Important Role In Embryo Development Research Articles

Identification and characterization of the WOX Gene Family revealed two WUS Clade Members associated with embryo development in Cunninghamia lanceolata

The WUSCHEL-related homeobox (WOX) gene family is vital for plant development and stress response. In this study, we conducted a comprehensive analysis of WOX genes in Cunninghamia lanceolata (C. lanceolata) and subsequently explored the potential roles of two ClWOX genes within the WUS clade. In total, six ClWOX genes were identified through a full-length transcriptome analysis. These genes, exhibiting conserved structural and functional motifs, were assigned to the ancient clade and Modern/WUS clade, respectively, through a phylogenetic analysis. Our expression analysis indicated that these ClWOX genes were highly expressed in the middle and late developmental stages of zygotic embryos in C. lanceolata. Moreover, only ClWOX5 and ClWOX6 within the Modern/WUS clade exhibited transcriptional activity, and their expressions were also induced in response to auxin and wounding. Overexpression of ClWOX5 and ClWOX6 in Arabidopsis caused a partially sterile phenotype, resulting in a very low seed setting rate. Transcriptomic analysis revealed that expressions of many embryo-defective (EMB) genes, phytohormone-related genes, and transcription factors (TFs) were dramatically altered in ClWOX5 and ClWOX6 transgenic plants, which suggested that ClWOX5 and ClWOX6 may play specific important roles in embryo development via complex gene networks. In addition, overexpression of ClWOX5 and ClWOX6 in leaf segments promoted shoot regeneration in tobacco, indicating that ClWOX5 and ClWOX6 can promote plant regeneration and could be used to improve genetic transformation. In conclusion, these results help to elucidate the function of the WOX gene and provide a valuable basis for future studies of the developmental regulation and applications of WOX genes in C. lanceolata.

Genome-wide analysis of the GLP gene family and overexpression of GLP1-5–1 to promote lignin accumulation during early somatic embryo development in Dimocarpus longan

Longan (Dimocarpus longan Lour.) is an economically important subtropical fruit tree. Its fruit quality and yield are affected by embryo development. As a plant seed germination marker gene, the germin-like protein (GLP) gene plays an important role in embryo development. However, the mechanism underlying the role of the GLP gene in somatic embryos is still unclear. Therefore, we conducted genome-wide identification of the longan GLP (DlGLP) gene and preliminarily verified the function of DlGLP1-5–1. Thirty-five genes were identified as longan GLP genes and divided into 8 subfamilies. Based on transcriptome data and qRT‒PCR results, DlGLP genes exhibited the highest expression levels in the root, and the expression of most DlGLPs was upregulated during the early somatic embryogenesis (SE) in longan and responded to high temperature stress and 2,4-D treatment; eight DlGLP genes were upregulated under MeJA treatment, and four of them were downregulated under ABA treatment. Subcellular localization showed that DlGLP5-8–2 and DlGLP1-5–1 were located in the cytoplasm and extracellular stroma/chloroplast, respectively. Overexpression of DIGLP1-5–1 in the globular embryos (GEs) of longan promoted the accumulation of lignin and decreased the H2O2 content by regulating the activities of ROS-related enzymes. The results provide a reference for the functional analysis of DlGLPs and related research on improving lignin accumulation in the agricultural industry through genetic engineering.

Synthetic apomixis with normal hybrid rice seed production

Synthetic apomixis with normal hybrid rice seed production

Extracellular vesicles from seminal plasma improved development of in vitro-fertilized mouse embryos.

In vitro fertilization (IVF) has wide application in human infertility and animal breeding. It is also used for research on reproduction, fertility and development. However, IVF embryos are still inferior to their in vivo counterparts. Some substances in seminal plasma appear to have important roles in embryo development, and during the traditional IVF procedure, the seminal plasma is washed away. In this study, extracellular vesicles (EVs) were concentrated from seminal plasma by ultracentrifugation, visualized using transmission electron microscopy, and particle size distributions and concentrations were determined with a NanoSight particle analyzer. We found particles of various sizes in the seminal plasma, the majority having diameters ranging from 100 to 200 nm and concentrations of 6.07 × 1010 ± 2.91 × 109 particles/ml. Addition of seminal plasma EVs (SP-EVs) to the IVF medium with mouse oocytes and sperm significantly increased the rate of blastocyst formation and the inner cell mass (ICM)/trophectoderm (TE) cell ratio, and reduced the apoptosis of blastocysts. Our findings provide new insights into the role of seminal plasma EVs in mediating embryo development and it suggests that SP-EVs may be used to improve the developmental competence of IVF embryos, which has important significance for assisted reproduction in animals and humans.

Sperm-borne small RNAs improve the developmental competence of pre-implantation cloned embryos in rabbit.

The low efficiency of somatic cell nuclear transfer (SCNT) greatly limits its application. Compared with the fertilized embryo, cloned embryos display abnormal epigenetic modification and other inferior developmental properties. In this study, small RNAs were isolated, and miR-34c and miR-125b were quantified by real-time PCR; results showed that these micro-RNAs were highly expressed in sperm. The test sample was divided into three groups: one was the fertilized group, one was the SCNT control group (NT-C group), and the third group consisted of SCNT embryos injected with sperm-borne small RNA (NT-T group). The level of tri-methylation of lysine 9 on histone H3 (H3K9me3) at the 8-cell stage was determined by immunofluorescence staining, and the cleavage ratio, blastocyst ratio, apoptotic cell index of the blastocyst and total cell number of blastocysts in each group were analyzed. Results showed that the H3K9me3 level was significantly higher in the NT-C group than in the fertilized group and the NT-T group. The apoptosis index of blastocysts in the NT-C group was significantly higher than that in the fertilized group and the NT-T group. The total cell number of SCNT embryos was significantly lower than that of fertilized embryos, and injecting sperm-borne small RNAs could significantly increase the total cell number of SCNT blastocysts. Our study not only demonstrates that sperm-borne small RNAs have an important role in embryo development, but also provides a new strategy for improving the efficiency of SCNT in rabbit.

Intrauterine Infusion of TGF-β1 Prior to Insemination, Alike Seminal Plasma, Influences Endometrial Cytokine Responses but Does Not Impact the Timing of the Progression of Pre-Implantation Pig Embryo Development.

Simple SummaryAlthough endometrial immune regulation in pigs during the early preimplantation period is poorly documented, particularly under conditions of embryo transfer (ET), it is recognized that seminal plasma (SP) induces molecular changes in the reproductive tract, influencing numerous reproductive functions. A principal constituent of SP is the cytokine transforming growth factor β1 (TGF-β1), which has an important role in embryo development, pregnancy establishment, and progression. The present study evaluated different intrauterine infusion treatments at estrus (40 mL of SP, porcine TGF-β1 in an extender, or an extender alone (control)) by mimicking an ET scenario in so-called “donor” (inseminated) and “recipient” (uninseminated) sows. We investigated the effects of these treatments on day 6 embryo development (“donors”) and endometrial explants’ cytokine production (“donors” and “recipients”). SP infusion positively influenced embryo development compared with TGF-β1 or extender infusions. Infusion treatments differentially affected endometrial cytokine production, with the effects being stronger in “donors” than in “recipients.” Increased knowledge of the effects of SP or some of its active components on the female immune system may help to develop strategies for increasing the reproductive efficiency for the benefit of pig ET.Seminal plasma (SP) in the female genital tract induces changes that affect multiple reproductive processes. One of the active components in SP is the transforming growth factor β1 (TGF-β1), which has major roles in embryo development and pregnancy. Embryo transfer (ET) technology is welcomed by the pig industry provided that embryo quality at embryo collection as well as the fertility and prolificacy of the recipients after the ET is increased. This study evaluated different intrauterine infusion treatments at estrus (40 mL of SP, TGF-β1 cytokine in the extender, or the extender alone (control)) by mimicking an ET scenario in so-called “donor” (inseminated) and “recipient” (uninseminated) sows. On day 6 (day 0—onset of estrus), all “donors” were laparotomized to determine their pregnancy status (presence and developmental stage of the embryos). In addition, endometrial explants were collected from pregnant “donors” and cyclic “recipients,” incubated for 24 h, and analyzed for cytokine production. SP infusions (unlike TGF-β1 infusions) positively influenced the developmental stage of day 6 embryos. Infusion treatments differentially influenced the endometrial cytokine production, mainly in donors. We concluded that SP infusions prior to AI not only impacted the porcine preimplantation embryo development but also influenced the endometrial cytokine production six days after treatment, both in donors and recipients.

Flutamide induces uterus and ovary damage in the mouse via apoptosis and excessive autophagy of cells following triggering of the unfolded protein response.

Intrauterine exposure to flutamide not only causes abnormal development of the reproductive organs in male offspring, but also damages ovaries and uteri. The unfolded protein response (UPR) is believed to play an important role in embryo development and teratogenic processes. In the present study, pregnant mice were administered either flutamide (300mg kg-1 day-1, p.o.) on an equivalent volume of soybean oil (control) on Days 12-18 of gestation. Eight weeks after birth, female offspring in the flutamide-treated group had a lower bodyweight and lower ovarian and uterine weights, but there was no significant difference in uterine and ovarian weights normalised by bodyweight between the flutamide-treated and control groups. Furthermore, histopathological changes were observed in all uteri and ovaries in the flutamide-treated group, with fewer and less-developed follicles in the ovaries. In both the uteri and ovaries, flutamide increased the expression of UPR members, although the expression of cell cycle-related genes remained unchanged compared with the control group. Flutamide increased the expression of all autophagy- and apoptosis-related genes evaluated in the uterus, as well as some in the ovary. The results suggest that the in utero exposure of mice to flutamide may contribute to uterine and ovarian damage in the offspring, with endoplasmic reticulum stress possibly triggered by the UPR leading to the induction of excessive autophagy and apoptosis.

LEUKEMIA INHIBITORY FACTOR IN RECURRENT PREGNANCY LOSS: IS THERE ANY ROLE OF LIFESTYLE INTERVENTION?

Leukemia inhibitory factor (LIF), has been cited to play important role in embryo development and implantation. This pleiotropic cytokine affects a range of events in implantation, such as, endometrial receptivity, embryo-endometrial interaction, stromal decidualization, trophoblast invasion and blastocyst development. Lower levels of LIF have been found in blood and endometrial tissue of women with recurrent pregnancy loss (RPL). The objective of this study was to assess the levels of LIF in plasma of women with RPL and healthy fertile females.

Induction of autophagy promotes porcine parthenogenetic embryo development under low oxygen conditions.

Autophagy plays an important role in embryo development; however, only limited information is available on how autophagy specifically regulates embryo development, especially under low oxygen culture conditions. In this study we used parthenogenetic activation (PA) of porcine embryos to test the hypothesis that a low oxygen concentration (5%) could promote porcine embryo development by activating autophagy. Immunofluorescence staining revealed that low oxygen tension activated autophagy and alleviated oxidative stress in porcine PA embryos. Development was significantly affected when autophagy was blocked by 3-methyladenine, even under low oxygen culture conditions, with increased reactive oxygen species levels and malondialdehyde content. Furthermore, the decreased expression of pluripotency-associated genes induced by autophagy inhibition could be recovered by treatment with the antioxidant vitamin C. Together, these results demonstrate that low oxygen-induced autophagy regulates embryo development through antioxidant mechanisms in the pig.

Effect of Maternal Age on Hippo Pathway Related Gene Expressions and Protein Localization Pattern in Human Embryos.

Objective The Hippo pathway plays an important role in embryo development, and separation of trophectoderm (TE) and inner cell mass (ICM) cell lines. Therefore, this study investigated effect of maternal age on activity of Hippo pathway in human embryos. Materials and Methods In this experimental study, the developed up embryos to the blastocyst stage and the embryos whose growth stopped at the morula stage were collected from women aged 20-30 years old (young group, 94 embryos) and >37 years (old group, 89 embryos). Expression of OCT4, SOX2, CDX2, GATA3, YAP genes and the relevant proteins, in the both groups were evaluated using respectively quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunofluorescence methods. Results There was no significant difference in the expression level of OCT4, SOX2, CDX2, GATA3 and YAP genes in blastocyst and morula stages, between the two groups. However, SOX2 and CDX2 gene expressions in morula stage embryos of the old group was statistically lower than that of the young group (P=0.007 and P=0.008, respectively). Additionally, in the embryos collected from women with >37 years of age, at the blastocyst stage, phospho-YAP (p-YAP) protein was found to be accumulated in the TE, but it was almost disappeared from the ICM. Additionally, in the old group, contrary to the expectation, YAP protein was expressed in the ICM, rather than TE. ConclusionThe results of this study showed that YAP and P-YAP among the Hippo signalling pathway may be altered by increasing age.

Oxidative stress in pregnant women with diabetes mellitus

Oxidative stress (OS) plays an important role in embryo development, implantation, placentation, fetal development and labour. Diabetes mellitus (DM) is associated with an increase in OS processes. However, the expression of OS biomarkers in pregnant women with DM remains unclear. Based on a literature review, the features of the pro- and anti-oxidant systems of pregnant women with different types of DM have been established. Pregnancy in patients with DM has been shown to be characterised by an activation of OS processes. This leads to an overexpression of free radicals (peroxynitrite), toxic derivatives (malonic dialdehyde, 8-isoprostane) and specific enzymes (asymmetric dimethylarginine, catalase) and a decrease in the synthesis of antioxidants (superoxide dismutase, glutathione peroxidase and uric acid). The modified expression of these biomarkers is observed both in the blood and the placenta of pregnant women. These disorders can cause an unfavourable course of pregnancy, abnormal development of the placenta and development of adverse perinatal outcomes in pregnant women with DM. Nevertheless, given the inconsistency of data obtained, further scientific studies are needed to clarify this issue.

Limb-bud and Heart Overexpression Inhibits the Proliferation and Migration of PC3M Cells.

Background: The limb-bud and heart gene (LBH) was discovered in the early 21st century and is specifically expressed in the mouse embryonic limb and heart development. Increasing evidences have indicated that LBH not only plays an important role in embryo development, it is also closely correlated with the occurance and progression of many tumors. However, its function in prostate cancer (PCa) is still not well understood. Here, we explored the effects of LBH on the proliferation and migration of the PCa cell line PC3M.Methods: LBH expression in tissues and cell lines of PCa was detected by immunohistochemistry and Western blotting. Lentivirus was used to transduct the LBH gene into the PC3M cells. Stable LBH-overexpressing PC3M-LBH cells and PC3M-NC control cells were obtained via puromycin screening. Cell proliferation was examined using the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cell cycle distribution and apoptosis rate were investigated using flow cytometry. Cell migration was studied using the Transwell assay.Results: LBH expression level was down-regulated in 3 different PCa cell lines, especially in PC3M cells, compared with the normal prostate epithelial cells(RWPE-1). Cell lines of LBH-upregulated PC3M-LBH and PC3M-NC control were successfully constructed. Significantly increased LBH expression level and decreased cyclin D1 and cyclin E2 expression level was found in PC3M-LBH cells as compared to the PC3M-NC cells. The overexpression of LBH significantly inhibited PC3M cell proliferation in vitro and tumor growth in nude mice. LBH overexpression in PC3M cell, also induced cell cycle G0/G1 phase arrest and decreased the migration of PC3M cells.Conclusions: Our results reveal that LBH expression is down-regulated in the tissue and cell lines of PCa. LBH overexpression inhibits PC3M cell proliferation and tumor growth by inducing cell cycle arrest through down-regulating cyclin D1and cyclin E2 expression. LBH might be a therapeutic target and potential diagnostic marker in PCa.

Protein Expression Landscape of Mouse Embryos during Pre-implantation Development

Pre-implantation embryo development is an intricate and precisely regulated process orchestrated by maternally inherited proteins and newly synthesized proteins following zygotic genome activation. Although genomic and transcriptomic studies have enriched our understanding of the genetic programs underlying this process, the protein expression landscape remains unexplored. Using quantitative mass spectrometry, we identified nearly 5,000 proteins from 8,000 mouse embryos of each stage (zygote, 2-cell, 4-cell, 8-cell, morula, and blastocyst). We found that protein expression in zygotes, morulas, and blastocysts is distinct from 2- to 8-cell embryos. Analysis of protein phosphorylation identified critical kinases and signal transduction pathways. We highlight key factors and their important roles in embryo development. Combined analysis of transcriptomic and proteomic data reveals coordinated control of RNA degradation, transcription, and translation and identifies previously undefined exon-junction-derived peptides. Our study provides an invaluable resource for further mechanistic studies and suggests core factors regulating pre-implantation embryo development.

Tissue distribution and functional analysis of vitellogenin-6 of Toxocara canis

Toxocara canis is an common intestinal nematode of canids and the principal causative agent of human toxocariasis. Vitellogenin (Vg), a source of amino acids and lipids in the eggs, are considered to play an important role in embryo development of a wide range of organisms. In the present study, the transcriptional levels of Tc-vit-6 gene in male and female adult T. canis were determined by quantitative real-time PCR, which indicated high transcription of Tc-vit-6 in the intestine, reproductive tract and body wall of male and female adult T. canis. The fragment of Tc-vit-6 encoding a vWD domain, was cloned and expressed to produce a rabbit anti-TcvWD polyclonal antibody. Tissue distribution of TcVg6 was detected by immunohistochemical assays, which showed predominant distribution of TcVg6 in the tissues of intestine, as well as reproductive tract (including some of the germ cells) and musculature of male and female adult worms. Collectively, these results indicated multiple biological roles of TcVg6 apart from that in the reproduction of T. canis.

Molecular response and association analysis of Megalobrama amblycephala fih-1 with hypoxia.

Hypoxia is one of the most important environmental factors which affect fish growth, development and survival, but regulation mechanisms of hypoxia in fish remain unclear. Therefore, to further understand molecular functions of factor inhibiting HIF-1 (Fih-1), an essential hypoxia sensor, the full-length cDNA of fih-1 was cloned from Megalobrama amblycephala, a hypoxia-sensitive cyprinid fish. The deduced amino acid sequence showed high homology with that of other vertebrates, and all structural and functional domains were highly conserved. The mRNA level in different tissues and developmental stages indicated that M. amblycephala fih-1 expression was higher in liver and muscle, followed by gill, intestine and spleen. During embryogenesis, the fih-1 mRNA was highly expressed in the early embryonic development, then decreased to a very low level, and maintained a relative high level of expression after hatching. In most tissues, the fih-1 mRNA was down-regulated at 2h but up-regulated at 4h after hypoxia treatment. In addition, the promoter sequence of M. amblycephala fih-1 was obtained using thermal asymmetric interlaced PCR. Three single nucleotide polymorphism (SNP) sites were found in the cDNA and promoter sequences, and identified significantassociationwith hypoxia trait by correlation analysis in hypoxia-sensitive group and hypoxia-tolerant group. These results demonstrated that M. amblycephala fih-1 plays important roles in embryo development and hypoxia response, which will contribute to systematic understanding of the molecular mechanisms of fish in response to hypoxia, and provide help for fish genetic breeding with hypoxia-tolerant strains or breeds.

Retarded Embryo Development 1 (RED1) regulates embryo development, seed maturation and plant growth in Arabidopsis

Plant seeds accumulate large amounts of protein and carbohydrate as storage reserves during maturation. Thus, understanding the genetic control of embryo and seed development may provide bioengineering tools for yield improvement. In this study, we report the identification of Retarded Embryo Development 1 (RED1) gene in Arabidopsis, whose two independent T-DNA insertion mutant lines, SALK_085642 (red1-1) and SALK_022583 (red1-2), show a retarded embryo development phenotype. The embryogenesis process ceases at the late heart stage in red1-1 and at the bent-cotyledon stage in red1-2, respectively, resulting in seed abortion in both lines. The retarded embryo development and seed abortion phenotypes reverted to normal when RED1 complementation constructs were introduced into mutant plants. Small red1-2 homozygous plants can be successfully rescued by culturing immature seeds, indicating that seed abortion likely results from compromised tolerance to the desiccation process associated with seed maturation. Consistent with this observation, red1-2 seeds accumulate less protein, and the expression of two late embryo development reporter transgenes, LEA::GUS and β-conglycinin::GUS, was significantly weak and started relatively late in the red1-2 mutant lines compared to the wild type. The RED1 gene encodes a plant specific novel protein that is localized in the nucleus. These results indicate that RED1 plays important roles in embryo development, seed maturation and plant growth.

Targeted Disruption of miR-17-92 Impairs Mouse Spermatogenesis by Activating mTOR Signaling Pathway.

The miR-17-92 cluster and its 6 different mature microRNAs, including miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92a, play important roles in embryo development, immune system, kidney and heart development, adipose differentiation, aging, and tumorigenicity. Currently, increasing evidence indicates that some members of miR-17-92 cluster may be critical players in spermatogenesis, including miR-17, miR-18a, and miR-20a. However, the roles and underlying mechanisms of miR-17-92 in spermatogenesis remain largely unknown. Our results showed that the targeted disruption of miR-17-92 in the testes of adult mice resulted in severe testicular atrophy, empty seminiferous tubules, and depressed sperm production. This phenotype is partly because of the reduced number of spermatogonia and spermatogonial stem cells, and the significantly increased germ cell apoptosis in the testes of miR-17-92-deficient mice. In addition, overactivation of the mammalian target of rapamycin signaling pathway and upregulation of the pro-apoptotic protein Bim, Stat3, c-Kit, and Socs3 were also observed in miR-17-92-deficient mouse testes, which might be, at least partially if not all, responsible for the aforementioned phenotypic changes in mutant testes. Taken together, these findings suggest that miR-17-92 is essential for normal spermatogenesis in mice.

Characterization of the IGF2 Imprinted Gene Methylation Status in Bovine Oocytes during Folliculogenesis.

DNA methylation reprogramming occurs during mammalian gametogenesis and embryogenesis. Sex-specific DNA methylation patterns at specific CpG islands controlling imprinted genes are acquired during this window of development. Characterization of the DNA methylation dynamics of imprinted genes acquired by oocytes during folliculogenesis is essential for understanding the physiological and genetic aspects of female gametogenesis and to determine the parameters for oocyte competence. This knowledge can be used to improve in vitro embryo production (IVP), specifically because oocyte competence is one of the most important aspects determining the success of IVP. Imprinted genes, such as IGF2, play important roles in embryo development, placentation and fetal growth. The aim of this study was to characterize the DNA methylation profile of the CpG island located in IGF2 exon 10 in oocytes during bovine folliculogenesis. The methylation percentages in oocytes from primordial follicles, final secondary follicles, small antral follicles, large antral follicles, MII oocytes and spermatozoa were 73.74 ± 2.88%, 58.70 ± 7.46%, 56.00 ± 5.58%, 65.77 ± 5.10%, 56.35 ± 7.45% and 96.04 ± 0.78%, respectively. Oocytes from primordial follicles showed fewer hypomethylated alleles (15.5%) than MII oocytes (34.6%) (p = 0.039); spermatozoa showed only hypermethylated alleles. Moreover, MII oocytes were less methylated than spermatozoa (p<0.001). Our results showed that the methylation pattern of this region behaves differently between mature oocytes and spermatozoa. However, while this region has a classical imprinted pattern in spermatozoa that is fully methylated, it was variable in mature oocytes, showing hypermethylated and hypomethylated alleles. Furthermore, our results suggest that this CpG island may have received precocious reprogramming, considering that the hypermethylated pattern was already found in growing oocytes from primordial follicles. These results may contribute to our understanding of the reprogramming of imprinted genes during bovine oogenesis.

Effect of species-specific sound stimulation on the development and hatching of broiler chicks

1. Previous research has reported that chicken embryos develop a functionary auditory system during incubation and that prenatal sound may play an important role in embryo development and alter the hatch time. In this study the effects of prenatal auditory stimulation on hatch process, hatch performance, the development of embryo and blood parameters were investigated.2. Four batches of Ross 308 broiler breeder eggs were incubated either in control or in sound-stimulated groups. The sound-stimulated embryos were exposed to a discontinuous sound of species-specific calls by means of a speaker at 72 dB for 16 h a day: maternal calls from d 10 to d 19 of incubation time and embryo/chick calls from d 19 until hatching. The species-specific sound was excluded from the control group.3. The onset of hatch was delayed in the sound-stimulated group compared to the controls. This was also supported by comparison of the exact hatching time of individual focal chicks within the two groups. However, the sound-stimulated embryos had a lower hatchability than the control group, mainly due to significantly increased numbers of late deaths.4. The embryos exhibited a similar growth pattern between the sound-stimulated group and the control group. Although sound exposure decreased body weight at d 16, no consistent effect of sound on body weight at incubation stage was observed. Species-specific sound stimulation also had no impact on chick quality, blood values and plasma corticosterone concentrations during hatch.

ROP3 GTPase contributes to polar auxin transport and auxin responses and is important for embryogenesis and seedling growth in Arabidopsis.

ROP GTPases are crucial for the establishment of cell polarity and for controlling responses to hormones and environmental signals in plants. In this work, we show that ROP3 plays important roles in embryo development and auxin-dependent plant growth. Loss-of-function and dominant-negative (DN) mutations in ROP3 induced a spectrum of similar defects starting with altered cell division patterning during early embryogenesis to postembryonic auxin-regulated growth and developmental responses. These resulted in distorted embryo development, defective organ formation, retarded root gravitropism, and reduced auxin-dependent hypocotyl elongation. Our results showed that the expression of AUXIN RESPONSE FACTOR5/MONOPTEROS and root master regulators PLETHORA1 (PLT1) and PLT2 was reduced in DN-rop3 mutant embryos, accounting for some of the observed patterning defects. ROP3 mutations also altered polar localization of auxin efflux proteins (PINs) at the plasma membrane (PM), thus disrupting auxin maxima in the root. Notably, ROP3 is induced by auxin and prominently detected in root stele cells, an expression pattern similar to those of several stele-enriched PINs. Our results demonstrate that ROP3 is important for maintaining the polarity of PIN proteins at the PM, which in turn ensures polar auxin transport and distribution, thereby controlling plant patterning and auxin-regulated responses.