Differentiation In Arabidopsis Research Articles

Proteomic analysis of xylem vessel cell differentiation in VND7-inducible tobacco BY-2 cells by two-dimensional gel electrophoresis.

The xylem vessel is an essential structure for water conduction in vascular plants. Xylem vessel cells deposit thick secondary cell walls and undergo programmed cell death, to function as water-conducting elements. Since the discovery of the plant-specific NAC domain-type VASCULAR-RELATED NAC-DOMAIN (VND) transcription factors, which function as master switches of xylem vessel cell differentiation in Arabidopsis, much has been learned about the transcriptional regulatory network of xylem vessel cell differentiation. However, little is known about proteome dynamics during xylem vessel cell differentiation. Here, we performed two-dimensional electrophoresis-based proteomic analysis of xylem vessel cell differentiation using a transgenic tobacco BY-2 cell line carrying the VND7-inducible system (BY-2/35S::VND7-VP16-GR), in which synchronous trans-differentiation into xylem vessel cells can be induced by the application of a glucocorticoid. Of the 47 spots revealed by gel electrophoresis, we successfully identified 40 proteins. Seventeen proteins, including several well-characterized proteins such as a cysteine protease and serine carboxypeptidase (involved in programmed cell death), were upregulated after 24 h of induction. However, previous transcriptomic analysis showed that only eight of these proteins are upregulated at the transcriptional level during xylem vessel cell differentiation in BY-2/35S::VND7-VP16-GR cells. These findings suggest that post-transcriptional regulation strongly affects proteomic dynamics during xylem vessel cell differentiation.

Characterization of Root Epidermal Cell Patterning and Differentiation in Arabidopsis.

The root epidermis of Arabidopsis thaliana has been established as a model system for elucidating the mechanisms which govern the spatial patterningAbstract and morphogenesis of plant cells. Investigations into root hairs focus on various aspects of the biology of epidermal cells, using methods specifically developed to dissect the biological question under study. Despite the large number of studies related to epidermal cell differentiation, a survey of methods to analyze the phenotypic readout resulting from environmental conditions or the genetic background of the plant has not been provided so far. This protocol describes how to analyze the spatial arrangement and morphologic characteristics of cells in the root epidermis based on whole mount roots or cross sections, using confocal, scanning electron and light microscopy. This comparison of methods aids in selecting the most suitable strategy to examine the differentiation of root epidermal cells at different developmental stages.

Protein S-Nitrosylation Regulates Xylem Vessel Cell Differentiation in Arabidopsis.

Post-translational modifications of proteins have important roles in the regulation of protein activity. One such modification, S-nitrosylation, involves the covalent binding of nitric oxide (NO)-related species to a cysteine residue. Recent work showed that protein S-nitrosylation has crucial functions in plant development and environmental responses. In the present study, we investigated the importance of protein S-nitrosylation for xylem vessel cell differentiation using a forward genetics approach. We performed ethyl methanesulfonate mutagenesis of a transgenic Arabidopsis 35S::VND7-VP16-GR line in which the activity of VASCULAR-RELATED NAC-DOMAIN7 (VND7), a key transcription factor involved in xylem vessel cell differentiation, can be induced post-translationally by glucocorticoid treatment, with the goal of obtaining suppressor mutants that failed to differentiate ectopic xylem vessel cells; we named these mutants suppressor of ectopic vessel cell differentiation induced by VND7 (seiv) mutants. We found the seiv1 mutant to be a recessive mutant in which ectopic xylem cell differentiation was inhibited, especially in aboveground organs. In seiv1 mutants, a single nucleic acid substitution (G to A) leading to an amino acid substitution (E36K) was present in the gene encoding S-NITROSOGLUTATHIONE REDUCTASE 1 (GSNOR1), which regulates the turnover of the natural NO donor, S-nitrosoglutathione. An in vitro S-nitrosylation assay revealed that VND7 can be S-nitrosylated at Cys264 and Cys320 located near the transactivation activity-related domains, which were shown to be important for transactivation activity of VND7 by transient reporter assay. Our results suggest crucial roles for GSNOR1-regulated protein S-nitrosylation in xylem vessel cell differentiation, partly through the post-translational modification of VND7.

Localization of the CAPRICE-ENHANCER OF TRY AND CPC1 chimera protein in Arabidopsis root epidermis.

The CAPRICE (CPC) encodes an R3-type MYB transcription factor, which promotes root-hair differentiation. Previously, we showed that the CPC protein moves from the non-hair cell to the neighboring cell and induces root-hair differentiation in Arabidopsis. In addition, we proposed two cell-to-cell movement signal sequences, S1 and S2, in CPC. However, an S1:2xGFP:S2 chimera protein did not move between root epidermal cells. Here, we show that the S1 and S2 sequences do not confer cell-to-cell movement or nuclear localization ability to a GFP protein. The ENHANCER OF TRY AND CPC1 (ETC1) gene encodes the CPC homolog R3 MYB; this protein does not possess cell-to-cell movement ability or the S1 sequence. To elucidate whether the S1 sequence can induce cell-to-cell movement ability in ETC1, CPCp:S1:ETC1:2xGFP was constructed and introduced into Arabidopsis. Our results indicate that the addition of the S1 sequence was not sufficient for ETC1 to acquire cell-to-cell movement ability.

WOX14 promotes bioactive gibberellin synthesis and vascular cell differentiation in Arabidopsis.

Procambial and cambial stem cells provide the initial cells that allow the formation of vascular tissues. WOX4 and WOX14 have been shown to act redundantly to promote procambial cell proliferation and differentiation. Gibberellins (GAs), which have an important role in wood formation, also stimulate cambial cell division. Here we show that the loss of WOX14 function phenocopies some traits of GA-deficient mutants that can be complemented by exogenous GA application, whereas WOX14 overexpression stimulates the expression of GA3ox anabolism genes and represses GA2ox catabolism genes, promoting the accumulation of bioactive GA. More importantly, our data clearly indicate that WOX14 but not WOX4 promotes vascular cell differentiation and lignification in inflorescence stems of Arabidopsis.

RIMA-Dependent Nuclear Accumulation of IYO Triggers Auxin-Irreversible Cell Differentiation in Arabidopsis.

The transcriptional regulator MINIYO (IYO) is essential and rate-limiting for initiating cell differentiation in Arabidopsis thaliana Moreover, IYO moves from the cytosol into the nucleus in cells at the meristem periphery, possibly triggering their differentiation. However, the genetic mechanisms controlling IYO nuclear accumulation were unknown, and the evidence that increased nuclear IYO levels trigger differentiation remained correlative. Searching for IYO interactors, we identified RPAP2 IYO Mate (RIMA), a homolog of yeast and human proteins linked to nuclear import of selective cargo. Knockdown of RIMA causes delayed onset of cell differentiation, phenocopying the effects of IYO knockdown at the transcriptomic and developmental levels. Moreover, differentiation is completely blocked when IYO and RIMA activities are simultaneously reduced and is synergistically accelerated when IYO and RIMA are concurrently overexpressed, confirming their functional interaction. Indeed, RIMA knockdown reduces the nuclear levels of IYO and prevents its prodifferentiation activity, supporting the conclusion that RIMA-dependent nuclear IYO accumulation triggers cell differentiation in Arabidopsis. Importantly, by analyzing the effect of the IYO/RIMA pathway on xylem pole pericycle cells, we provide compelling evidence reinforcing the view that the capacity for de novo organogenesis and regeneration from mature plant tissues can reside in stem cell reservoirs.

Transcriptome-wide high-throughput deep m(6)A-seq reveals unique differential m(6)A methylation patterns between three organs in Arabidopsis thaliana.

Backgroundm6A is a ubiquitous RNA modification in eukaryotes. Transcriptome-wide m6A patterns in Arabidopsis have been assayed recently. However, differential m6A patterns between organs have not been well characterized.ResultsOver two-third of the transcripts in Arabidopsis are modified by m6A. In contrast to a recent observation of m6A enrichment in 5′ mRNA, we find that m6A is distributed predominantly near stop codons. Interestingly, 85 % of the modified transcripts show high m6A methylation extent compared to their transcript level. The 290 highly methylated transcripts are mainly associated with transporters, stress responses, redox, regulation factors, and some non-coding RNAs. On average, the proportion of transcripts showing differential methylation between two plant organs is higher than that showing differential transcript levels. The transcripts with extensively higher m6A methylation in an organ are associated with the unique biological processes of this organ, suggesting that m6A may be another important contributor to organ differentiation in Arabidopsis. Highly expressed genes are relatively less methylated and vice versa, and different RNAs have distinct m6A patterns, which hint at mRNA fate. Intriguingly, most of the transposable element transcripts maintained a fragmented form with a relatively low transcript level and high m6A methylation in the cells.ConclusionsThis is the first study to comprehensively analyze m6A patterns in a variety of RNAs, the relationship between transcript level and m6A methylation extent, and differential m6A patterns across organs in Arabidopsis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0839-2) contains supplementary material, which is available to authorized users.

TRANSPARENT TESTA GLABRA1 and GLABRA1 Compete for Binding to GLABRA3 in Arabidopsis.

The MBW (for R2R3MYB, basic helix-loop-helix [bHLH], and WD40) genes comprise an evolutionarily conserved gene cassette that regulates several traits such as (pro)anthocyanin and anthocyanin biosynthesis and epidermal cell differentiation in plants. Trichome differentiation in Arabidopsis (Arabidopsis thaliana) is governed by GLABRA1 (GL1; R2R3MYB), GL3 (bHLH), and transparent TESTA GLABRA1 (TTG1; WD40). They are thought to form a trimeric complex that acts as a transcriptional activation complex. We provide evidence that these three MBW proteins form either GL1 GL3 or GL3 TTG1 dimers. The formation of each dimer is counteracted by the respective third protein in yeast three-hybrid assays, pulldown experiments (luminescence-based mammalian interactome), and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer studies. We further show that two target promoters, Triptychon (TRY) and CAPRICE (CPC), are differentially regulated: GL1 represses the activation of the TRY promoter by GL3 and TTG1, and TTG1 suppresses the activation of the CPC promoter by GL1 and GL3. Our data suggest that the transcriptional activation by the MBW complex involves alternative complex formation and that the two dimers can differentially regulate downstream genes.

Arabidopsis CAPRICE (MYB) and GLABRA3 (bHLH) control tomato (Solanum lycopersicum) anthocyanin biosynthesis.

In Arabidopsis thaliana the MYB transcription factor CAPRICE (CPC) and the bHLH transcription factor GLABRA3 (GL3) are central regulators of root-hair differentiation and trichome initiation. By transforming the orthologous tomato genes SlTRY (CPC) and SlGL3 (GL3) into Arabidopsis, we demonstrated that these genes influence epidermal cell differentiation in Arabidopsis, suggesting that tomato and Arabidopsis partially use similar transcription factors for epidermal cell differentiation. CPC and GL3 are also known to be involved in anthocyanin biosynthesis. After transformation into tomato, 35S::CPC inhibited anthocyanin accumulation, whereas GL3::GL3 enhanced anthocyanin accumulation. Real-time reverse transcription PCR analyses showed that the expression of anthocyanin biosynthetic genes including Phe-ammonia lyase (PAL), the flavonoid pathway genes chalcone synthase (CHS), dihydroflavonol reductase (DFR), and anthocyanidin synthase (ANS) were repressed in 35S::CPC tomato. In contrast, the expression levels of PAL, CHS, DFR, and ANS were significantly higher in GL3::GL3 tomato compared with control plants. These results suggest that CPC and GL3 also influence anthocyanin pigment synthesis in tomato.

Regulation of root hair cell differentiation by R3 MYB transcription factors in tomato and Arabidopsis

CAPRICE (CPC) encodes a small protein with an R3 MYB motif and regulates root hair and trichome cell differentiation in Arabidopsis thaliana. Six additional CPC-like MYB proteins including TRIPTYCHON (TRY), ENHANCER OF TRY AND CPC1 (ETC1), ENHANCER OF TRY AND CPC2 (ETC2), ENHANCER OF TRY AND CPC3/CPC-LIKE MYB3 (ETC3/CPL3), TRICHOMELESS1 (TCL1), and TRICHOMELESS2/CPC-LIKE MYB4 (TCL2/CPL4) also have the ability to regulate root hair and/or trichome cell differentiation in Arabidopsis. In this review, we describe our latest findings on how CPC-like MYB transcription factors regulate root hair cell differentiation. Recently, we identified the tomato SlTRY gene as an ortholog of the Arabidopsis TRY gene. Transgenic Arabidopsis plants harboring SlTRY produced more root hairs, a phenotype similar to that of 35S::CPC transgenic plants. CPC is also known to be involved in anthocyanin biosynthesis. Anthocyanin accumulation was repressed in the SlTRY transgenic plants, suggesting that SlTRY can also influence anthocyanin biosynthesis. We concluded that tomato and Arabidopsis partially use similar transcription factors for root hair cell differentiation, and that a CPC-like R3 MYB may be a key common regulator of plant root-hair development.

Leaf Photosynthesis and Its Genetic Improvement from the Perspective of Energy Flow and CO2 Diffusion

Single-leaf photosynthesis is a fundamental process in plant biomass production, and is a major research topic in crop physiology. This paper reviews the recent achievements of research on the physiological determinants of the photosynthetic capacity from the perspective of energy flow and CO2 diffusion. Measurement of chlorophyll fluorescence is a popular method to diagnose the function of photosystem II, and is useful to assess the susceptibility to photoinhibition and allocation of energy, which are keys to improving both stress resistance and photosynthetic productivity. Mesophyll conductance (gm) is the conductance to CO2 diffusion from intercellular airspaces to the chloroplast, and was long thought to be determined by leaf anatomical properties. However, recent studies showed that environmental conditions affect g m. It is possible that g m is affected by the gating of the CO2-permeable aquaporins (cooporins). Stomatal morphology is revealed to be an important factor affecting gas exchange both in crop plants and inArabidopsis thaliana.The knowledge of the stomatal differentiation in Arabidopsis will be applicable to various crops. gm, stomatal conductance (gs) and leaf nitrogen content are the main factors to cause difference in leaf photosynthesis among rice lines, and recent activities are conducted to find genes to manipulate these factors. Although the association of leaf photosynthesis with crop productivity still has a large ‘missing link’, these achievements strongly suggest that the leaf photosynthetic capacity can be genetically improved in crop species.

Ectopic Expression of WUS in Hypocotyl Promotes Cell Division via GRP23 in Arabidopsis

WUSCHEL (WUS) is essential for preventing stem cell differentiation in Arabidopsis . Here we report that in addition to its functions in meristematic stem cell maintenance, WUS is involved in the regulation of cell division. The WUS gain-of-function mutant, stem ectopic flowers (sef), displayed elongated hypocotyls, whereas the loss-of-function wus-1 mutant had shortened hypocotyls. The long hypocotyl in sef was due to the presence of more cells, rather than increased cell elongation. Microscopic observation, flow cytometry assays, quantitative RT-PCR (qRT-PCR), and histochemical staining of CycB1;1::GUS supported the hypothesis that ectopic cell division occurred in the sef hypocotyls after germination. Both immunoblot and qRT-PCR results showed that WUS was ectopically expressed in sef hypocotyls. Luciferase activity, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) showed that GLUTAMINE-RICH PROTEIN 23 (GRP23) expression can be activated by WUS and that GRP23 is a direct target gene of WUS. The phenotypes of 35S::GRP23 plants and GRP23 knockdown lines supported the notion that GRP23 mediates the effects of WUS on hypocotyl length. Together, our data suggest that ectopic expression of WUS in hypocotyl controls cell division through its target gene GRP23.

Functional Divergence of MYB-Related Genes,WEREWOLFandAtMYB23inArabidopsis

Epidermal cell differentiation in Arabidopsis is studied as a model system to understand the mechanisms that determine the developmental end state of plant cells. MYB-related transcription factors are involved in cell fate determination. To examine the molecular basis of this process, we analyzed the functional relationship of two R2R3-type MYB genes, AtMYB23 (MYB23) and WEREWOLF (WER). MYB23 is involved in leaf trichome formation. WER represses root-hair formation. Swapping domains between MYB23 and WER, we found that a low homology region of MYB23 might be involved in ectopic trichome initiation on hypocotyls. MYB23 and all MYB23-WER (MW) chimeric transgenes rescued the increased root-hair phenotype of the wer-1 mutant. Although WER did not rescue the gl1-1 no-trichome phenotype, MYB23 and all MW chimeric transgenes rescued gl1-1. These results suggest that MYB23 acquired a specific function for trichome differentiation during evolution.

A gene regulatory network for root epidermis cell differentiation in Arabidopsis.

The root epidermis of Arabidopsis provides an exceptional model for studying the molecular basis of cell fate and differentiation. To obtain a systems-level view of root epidermal cell differentiation, we used a genome-wide transcriptome approach to define and organize a large set of genes into a transcriptional regulatory network. Using cell fate mutants that produce only one of the two epidermal cell types, together with fluorescence-activated cell-sorting to preferentially analyze the root epidermis transcriptome, we identified 1,582 genes differentially expressed in the root-hair or non-hair cell types, including a set of 208 “core” root epidermal genes. The organization of the core genes into a network was accomplished by using 17 distinct root epidermis mutants and 2 hormone treatments to perturb the system and assess the effects on each gene's transcript accumulation. In addition, temporal gene expression information from a developmental time series dataset and predicted gene associations derived from a Bayesian modeling approach were used to aid the positioning of genes within the network. Further, a detailed functional analysis of likely bHLH regulatory genes within the network, including MYC1, bHLH54, bHLH66, and bHLH82, showed that three distinct subfamilies of bHLH proteins participate in root epidermis development in a stage-specific manner. The integration of genetic, genomic, and computational analyses provides a new view of the composition, architecture, and logic of the root epidermal transcriptional network, and it demonstrates the utility of a comprehensive systems approach for dissecting a complex regulatory network.

A full-length R-like basic-helix-loop-helix transcription factor is required for anthocyanin upregulation whereas the N-terminal region regulates epidermal hair formation

Earlier studies have shown that the Lc gene of maize, a member of the R gene family that encode basic-helix-loop-helix (bHLH) transcription factors, is involved with anthocyanin production and trichome formation in Arabidopsis. We previously reported that the N-terminus of R protein interacts with CAPRICE (CPC), a regulatory protein, in triggering epidermal hair differentiation in Arabidopsis. In this study, we investigated the roles of full-length R, the N-terminal region of R (RN) and the C-terminal region of R (RC) in epidermal cell differentiation and anthocyanin production. We found that the N-terminal region was responsible for leaf trichome and root hair differentiation, whereas full-length R was required for anthocyanin upregulation. Yeast two-hybrid analysis showed that the C-terminal region was the binding site for the formation of homo- or hetero-dimers of the R-like bHLH transcription factor. To stimulate anthocyanin production, full-length R is required.

The Cytosolic Kinases STY8, STY17, and STY46 Are Involved in Chloroplast Differentiation in Arabidopsis

In Arabidopsis (Arabidopsis thaliana), transit peptides for chloroplast-destined preproteins can be phosphorylated by the protein kinases STY8, STY17, and STY46. In this study, we have investigated the in vitro properties of these plant-specific kinases. Characterization of the mechanistic functioning of STY8 led to the identification of an essential threonine in the activation segment, which is phosphorylated by an intramolecular mechanism. STY8 is inhibited by specific tyrosine kinase inhibitors, although it lacked the ability to phosphorylate tyrosine residues in vitro. In vivo analysis of sty8, sty17, and sty46 Arabidopsis knockout/knockdown mutants revealed a distinct function of the three kinases in the greening process and in the efficient differentiation of chloroplasts. Mutant plants displayed not only a delayed accumulation of chlorophyll but also a reduction of nucleus-encoded chloroplast proteins and a retarded establishment of photosynthetic capacity during the first 6 h of deetiolation, supporting a role of cytosolic STY kinases in chloroplast differentiation.

Female gametophytic cell specification and seed development require the function of the putativeArabidopsisINCENP orthologWYRD

In plants, gametes, along with accessory cells, are formed by the haploid gametophytes through a series of mitotic divisions, cell specification and differentiation events. How the cells in the female gametophyte of flowering plants differentiate into gametes (the egg and central cell) and accessory cells remains largely unknown. In a screen for mutations that affect egg cell differentiation in Arabidopsis, we identified the wyrd (wyr) mutant, which produces additional egg cells at the expense of the accessory synergids. WYR not only restricts gametic fate in the egg apparatus, but is also necessary for central cell differentiation. In addition, wyr mutants impair mitotic divisions in the male gametophyte and endosperm, and have a parental effect on embryo cytokinesis, consistent with a function of WYR in cell cycle regulation. WYR is upregulated in gametic cells and encodes a putative plant ortholog of the inner centromere protein (INCENP), which is implicated in the control of chromosome segregation and cytokinesis in yeast and animals. Our data reveal a novel developmental function of the conserved cell cycle-associated INCENP protein in plant reproduction, in particular in the regulation of egg and central cell fate and differentiation.

A Molecular Switch for Initiating Cell Differentiation in Arabidopsis

The onset of differentiation entails modifying the gene expression state of cells, to allow activation of developmental programs that are maintained repressed in the undifferentiated precursor cells [1, 2]. This requires a mechanism to change gene expression on a genome-scale. Recent evidence suggests that in mammalian stem cells, derepression of developmental regulators during differentiation involves a shift from stalled to productive elongation of their transcripts [3-5], but factors mediating this shift have not been identified and the evidence remains correlative. We report the identification of the MINIYO (IYO) gene, a positive regulator of transcriptional elongation that is essential for cells to initiate differentiation in Arabidopsis. IYO interacts with RNA polymerase II and the Elongator complex and is required to sustain global levels of transcriptional elongation activity, specifically in differentiating tissues. Accordingly, IYO is expressed in embryos, meristems, and organ primordia and not in mature tissues. Moreover, differential subcellular protein distribution further refines the domain of IYO function by directing nuclear accumulation, and thus its transcriptional activity, to cells initiating differentiation. Importantly, IYO overexpression induces premature cell differentiation and leads to meristem termination phenotypes. These findings identify IYO as a necessary and sufficient factor for initiating differentiation in Arabidopsis and suggest that the targeted nuclear accumulation of IYO functions as a transcriptional switch for this fate transition.

Arabidopsis brassinosteroid biosynthetic mutant dwarf7-1exhibits slower rates of cell division and shoot induction

BackgroundPlant growth depends on both cell division and cell expansion. Plant hormones, including brassinosteroids (BRs), are central to the control of these two cellular processes. Despite clear evidence that BRs regulate cell elongation, their roles in cell division have remained elusive.ResultsHere, we report results emphasizing the importance of BRs in cell division. An Arabidopsis BR biosynthetic mutant, dwarf7-1, displayed various characteristics attributable to slower cell division rates. We found that the DWARF4 gene which encodes for an enzyme catalyzing a rate-determining step in the BR biosynthetic pathways, is highly expressed in the actively dividing callus, suggesting that BR biosynthesis is necessary for dividing cells. Furthermore, dwf7-1 showed noticeably slower rates of callus growth and shoot induction relative to wild-type control. Flow cytometric analyses of the nuclei derived from either calli or intact roots revealed that the cell division index, which was represented as the ratio of cells at the G2/M vs. G1 phases, was smaller in dwf7-1 plants. Finally, we found that the expression levels of the genes involved in cell division and shoot induction, such as PROLIFERATING CELL NUCLEAR ANTIGEN2 (PCNA2) and ENHANCER OF SHOOT REGENERATION2 (ESR2), were also lower in dwf7-1 as compared with wild type.ConclusionsTaken together, results of callus induction, shoot regeneration, flow cytometry, and semi-quantitative RT-PCR analysis suggest that BRs play important roles in both cell division and cell differentiation in Arabidopsis.

The Rab GTPase RabG3b functions in autophagy and contributes to tracheary element differentiation in Arabidopsis

The tracheary elements (TEs) of the xylem serve as the water-conducting vessels of the plant vascular system. To achieve this, TEs undergo secondary cell wall thickening and cell death, during which the cell contents are completely removed. Cell death of TEs is a typical example of developmental programmed cell death that has been suggested to be autophagic. However, little evidence of autophagy in TE differentiation has been provided. The present study demonstrates that the small GTP binding protein RabG3b plays a role in TE differentiation through its function in autophagy. Differentiating wild type TE cells were found to undergo autophagy in an Arabidopsis culture system. Both autophagy and TE formation were significantly stimulated by overexpression of a constitutively active mutant (RabG3bCA), and were inhibited in transgenic plants overexpressing a dominant negative mutant (RabG3bDN) or RabG3b RNAi (RabG3bRNAi), a brassinosteroid insensitive mutant bri1-301, and an autophagy mutant atg5-1. Taken together, our results suggest that autophagy occurs during TE differentiation, and that RabG3b, as a component of autophagy, regulates TE differentiation.