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eIF4E1 Regulates Arabidopsis Embryo Development and Root Growth by Interacting With RopGEF7

Eukaryotic translation initiation factor 4E1 (eIF4E1) is required for the initiation of protein synthesis. The biological function of eIF4E1 in plant–potyvirus interactions has been extensively studied. However, the role of eIF4E1 in Arabidopsis development remains unclear. In this study, we show that eIF4E1 is highly expressed in the embryo and root apical meristem. In addition, eIF4E1 expression is induced by auxin. eIF4E1 mutants show embryonic cell division defects and short primary roots, a result of reduced cell divisions. Furthermore, our results show that mutation in eIF4E1 severely reduces the accumulation of PIN-FORMED (PIN) proteins and decreases auxin-responsive gene expression at the root tip. Yeast two-hybrid assays identified that eIF4E1 interacts with an RAC/ROP GTPase activator, RopGEF7, which has been previously reported to be involved in the maintenance of the root apical meristem. The interaction between eIF4E1 and RopGEF7 is confirmed by protein pull-down and bimolecular fluorescent complementation assays in plant cells. Taken together, our results demonstrated that eIF4E1 is important for auxin-regulated embryo development and root growth. The eIF4E1–RopGEF7 interaction suggests that eIF4E1 may act through ROP signaling to regulate auxin transport, thus regulating auxin-dependent patterning.


INTRODUCTION
RAC/ROP GTPases are multi-functional signaling molecules which regulate a wide range of processes during plant growth and development, including pollen tube growth, leaf epidermal cell morphogenesis, root hair development, embryonic development, seedling growth, stress tolerance, and hormone response (Yang and Fu, 2007;Wu et al., 2011;Craddock et al., 2012;Bloch and Yalovsky, 2013;Li et al., 2021). RAC/ROPs undergo exchange reactions between an active GTP-bound form and an inactive GDP-bound form (Nibau et al., 2013;Akamatsu et al., 2015). The cycling of GTP-and GDP-bound forms is regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). In short, GAPs catalyze GTP hydrolysis and convert them back to GDP-bound forms. Oppositely, GEFs activate RAC/ROPs by stimulating the exchange of GDP-to GTP-bound forms (Yalovsky, 2015). The plant-specific family of GEFs (i.e., RopGEFs) is utilized predominantly by RAC/ROPs to activate the signaling pathway to regulate plant development (Berken et al., 2005;Chen et al., 2011;Huang et al., 2018).
Arabidopsis possesses a distinct small GTPase family of RopGEFs (Gu et al., 2006;Liu et al., 2017). There are 14 RopGEF genes in Arabidopsis, namely, RopGEF1 to RopGEF14, which are versatile signaling molecules in plant growth and development, as well as responses to various stresses (Wu et al., 2011;Nibau et al., 2013;Liu et al., 2017;Ashraf and Rahman, 2019). The transient expression of RopGEF1 and RopGEF12 suggested that these genes mediate polarized pollen tube growth (Gu et al., 2006;Zhang and McCormick, 2007;Chang et al., 2013). In addition, studies of ropgef1 ropgef4 double mutants and overexpression transgenic lines showed that RopGEF1 and RopGEF4 are involved in ABA-mediated stomatal closure in response to drought stress (Li and Liu, 2012). A recent study reported that RopGEF1 participates in the ABA-mediated inhibition of lateral root growth via ABAmediated protein degradation (Li et al., 2016). Moreover, ABAinduced trafficking and degradation of RopGEF1 are disrupted in a cpk3/4/6/11 quadruple mutant of calcium-dependent protein kinase (CPK) genes in Arabidopsis, suggesting that RopGEF1 cooperates with CPKs and plays crucial roles in root hair polar growth and seedling growth (Li Z. et al., 2018). In our previous study, we have shown that RopGEF1 plays essential roles in the early embryonic and seedling development through regulating polar auxin transport and cell polarity . Another study reported that phytochrome B (phyB) acts genetically upstream of RopGEF2 and RopGEF4 to activate RopGEF2 regulating stomatal opening (Wang et al., 2017). Our group reported that RopGEF7 participates in regulating auxin-dependent PLETHORA1 (PLT1)-and PLT2-mediated maintenance of root stem cell niches (Chen et al., 2011). In addition, we found that ROP3, which interacts with RopGEF7 (Chen et al., 2011), plays a critical role in maintaining the polarity of auxin efflux proteins (PINs) at the plasma membrane (PM), and whereby regulates embryonic development and postembryonic growth (Huang et al., 2014). To further investigate the role of RopGEF7, we used RopGEF7 as a bait in the screen of Arabidopsis seedling cDNA library and identified a potential interaction protein, a translation initiation factor, eIF4E1.
Extensive studies have shown that eIF4E genes play an essential role in plant virus infection. Natural mutations in eIF4E1 and eIF(iso)4E enhance the resistance against plant viruses in diverse plant species (Li G. et al., 2018;Liu and Goss, 2018). ncbp-1/ncbp-2, a double mutant of the cassava eIF4E isoforms nCBP-1 and nCBP-2, displayed delayed and attenuated cassava brown streak disease symptom (Gomez et al., 2019). However, how exactly eIF4E mediates plant growth and development has not been well-studied. Downregulation of both eIF4E1 and eIF(iso)4E in antisense tobacco plants caused a semi-dwarf phenotype (Combe et al., 2005). Similarly, silencing of both eIF4E1 and eIF4E2 with RNAi strategy in tomato resulted in dwarf plants with fewer seeds and smaller fruits (Mazier et al., 2011). A more pronounced phenotype was observed in the tomato transgenic lines silenced for eIF4E1, eIF4E2, and eIF(iso)4E (Mazier et al., 2011). The knockout of eIF(iso)4E and overexpression of transgenic plants were associated with phosphate-regulated root growth (Martínez-Silva et al., 2012). Arabidopsis eif4e1 knockout mutants displayed a consistent 7-day bolting delay compared with the wild-type Columbia (Bastet et al., 2018). In this article, we report a specific role for eIF4E1 in embryo development and root growth through interactions with RopGEF7 in the regulation of the auxin pathway.

eIF4E1 Is Expressed in Embryos and Seedlings and Is Induced by Auxin
To investigate the expression patterns of eIF4E1, we generated the transgenic plants of eIF4E1 pro :YFP-eIF4E1 with a YFP reporter, and eIF4E1 pro :GUS with a β-glucuronidase (GUS) reporter. Analysis of several independent eIF4E1 pro :YFP-eIF4E1 transgenic lines showed that eIF4E1 pro :YFP-eIF4E1 was ubiquitously expressed in both embryo proper and suspensor during embryogenesis. In detail, the YFP signal was detected in 8-cell, 16-cell, 32-cell, globular, triangle, early heart, and heart and mature embryo stages (Figure 1, A1-9), compared to the wild-type embryos which did not show any GFP fluorescence (Supplementary Figure S1). Similarly, eIF4E1 pro :GUS transgenic lines displayed that GUS activities were expressed in embryogenesis. The GUS activities were detected in early globular, globular, early heart, and heart and mature embryo stages (Supplementary Figure S2). In postembryonic stages, eIF4E1 was expressed in primary roots (Figure 1, B1, 2; Supplementary Figure S3), lateral root primordia and lateral roots (Figure 1, B3, 4), and anthers (Figure 1, B5, 6), especially with strong expression in pollens (Figure 1, B6). To investigate how eIF4E1 expression is regulated, we treated the roots of 7-day-old eIF4E1 pro :GUS seedlings with exogenous auxin NAA. The results indicated that GUS activity was markedly induced FIGURE 1 | Expression profiles of eIF4E1 in Arabidopsis. (A) eIF4E1 pro :YFP-eIF4E1 was expressed in embryos. The confocal images were merged by the DIC and the GFP channels. 8-cell (A1), 16-cell (A2), 32-cell (A3), globular (A4), triangle (A5), early heart (A6, 7), heart (A8), and mature (A9) embryo stages. (B) Detection of GUS activity in the primary roots (B1, 2) of 7-day-old eIF4E1 pro :GUS seedlings, the early stage (up panel) and the late stage (down panel) of lateral root primordia (B3), and lateral roots (B4), as well as in anthers and pollens (B5, 6). (C) Detection of GUS activity under exogenous 10 µM NAA treatment. To display the meristem zone and entire elongation zone, (C) is generated by three field photographs (using the Photoshop6 software overlap function). The region between two white or two black arrowheads indicates the meristem or elongation zone, respectively. CK, a control without NAA treatment. (D,E) Expression level of eIF4E1 was significantly induced in 7-day-old wild-type seedlings by 10 µM NAA treatment. Transcription levels were normalized to ACTIN 2 (D) or TUBULIN 2 (E), respectively. Data are presented as mean values of three biological repeats. Scale bars: (A) 10 µm; (B1-4) 2 mm; (B5, 6; C) 50 µm. by NAA treatment, especially in the meristem region. Compared to untreated control, NAA enlarged the expression area of eIF4E1 and enhanced GUS activity in the elongation zone of roots ( Figure 1C). To confirm such observations, we analyzed the eIF4E1 relative expression levels by quantitative real-time PCR (qRT-PCR) in 7-day-old wild-type seedling roots. eIF4E1 expression showed an increase following NAA treatment to up to 24 h; 24-h NAA treatment induced over a two-fold increase in eIF4E1 relative expression (Figures 1D,E).

Disruption of eIF4E1 Results in Developmental Defects During Embryogenesis and Seedling Growth
The expression patterns of eIF4E1 suggested that eIF4E1 may be functionally important for embryogenesis. To examine more preciously the role of eIF4E1, eif4e1-1 carrying a single base replacement mutation which caused a nonsense mutation from Trp (TGG) to a stop codon (TGA), and a T-DNA insertion mutant eif4e1-2 were used (Supplementary Figure S4A). The relative expression of eIF4E1 was undetectable in eif4e1-1 or reduced to <20% in eif4e1-2, respectively, compared to those of wild-type Col-0 (Supplementary Figures S4B,C).

eIF4E1 Is Necessary for Primary Root Growth
To investigate the roles of eIF4E1 in plant root development, we used eif4e1-1 and eif4e1-2 to analyze the root growth. The results indicated that the length of primary root of both mutants is remarkably shorter than that of the wild type ( Figures 3A,C). To confirm that these phenotypes were caused by the mutation of eIF4E1, we generated the complementation transgenic lines by crossing eif4e1-1 and eIF4E1 pro :YFP-eIF4E1. The complementary line rescued the shorter primary root phenotype (Figures 3A,C), suggesting that root growth defects were due to loss of eIF4E1. To further investigate how eIF4E1 mutation affects root growth, we measured the length of the root meristem and elongation zones and quantified their cell numbers. The length of root meristem (RM) and elongation zone of the eif4e1 mutants were significantly shorter than those of the wild type. However, complementary line was able to rescue the short root phenotype of eif4e1-1 (Figures 3B,D; Supplementary Figure S5). The cell numbers in the meristem and elongation zones of mutants were less than those of the wild type but recovered in the complementary transgenic line ( Figure 3E; Supplementary Figure S5). We could not, however, see any difference in the cell size of the root apical meristem between eif4e1 mutants and wild type ( Figure 3F), suggesting that eIF4E1 may regulate root growth by affecting cell division of root cells.

eIF4E1 Regulates Auxin-Responsive Report Gene Expression and the Accumulation of PIN3 and PIN7 Proteins
The developmental phenotypes of the embryo and seedling of eif4e1 mutants (Figures 2A,B), and the expression levels of eIF4E1 were induced by exogenous NAA application ( Figures 1D,E), suggesting that eIF4E1 may participate in the regulation and/or expression of auxin-mediated processes. To test our speculation, we examined whether auxin reporter DR5 rev :GFP and auxin efflux carrier reporters were altered in eif4e1 mutants. First, we crossed DR5 rev :GFP transgenic plants with eif4e1-1 mutants. We then examined DR5 activity during root growth in eif4e1-1 andcompared with that of the wild type. Two independent lines, L1 ( Figure 4B) and L2 ( Figure 4C), showed markedly reduction in GFP fluorescent signals in contrast to the wild type ( Figure 4A), suggesting that eIF4E1 participates in auxin-regulated root growth.
Auxin efflux carrier PIN proteins are major players to generate auxin gradients in root tips (Adamowski and Friml, 2015;Sauer and Kleine-Vehn, 2019). To investigate the PIN expression in eif4e1 mutants, we crossed the PIN3 pro :PIN3-GFP and PIN7 pro :PIN7-GFP into eif4e1-1, respectively. We next analyzed the expression of PIN3 pro :PIN3-GFP and PIN7 pro :PIN7-GFP in the roots of the eif4e1-1 mutant. In wild-type roots, PIN3-GFP is localized in root stele cells and the root cap, while the PIN3-GFP signal was strongly  Compared with the wild type, PIN3-GFP was even absent in the root cap ( Figure 4D). We also found that PIN7-GFP was reduced in the roots of two independent eif4e1-1 lines (Figures 4H,I) compared to the wild type ( Figure 4G), which further supports the importance of PIN3 and PIN7 in forming auxin gradients. We also examined the transcription level of PIN3 and PIN7 in eif4e1 mutants, and a slight elevation in the mutant background was observed compared to the wild type (Supplementary Figure S6). However, the location and accumulation of PIN1-GFP (Supplementary Figures S7B,C), PIN2-GFP (Supplementary Figures S7E,F), and AUX1-YFP (Supplementary Figures S7H,I) were not affected in eif4e1-1 in contrast to the wild type (Supplementary Figures S7A,D,G). Taken together, the loss-of-function mutation of eIF4E1 obviously reduced the expression of DR5 rev :GFP and the abundance of PIN3 and PIN7 proteins but has no effect on the localization and accumulation of PIN1, PIN2, and AUX1.

Treatment of CHX Can Mimic the Phenotypes of eif4e1
Considering that eIF4E1 is a eukaryotic translation initiation factor, we examined whether the treatment of translation inhibitor cycloheximide (CHX) can mimic the phenotypes of eif4e1 mutants. eif4e1 mutants and the wild type exhibit significant inhibition in root elongation in the presence of 50 nM CHX, compared to those without CHX treatment (Figures 5A,B), and eif4e1 mutants were less sensitive to CHX than the wild type (Figures 5A,B). In view of the decline of PIN3 and PIN7 protein abundance in eif4e1 mutants, we investigated whether the CHX treatment affects PIN accumulation. Seedlings harboring PIN3 pro :PIN3-GFP or PIN7 pro :PIN7-GFP display inhibited root growth similar to the wild type (Figures 5C,D). Moreover, the abundance of PIN3 and PIN7 proteins is reduced in the presence of CHX (Figures 5F,H), compared to that of CHX treatment (Figures 5E,G). Meanwhile, we examined the abundance of PIN1, PIN2, and AUX1 proteins after  CHX treatment, and we also could see slight alterations on protein abundance compared to those without CHX treatment (Supplementary Figure S8). Taken together, our observations showed that the treatment of CHX can mimic the phenotypes of eif4e1, suggesting that root growth defects of eif4e1 mutants are attributed to altered polar auxin transport.

eIF4E1 Interacts With RopGEF7
AtRopGEF7 is an activator of ROP GTPases, molecular switches function in multiple signaling pathways (Chen et al., 2011;Yalovsky, 2015). Previously, our group reported that AtRopGEF7 regulates the maintenance of the root apical meristem via affecting PIN-dependent polar auxin transport (Chen et al., 2011;Huang et al., 2014). Interestingly, we found a potential interacting eIF4E1, when we used RopGEF7 as bait in the screen of Arabidopsis seedling cDNA library. The capbinding translation initiation factor, eIF4E1, could be involved in a potential RopGEF7 regulatory mechanism. To further confirm the interaction between eIF4E1 and RopGEF7, we performed the Y2H direct interaction assay. eIF4E1 strongly interacted with full-length protein of RopGEF7 (RopGEF7-CDS) and truncated proteins containing the PRONE domain (RopGEF7-N and RopGEF7-PRONE), respectively, whereas eIF4E1 could not interact with the C terminus of RopGEF7 (RopGEF7-C) (Figures 6A,B), suggesting that the interaction requires a functional PRONE structure domain. Furthermore, we found eIF(iso)4E, a homolog of eIF4E1, also interacted with the PRONE domain of RopGEF7 but only weakly interacted with the full length of RopGEF7 in yeast cells (Figure 6C), suggesting this interaction may differ between these two forms of the cap-binding protein. Additionally, considering the conserved PRONE domain of RopGEFs, we tested whether the three homologs RopGEFs (RopGEF1, RopGEF4, and RopGEF6) closely related to RopGEF7 interact with eIF4E1 in yeast cells. However, eIF4E1 does not interact with them ( Figure 6C), suggesting that eIF4E1 may predominantly interact with RopGEF7. The interaction between eIF4E1 and RopGEF7 was further confirmed by pull-down assays. In these experiments, the recombinant GST-tagged eIF4E1 protein and the YFP-RopGEF7 total plant protein were used as bait and prey, respectively. GFP antibody was used to detect the interaction between eIF4E1-and YFP-tagged RopGEF7 proteins. The results further support the interaction between eIF4E1 and RopGEF7 ( Figure 6D). Moreover, to further investigate the relationship between RopGEF7 and eIF4E1, by using RopGEF7 antibody, the immunoblotting analysis showed that the abundance of RopGEF7 protein in eif4e1-1 and eif4e1-2 was markedly reduced (Figure 6F). On the contrary, the level of eIF4E1 protein in RopGEF7 RNAi transgenic lines was not affected (Figure 6E). To further verify the interactions between eIF4E1 and RopGEF7 in plant cells, we performed bimolecular fluorescence complementation (BiFC) assays in Arabidopsis Anti-eIF4E1 was designated as eIF4E1 antibody. (F) Abundance of RopGEF7 protein in eif4e1-1 and eif4e1-2 was detected by Western blotting. Anti-RopGEF7 was designated as RopGEF7 antibody. The protein amount was quantified by its loading control for each sample, and the protein value of the wild type was set as one. The values are shown up the bands in (E,F). The black arrowheads indicate the aim protein bands in (D,F).

protoplasts. The BiFC constructs of eIF4E1 in combination with
RopGEF7 were co-transformed into Arabidopsis protoplasts, and the BiFC-generated yellow fluorescent protein (YFP) signal was observed in the cortical cytoplasm (Figure 7D), whereas the controls showed only the background signal (Figures 7A-C). We next observed the subcellular localization of RopGEF7 and eIF4E1. The results indicated that GFP-tagged eIF4E1 fusion protein was localized at the cytoplasm (Figure 8B), and mCherry-labeled RopGEF7 was localized at the cytoplasm and PM (Figure 8D), compared to their GFP or mCherry control vectors which are widely expressed at nucleus, cytoplasm, and PM (Figures 8A,C). Moreover, subcellular localization of YFP-eIF4E1 in roots of eIF4E1 pro :YFP-eIF4E1 transgenic plants displayed that YFP-eIF4E1 is predominantly localized at the cytoplasm (Supplementary Figure S3). Meanwhile, cotransformation of 35S:eIF4E1-eGFP and 35S:mCherry-RopGEF7 in Arabidopsis protoplasts showed obviously merged signals in the cytoplasm (Figure 8E), indicating that eIF4E1 and RopGEF7 are colocalized at the cytoplasm. Taken together, these results show that eIF4E1 interacts with RopGEF7 and may act on the upstream of RopGEF7 to regulate RopGEF7 protein translation.

DISCUSSION
Previous investigations revealed the important role of eIF4E1 in plant-potyvirus interactions (Wang, 2015). Little is known about its function in plant growth and development. In this work, we show that mutations in eIF4E1 resulted in embryo defects and reduced root growth (Figures 2, 3). The developmental abnormalities of eif4e1 might be attributed to altered polar auxin transport based on the auxin marker analysis (Figure 4). Furthermore, we discovered that eIF4E1 interacts directly with RopGEF7, an activator of ROP GTPases to regulate the abundance of RopGEF7 protein (Figure 6). Our  results provide compelling evidence that eIF4E1 regulates auxindependent development and also establishes a link between protein translation and ROP signaling.
We found that eIF4E1 is broadly expressed in embryos from the early embryo stage to the mature embryo stage (Figure 1, A1-9), and the eif4e1 mutant embryos showed cell division defects at embryo proper, basal region, and suspensor from the 8-cell stage to the mature embryo stage (Figure 2A; Supplementary Table S2). RopGEF7 is also expressed in embryos, but its expression did not fully overlap with eIF4E1; it is rather restricted to the precursor cells of the quiescent center (QC) during the early heart stage to the mature stage of embryogenesis (Chen et al., 2011). The knockdown of RopGEF7 by RNA interference technique caused the defects in the root pole and suspensor (Chen et al., 2011). Interestingly, the expression pattern for RopGEF1, a close homolog of RopGEF7, is comparable to that of eIF4E1 in the embryo . Moreover, the phenotypes of the embryo of ropgef1 resemble to those of eif4e1 mutants .
To further understand the role of eIF4E1 in plant early development, we characterized a point mutation mutant eif4e1-1 (cum1) and a T-DNA insertion mutant eif4e1-2 (Supplementary Figure S4). Both mutant lines displayed embryo phenotypes and seedling cotyledon phenotypes (Figure 2), which partially originated from defective embryo development. These data indicates that eIF4E1 is important for embryo development. Further study to examine whether eIF(iso)4E, an isoform of eIF4E1, contributes to embryo development would provide more information. Several genetic studies failed to generate eif4e1 eif(iso)4e double mutants but only obtained plants homozygous for one locus and heterozygous for the other locus, due to embryo or gametophyte lethality (Callot and Gallois, 2014;Patrick et al., 2014).
The strong expression of eIF4E1 in the root apical meristem (Figure 1, B1; Supplementary Figure S3) supports its importance in root development. The expression pattern in the root apical meristem shown by eIF4E1 pro :GUS reporter (Figure 1) is consistent with previous investigation by mRNA in situ hybridization (Rodriguez et al., 1998). eIF4E1 expression is induced by auxin (Figures 1C-E), suggesting that it might function in the auxin-regulated pathway. eif4e1 mutants developed shorter roots with small size of root apical meristems and elongation zones than those of the wild type (Figure 3;  Supplementary Figure S5). Auxin maxima were reduced in the eif4e1 root tips (Figures 4A-C). Auxin efflux carrier PIN proteins are major contributors to establish the auxin maxima in the root tips. We also observed the abundance of PIN3 and PIN7 to be reduced in eif4e1 mutants (Figures 4D-I)

.
However, the transcript level of PIN3 and PIN7 did not decrease in eif4e1 mutants (Supplementary Figure S6). We cannot exclude the possibility that eIF4E1 directly affects PIN protein translation. CHX treatment resulted in reduced root growth (Figures 5C,D) and a decreased abundance of PIN3 and PIN7 proteins (Figures 5E-H). Further study is required to decipher the precise mechanism how eIF4E1 affects the PIN accumulation and auxin-dependent development.
Our previous work showed that the knockdown of RopGEF7 by RNA interference technique caused defects in root growth and failure to maintain root stem cell niche (Chen et al., 2011). RopGEF7 could activate ROP GTPases, including ROP3, which is required for the localization of several PIN proteins in the plasma membrane of root cells (Chen et al., 2011;Huang et al., 2014). RopGEF7RNAi plants and ROP3-dominant negative plants showed reduced auxin maxima reflected by the DR5 rev :GFP auxin reporter in the root tips (Chen et al., 2011;Huang et al., 2014). An ROP effector ICR1 is required for the recruitment of PIN proteins to the PM, and loss of function in ICR1 resulted in the reduced auxin maxima in the root tips and caused the collapse of the root apical meristem (Hazak et al., 2010). Interestingly, ICR1 induced by auxin is essential for auxin transport. When auxin reaches certain levels, ICR1 is degraded to result in auxin accumulation in the embryo hypophysis and the root tips (Hazak et al., 2014). In eif4e1 mutants, the perturbed auxin maxima in the root tips (Figures 4A-C) likely led to the retarded root growth. At present, it remains unclear how eIF4E1 acts through ROP signaling to affect auxin transport; additional work will be needed to elucidate how eIF4E1 regulates RopGEF7 to activate ROP in the plant cells. A recent study has shown that RALF1 peptide hormone promotes FERONIA receptor kinase-mediated phosphorylation of eIF4E1, which modulates mRNA translation and root hair polar growth (Zhu et al., 2020). FERONIA is also known to activate ROP signaling by interacting with several RopGEF1/4/7/10 to regulate auxindependent root hair development (Duan et al., 2010). Our recent study showed that FERONIA targeted PIN2 polarity to regulate lateral root growth and root gravitropic response (Dong et al., 2019). It is likely that FERONIA, eIF4E1, and RopGEF7 may act on the same pathway to regulate PINdependent auxin transport, which is essential for embryo and root development. Taken together, we provide evidence that eIF4E1 plays a critical role in embryo development and root growth in Arabidopsis. Mutations in eIF4E1 caused aberrant cell divisions in embryos and reduced cell numbers of the root apical meristem that might be attributed in part to the altered polar auxin transport. Moreover, eIF4E1 physically interacts with RopGEF7 (Figures 6B-D) to regulate the accumulation of RopGEF7 protein ( Figure 6F) for targetingauxin-mediated embryo and root development in Arabidopsis.

Plasmid Construction and Plant Transformation
To construct a vector for the eIF4E1 pro :GUS transgenic plants, a fragment of the 3,926-bp eIF4E1 promoter sequence was cloned into pBI101.1 at HindIII and BamHI sites. To generate 35S pro :YFP-RopGEF7 transgenic plants, a constitutive promoter from cauliflower mosaic virus (CaMV) was used, and two independently transgenic lines, 35S pro :YFP-RopGEF7 L1 and L11 with high levels of YFP-RopGEF7 expression, were obtained. eIF4E1 pro :YFP-eIF4E1 vector contains a YFP coding sequence (CDS) fused in-frame with an eIF4E1 CDS under the control of the same 3,926-bp eIF4E1 promoter region.
The aforementioned constructs were introduced into Agrobacterium tumefaciens strain GV3101 and subsequently used to transform Arabidopsis (Col-0) by using the floral dip method (Clough and Bent, 1998). The transgenic plants were screened on half-strength MS agar medium containing 25 µg/mL kanamycin or 15 µg/mL hygromycin. The primers used for generating the constructs are listed in Supplementary Table S1.

Quantitative RT-PCR Analysis
For qRT-PCR analysis, total RNA was extracted from 7-dayold seedlings using the RNeasy plant mini kit (Qiagen), and the first-strand cDNA was synthesized using the Takara PrimeScript First-strand cDNA synthesis kit (TAKARA). Next, PCR was performed on an Illumine Eco (Illumina) System with an SYBR probe (TAKARA). Expression levels of eIF4E1 were normalized to the expression level of two reference genes (ACTIN 2 and TUBULIN 2), respectively. The primers used are listed in Supplementary Table S1. Results obtained from three biological repeats with standard deviation (SD).
Length Analysis of Primary Root, Root Meristem, and Elongation Zone eif4e1-1 (CS6552), which was also reported as the cum1 mutant by Yoshii et al. (2004), and eif4e1-2 (SALK_145583C) mutants; the complementary line eif4e1-1/eIF4E1 pro :YFP-eIF4E1, which was generated by the cross of eif4e1-1, eIF4E1 pro :YFP-eIF4E1 transgenic line; and the wild-type seeds were surfaced sterilized and germinated on half-strength MS medium, and then grown under normal growth conditions. The length of the primary root of 6-day-old seedlings was measured. The length of RM and elongation zone were measured using 6-day-old seedlings. Data were presented as averages of over 60 seedlings from at least three biological replicates with SD.

Auxin Treatment
Wild-type (Col-0) seeds were surface-sterilized and germinated on the half-strength MS medium. The total RNA of 7-dayold wild-type seedlings was extracted after 0, 2, 4, and 24 h of treatment with exogenous 10 µM NAA. The expression levels of eIF4E1 were analyzed by qRT-PCR. In addition, 5-dayold eIF4E1 pro :GUS transgenic seedlings were transferred to the 1/2 MS liquid medium with or without 10 µM NAA treatment for 12 h, and GUS staining samples were observed under an Olympus BX51 microscope.

CHX Treatment
Four-day-old seedlings were transferred onto half-strength MS medium containing 50 or 100 nM CHX (Cat: AC466, Genview) for another 3-day growth and then observed or took photos under a confocal microscope.

Microscopy
To identify the phenotype of embryos and RM of eif4e1-1 and eif4e1-2, the ovules and seedling roots were cleared in modified Hoyer's solution and an HCG solution as previously described (Chen et al., 2011). The samples were observed by using an Olympus BX51 microscope connected to a Ritiga 2000R camera. Seedlings were observed under a Nikon SMZ1000 microscope equipped with a Nikon digital sight Ds-Fi1 camera.

Yeast Two-Hybrid Library Screening
Screening for interacting proteins of RopGEF7 was performed using a MatchmakerTM Library Construction & Screen Kit (Cat: 630445, Clontech). The cDNA library was generated by using 7day-old Arabidopsis seedlings. The full-length coding sequence of RopGEF7 was constructed and used as a bait to screen the cDNA seedling library. The cDNA fragments coding for the RopGEF7 were amplified with primer pairs for RopGEF7-F and RopGEF7-R and then cloned into pGBKT7 (BD) to generate the bait of pGBKT7-GEF7. The primers used to generate the constructs are listed in Supplementary Table S1. Seedling cDNA was cloned with pGADT7 (AD) to generate the library. Next, the plasmids of pGBKT7-GEF7 and library were co-transformed into yeast (AH109) following the modified lithium acetate method as directed by the manufacturer. More than 2,000 transformants were screened, and the positive colonies were re-screened on the SD medium lacking Leu, Trp, Ade, and His. The selected clones were further tested on the same medium supplemented with 0 or 10 µM 3-amino-1,2,4-triazole. DNA was extracted from the positive clones and transformed into the Escherichia coli strain DH5α to amplify the cDNA inserts, and the plasmids were sequenced.

Direct Interaction Assays in Yeast Cells
The full-length cDNA, the N-terminal deleted fragment (GEF7-N, aa 60-546), the PRONE domain (GEF7-PRONE, aa 60-447), and the C-terminal fragment (GEF7-C, aa 448-546) of RopGEF7 were cloned into vector pGBKT7, respectively. The cDNA of eIF4E1 was cloned into vector pGADT7, respectively. The primers used to generate the Y2H constructs are listed in Supplementary Table S1. The bait and prey constructs were co-transformed by pairs into the yeast AH109, and the transformants were selected on synthetic dextrose (SD) double dropout medium lacking tryptophan and leucine (SD/-Trp-Leu) following incubation at 28 • C for 3 days. Single co-transformed yeast clones of double dropout plates were transferred to quadruple dropout medium (SD/-Trp-Leu-Ade-His). The positive (pGBKT7-53/pGADT7-T) and negative (pGBKT7-Lam/pGADT7-T) controls were used.

Pull-Down Assay
To generate the chimeric eIF4E1 protein, we cloned the fulllength cDNA of eIF4E1 to the pGEX-4T-1 vector, which contains a GST tag before the multiple cloning sites (MCS). Next, the fused GST-eIF4E1 protein was expressed in E. coli and purified on glutathione affinity resin. The purified fusion protein GST-eIF4E1 was mixed with YFP-tagged RopGEF7 protein (extracted from 35Spro:YFP-RopGEF7 transgenic plants) and then incubated at 4 • C for 2 h. The amino acid sequences of GFP and YFP differ only one amino acid, and GFP antibody (Abcam) was used as a substitute for the YFP antibody to detect the YFP-RopGEF7 plant protein. The primers used for plasmid construction are listed in Supplementary Table S1.

Subcellular Localization and Colocalization of eIF4E1 and RopGEF7
For subcellular localization, the eIF4E1 coding sequence was inserted into a modified pCAMBIA-1300 vector (35S:eGFP) at the XbaI site by using a homologous recombination kit (Multis One Step Cloning Kit, VazymE). The RopGEF7 coding sequence was inserted into the pSAT6-mCherry-C1-B (pE3275) vector containing 35S:mCherry at the SalI and BamHI sites. Then the resulting constructs were transformed into Arabidopsis protoplasts, respectively. GFP and RFP were visualized by using a confocal microscope.
For subcellular colocalization, the aforementioned constructs of 35S:eIF4E1-GFP and 35S:mCherry-RopGEF7 were co-transformed into Arabidopsis protoplasts and then observed by using a confocal microscope.

Bimolecular Fluorescence Complement Analysis in Arabidopsis Protoplasts
To construct the fusion proteins of nYFP-eIF4E1 and cYFP-RopGEF7, the eIF4E1 coding sequence was inserted into the pSAT6-nEYFP-C1 vector, and the RopGEF7 coding sequence was inserted into the pSAT6-cEYFP-C1 vector (Chen et al., 2011). The resulting constructs were co-transformed into Arabidopsis protoplasts for BiFC assays. Protoplast isolation and transfection were performed as described (Tao et al., 2005;Chen et al., 2011). YFP was visualized by using an Olympus BX51 microscope using YFP filter sets as previously described (Chen et al., 2011;Huang et al., 2018). The FM4-64 dye labels the plasma membrane. The primers used for plasmid construction are listed in Supplementary Table S1.

Statistics
All the data were represented the average with SD from at least three biological replicates. A significant difference was determined by paired two-tailed Student's t-tests. P < 0.05 was considered significant.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.