4-Phenylbutyric acid promotes plant regeneration as an auxin by being converted to phenylacetic acid via an IBR3-independent pathway.

4-Phenylbutyric acid (4PBA) is utilized as a drug to treat urea cycle disorders and is also being studied as a potential anticancer drug that acts via its histone deacetylase (HDAC) inhibitor activity. During a search to find small molecules that affect plant regeneration in Arabidopsis, we found that 4PBA treatment promotes this process by mimicking the effect of exogenous auxin. Specifically, plant tissue culture experiments revealed that a medium containing 4PBA enhances callus formation and subsequent shoot regeneration. Analyses with auxin-responsive or cytokinin-responsive marker lines demonstrated that 4PBA specifically enhances AUXIN RESPONSE FACTOR (ARF)-dependent auxin responses. Our western blot analyses showed that 4PBA treatment does not enhance histone acetylation in Arabidopsis, in contrast to butyric acid and trichostatin A, other chemicals often used as HDAC inhibitors, suggesting this mechanism of action does not explain the observed effect of 4PBA on regeneration. Finally, mass spectroscopic analysis and genetic approaches uncovered that 4PBA in Arabidopsis plants is converted to phenylacetic acid (PAA), a known natural auxin, in a manner independent of peroxisomal IBR3-related ß-oxidation. This study demonstrates that 4PBA application promotes regeneration in explants via its auxin activity and has potential applications to not only plant tissue culture engineering but also research on the plant ß-oxidation pathway.

An efficient DNA- and selectable-marker-free genome-editing system using zygotes in rice.

Technology involving the targeted mutagenesis of plants using programmable nucleases has been developing rapidly and has enormous potential in next-generation plant breeding. Notably, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) (CRISPR-Cas9) system has paved the way for the development of rapid and cost-effective procedures to create new mutant populations in plants1,2. Although genome-edited plants from multiple species have been produced successfully using a method in which a Cas9-guide RNA (gRNA) expression cassette and selectable marker are integrated into the genomic DNA by Agrobacterium tumefaciens-mediated transformation or particle bombardment3, CRISPR-Cas9 integration increases the chance of off-target modifications4, and foreign DNA sequences cause legislative concerns about genetically modified organisms5. Therefore, DNA-free genome editing has been developed, involving the delivery of preassembled Cas9-gRNA ribonucleoproteins (RNPs) into protoplasts derived from somatic tissues by polyethylene glycol-calcium (PEG-Ca2+)-mediated transfection in tobacco, Arabidopsis, lettuce, rice6, Petunia7, grapevine, apple8 and potato9, or into embryo cells by biolistic bombardment in maize10 and wheat11. However, the isolation and culture of protoplasts is not feasible in most plant species and the frequency of obtaining genome-edited plants through biolistic bombardment is relatively low. Here, we report a genome-editing system via direct delivery of Cas9-gRNA RNPs into plant zygotes. Cas9-gRNA RNPs were transfected into rice zygotes produced by in vitro fertilization of isolated gametes12 and the zygotes were cultured into mature plants in the absence of selection agents, resulting in the regeneration of rice plants with targeted mutations in around 14-64% of plants. This efficient plant-genome-editing system has enormous potential for the improvement of rice as well as other important crop species.

Cell dedifferentiation and organogenesis in vitro require more snRNA than does seedling development in Arabidopsis thaliana.

Small nuclear RNA (snRNA) is a class of non-coding RNAs that processes pre-mRNA and rRNA. Transcription of abundant snRNA species is regulated by the snRNA activating protein complex (SNAPc), which is conserved among multicellular organisms including plants. SRD2, a putative subunit of SNAPc in Arabidopsis thaliana, is essential for development, and the point mutation srd2-1 causes severe defects in hypocotyl dedifferentiation and de novo meristem formation. Based on phenotypic analysis of srd2-1 mutant plants, we previously proposed that snRNA content is a limiting factor in dedifferentiation in plant cells. Here, we performed functional complementation analysis of srd2-1 using transgenic srd2-1 Arabidopsis plants harboring SRD2 homologs from Populus trichocarpa (poplar), Nicotiana tabacum (tobacco), Oryza sativa (rice), the moss Physcomitrella patens, and Homo sapiens (human) under the control of the Arabidopsis SRD2 promoter. Only rice SRD2 suppressed the faulty tissue culture responses of srd2-1, and restore the snRNA levels; however, interestingly, all SRD2 homologs except poplar SRD2 rescued the srd2-1 defects in seedling development. These findings demonstrated that cell dedifferentiation and organogenesis induced during tissue culture require higher snRNA levels than does seedling development.