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Transcriptomics analysis of developing wild type and val1 mutant Arabidopsis embryos

ABSTRACT: Developing Arabidopsis seeds accumulate oils and seed storage proteins synthesized by the pathways of primary metabolism. Seed development and metabolism are positively regulated by transcription factors belonging to the LAFL regulatory network. The VAL gene family encodes repressors of the seed maturation program in germinating seeds, although they are also expressed during seed maturation. VAL1 functions as a repressor of seed metabolism, as val1 mutant seeds accumulated elevated levels of storage proteins compared to the wild type. Two VAL1 splice variants were identified through RNA sequencing analysis: a full-length and a truncated form lacking the plant-homeodomain-like domain associated with epigenetic repression. None of the transcripts encoding the core LAFL network transcription factors were affected in val1 embryos. Instead, activation of VAL1 by FUSCA3 appears to result in repression of a subset of seed maturation genes downstream of core LAFL regulators as 39% of transcripts in the FUSCA3 regulon were de-repressed in the val1 mutant. The LEC1 and LEC2 regulons also responded but to a lesser extent. Additional 832 transcripts that were not LAFL targets were de-repressed in val1 mutant embryos. These transcripts are candidate targets of VAL1, acting through epigenetic and/or transcriptional repression.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE74692 | GEO | 2016/02/19

SECONDARY ACCESSION(S): PRJNA301162

REPOSITORIES: GEO

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Project description:Plant embryos can survive years in a desiccated, quiescent state within seeds. In many species, seeds are dormant and unable to germinate at maturity. They acquire the capacity to germinate through a period of dry storage called after-ripening (AR), a biological process that occurs at 5-15% moisture when most metabolic processes cease. Because stored transcripts will be among the first proteins translated upon water uptake, they likely impact germination potential. Transcriptome changes associated with the increased seed dormancy of the GA-insensitive sly1-2 mutant, and with dormancy loss through sly1-2 after-ripening or constitutive overexpression of the GA receptor (GID1b) were characterized in dry seeds. This experiment used the same seed batches as a previous experiment (E-MTAB-4782) to characterize transcriptional changes associated with the increased seed dormancy and dormancy loss in imbibing seeds. The SLY1 gene encodes the F-box subunit of an SCF E3 ubiquitin ligase needed for GA-triggered proteolysis of DELLA repressors of seed germination. In the sly1-2 mutant, GA-directed DELLA proteolysis cannot occur leading to DELLA protein accumulation and increased dormancy. sly1-2 mutant seeds are fully dormant at 2 weeks of dry storage (0% germination), but germinate well with very long after-ripening (51% germination after 19 months). sly1-2 seed germination can also be rescued by overexpression of the GA receptor, GA-INSENSITIVE DWARF1b (GID1b-OE), which resulted in 74% germination at 2 weeks of dry storage. In this experiment, we sampled dry seeds of wild-type Ler at 2 weeks of dry storage (non-dormant), dormant sly1-2 (2 weeks of dry storage; sly1-2(D)), long after-ripened sly1-2 (non-dormant, 19 months of dry storage; sly1-2(AR)), and sly1-2 GID1b-OE (non-dormant, 2 weeks of dry storage). This experimental design allowed comparison between these transcriptomes in dry seeds to determine if dry seed stored mRNA differences contribute to the dormancy phenotypes observed once seeds are imbibed. Seeds for Ler wt, sly1-2(D), and sly1-2 GID1b-OE were grown alongside each other under the same conditions and after-ripened for 2 weeks. Seeds from sly1-2(AR) were grown under the same conditions in advance of the other lines to allow for the long after-ripening requirement. RNA was extracted using a phenol-chloroform-based extraction from three biological replicates per treatment.

Project description:The FUSCA3 (FUS3) transcription factor is considered to be a master regulator of seed maturation because a wide range of seed maturation events are impaired in its defective mutant. To comprehensively identify genes under the control of FUS3, two types of microarray experiments were performed. First, transgenic plants in which FUS3 expression could be induced by the application of estrogen (ESTR) were used to identify the genes up-regulated in young seedlings of Arabidopsis in response to ectopic expression of FUS3. Second, transcriptomes were compared between fus3 mutant and wild type developing seeds. Combining the results of these experiments identified genes under relatively immediate and robust control of FUS3 during seed development. The analyses expanded the range of types of genes under the control of FUS3. The genes positively controlled by FUS3 are not confined to previously known seed maturation-related genes and include those for production of secondary metabolites such as glucosinolates, phenylpropanoids and falvonoids, and primary metabolism such as photosynthesis and fatty acid biosynthesis. Furthermore, several different patterns were identified in the manner of ectopic activation by FUS3 including the kinetics and abscisic acid (ABA) requirement of downstream genes depending on their natures of developmental regulation, suggesting mechanistic diversity of gene regulation by FUS3. This Series contains two different types of microarray experiments. First, seven-day-old seedlings of the ESTR-inducible FUS3 transgenic plants (ER-FUS3) were cultured for 3, 6, 12, 24, 36 or 48 h in a standard liquid medium or in that containing ESTR, ABA or both. RNA was prepared from each time point sample of each treatment and used to prepare Cy5-labeled cRNA, which was subsequently probed by DNA array with the reference Cy3-labeled cRNA prepared from the time zero untreated seedlings. Two biological replicates per treatment. Second, transcriptomes were compared between developing seeds of the wild-type (Col-0) and the fus3-3 mutant. This experiment contains two seed developmental stages, 8 days after flowering (DAF; bent cotyledon stage) and at 12 DAF (green cotyledon stage). Sample pairs in three biological replicates were analyzed by the two-color method. Dye-swapped hybridizations were performed in every replicate.

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Transcriptomics analysis of developing wild type and val1 mutant Arabidopsis embryos

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