RESUMO
Understanding how flowers form is an important problem in plant biology, as human food supply depends on flower and seed production. Flower development also provides an excellent model for understanding how cell division, expansion and differentiation are coordinated during organogenesis. In the model plant Arabidopsis thaliana, floral organogenesis requires AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE 6 (AIL6)/PLETHORA 3 (PLT3), two members of the Arabidopsis AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) transcription factor family. Together, ANT and AIL6/PLT3 regulate aspects of floral organogenesis, including floral organ initiation, growth, identity specification and patterning. Previously, we used RNA-Seq to identify thousands of genes with disrupted expression in ant ail6 mutant flowers, indicating that ANT and AIL6/PLT3 influence a vast transcriptional network. The immediate downstream targets of ANT and AIL6/PLT3 in flowers are unknown, however. To identify direct targets of ANT regulation, we performed an RNA-Seq time-course experiment in which we induced ANT activity in transgenic plants bearing an ANT-glucocorticoid receptor fusion construct. In addition, we performed a ChIP-Seq experiment that identified ANT binding sites in developing flowers. These experiments identified 200 potential ANT target genes based on their proximity to ANT binding sites and differential expression in response to ANT. These 200 candidate target genes were involved in functions such as polarity specification, floral organ development, meristem development and auxin signaling. In addition, we identified several genes associated with lateral organ growth that may mediate the role of ANT in organ size control. These results reveal new features of the ANT transcriptional network by linking ANT to previously unknown regulatory targets.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Flores/crescimento & desenvolvimento , Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Fatores de Transcrição/fisiologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Flores/anatomia & histologia , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Reguladores de Crescimento de Plantas/fisiologia , Plantas Geneticamente Modificadas , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6) are two related transcription factors in Arabidopsis (Arabidopsis thaliana) that have partially overlapping roles in several aspects of flower development, including floral organ initiation, identity specification, growth, and patterning. To better understand the biological processes regulated by these two transcription factors, we performed RNA sequencing (RNA-Seq) on ant ail6 double mutants. We identified thousands of genes that are differentially expressed in the double mutant compared with the wild type. Analyses of these genes suggest that ANT and AIL6 regulate floral organ initiation and growth through modifications to the cell wall polysaccharide pectin. We found reduced levels of demethylesterified homogalacturonan and altered patterns of auxin accumulation in early stages of ant ail6 flower development. The RNA-Seq experiment also revealed cross-regulation of AIL gene expression at the transcriptional level. The presence of a number of overrepresented Gene Ontology terms related to plant defense in the set of genes differentially expressed in ant ail6 suggest that ANT and AIL6 also regulate plant defense pathways. Furthermore, we found that ant ail6 plants have elevated levels of two defense hormones: salicylic acid and jasmonic acid, and show increased resistance to the bacterial pathogen Pseudomonas syringae These results suggest that ANT and AIL6 regulate biological pathways that are critical for both development and defense.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Parede Celular/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/citologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Parede Celular/genética , Ciclopentanos/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Inflorescência/genética , Inflorescência/crescimento & desenvolvimento , Meristema/genética , Meristema/metabolismo , Mutação , Oxilipinas/metabolismo , Pectinas/genética , Pectinas/metabolismo , Doenças das Plantas/microbiologia , Pseudomonas syringae/patogenicidade , Ácido Salicílico/metabolismo , Análise de Sequência de RNA , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Cytokinin activates transcriptional cascades important for development and the responses to biotic and abiotic stresses. Most of what is known regarding cytokinin-regulated gene expression comes from studies of the dicotyledonous plant Arabidopsis thaliana. To expand the understanding of the cytokinin-regulated transcriptome, we employed RNA-Seq to analyze gene expression in response to cytokinin in roots and shoots of the monocotyledonous plant rice. RESULTS: We identified over 4,600 and approximately 2,400 genes differentially expressed in response to cytokinin in roots and shoots respectively. There were some similarities in the sets of cytokinin-regulated genes identified in rice and Arabidopsis, including an up-regulation of genes that act to reduce cytokinin function. Consistent with this, we found that the preferred DNA-binding motif of a rice type-B response regulator is similar to those from Arabidopsis. Analysis of the genes regulated by cytokinin in rice revealed a large number of transcription factors, receptor-like kinases, and genes involved in protein degradation, as well as genes involved in development and the response to biotic stress. Consistent with the over-representation of genes involved in biotic stress, there is a substantial overlap in the genes regulated by cytokinin and those differentially expressed in response to pathogen infection, suggesting that cytokinin plays an integral role in the transcriptional response to pathogens in rice, including the induction of a large number of WRKY transcription factors. CONCLUSIONS: These results begin to unravel the complex gene regulation after cytokinin perception in a crop of agricultural importance and provide insight into the processes and responses modulated by cytokinin in monocots.
Assuntos
Citocininas/farmacologia , Oryza/genética , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Transcriptoma/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Oryza/efeitos dos fármacos , Oryza/metabolismo , Proteínas de Plantas/metabolismoRESUMO
BACKGROUND: Soybean (Glycine max) has been bred for thousands of years to produce seeds rich in protein for human and animal consumption, making them an appealing bioreactor for producing valuable recombinant proteins at high levels. However, the effects of expressing recombinant protein at high levels on bean physiology are not well understood. To address this, we investigated whether gene expression within transgenic soybean seed tissue is altered when large amounts of recombinant proteins are being produced and stored exclusively in the seeds. We used RNA-Seq to survey gene expression in three transgenic soybean lines expressing recombinant protein at levels representing up to 1.61 % of total protein in seed tissues. The three lines included: ST77, expressing human thyroglobulin protein (hTG), ST111, expressing human myelin basic protein (hMBP), and 764, expressing a mutant, nontoxic form of a staphylococcal subunit vaccine protein (mSEB). All lines selected for analysis were homozygous and contained a single copy of the transgene. METHODS: Each transgenic soybean seed was screened for transgene presence and recombinant protein expression via PCR and western blotting. Whole seed mRNA was extracted and cDNA libraries constructed for Illumina sequencing. Following alignment to the soybean reference genome, differential gene expression analysis was conducted using edgeR and cufflinks. Functional analysis of differentially expressed genes was carried out using the gene ontology analysis tool AgriGO. RESULTS: The transcriptomes of nine seeds from each transgenic line were sequenced and compared with wild type seeds. Native soybean gene expression was significantly altered in line 764 (mSEB) with more than 3000 genes being upregulated or downregulated. ST77 (hTG) and ST111 (hMBP) had significantly less differences with 52 and 307 differentially expressed genes respectively. Gene ontology enrichment analysis found that the upregulated genes in the 764 line were annotated with functions related to endopeptidase inhibitors and protein synthesis, but suppressed expression of genes annotated to the nuclear pore and to protein transport. No significant gene ontology terms were detected in ST77, and only a few genes involved in photosynthesis and thylakoid functions were downregulated in ST111. Despite these differences, transgenic plants and seeds appeared phenotypically similar to non-transgenic controls. There was no correlation between recombinant protein expression level and the quantity of differentially expressed genes detected. CONCLUSIONS: Measurable unscripted gene expression changes were detected in the seed transcriptomes of all three transgenic soybean lines analyzed, with line 764 being substantially altered. Differences detected at the transcript level may be due to T-DNA insert locations, random mutations following transformation or direct effects of the recombinant protein itself, or a combination of these. The physiological consequences of such changes remain unknown.
Assuntos
Regulação da Expressão Gênica de Plantas/genética , Glycine max/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Sementes/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma/genética , Perfilação da Expressão Gênica , Plantas Geneticamente Modificadas/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA de Plantas/análise , RNA de Plantas/genética , Sementes/química , Sementes/genética , Glycine max/genéticaRESUMO
INTRODUCTION: Microscopic colitis is a relatively common cause of chronic diarrhea and may be linked to luminal factors. Given the essential role of the microbiome in human gut health, analysis of microbiome changes associated with microscopic colitis could provide insights into the development of the disease. METHODS: We enrolled patients who underwent colonoscopy for diarrhea. An experienced pathologist classified patients as having microscopic colitis (n = 52) or controls (n = 153). Research biopsies were taken from the ascending (ASC) and descending (DES) colon, and the microbiome was characterized with Illumina sequencing. We analyzed the associations between microscopic colitis and microbiome with a series of increasingly complex models adjusted for a range of demographic and health factors. RESULTS: We found that alpha diversity was significantly lower in cases with microscopic colitis compared with that in controls in the DES colon microbiome. In the DES colon, a series of models that adjusted for an increasing number of covariates found taxa significantly associated with microscopic colitis, including Proteobacteria that was enriched in cases and Collinsella that was enriched in controls. While the alpha diversity and taxa were not significantly associated with microscopic colitis in the ASC colon microbiome, the inference P values based on ASC and DES microbiomes were highly correlated. DISCUSSION: Our study demonstrates an altered microbiome in cases with microscopic colitis compared with that in controls. Because both the cases and controls experienced diarrhea, we have identified candidate taxa that could be mechanistically responsible for the development of microscopic colitis independent of changes to the microbial community caused by diarrhea.
Assuntos
Colite Microscópica , Microbiota , Humanos , Colite Microscópica/epidemiologia , Colite Microscópica/complicações , Colonoscopia/efeitos adversos , Diarreia/etiologia , Diarreia/patologia , Biópsia/efeitos adversosRESUMO
Previous studies have investigated the associations between the vaginal microbiome and preterm birth, with the aim of determining whether differences in community patterns meaningfully alter risk and could therefore be the target of intervention. We report on vaginal microbial analysis of a nested case-control subset of the Pregnancy, Infection, and Nutrition (PIN) Study, including 464 White women (375 term birth and 89 spontaneous preterm birth, sPTB) and 360 Black women (276 term birth and 84 sPTB). We found that the microbiome of Black women has higher alpha-diversity, higher abundance of Lactobacillus iners, and lower abundance of Lactobacillus crispatus. However, among women who douche, there were no significant differences in microbiome by race. The sPTB-associated microbiome exhibited a lower abundance of L. crispatus, while alpha diversity and L. iners were not significantly associated with sPTB. For each order of magnitude increase in the normalized relative abundance of L. crispatus, multivariable adjusted odds of sPTB decreased by approximately 20% (odds ratio, 0.81; 95% confidence interval, 0.70, 0.94). When we considered the impact of douching, associations between the microbiome and sPTB were limited to women who do not douche. We also observed strong intercorrelations between a range of maternal factors, including poverty, education, marital status, age, douching, and race, with microbiome effect sizes in the range of 1.8 to 5.2% in univariate models. Therefore, race may simply be a proxy for other socially driven factors that differentiate microbiome community structures. Future work will continue to refine reliable microbial biomarkers for preterm birth across diverse cohorts. IMPORTANCE Approximately 10% of all pregnancies in the United States end in preterm birth, and over 14% of pregnancies end in preterm birth among Black women. Knowledge on the associations between vaginal microbiome and preterm birth is important for understanding the potential cause and assessing risk of preterm birth. Our study is one of the largest studies performed to date to investigate the associations between vaginal microbiome and spontaneous preterm birth (sPTB), with stratified design for Black and White women. We found that the vaginal microbiome was different between Black and White women. The vaginal microbiome was associated with sPTB, and a lower abundance of L. crispatus increased the risk of sPTB independent of racial differences in microbial community structures. Furthermore, we also found that vaginal douching obscured the associations between vaginal microbiome, race, and preterm birth, suggesting that vaginal douching is an important factor to consider in future studies.
Assuntos
Lactobacillus crispatus , Microbiota , Nascimento Prematuro , Gravidez , Feminino , Recém-Nascido , Humanos , Estados Unidos , Nascimento Prematuro/etiologia , Vagina , População NegraRESUMO
The gut microbiota plays an important role in human health and disease. Stool, rectal swab and rectal mucosal tissue samples have been used in individual studies to survey the microbial community but the consequences of using these different sample types are not completely understood. In this study, we report differences in stool, rectal swab and rectal mucosal tissue microbial communities with shotgun metagenome sequencing of 1397 stool, swab and mucosal tissue samples from 240 participants. The taxonomic composition of stool and swab samples was distinct, but less different to each other than mucosal tissue samples. Functional profile differences between stool and swab samples are smaller, but mucosal tissue samples remained distinct from the other two types. When the taxonomic and functional profiles were used for inference in association with host phenotypes of age, sex, body mass index (BMI), antibiotics or non-steroidal anti-inflammatory drugs (NSAIDs) use, hypothesis testing using either stool or rectal swab gave broadly significantly correlated results, but inference performed on mucosal tissue samples gave results that were generally less consistent with either stool or swab. Our study represents an important resource for determination of how inference can change for taxa and pathways depending on the choice of where to sample within the human gut.
Assuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Fezes/microbiologia , Microbioma Gastrointestinal , Mucosa Intestinal/microbiologia , Microbiota , Reto/microbiologia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Antibacterianos , Anti-Inflamatórios não Esteroides , Índice de Massa Corporal , Feminino , Humanos , Masculino , Metagenômica/métodos , Pessoa de Meia-Idade , Fatores SexuaisRESUMO
Animal work indicates exposure to air pollutants may alter the composition of the gut microbiota. This study examined relationships between air pollutants and the gut microbiome in young adults residing in Southern California. Our results demonstrate significant associations between exposure to air pollutants and the composition of the gut microbiome using whole-genome sequencing. Higher exposure to 24-hour O3 was associated with lower Shannon diversity index, higher Bacteroides caecimuris, and multiple gene pathways, including L-ornithine de novo biosynthesis as well as pantothenate and coenzyme A biosynthesis I. Among other pollutants, higher NO2 exposure was associated with fewer taxa, including higher Firmicutes. The percent variation in gut bacterial composition that was explained by air pollution exposure was up to 11.2% for O3 concentrations, which is large compared to the effect size for many other covariates reported in healthy populations. This study provides the first evidence of significant associations between exposure to air pollutants and the compositional and functional profile of the human gut microbiome. These results identify O3 as an important pollutant that may alter the human gut microbiome.
Assuntos
Poluentes Atmosféricos , Poluição do Ar , Microbioma Gastrointestinal , Poluentes Atmosféricos/análise , Poluentes Atmosféricos/toxicidade , Poluição do Ar/efeitos adversos , Poluição do Ar/análise , Animais , Bacteroides , Humanos , Metagenoma , Adulto JovemRESUMO
Alternatively spliced genes produce multiple spliced isoforms, called transcript variants. In differential alternative splicing, transcript variant abundance differs across sample types. Differential alternative splicing is common in animal systems and influences cellular development in many processes, but its extent and significance is not as well known in plants. To investigate differential alternative splicing in plants, we examined RNA-Seq data from rice seedlings. The data included three biological replicates per sample type, approximately 30 million sequence alignments per replicate, and four sample types: roots and shoots treated with exogenous cytokinin delivered hydroponically or a mock treatment. Cytokinin treatment triggered expression changes in thousands of genes but had negligible effect on splicing patterns. However, many genes were differentially spliced between mock-treated roots and shoots, indicating that our methods were sufficiently sensitive to detect differential splicing between data sets. Quantitative fragment analysis of reverse transcriptase-PCR products made from newly prepared rice samples confirmed 9 of 10 differential splicing events between rice roots and shoots. Differential alternative splicing typically changed the relative abundance of splice variants that co-occurred in a data set. Analysis of a similar (but less deeply sequenced) RNA-Seq data set from Arabidopsis showed the same pattern. In both the Arabidopsis and rice RNA-Seq data sets, most genes annotated as alternatively spliced had small minor variant frequencies. Of splicing choices with abundant support for minor forms, most alternative splicing events were located within the protein-coding sequence and maintained the annotated reading frame. A tool for visualizing protein annotations in the context of genomic sequence (ProtAnnot) together with a genome browser (Integrated Genome Browser) were used to visualize and assess effects of differential splicing on gene function. In general, differentially spliced regions coincided with conserved protein domains, indicating that differential alternative splicing is likely to affect protein function between root and shoot tissue in rice.
RESUMO
Alternative splicing enables a single gene to produce multiple mRNA isoforms by varying splice site selection. In animals, alternative splicing of mRNA isoforms between cell types is widespread and supports cellular differentiation. In plants, at least 20% of multi-exon genes are alternatively spliced, but the extent and significance of tissue-specific splicing is less well understood, partly because it is difficult to isolate cells of a single type. Pollen is a useful model system to study tissue-specific splicing in higher plants because pollen grains contain only two cell types and can be collected in large amounts without damaging cells. Previously, we identified pollen-specific splicing patterns by comparing RNA-Seq data from Arabidopsis pollen and leaves. Here, we used semi-quantitative PCR to validate pollen-specific splicing patterns among genes where RNA-Seq data analysis indicated splicing was most different between pollen and leaves. PCR testing confirmed eight of nine alternative splicing patterns, and results from the ninth were inconclusive. In four genes, alternative transcriptional start sites coincided with alternative splicing. This study highlights the value of the low-cost PCR assay as a method of validating RNA-Seq results.