Transcriptomic Analysis of Arabidopsis Seedlings in Response to an Agrobacterium-Mediated Transformation Process.

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease. This pathogen is capable of transferring the T-DNA from its Ti plasmid to the host cell and, then, integrating it into the host genome. To date, this genetic transformation ability has been harnessed as the dominant technology to produce genetically modified plants for both basic research and crop biotechnological applications. However, little is known about the interaction between Agrobacterium tumefaciens and host plants, especially the host responses to Agrobacterium infection and its associated factors. We employed RNA-seq to follow the time course of gene expression in Arabidopsis seedlings infected with either an avirulent or a virulent Agrobacterium strain. Gene Ontology analysis indicated many biological processes were involved in the Agrobacterium-mediated transformation process, including hormone signaling, defense response, cellular biosynthesis, and nucleic acid metabolism. RNAseq and quantitative reverse transcription-polymerase chain reaction results indicated that expression of genes involved in host plant growth and development were repressed but those involved in defense response were induced by Agrobacterium tumefaciens. Further analysis of the responses of transgenic Arabidopsis lines constitutively expressing either the VirE2 or VirE3 protein suggested Vir proteins act to enhance plant defense responses in addition to their known roles facilitating T-DNA transformation.

The novel cyst nematode effector protein 30D08 targets host nuclear functions to alter gene expression in feeding sites.

Cyst nematodes deliver effector proteins into host cells to manipulate cellular processes and establish a metabolically hyperactive feeding site. The novel 30D08 effector protein is produced in the dorsal gland of parasitic juveniles, but its function has remained unknown. We demonstrate that expression of 30D08 contributes to nematode parasitism, the protein is packaged into secretory granules and it is targeted to the plant nucleus where it interacts with SMU2 (homolog of suppressor of mec-8 and unc-52 2), an auxiliary spliceosomal protein. We show that SMU2 is expressed in feeding sites and an smu2 mutant is less susceptible to nematode infection. In Arabidopsis expressing 30D08 under the SMU2 promoter, several genes were found to be alternatively spliced and the most abundant functional classes represented among differentially expressed genes were involved in RNA processing, transcription and binding, as well as in development, and hormone and secondary metabolism, representing key cellular processes known to be important for feeding site formation. In conclusion, we demonstrated that the 30D08 effector is secreted from the nematode and targeted to the plant nucleus where its interaction with a host auxiliary spliceosomal protein may alter the pre-mRNA splicing and expression of a subset of genes important for feeding site formation.

Consumption of a high-fat diet alters the seminal fluid and gut microbiomes in male mice.

Our prior work showed that a novel microbiome resides in the seminal vesicles of wild-type and oestrogen receptor α (Esr1) knock-out mice and is impacted by the presence of functional Esr1 genes. The seminal fluid microbiome (SFM) may influence the health and reproductive status of the male, along with that of his partner and offspring. A high-fat diet (HFD) alters metabolites and other factors within seminal fluid and might affect the SFM. Adult (~15 weeks old) male mice were placed for 4 weeks on a control or high-fat diet and seminal fluid and fecal samples were collected, bacterial DNA isolated and subjected to 16s rRNA sequencing. Corynebacterium spp. were elevated in the seminal fluid of HFD males; however, Acinetobacter johnsonii, Streptophyta, Ammoniphilus spp., Bacillus spp. and Propionibacterium acnes were increased in control males. Rikenellaceae was more abundant in the fecal samples from HFD males. However, Bacteroides ovatus and another Bacteroides species, Bilophila, Sutterella spp., Parabacteroides, Bifidobacterium longum, Akkermansia muciniphila and Desulfovibrio spp. were greater in control males. Thus, short-term consumption of a HFD influences the seminal fluid and fecal microbiomes, which may have important health consequence for males and developmental origins of health and disease effects in resulting offspring.

Transcriptional profiling by RNA-Seq of peri-attachment porcine embryos generated by a variety of assisted reproductive technologies.

Substantial mortality of in vitro manipulated porcine embryos is observed during peri-attachment development. Herein we describe our efforts to characterize the transcriptomes of embryonic disc (ED) and trophectoderm (TE) cells from porcine embryos derived from in vivo fertilization, in vitro fertilization (IVF), parthenogenetic oocyte activation (PA), and somatic cell nuclear transfer (SCNT) on days 10, 12, and 14 of gestation. The IVF, PA, and SCNT embryos were generated with in vitro matured oocytes and were cultured overnight in vitro before being transferred to recipient females. Sequencing of cDNA from the resulting embryonic samples was accomplished with the Genome Analyzer IIx platform from Illumina. Reads were aligned to a custom-built swine transcriptome. A generalized linear model was fit for ED and TE samples separately, accounting for embryo type, gestation day, and their interaction. Those genes with significant differences between embryo types were characterized in terms of gene ontologies and KEGG pathways. Transforming growth factor-ß signaling was downregulated in the EDs of IVF embryos. In TE cells from IVF embryos, ubiquitin-mediated proteolysis and ErbB signaling were aberrantly regulated. Expression of genes involved in chromatin modification, gene silencing by RNA, and apoptosis was significantly disrupted in ED cells from SCNT embryos. In summary, we have used high-throughput sequencing technologies to compare gene expression profiles of various embryo types during peri-attachment development. We expect that these data will provide important insight into the root causes of (and possible opportunities for mitigation of) suboptimal development of embryos derived from assisted reproductive technologies.

Transcriptional profiling by deep sequencing identifies differences in mRNA transcript abundance in in vivo-derived versus in vitro-cultured porcine blastocyst stage embryos.

In vitro embryo culture systems promote development at rates lower than in vivo systems. The goal of this project was to discover transcripts that may be responsible for a decrease of embryo competency in blastocyst-stage embryos cultured in vitro. Gilts were artificially inseminated on the first day of estrus, and on Day 2, one oviduct and the tip of a uterine horn were flushed and the recovered embryos were cultured in porcine zygote medium 3 for 4 days. On Day 6, the gilts were euthanized and the contralateral horn was flushed to obtain in vivo derived embryos. Total RNA was extracted from three pools of 10 blastocysts from each treatment. First and second strand cDNA was synthesized and sequenced using Illumina sequencing. The reads generated were aligned to a custom-built database designed to represent the known porcine transcriptome. A total of 1170 database members were different between the two groups (P < 0.05), and 588 of those had at least a 2-fold difference. Eleven transcripts were subjected to real-time PCR that validated the sequencing. There was an overall decrease in inner cell mass (ICM) and trophectodermal (TE) cell numbers in embryos cultured in vitro; however, no difference in the ICM:TE ratio was found. Interestingly, the transcript SLC7A1 was higher in the in vitro cultured group. This difference disappeared after addition of arginine to the 4-day culture. Illumina sequencing and alignment to a custom transcriptome identified a large number of genes that yield clues on ways to manipulate the culture media to mimic the in vivo environment.

The role of cytoplasmic polyadenylation element sequence on mRNA abundance during porcine embryogenesis and parthenogenetic development.

Development of a porcine germinal vesicle oocyte (GVO) to a 4-cell stage embryo occurs during a transcriptionally silent period when the oocyte/embryo relies on maternally derived mRNA to encode proteins required for development. Regulation of translation and degradation of maternal mRNA is thought to be partially dependent on cytoplasmic polyadenylation elements (CPEs) within the 3' untranslated region of the mRNA. The goal of this study was to determine how CPE sites affect the abundance of mRNA during embryogenesis and parthenogenetic development, and how cordycepin, a 3'-deooxyadenosine (3'-dA) that inhibits poly-(A) tail formation, affects polyadenylation and transcript abundance. Expressed sequence tags (ESTs) from oocytes and 4-cell stage embryos were scanned for the presence of five consensus CPEs. Nineteen different transcripts containing one to three CPEs were selected, and transcript abundance was determined in GVO, metaphase II, 2-cell, and 4-cell stage embryos via real-time PCR while the length of the poly-(A) tail was determined by using a poly-(A) tail PCR (PAT) assays. Real-time PCR was performed on three biological and two technical replicates for each stage. There was no direct correlation between poly-(A) tail length, transcript abundance, and the CPE. In addition, the abundance of some messages was different if the embryo was the result of parthenogenetic activation. Cordycepin prevented polyadenylation of transcripts that normally undergo noticeable polyadenylation. Thus, CPEs may not be the only factors that regulate message stability, and parthenogenetic activation does not result in changes in transcript abundance that mimic in vitro fertilization.

Transcriptional profiling of day 12 porcine embryonic disc and trophectoderm samples using ultra-deep sequencing technologies.

cDNA derived from trophectoderm (TE) and embryonic disc (ED) of a single day 12 porcine embryo was subjected to next-generation sequencing using the Illumina platform. The short sequencing reads from triplicate sequencing runs were aligned to a custom database designed to represent the known porcine transcriptome. As expected, genes involved in epithelial cell function and steroid biosynthesis were more abundant in cells from the TE; genes involved in maintenance of pluripotency and chromatin remodeling were more highly expressed in cells from the ED. Quantitative real-time PCR was used to confirm the validity of the approach. We conclude that gene expression profiles of even extremely small samples (

A seed germination transcriptomic study contrasting two soybean genotypes that differ in terms of their tolerance to the deleterious impacts of elevated temperatures during seed fill.

OBJECTIVE: Soybean seed development is negatively impacted by elevated temperatures during seed fill, which can decrease seed quality and economic value. Prior germplasm screens identified an exotic landrace able to maintain ~ 95% seed germination under stress conditions that reduce germination dramatically (> 50%) for typical soybean seeds. Seed transcriptomic analysis was performed for two soybean lines (a heat-tolerant landrace and a typical high-yielding adapted line) for dry, mature seed, 6-h imbibed seed and germinated seed. Seeds were produced in two environments: a typical Midwestern field and a heat stressed field located in the Midsouth soybean production region. RESULTS: Transcriptomic analysis revealed 23-30K expressed genes in each seed tissue sample, and differentially expressed genes (DEGs) with ≥ twofold gene expression differences (at q-value < 0.05) comprised ~ 5-44% of expressed genes. Gene ontology (GO) enrichment analysis on DEGs revealed enrichment in heat-tolerant seeds for genes annotated for general and temperature-specific stress, as well as protein-refolding. DEGs were also clustered in modules using weighted co-expressed gene network analysis, which were examined for enrichment of GO biological process terms. Collectively, our results provide new and valuable insights into this unique form of genetic abiotic stress tolerance and to soybean seed physiological responses to elevated temperatures.

Spatial distribution of transcript changes in the maize primary root elongation zone at low water potential.

BACKGROUND: Previous work showed that the maize primary root adapts to low Psiw (-1.6 MPa) by maintaining longitudinal expansion in the apical 3 mm (region 1), whereas in the adjacent 4 mm (region 2) longitudinal expansion reaches a maximum in well-watered roots but is progressively inhibited at low Psiw. To identify mechanisms that determine these responses to low Psiw, transcript expression was profiled in these regions of water-stressed and well-watered roots. In addition, comparison between region 2 of water-stressed roots and the zone of growth deceleration in well-watered roots (region 3) distinguished stress-responsive genes in region 2 from those involved in cell maturation. RESULTS: Responses of gene expression to water stress in regions 1 and 2 were largely distinct. The largest functional categories of differentially expressed transcripts were reactive oxygen species and carbon metabolism in region 1, and membrane transport in region 2. Transcripts controlling sucrose hydrolysis distinguished well-watered and water-stressed states (invertase vs. sucrose synthase), and changes in expression of transcripts for starch synthesis indicated further alteration in carbon metabolism under water deficit. A role for inositols in the stress response was suggested, as was control of proline metabolism. Increased expression of transcripts for wall-loosening proteins in region 1, and for elements of ABA and ethylene signaling were also indicated in the response to water deficit. CONCLUSION: The analysis indicates that fundamentally different signaling and metabolic response mechanisms are involved in the response to water stress in different regions of the maize primary root elongation zone.

The influence of caging, bedding, and diet on the composition of the microbiota in different regions of the mouse gut.

Countless studies have identified differences between the gut microbiota of humans affected with myriad conditions and healthy individuals, and animal models are commonly used to determine whether those differences are causative or correlative. Recently, concerns have arisen regarding the reproducibility of animal models between institutions and across time. To determine the influence of three common husbandry-associated factors that vary between institutions, groups of weanling mice were placed in either static or ventilated microisolator caging, with either aspen or paperchip bedding, and with one of three commonly used rodent chows, in a fully crossed study design. After thirteen weeks, samples were collected from multiple regions of the gastrointestinal tract and characterized using culture-independent sequencing methods. Results demonstrated that seemingly benign husbandry factors can interact to induce profound changes in the composition of the microbiota present in certain regions of the gut, most notably the cecum, and that those changes are muted during colonic transit. These findings indicate that differences in factors such as caging and bedding can interact to modulate the gut microbiota that in turn may affect reproducibility of some animal models, and that cecal samples might be optimal when screening environmental effects on the gut microbiota.

Hypothalamic transcriptomic alterations in male and female California mice (Peromyscus californicus) developmentally exposed to bisphenol A or ethinyl estradiol.

Bisphenol A (BPA) is an endocrine-disrupting chemical (EDC) prevalent in many household items. Rodent models and human epidemiological studies have linked this chemical to neurobehavior impairments. In California mice, developmental exposure to BPA results in sociosexual disorders at adulthood, including communication and biparental care deficits, behaviors that are primarily regulated by the hypothalamus. Thus, we sought to examine the transcriptomic profile in this brain region of juvenile male and female California mice offspring exposed from periconception through lactation to BPA or ethinyl estradiol (EE, estrogen present in birth control pills and considered a positive estrogen control for BPA studies). Two weeks prior to breeding, P0 females were fed a control diet, or this diet supplemented with 50 mg BPA/kg feed weight or 0.1 ppb EE, and continued on the diets through lactation. At weaning, brains from male and female offspring were collected, hypothalamic RNA isolated, and RNA-seq analysis performed. Results indicate that BPA and EE groups clustered separately from controls with BPA and EE exposure leading to unique set of signature gene profiles. Kcnd3 was downregulated in the hypothalamus of BPA- and EE-exposed females, whereas Tbl2, Topors, Kif3a, and Phactr2 were upregulated in these groups. Comparison of transcripts differentially expressed in BPA and EE groups revealed significant enrichment of gene ontology terms associated with microtubule-based processes. Current results show that perinatal exposure to BPA or EE can result in several transcriptomic alterations, including those associated with microtubule functions, in the hypothalamus of California mice. It remains to be determined whether these genes mediate BPA-induced behavioral disruptions.

Gut Dysbiosis and Neurobehavioral Alterations in Rats Exposed to Silver Nanoparticles.

Due to their antimicrobial properties, silver nanoparticles (AgNPs) are being used in non-edible and edible consumer products. It is not clear though if exposure to these chemicals can exert toxic effects on the host and gut microbiome. Conflicting studies have been reported on whether AgNPs result in gut dysbiosis and other changes within the host. We sought to examine whether exposure of Sprague-Dawley male rats for two weeks to different shapes of AgNPs, cube (AgNC) and sphere (AgNS) affects gut microbiota, select behaviors, and induces histopathological changes in the gastrointestinal system and brain. In the elevated plus maze (EPM), AgNS-exposed rats showed greater number of entries into closed arms and center compared to controls and those exposed to AgNC. AgNS and AgNC treated groups had select reductions in gut microbiota relative to controls. Clostridium spp., Bacteroides uniformis, Christensenellaceae, and Coprococcus eutactus were decreased in AgNC exposed group, whereas, Oscillospira spp., Dehalobacterium spp., Peptococcaeceae, Corynebacterium spp., Aggregatibacter pneumotropica were reduced in AgNS exposed group. Bacterial reductions correlated with select behavioral changes measured in the EPM. No significant histopathological changes were evident in the gastrointestinal system or brain. Findings suggest short-term exposure to AgNS or AgNC can lead to behavioral and gut microbiome changes.

Bisphenol A (BPA) in the serum of pet dogs following short-term consumption of canned dog food and potential health consequences of exposure to BPA.

Bisphenol A (BPA) is a widely present endocrine disruptor chemical found in many household items. Moreover, this chemical can bioaccumulate in various terrestrial and aquatic sources; thereby ensuring continual exposure of animals and humans. For most species, including humans, diet is considered the primary route of exposure. However, there has been little investigation whether commercial-brands of dog foods contain BPA and potential health ramifications of BPA-dietary exposure in dogs. We sought to determine BPA content within dog food, whether short-term consumption of these diets increases serum concentrations of BPA, and potential health consequences, as assessed by potential hematological, serum chemistry, cortisol, DNA methylation, and gut microbiome changes, in dogs associated with short-term dietary exposure to BPA. Fourteen healthy privately-owned dogs were used in this study. Blood and fecal samples were collected prior to dogs being placed for two-weeks on one of two diets (with one considered to be BPA-free), and blood and fecal samples were collected again. Serum/plasma samples were analyzed for chemistry and hematology profiles, cortisol concentrations, 5-methylcytosine in lymphocytes, and total BPA concentrations. Fecal samples were used for microbiome assessments. Both diets contained BPA, and after two-weeks of being on either diet, dogs had a significant increase in circulating BPA concentrations (pre-samples=0.7±0.15ng/mL, post-samples=2.2±0.15ng/mL, p<0.0001). Elevated BPA concentrations positively correlated with increased plasma bicarbonate concentrations and associated with fecal microbiome alterations. Short-term feeding of canned dog food increased circulating BPA concentrations in dogs comparable to amounts detected in humans, and greater BPA concentrations were associated with serum chemistry and microbiome changes. Dogs, who share our internal and external environments with us, are likely excellent indicators of potential human health concerns to BPA and other environmental chemicals. These findings may also have relevance to aquatic and terrestrial wildlife.

Effects of exposure to bisphenol A and ethinyl estradiol on the gut microbiota of parents and their offspring in a rodent model.

Gut dysbiosis may result in various diseases, such as metabolic and neurobehavioral disorders. Exposure to endocrine disrupting chemicals (EDCs), including bisphenol A (BPA) and ethinyl estradiol (EE), especially during development, may also increase the risk for such disorders. An unexplored possibility is that EDC-exposure might alter the gut microbial composition. Gut flora and their products may thus be mediating factors for the disease-causing effects of these chemicals. To examine the effects of EDCs on the gut microbiome, female and male monogamous and biparental California mice (Peromyscus californicus) were exposed to BPA (50 mg/kg feed weight) or EE (0.1 ppb) or control diet from periconception through weaning. 16s rRNA sequencing was performed on bacterial DNA isolated from fecal samples, and analyses performed for P0 and F1 males and females. Both BPA and EE induced generational and sex-dependent gut microbiome changes. Many of the bacteria, e.g. Bacteroides, Mollicutes, Prevotellaceae, Erysipelotrichaceae, Akkermansia, Methanobrevibacter, Sutterella, whose proportions increase with exposure to BPA or EE in the P0 or F1 generation are associated with different disorders, such as inflammatory bowel disease (IBD), metabolic disorders, and colorectal cancer. However, the proportion of the beneficial bacterium, Bifidobacterium, was also elevated in fecal samples of BPA- and EE-exposed F1 females. Intestinal flora alterations were also linked to changes in various metabolic and other pathways. Thus, BPA and EE exposure may disrupt the normal gut flora, which may in turn result in systemic effects. Probiotic supplementation might be an effective means to mitigate disease-promoting effects of these chemicals.

Discovery of a Novel Seminal Fluid Microbiome and Influence of Estrogen Receptor Alpha Genetic Status.

Bacteria harbored in the male reproductive system may influence reproductive function and health of the male and result in developmental origins of adult health and disease (DOHaD) effects in his offspring. Such effects could be due to the seminal fluid, which is slightly basic and enriched with carbohydrates; thereby, creating an ideal habitat for microbes or a potential seminal fluid microbiome (SFM). Using wild-type (WT) and estrogen receptor-alpha (ESR1) knockout (KO) male mice, we describe a unique SFM whose inhabitants differ from gut microbes. The bacterial composition of the SFM is influenced according to whether mice have functional Esr1 genes. Propionibacterium acnes, causative agent of chronic prostatitis possibly culminating in prostate cancer, is reduced in SFM of ESR1 KO compared to WT mice (P ≤ 0.0007). In certain genetic backgrounds, WT mice show a greater incidence of prostate cancer than ESR1 KO, which may be due to increased abundance of P. acnes. Additionally, select gut microbiome residents in ESR1 KO males, such as Lachnospiraceae and Christensenellaceae, might contribute to previously identified phenotypes, especially obesity, in these mutant mice. Understanding how genetics and environmental factors influence the SFM may provide the next frontier in male reproductive disorders and possibly paternal-based DOHaD diseases.

Transcriptome Analysis of Pig In Vivo, In Vitro-Fertilized, and Nuclear Transfer Blastocyst-Stage Embryos Treated with Histone Deacetylase Inhibitors Postfusion and Activation Reveals Changes in the Lysosomal Pathway.

Genetically modified pigs are commonly created via somatic cell nuclear transfer (SCNT). Treatment of reconstructed embryos with histone deacetylase inhibitors (HDACi) immediately after activation improves cloning efficiency. The objective of this experiment was to evaluate the transcriptome of SCNT embryos treated with suberoylanilide hydroxamic acid (SAHA), 4-iodo-SAHA (ISAHA), or Scriptaid as compared to untreated SCNT, in vitro-fertilized (IVF), and in vivo (IVV) blastocyst-stage embryos. SAHA (10 µM) had the highest level of blastocyst development at 43.9%, and all treatments except 10 µM ISAHA had the same percentage of blastocyst development as Scriptaid (p<0.05). Two treatments, 1.0 µM ISAHA and 1.0 µM SAHA, had higher mean cell number than No HDACi treatment (p<0.021). Embryo transfers performed with 10 µM SAHA- and 1 µM ISAHA-treated embryos resulted in the birth of healthy piglets. GenBank accession numbers from up- and downregulated transcripts were loaded into the Database for Annotation, Visualization and Integrated Discovery to identify enriched biological themes. HDACi treatment yielded the highest enrichment for transcripts within the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway, lysosome. The mean intensity of LysoTracker was lower in IVV embryos compared to IVF and SCNT embryos (p<0.0001). SAHA and ISAHA can successfully be used to create healthy piglets from SCNT.

Identification of small RNAs associated with meiotic silencing by unpaired DNA.

In Neurospora crassa, unpaired genes are silenced by a mechanism called meiotic silencing by unpaired DNA (MSUD). Although some RNA interference proteins are necessary for this process, its requirement of small RNAs has yet to be formally established. Here we report the characterization of small RNAs targeting an unpaired region, using Illumina sequencing.

Computational analysis of RNA-seq.

Using High-Throughput DNA Sequencing (HTS) to examine gene expression is rapidly becoming a -viable choice and is typically referred to as RNA-seq. Often the depth and breadth of coverage of RNA-seq data can exceed what is achievable using microarrays. However, the strengths of RNA-seq are often its greatest weaknesses. Accurately and comprehensively mapping millions of relatively short reads to a reference genome sequence can require not only specialized software, but also more structured and automated procedures to manage, analyze, and visualize the data. Additionally, the computational hardware required to efficiently process and store the data can be a necessary and often-overlooked component of a research plan. We discuss several aspects of the computational analysis of RNA-seq, including file management and data quality control, analysis, and visualization. We provide a framework for a standard nomenclature -system that can facilitate automation and the ability to track data provenance. Finally, we provide a general workflow of the computational analysis of RNA-seq and a downloadable package of scripts to automate the processing.

Maintenance of shoot growth by endogenous ABA: genetic assessment of the involvement of ethylene suppression.

Previous work demonstrated that normal levels of endogenous abscisic acid (ABA) are required to maintain shoot growth in well-watered tomato plants independently of effects of hormone status on plant water balance. The results suggested that the impairment of shoot growth in ABA-deficient mutants is at least partly attributable to increased ethylene production. To assess the extent to which ABA maintains shoot growth by ethylene suppression, the growth of ABA-deficient (aba2-1) and ethylene-insensitive (etr1-1) single- and double-mutants of Arabidopsis was examined. To ensure that the results were independent of effects of hormone status on plant water balance, differential relative humidity regimes were used to achieve similar leaf water potentials in all genotypes and treatments. In aba2-1, shoot growth was substantially inhibited and ethylene evolution was doubled compared with the wild type, consistent with the results for tomato. In the aba2-1 etr1-1 double mutant, in which ABA was equally as deficient as in aba2-1 and shoot growth was shown to be insensitive to ethylene, shoot growth was substantially, although incompletely, restored relative to etr1-1. Treatment with ABA resulted in the complete recovery of shoot growth in aba2-1 relative to the wild type, and also significantly increased the growth of aba2-1 etr1-1 such that total leaf area and shoot fresh weight were not significantly lower than in etr1-1. In addition, ABA treatment of aba2-1 etr1-1 restored the wider leaf morphology phenotype exhibited by etr1-1. The results demonstrate that normal levels of endogenous ABA maintain shoot development, particularly leaf expansion, in well-watered Arabidopsis plants, partly by suppressing ethylene synthesis and partly by another mechanism that is independent of ethylene.