquid IM medium (MS liquid medium, 2 mg/L 6-BA, 1 mg/L ABA, 200 μM AS, MES 1.25 mM pH 5.2), and co-cultured in solid IM medium for 3 days.The explants were then transferred into SRM medium (MS solid medium, 2 mg/L 6-BA, 1 mg/L ABA, 300 mg/L Timentin) for 30 days, and the presence of GFP f luorescent buds was detected using a LUYOR-3260 GFP f luorescent f lashlight (LUYOR, USA) [70,71].

Yeast one-hybrid LacZi reporter system assay
According to the results of Busch et al. and Sloan et al., three DNA cis-element motifs (TGAA, TAAT, and G-box like) that might be bound by CsWOX3 were verified by yeast one-hybrid LacZi reporter system.These motifs were ligated into pLacZi vectors (EcoRI and XhoI) by SolutionI ligase [46,47].
Through promoter sequence alignment analysis, it was discovered that CsIAA4, CsYUC10, and CsARF18 contain TAAT-box DNA cis-elements.Additionally, CsARF18 also contains a G-box like DNA cis-element.Each box motif was ligated into the pLacZi vector using EcoRI and XhoI, and the interaction between CsWOX3 and these auxin-related genes was verified by yeast one-hybrid LacZi reporter system.

Dual-luciferase reporter assay
The CDS of CsSPL15 or CsWOX3 was recombined into pGreenII-62SK vectors between BamHI and HindIII as effector vectors.The full-length (2.5 kb) of CsWOX3 promoter or the full-length (2.0 kb) of putative downstream auxin-related genes' promoter was recombined into the pGreenII-0800 vector between BamHI and HindIII as a reporter vector.The fusion plasmid was transformed into Agrobacterium strain GV3101 (pSoup-P19), and N. benthamiana leaves were collected after infection to calculate the relative ratio of f luorescence.All the interaction between CsSPL15 and the full length of CsWOX3 promoter, or between CsWOX3 and the full-length of putative downstream auxin-related genes' promoter were verified by dual-luciferase reporter system.

Yeast-two-hybrid library screening and verification of protein-protein interaction
Because CsWOX3 was a transcriptional repressor and lacked selfactivation activity, the yeast mating method was used directly to screen the two-hybrid library.The fusion plasmid pGBKT7-CsWOX3 was transformed into yeast strain Y2H Gold, which would mate with the yeast library strain Y187 to form Mickey head or clover complexes.Finally, sequencing was performed on the monoclonal plaques appearing in the SD/−Trp-Leu-His-Ade medium to obtain protein sequence information.The yeast library is a cDNA library of the temporal development of cucumber cotyledon trichomes, and the library screening assay was repeated twice.
Next, the screened target protein CDS was recombined into the pGADT7 vector using EcoRI and SacI, and co-transformed with the pGBKT7-CsWOX3 fusion plasmid into the yeast strain AH109 to verify the interaction using yeast-two-hybrid assay.

Firefly luciferase complementation imaging assay
CsWOX3 CDS (without the termination codon) was recombined into pCAMBIA-nLUC vector between BamHI and SalI to form the CsWOX3-nLUC fusion protein.CsMIEL1-like CDS was recombined into pCAMBIA-cLUC vector between BamHI and SalI to generate the cLUC-CsMIEL1-like fusion protein.The VILBER Fusion FX7 multifunctional imaging system (VILBER, France) was used to measure the bioluminescence of f luorescein released after Agrobacterium-mediated transformation and injection into N. benthamiana leaves.Approximately 20 minutes before the measurement, an appropriate amount of 200 μM D-f luorescein potassium solution was injected into the N. benthamiana leaves and treated in dark conditions.

Bimolecular fluorescence complementation assay
The CsWOX3 CDS (without the termination codon) was recombined into the pSPY35S-CE vector using BamHI and SmaI to form the CsWOX3-cYFP fusion protein and the CsMIEL1-like CDS (without the termination codon) was recombined into the pSPY35S-NE vector between BamHI and SmaI to form the CsMIEL1-like-nYFP fusion protein.After the fusion plasmid was transformed into Agrobacterium strain and injected into N. benthamiana leaves, GFP f luorescence was observed with a Nikon A1 confocal microscope (Japan) under 488 nm excitation light.

Transcriptome profiling and data analysis
3661 (Xintai-Mici) and CsWOX3 DOE lines were used for RNAsequencing.First, total RNA was extracted from the ovary exocarp of cucumber female f lowers 2 days before f lowering.In order to obtain RNA with good quality for following procedures, samples were examined by NanoDrop2000 and Agient2100/LabChip GX.The qualified cDNA library was pooled and sequenced on the Illumina NovaSeq6000 sequencing platform.All the sequencing work was completed using Biomarker Technologies, and the raw data was analysed on BMKCloud (www.biocloud.net).In this project, differentially expressed genes (DEGs) were identified using the criteria of Fold Change≥2 and FDR < 0.01.Then GO and KEGG enrichment were analysed.DEGs are shown in Supplemental Table S2 (see online supplementary material).

Determination of endogenous IAA content
We sampled the outer peel containing fruit spines of cucumber young female f loral ovaries.These samples were ground into powder using liquid nitrogen, and the endogenous auxin content was determined using plate direct competition enzyme-linked immunosorbent assay (ELISA).

Figure 2 .
Figure 2. CsWOX3 is a typical WOX transcriptional repressor.A Subcellular localization of CsWOX3-GFP in Nicotiana benthamiana leaf cells 60 h post inoculation (A1, bar = 100 μm) and cucumber protoplasts 20 h post transfection (A2, bar = 10 μm).B Verification of downstream DNA cis-elements bound by CsWOX3 using yeast one-hybrid LacZi reporter system.C Validation of transcriptional activation activity of different CsWOX3 domain truncations fused with VP16 protein in yeast.Left: Schematic diagram of vectors with different protein domain truncations.Right: The result of transcriptional activity of different CsWOX3 domain truncations in yeast.D Transient infection of Nicotiana benthamiana to examine the transcriptional inhibition efficiency of different CsWOX3 domain truncations.Left: Schematic diagram of different vectors.Right: The relative ratio of firef ly luciferase (LUC) to renilla luciferase (REN).Error bars represent SD from three biological replicates; * P < 0.05.

Figure 4 .
Figure 4. Critical role of CsWOX3 in the morphogenesis of cucumber fruit spines.A Genetic modifications within the CsWOX3 knockout (Cswox3 CRISPR ) lines.There are two types of genetic modifications in the Cswox3 CRISPR 1 line: one involves 1 bp deletion from target 1 (highlighted in green) and a small fragment deletion from target 2 (marked in blue), while another type is the large fragment deletion between target 1 and target 2.In the Cswox3 CRISPR 3 line, both target 1 and target 2 were deleted from the baseline.In addition, the base sequence marked in red is the PAM region.B to C Phenotypes of cucumber fruit spines within CsWOX3 knockout (Cswox3 CRISPR ) (B, bar = 1 cm) and overexpression (CsWOX3 DOE ) (C, bar = 5 mm) lines.D to E Scanning electron microscope observation of spines attached on the female f loral ovary within CsWOX3 knockout (Cswox3 CRISPR ) (D) and overexpression (CsWOX3 DOE ) (E) lines, in comparison to their respective wild-type lines 6101-4 and 3661.Bar = 500 μm, and red arrows indicate these differences.F The relative transcript level of CsWOX3 within overexpression (CsWOX3 DOE ) lines.Significance compared to WT was determined by Student's t test; * P < 0.05; N = 3. G to H Average diameter of cucumber fruit spines base was determined within CRISPR lines (G) and overexpression lines (H).Significance compared to WT was determined by Student's t test; * P < 0.05; N = 3.

Figure 5 .
Figure 5. Transcriptome analysis of the CsWOX3 overexpression lines.GO enrichment analysis of differentially expressed genes identified from transcriptome profiling of CsWOX3 overexpression lines.GO terms are presented as biological processes (A) and cellular component (B).The x-axis represents GeneRatio, indicating the proportion of genes annotated in a specific item relative to all differentially expressed genes.The y-axis lists each GO annotation item.The point size represents the number of differentially expressed genes annotated in the pathway, and the point color represents the q value of the hypergeometric test.Differentially expressed genes were identified using the criteria of Fold Change ≥2 and FDR <0.01 (N = 3).Heatmap showing differentially expressed genes related to auxin signaling (C) and ubiquitin ligases (D).E Determination of endogenous IAA content within CsWOX3 overexpression (CsWOX3 DOE ) and knockout (Cswox3 CRISPR ) lines.Significance compared to WT was determined by Student's t test; * P < 0.05; N = 3.

Figure 6 .
Figure 6.Conserved CsSPL15-CsWOX3-IAA/AUX module in the development of cucumber fruit spines.A Phylogenetic analysis of SPL family proteins in cucumber using maximum likelihood (ML).B Dual-luciferase reporter assay verification of the interaction between partial SPL proteins and CsWOX3.Error bars represent SD from 3 biological replicates; * P < 0.05.C Verification of interaction between CsSPL15 and some SBP-box DNA cis-elements presented in the promoter of CsWOX3 using yeast-one-hybrid LacZi reporter assay.D Dual-luciferase reporter assay validation of the interaction between CsWOX3 and the promoters of genes involved in auxin signaling.Error bars represent SD from 3 biological replicates; * P < 0.05.E Verification of interaction between CsWOX3 and some G-box like or TAAT-box DNA cis-elements presented in the promoter of partial auxin-related genes using yeast-one-hybrid LacZi reporter assay.Within two independent CsSPL15 TRSV-VIGS lines (TRSV2::Csspl15-1 and − 2), the phenotypic observation of trichomes attached to the surface of abaxial leaf (F1, G1 and H1), petiole (F2, G2 and H2) and ovary (F3, G3 and H3), and the details of cucumber fruit spines (F4, G4 and H4).Bar = 1 mm.Based on 6101-4 wild-type plants, TRSV2 empty vector was used as a negative control (TRSV2::00), and TRSV2::Cspds was used as a positive control.Red arrows indicate these differences.I The mRNA transcriptional level of CsSPL15 within TRSV-VIGS lines.Significance compared to TRSV2::00 was determined by Student's t test; * P < 0.05; N = 2, n = 3. J Average diameter of cucumber fruit spines base within TRSV-VIGS lines.Significance compared to TRSV2::00 was determined by Student's t test; * P < 0.05; N = 2, n = 25.K The relative transcript level of CsWOX3 within CsSPL15 TRSV-VIGS lines.Significance compared to TRSV2::00 was determined by Student's t test; * P < 0.05; N = 2, n = 3. L The relative transcript level of CsARF18 within CsWOX3 transgenic lines.Significance compared to WT was determined by Student's t test; * P < 0.05; N = 3.

Figure 7 .
Figure 7. Physical interaction of CsWOX3 with the RING-finger type E3 ubiquitin ligase CsMIEL1-like.A Investigation of the interactions between CsWOX3 and the E3 ubiquitin ligases from the screening results using a yeast-two-hybrid assay.B Verifying the interaction between CsWOX3 and CsMIEL1-like through a firef ly luciferase complementation imaging assay after transient Nicotiana benthamiana infection.C Bimolecular f luorescence complementation assay is used for verifying the interaction between CsWOX3 and CsMIEL1-like in Nicotiana benthamiana leaf cells 50 h post inoculation.Bar = 100 μm.D Verification of interaction between different protein domain truncations of CsWOX3 and CsMIEL1-like.E The relative transcript level of CsMIEL1-like in various cucumber tissues.FE, fruit f lesh of the female f loral ovary only; FFBC, female f lower bud corolla; FFBO, female f lower bud ovary; LE, leaf; MF, male f lower; PE/S, the exocarp of female f loral ovary containing fruit spines and glandular trichomes; ST, stem; TE, tendril.Error bars represent SD from three biological replicates.F The relative transcript level of CsMIEL1-like within transgenic lines of CsWOX3 (Cswox3 CRISPR and CsWOX3 DOE lines).Significance compared to WT was determined by Student's t test; * P < 0.05; N = 3.