Binding of transcription factors to their target sequences is a primary step in the regulation of gene expression and largely determines gene regulatory networks. Chromatin immunoprecipitation (ChIP) is an indispensable tool used to investigate the binding of DNA-binding proteins (e.g., transcription factors) to their target sequences in vivo. ChIP assays require specific antibodies that recognize endogenous target transcription factors; however, in most cases, such specific antibodies are unavailable. To overcome this problem, many ChIP assays use transgenic plants that express epitope-tagged transcription factors and immunoprecipitate the protein with a tag-specific antibody. However, generating transgenic plants that stably express epitope-tagged proteins is difficult and time-consuming. Here, we present a rapid, efficient ChIP protocol using transient expression in Arabidopsis mesophyll protoplasts that can be completed in 4 days. We provide optimized experimental conditions, including the amount of transfected DNA and the number of protoplasts. We also show that the efficiency of our ChIP protocol using protoplasts is comparable to that obtained using transgenic Arabidopsis plants. We propose that our ChIP method can be used to analyze in vivo interactions between tissue-specific transcription factors and their target sequences, to test the effect of genotype on the binding of a transcription factor within a protein complex to its target sequences, and to measure temperature-dependent binding of a transcription factor to its target sequence. The rapid and simple nature of our ChIP assay using Arabidopsis mesophyll protoplasts facilitates the investigation of in vivo interactions between transcription factors and their target genes.

During pre-mRNA splicing, U2 small nuclear ribonucleoprotein auxiliary factor 65 (U2AF65) interacts with U2AF35 and splicing factor 1 (SF1), allowing for the recognition of the 3′-splice site by the ternary complex. The functional characterization of U2AF65 homologs has not been performed in Arabidopsis thaliana yet. Here, we show that normal plant development, including floral transition, and male gametophyte development, requires two Arabidopsis U2AF65 isoforms (AtU2AF65a and AtU2AF65b). Loss-of-function mutants of these two isoforms displayed opposite flowering phenotypes: atu2af65a mutants showed late flowering, whereas atu2af65b mutants were characterized by slightly early flowering, as compared to that in the wild-type (Col-0) plants. These abnormal flowering phenotypes were well-correlated with the expression patterns of the flowering time genes such as FLOWERING LOCUS C (FLC) and FLOWERING LOCUS T (FT). However, the two atu2af65 mutants did not display any morphological abnormalities or alterations in abiotic stress tests. Double mutation of the AtU2AF65a and AtU2AF65b genes resulted in non-viable seeds due to defective male gametophyte. In vitro pollen germination test revealed that mutations in both AtU2AF65a and AtU2AF65b genes significantly impaired pollen tube growth. Collectively, our findings suggest that two protein isoforms of AtU2AF65 are differentially involved in regulating flowering time and display a redundant role in pollen tube growth.

Cannabis (Cannabis sativa L.) is widely cultivated and studied for its psychoactive and medicinal properties. As the major cannabinoids are present in acidic forms in Cannabis plants, non-enzymatic processes, such as decarboxylation, are crucial for their conversion to neutral active cannabinoid forms. Herein, we detected the levels of cannabidivarin (CBDV), cannabidiol (CBD), cannabichromene (CBC), and Δ9-tetrahydrocannabinol (Δ9-THC) in the leaves and vegetative shoots of five commercial Cannabis cultivars using a combination of relatively simple extraction, decarboxylation, and high-performance liquid chromatography analyses. The CBDV, CBC, and Δ9-THC levels were 6.3–114.9, 34.4–187.2, and 57.6–407.4 μg/g, respectively, and the CBD levels were the highest, ranging between 1.2–8.9 μg/g in leaf and vegetative shoot tissues of Cannabis cultivars. Additionally, correlations were observed between cannabinoid accumulation and transcription levels of genes encoding key enzymes for cannabinoid biosynthesis, including CsCBGAS, CsCBDAS, CsCBCAS, and CsTHCAS. These data suggest that the high accumulation of cannabinoids, such as CBC, Δ9-THC, and CBD, might be derived from the transcriptional regulation of CsCBGAS and CsCBDAS in Cannabis plants.

Small changes in temperature affect plant ecological and physiological factors that impact agricultural production. Hence, understanding how temperature affects flowering is crucial for decreasing the effects of climate change on crop yields. Recent reports have shown that FLM-β, the major spliced isoform of FLOWERING LOCUS M (FLM)—a flowering time gene, contributes to temperature-responsive flowering in Arabidopsis thaliana. However, the molecular mechanism linking pre-mRNA processing and temperature-responsive flowering is not well understood. Genetic and molecular analyses identified the role of an Arabidopsis splicing factor SF1 homolog, AtSF1, in regulating temperature-responsive flowering. The loss-of-function AtSF1 mutant shows temperature insensitivity at different temperatures and very low levels of FLM-β transcript, but a significantly increased transcript level of the alternative splicing (AS) isoform, FLM-δ. An RNA immunoprecipitation (RIP) assay revealed that AtSF1 is responsible for ambient temperature-dependent AS of FLM pre-mRNA, resulting in the temperature-dependent production of functional FLM-β transcripts. Moreover, alterations in other splicing factors such as ABA HYPERSENSITIVE1/CBP80 (ABH1/CBP80) and STABILIZED1 (STA1) did not impact the FLM-β/FLM-δ ratio at different temperatures. Taken together, our data suggest that a temperature-dependent interaction between AtSF1 and FLM pre-mRNA controls flowering time in response to temperature fluctuations.