ABSTRACT: The aim of this study was to examine the contribution of ARF6 and ARF8 to flower gene expression. Flowers from arf6 arf8 plants undergo a developmental arrest at approximately stage 12, just prior to flower opening. Flowers from wild-type, ARF6/arf6 arf8/arf8, and arf6 arf8 plants were separated into stage 1-10 flowers, stage 11+12 flowers, and stage 13-14 flowers to define the developmental stages at which ARF6 and ARF8 are required for gene expression. Keywords: comparison of wild type and arf6 arf8 mutants
Project description:The aim of this study was to examine the contribution of ARF6 and ARF8 to flower gene expression. Flowers from arf6 arf8 plants undergo a developmental arrest at approximately stage 12, just prior to flower opening. Flowers from wild-type, arf6/arf6 ARF8/arf8, and arf6 arf8 plants were separated into stage 1-10 flowers, stage 11+12 flowers, and stage 13-14 flowers to define the developmental stages at which ARF6 and ARF8 are required for gene expression.
Project description:The aim of this study was to examine the roles of Auxin Response Factors (ARFs) in flower gene expression. Flowers from arf6 arf8 plants undergo a developmental arrest at approximately stage 12, just prior to flower opening. Wild-type, ARF6/arf6 arf8/arf8, and arf6 arf8 plants were treated with 10 uM indole-3-acetic acid for thirty minutes to identify genes that respond rapidly to auxin in an ARF6/ARF8-dependent manner. Keywords: auxin response; comparison of wild type and arf6 arf8 mutants
Project description:The aim of this study was to examine the roles of Auxin Response Factors (ARFs) in flower gene expression. Flowers from arf6 arf8 plants undergo a developmental arrest at approximately stage 12, just prior to flower opening. Wild-type, ARF6/arf6 arf8/arf8, and arf6 arf8 plants were treated with 10 uM indole-3-acetic acid for thirty minutes to identify genes that respond rapidly to auxin in an ARF6/ARF8-dependent manner. Experiment Overall Design: Wild-type, ARF6/arf6 arf8/arf8, and arf6 arf8 plants were sprayed with 10 mM indole-3-acetic acid in 1% methanol, 0.05% Tween-20. Thirty minutes after treatment, flowers (stage 1-14) were collected, frozen in liquid nitrogen, and used for RNA extraction.
Project description:Flower maturation consists of several events that contribute to reproductive success as flowers open, including petal expansion, stamen filament elongation, pollen release, nectary maturation, stigma growth, and gynoecium maturation to support pollen tube growth. The Arabidopsis transcription factors ARF6 (Auxin Response Factor 6) and ARF8 regulate all of these processes, in part by activating jasmonate biosynthesis. Jasmonates in turn activate genes encoding the transcription factors MYB21 and MYB24, which mediate a subset of the processes controlled by ARF6 and ARF8. This experiment was designed to characterize gene expression in flowers before and after they open, and to determine how arf6 arf8 and myb21 myb24 mutation combinations affect these gene expression patterns. Three biological replicates were prepared at each of two developmental stages, stage 12 (oldest closed buds) and stage 13 (youngest open flowers), for three genotypes (Wild type, arf6-2 arf8-3, and myb21-5 myb24-5). For the mutant genotypes, stage 13 flowers do not actually open, so corresponding flowers of equivalent age were chosen based on the position of open flowers in wild-type inflorescences.
Project description:Flower maturation consists of several events that contribute to reproductive success as flowers open, including petal expansion, stamen filament elongation, pollen release, nectary maturation, stigma growth, and gynoecium maturation to support pollen tube growth. The Arabidopsis transcription factors ARF6 (Auxin Response Factor 6) and ARF8 regulate all of these processes, in part by activating jasmonate biosynthesis. Jasmonates in turn activate genes encoding the transcription factors MYB21 and MYB24, which mediate a subset of the processes controlled by ARF6 and ARF8. This experiment was designed to characterize gene expression in flowers before and after they open, and to determine how arf6 arf8 and myb21 myb24 mutation combinations affect these gene expression patterns.
Project description:In flowering plants, anther dehiscence and pollen release are essential for sexual reproduction. Anthers dehisce after cell wall degradation weakens stomium cell junctions in each anther locule, and desiccation creates mechanical forces that open the locules. Either effect or both together may break stomium cell junctions. The microRNA miR167 negatively regulates ARF6 and ARF8, which encode Auxin Response transcription Factors. Arabidopsis mARF6 or mARF8 plants with mutated miR167 target sites have defective anther dehiscence and ovule development. Null mir167a mutations recapitulated mARF6 and mARF8 anther and ovule phenotypes, indicating that MIR167a is the main miR167 precursor gene that delimits ARF6 and ARF8 expression in these organs. Anthers of mir167a or mARF6/8 plants overexpressed genes encoding cell wall loosening functions associated with cell expansion, and grew too large starting at flower stage 11. Experimental desiccation enabled dehiscence of miR167-deficient anthers, indicating competence to dehisce. Conversely, high humidity conditions delayed anther dehiscence in wild-type flowers. These results support a model in which miR167-mediated anther growth arrest permits anther dehiscence. Without miR167 regulation, excess anther growth delays dehiscence by prolonging desiccation.
Project description:The phytohormone auxin plays a pivotal role in promoting fruit initiation and growth upon fertilization in flowering plants. Upregulation of auxin signaling by genetic mutations or exogenous auxin treatment can induce seedless fruit formation from unpollinated ovaries, termed parthenocarpy. Recent studies suggested that the class A AUXIN RESPONSE FACTOR6 (ARF6) and ARF8 play dual functions by first inhibiting fruit initiation when complexed with unidentified corepressor IAA protein(s) before pollination, and later promoting fruit growth after fertilization as ARF dimers. However, whether and how posttranslational modification(s) regulate ARF6- and ARF8-mediated fruit growth were unknown. In this study, we reveal that both ARF6 and ARF8 are O-fucosylated in their middle region (MR) by SPINDLY (SPY), a novel nucleocytoplasmic protein O-fucosyltransferase, which catalyzes the addition of a fucose moiety to specific Ser/Thr residues of target proteins. Epistasis, biochemical and transcriptome analyses indicated that ARF6 and ARF8 are downstream of SPY, but ARF8 plays a more predominant role in parthenocarpic fruit growth. Intriguingly, two ARF6/8 interacting proteins, the co-repressor IAA9 and MED8, a subunit of the coactivator Mediator complex, were also O-fucosylated by SPY. Biochemical assays demonstrated that SPY-mediated O-fucosylation of these proteins reduced ARF-MED8 interaction, which led to enhanced transcription repression activity of the ARF6/8-IAA9 complex but impaired transactivation activities of ARF6/8. Our study unveils the role of protein O-fucosylation by SPY in attenuating auxin-triggered fruit growth through modulation of activities of key transcription factors, co-repressor and the coactivator MED complex.
Project description:Papaya (Carica papaya) is a trioecious species, with female, male, and hermaphrodite plants. Because of sex segregation, selecting hermaphroditic plants is vital for orchard establishment due to their higher commercial value. In addition to the costly sexing step, environmental stresses can result in abnormal flower development. However, molecular mechanisms that regulate sex differentiation in papaya are still poorly known. Thus, this study aimed to identify proteins associated with sex development in female and hermaphrodite flowers of papaya through comparative proteomic analysis. Proteins from flower buds at the early and late developmental stages of three papaya genotypes (UENF-CALIMAN 01, JS12, and Sunrise Solo 72/12) were studied via proteomic analysis via the combination of the shotgun method and nanoESI-HDMSE technology. In buds at an early stage of development, 496 proteins exhibited significantly different abundances between sexes for the SS72/12 genotype, 139 for the JS12 genotype, and 165 for the UC-01 genotype. At the final stage of development, there were 181 for SS72/12, 113 for JS12, and 125 for UC-01. The large group of differentially accumulated proteins (DAPs) between the sexes was related to metabolism, as shown by the observation of only the proteins that exhibited the same pattern of accumulation in the three genotypes. Specifically, carbohydrate metabolism proteins were up-regulated in hermaphrodite flower buds early in development, while those linked to monosaccharide and amino acid metabolism increased during late development. Enrichment of sporopollenin and phenylpropanoid biosynthesis pathways characterizes hermaphrodite samples across developmental stages, with predicted protein interactions highlighting the crucial role of phenylpropanoids in sporopollenin biosynthesis for pollen wall formation. Most of the DAPs played key roles in pectin, cellulose, and lignin synthesis and were essential for cell wall formation and male flower structure development, notably in the pollen coat. These findings suggest that hermaphrodite flowers require more energy for development, likely due to complex pollen wall formation. Overall, these insights illuminate the molecular mechanisms of papaya floral development, revealing complex regulatory networks and energetic demands in the formation of male reproductive structures.
Project description:Gene expression profile of flower buds at stage 13, open flowers at stage 14 and siliques at stages 15/16, according to Smyth et al., 1990) of ABAP1 overexpressing (ABAP1OE) plants compared to wild type flower buds, open flowers and siliques (Col-0) using a a whole-genome oligonucleotide array (Operon) (platform accession number accession number GPL1077). ABAP1 is a negative regulator of the cell cycle that binds to transcription factors and represses their target gene expression, including pre-replication complex genes. All experiments were performed in triplicate.
Project description:Establishing the global proteomes of Petunia inflata flower protoplasts at S3 and S5 developmental stages to assess the presence of anthocyanin biosynthetic enzymes and determine the suitability of flowers in different developmental stages as material for transient expression of TurboID-tagged bait proteins for proximity labeling.