Katsutomo Sasaki

Diverse expression profiles of 21 rice peroxidase genes

Secretory class III plant peroxidases (POXs) catalyze the oxidation of various reductants, and are encoded by a large multigene family. In rice, 42 independent expressed sequence tags for POXs have been identified. By RNA gel blot analysis using specific probes, we show here that 21 rice POX genes are unique in their developmental, organ specific and external stimuli‐responsive expression. This would suggest that encoded POX isoenzymes are involved in a broad range of physiological processes in rice plants, individually.

Role of hydroperoxide lyase in white‐backed planthopper (Sogatella furcifera Horváth)‐induced resistance to bacterial blight in rice, Oryza sativa L.

A pre-infestation of the white-backed planthopper (WBPH), Sogatella furcifera Horváth, conferred resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) in rice (Oryza sativa L.) under both laboratory and field conditions. The infestation of another planthopper species, the brown planthopper (BPH) Nilaparvata lugens Stål, did not significantly reduce the incidence of bacterial blight symptoms. A large-scale screening using a rice DNA microarray and quantitative RT-PCR revealed that WBPH infestation caused the upregulation of more defence-related genes than did BPH infestation. Hydroperoxide lyase 2 (OsHPL2), an enzyme for producing C(6) volatiles, was upregulated by WBPH infestation, but not by BPH infestation. One C(6) volatile, (E)-2-hexenal, accumulated in rice after WBPH infestation, but not after BPH infestation. A direct application of (E)-2-hexenal to a liquid culture of Xoo inhibited the growth of the bacterium. Furthermore, a vapour treatment of rice plants with (E)-2-hexenal induced resistance to bacterial blight. OsHPL2-overexpressing transgenic rice plants exhibited increased resistance to bacterial blight. Based on these data, we conclude that OsHPL2 and its derived (E)-2-hexenal play some role in WBPH-induced resistance in rice.

Characterization of two rice peroxidase promoters that respond to blast fungus-infection

Peroxidase (POX) genes consist of a large gene family possibly contributing to self-defense, however constitutive and stress-induced expression patterns of individual gene were poorly understood in rice. We studied here the characteristic expression of two representative rice POX genes, R2329 and R2184, which are blast fungus-inducible (Sasaki et al. in Plant Cell Physiol 45:1442–1452, 2004). Basal GUS activity in R2329 promoter::GUS rice plants was 100-fold higher than that in R2184 promoter::GUS plants, and these levels reflected the transcript levels monitored by quantitative real-time RT-PCR. R2329 promoter was activated by blast fungus-infection and wounding, and R2184 promoter was activated by the fungal-infection and methyl jasmonate (MeJA)-treatment. By histochemical GUS staining analysis, constitutive R2329 and R2184 expression was commonly found in vascular bundle and exodermis in leaves and roots, while the precise expression profile was characteristic. In blast fungus inoculated R2329 promoter::GUS leaves, GUS staining was induced just around fungus-induced local lesions. Analysis of the 5′ deleted promoters suggests the presence of many kinds of stress-responsive elements in the regions between −1798 and −748 of R2329 promoter and between −1975 and −548 of R2184 promoter. These results revealed the stress-responsive characteristics of R2329 and R2184 promoters, and indicated the possible use for generation of useful transgenic plants.

Generation of Gene-Edited Chrysanthemum morifolium Using Multi-Copy Transgenes as Targets and Markers

The most widely used gene editing technology-the CRISPR/Cas9 system-employs a bacterial monomeric DNA endonuclease known as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) and single-guide RNA (sgRNA) that directs Cas9 to a complementary target DNA. However, introducing mutations into higher polyploid plant species, especially for species without genome information, has been difficult. Chrysanthemum morifolium (chrysanthemum) is one of the most important ornamental plants, but it is a hexaploid with a large genome; moreover, it lacks whole-genome information. These characteristics hinder genome editing in chrysanthemum. In the present study, we attempted to perform gene editing using the CRISPR/Cas9 system to introduce mutations into chrysanthemum. We constructed transgenic chrysanthemum plants expressing the yellowish-green fluorescent protein gene from Chiridius poppei (CpYGFP) and targeted CpYGFP for gene editing. We compared the activity of a Cauliflower mosaic virus (CaMV) 35S promoter and parsley ubiquitin promoter in chrysanthemum calli and chose the parsley ubiquitin promoter to drive Cas9. We selected two sgRNAs to target different positions in the CpYGFP gene and obtained transgenic calli containing mutated CpYGFP genes (CRISPR-CpYGFP-chrysanthemum). A DNA sequencing analysis and fluorescence observations indicated that cells containing the mutated CpYGFP gene grew independently of cells containing the original CpYGFP gene in one callus. We finally obtained the CRISPR-CpYGFP-chrysanthemum shoot containing a mutation in the CpYGFP sequence. This is the first report of gene editing using the CRISPR/Cas9 system in chrysanthemum and sheds light on chrysanthemum genome editing.

Mutation in Torenia fournieri Lind. UFO homolog confers loss of TfLFY interaction and results in a petal to sepal transformation

We identified a Torenia fournieri Lind. mutant (no. 252) that exhibited a sepaloid phenotype in which the second whorls were changed to sepal-like organs. This mutant had no stamens, and the floral organs consisted of sepals and carpels. Although the expression of a torenia class B MADS-box gene, GLOBOSA (TfGLO), was abolished in the 252 mutant, no mutation of TfGLO was found. Among torenia homologs such as APETALA1 (AP1), LEAFY (LFY), and UNUSUAL FLORAL ORGANS (UFO), which regulate expression of class B genes in Arabidopsis, only accumulation of the TfUFO transcript was diminished in the 252 mutant. Furthermore, a missense mutation was found in the coding region of the mutant TfUFO. Intact TfUFO complemented the mutant phenotype whereas mutated TfUFO did not; in addition, the transgenic phenotype of TfUFO-knockdown torenias coincided with the mutant phenotype. Yeast two-hybrid analysis revealed that the mutated TfUFO lost its ability to interact with TfLFY protein. In situ hybridization analysis indicated that the transcripts of TfUFO and TfLFY were partially accumulated in the same region. These results clearly demonstrate that the defect in TfUFO caused the sepaloid phenotype in the 252 mutant due to the loss of interaction with TfLFY.

Combination of Cyclamen persicum Mill. floral gene promoters and chimeric repressors for the modification of ornamental traits in Torenia fournieri Lind.

Although chimeric repressors such as the Arabidopsis TCP3 repressor are known to have significant effects on flower morphology and color, their cellular-level effects on flower petals are not understood. The promoter sequences of the genes expressed in the flowers of cyclamen, a representative potted flower grown during the winter season, are also unknown. Here, we isolated eight promoters from cyclamen genes that are reportedly expressed in the petals. These promoters were then fused to four chimeric repressors and introduced into the model flower torenia to screen for effective combinations of promoters and repressors for flower breeding. As expected, some of the constructs altered flower phenotypes upon transformation. We further analyzed the effects of chimeric repressors at the cellular level. We observed that complicated petal and leaf serrations were accompanied by excessive vascular branching. Dichromatism in purple anthocyanin was inferred to result in bluish flowers, and imbalanced cell proliferation appeared to result in epinastic flowers. Thus, the genetic constructs and phenotypic changes described in this report will benefit the future breeding and characterization of ornamental flowers.