Original Papers

Characterization and fine mapping of nonstop glumes 2 (nsg2) mutant in rice (Oryza sativa L.)

Yunfeng Li, Xiaoqin Zeng, Hui Zhuang, Huan Chen, Ting Zhang, Jun Zhang, Hao Zheng, Jun Tang, Honglei Wang, Suxian Ren, Yinghua Ling, Guanghua He . . . . . . . 125 [PDF] [Supplement]

In cereal crops, the grain number per panicle and the grain yield are greatly affected by the number of florets in a spikelet. In wild-type rice, a spikelet only produces one fertile floret and beneath the floret are a pair of sterile lemmas and a pair of rudimentary glumes. This study characterized a rice spikelet mutant nonstop glumes 2 (nsg2). In the nsg2 mutant, both the sterile lemmas and rudimentary glumes were elongated, and part of sterile lemma looked like a lemma in appearance, shape and size. Detailed histological analysis and qPCR analysis revealed that the sterile lemmas in the nsg2 mutant had homeotically transformed into lemma-like organs. This phenotype indicates that NSG2 is involved in the regulation of spikelet development and supports the long-held view that sterile lemmas were derived from the lemmas of the two lateral florets. This implies that the rice spikelet has the potential to be restored to the “three florets spikelet”, which may have existed in its ancestors. Genetic analysis reveals that the nsg2 trait is controlled by a single recessive gene. The NSG2 gene was finally mapped between markers R-20 and R-39 on chromosome 7 with a physical region of 180 kb. The two MYB family factors LOC_Os07g44030 and LOC_Os07g44090 might be involved in the development of the spikelet and floral organ, and they were considered as candidate genes of NSG2. These results provide a foundation for map-based cloning and function analysis of NSG2, as well as evidence to support “three-florets spikelet” breeding in rice.

Copper treatment of peach leaves causes lesion formation similar to the biotic stress response

Fumiyuki Goto, Yusuke Enomoto, Kazuhiro Shoji, Hiroaki Shimada, Toshihiro Yoshihara . . . . . . . 135 [PDF]

Peach (Prunus persica (L.) Batsch) leaves are sensitive to copper (Cu) exposure. The symptoms of Cu exposure are similar to those of bacterial spot disease; however, the mechanism underlying lesion formation caused by Cu exposure is not clear. Here, we investigated whether lesion formation caused by Cu exposure was related to the mechanism underlying plant resistance to microbial pathogens. When Cu was applied to the centre of a pinhole on peach leaves, a two-step process was observed. A pale green section in the shape of a doughnut, located far from a Cu treatment point, first appeared on a leaf treated with 2 mM CuSO4. Next, a yellow–white section gradually spread from the Cu treatment point to the pale green section. Finally, a gap was formed in the middle of the pale green section. The inner part of the pale green section contained 96% of the Cu applied, indicating that Cu is retained in the lesion area. Real-time PCR analysis of the expression of genes encoding pathogenesis-related proteins and enzymes involved in phytoalexin synthesis revealed that three genes (encoding chitinase, pathogenesis-related protein 4, and β-1,3-glucanase-3) of the eight tested were upregulated by Cu treatment. Furthermore, treatment with caspase-1 inhibitors reduced lesion formation. These results show that Cu treatment of peach leaves causes cell death similar to that occurring during the biotic stress response.

Comparative co-expression network analysis extracts the SlHSP70 gene affecting to shoot elongation of tomato

Nam Tuan Vu, Ken Kamiya, Atsushi Fukushima, Shuhei Hao, Wang Ning, Tohru Ariizumi, Hiroshi Ezura, Miyako Kusano . . . . . . . 143 [PDF] [Supplement]

Tomato is one of vegetables crops that has the highest value in the world. Thus, researchers are continually improving the agronomical traits of tomato fruits. Auxins and gibberellins regulate plant growth and development. Aux/indole-3-acetic acid 9 (SlIAA9) and the gene encoding the DELLA protein (SlDELLA) are well-known genes that regulate plant growth and development, including fruit set and enlargement by cell division and cell expansion. The absence of tomato SlIAA9 and SlDELLA results in abnormal shoot growth and leaf shape and giving rise to parthenocarpy. To investigate the key regulators that exist up- or downstream of SlIAA9 and SlDELLA signaling pathways for tomato growth and development, we performed gene co-expression network analysis by using publicly available microarray data to extract genes that are directly connected to the SlIAA9 and SlDELLA nodes, respectively. Consequently, we chose a gene in the group of heat-shock protein (HSP)70s that was connected with the SlIAA9 node and SlDELLA node in each co-expression network. To validate the extent of effect of SlHSP70-1 on tomato growth and development, overexpressing lines of the target gene were generated. We found that overexpression of the targeted SlHSP70-1 resulted in internode elongation, but the overexpressing lines did not show abnormal leaf shape, fruit set, or fruit size when compared with that of the wild type. Our study suggests that the targeted SlHSP70-1 is likely to function in shoot growth, like SlIAA9 and SlDELLA, but it does not contribute to parthenocarpy as well as fruit set. Our study also shows that only a single SlHSP70 out of 25 homologous genes could change the shoot length.

Association analysis of phenotypic and metabolomic changes in Arabidopsis accessions and their F1 hybrids affected by different photoperiod and sucrose supply

Quynh Thi Ngoc Le, Naoya Sugi, Jun Furukawa, Makoto Kobayashi, Kazuki Saito, Miyako Kusano, Hiroshi Shiba . . . . . . . 155 [PDF] [Supplement1] [Supplement2]

Photoperiod and sucrose (Suc) assimilation play important roles in the regulation of plant growth and development. However, it remains unclear how natural variation of plants could contribute to metabolic changes under various growth conditions. Here, we investigated the developmental and metabolomic responses of two natural accessions of Arabidopsis thaliana, Columbia (Col) and C24, and their reciprocal F1 hybrids grown under four carbon source regimens, i.e., two different photoperiods and the presence or absence of exogenous Suc supply. The effect of exogenous Suc clearly appeared in the growth of Col and the F1 hybrid but not in C24, whereas long-day conditions had significant positive effects on the growth of all lines. Comparative metabolite profiling of Col, C24, and the F1 hybrid revealed that changes in metabolite levels, particularly sugars, were highly dependent on genotype-specific responses rather than growth conditions. The presence of Suc led to over-accumulation of seven metabolites, including four sugars, a polyamine, and two amino acids in C24, whereas no such accumulation was observed in the profiles of Col and the F1 hybrid. Thus, the comparative metabolite profiling revealed that the two parental lines of the hybrid show a distinct difference in sugar metabolism.

Efficient genome engineering using Platinum TALEN in potato

Shuhei Yasumoto, Naoyuki Umemoto, Hyoung Jae Lee, Masaru Nakayasu, Satoru Sawai, Tetsushi Sakuma, Takashi Yamamoto, Masaharu Mizutani, Kazuki Saito, Toshiya Muranaka . . . . . . . 167 [PDF] [Supplement]

Potato (Solanum tuberosum) is one of the most important crops in the world. However, it is generally difficult to breed a new variety of potato crops because they are highly heterozygous tetraploid. Steroidal glycoalkaloids (SGAs) such as α-solanine and α-chaconine found in potato are antinutritional specialized metabolites. Because of their toxicity following intake, controlling the SGA levels in potato varieties is critical in breeding programs. Recently, genome-editing technologies using artificial site-specific nucleases such as TALEN and CRISPR-Cas9 have been developed and used in plant sciences. In the present study, we developed a highly active Platinum TALEN expression vector construction system, and applied to reduce the SGA contents in potato. Using Agrobacterium-mediated transformation, we obtained three independent transgenic potatoes harboring the TALEN expression cassette targeting SSR2 gene, which encodes a key enzyme for SGA biosynthesis. Sequencing analysis of the target sequence indicated that all the transformants could be SSR2-knockout mutants. Reduced SGA phenotype in the mutants was confirmed by metabolic analysis using LC-MS. In vitro grown SSR2-knockout mutants exhibited no differences in morphological phenotype or yields when compared with control plants, indicating that the genome editing of SGA biosynthetic genes such as SSR2 could be a suitable strategy for controlling the levels of toxic metabolites in potato. Our simple and powerful plant genome-editing system, developed in the present study, provides an important step for future study in plant science.

Production and characterization of intersectional hybrids between Tricyrtis sect. Brachycyrtis and sect. Hirtae via ovule culture

Toshiya Inamura, Manami Nakazawa, Mitsuyo Ishibe, Masahiro Otani, Masaru Nakano . . . . . . . 175 [PDF]

The liliaceous perennial plants, Tricyrtis spp., have recently become popular as ornamental plants for pot and garden uses. In order to broaden the variability in plant form, flower form and flower color of Tricyrtis spp., intersectional hybridization was examined between four T. formosana cultivars or T. hirta var. albescens (sect. Hirtae) and T. macranthopsis (sect. Brachycyrtis). After cross-pollination, ovary enlargement was observed only when T. macranthopsis was used as a pollen parent. Ovules with placental tissues were excised from enlarged ovaries and cultured on half-strength MS medium without plant growth regulators. From five cross-combinations, 31 ovule culture-derived plantlets were obtained and 20 of them were confirmed to be intersectional hybrids by flow cytometry and inter-simple sequence repeat analyses. Almost all hybrids grew well and produced flowers 1–2 years after transplantation to the greenhouse. Hybrids had semi-cascade-type shoots, which was intermediate between T. formosana cultivars and T. hirta var. albescens (erect-type shoots) and T. macranthopsis (cascade-type shoots). They produced flowers with novel forms and colors compared with the corresponding parents, and some were horticulturally attractive. The results obtained in the present study indicate the validity of intersectional hybridization via ovule culture for breeding of Tricyrtis spp.

Environmental biosafety assessment on transgenic Oncidium orchid modified by RNA interference of Phytoene Synthase genes

Swee-Suak Ko, Yao-Chung Liu, Mei-Chu Chung, Ming-Che Shih, Habibi Mohammadmehdi, Taichi Oguchi, Kazuo N. Watanabe, Kai-Wun Yeh . . . . . . . 181 [PDF] [Supplement]

Hybrid Oncidium orchids, such as Oncidium Gower Ramsey and Oncidium “Honey Angel,” are popular cut flowers in Japan and Taiwan. Due to pollen sterility, no new varieties have been created by conventional breeding methods. Recently, we employed RNA interference (RNAi) technology to suppress phytoene synthase and successfully modified floret hue from yellow to white (Liu et al. 2019). Transgenic white Oncidium orchids, Honey Snow MF-1, have been grown to test their genetic stability, and their environmental biosafety was assessed for approximately one year under government regulatory instructions from the Council of Agriculture, Taiwan. In the present study, pollen sterility was demonstrated by cytological observation of the microsporogenesis step, pollen morphology abortion, and failure of pollen germination. Assays on allelopathic effect on the other plants and the soil rhizospheric microbial flora-revealed that transgenic Oncidium orchids are potentially safe with regard to environmental biodiversity. Therefore, the general release permissions have been granted and an application for licensing for commercial production is under way.


Monitoring single-cell bioluminescence of Arabidopsis leaves to quantitatively evaluate the efficiency of a transiently introduced CRISPR/Cas9 system targeting the circadian clock gene ELF3

Yuki Kanesaka, Masaaki Okada, Shogo Ito, Tokitaka Oyama . . . . . . . 187 [PDF] [Supplement]

The rapid assessment of gene function is crucial in biological research. The CRISPR/Cas9 system is widely used as a tool for targeted gene editing in many organisms including plants. Previously, we established a transient gene expression system for investigating cellular circadian rhythms in duckweed. In this system, circadian reporters and clock gene effectors—such as overexpressors, RNA interference (RNAi), and CRISPR/Cas9—were introduced into duckweed cells using a particle bombardment method. In the present study, we applied the CRISPR/Cas9 system at a single cell level to Arabidopsis thaliana, a model organism in plant biology. To evaluate the mutation induction efficiency of the system, we monitored single-cell bioluminescence after application of the CRISPR/Cas9 system targeting the ELF3 gene, which is essential for robust circadian rhythmicity. We evaluated the mutation induction efficiency by determining the proportion of cells with impaired circadian rhythms. Three single guide RNAs (sgRNAs) were designed, and the proportion of arrhythmic cells following their use ranged from 32 to 91%. A comparison of the mutation induction efficiencies of diploid and tetraploid Arabidopsis suggested that endoreduplication had a slight effect on efficiency. Taken together, our results demonstrate that the transiently introduced CRISPR/Cas9 system is useful for rapidly assessing the physiological function of target genes in Arabidopsis cells.

Temporal restriction of salt inducibility in expression of salinity-stress related gene by the circadian clock in Solanum lycopersicum

Kelsey Coyne, Melissa Mullen Davis, Tsuyoshi Mizoguchi, Ryosuke Hayama . . . . . . . 195 [PDF]

Exposure to salinity causes plants to trigger transcriptional induction of a particular set of genes for initiating salinity-stress responses. Recent transcriptome analyses reveal that expression of a population of salinity-inducible genes also exhibits circadian rhythms. However, since the analyses were performed independently from those with salinity stress, it is unclear whether the observed circadian rhythms simply represent their basal expression levels independently from their induction by salinity, or these rhythms demonstrate the function of the circadian clock to actively limit the timing of occurrence of the salinity induction to particular times in the day. Here, by using tomato, we demonstrate that salt inducibility in expression of particular salinity-stress related genes is temporally controlled in the day. Occurrence of salinity induction in expression of SlSOS2 and P5CS, encoding a sodium/hydrogen antiporter and an enzyme for proline biosynthesis, is limited specifically to the morning, whereas that of SlDREB2, which encodes a transcription factor involved in tomato responses to several abiotic stresses such as salinity and drought, is restricted specifically to the evening. Our findings not only demonstrate potential importance in further investigating the basis and significance of circadian gated salinity stress responses under fluctuating day/night conditions, but also provide the potential to exploit an effective way for improving performance of salinity resistance in tomato.

A gain-of-function T-DNA insertion mutant of Marchantia polymorpha hyper-accumulates flavonoid riccionidin A

Noriko Hamashima, Xiaonan Xie, Mio Hikawa, Tomohiro Suzuki, Yutaka Kodama . . . . . . . 201 [PDF] [Supplement]

Marchantia polymorpha is a model liverwort for which many molecular biological techniques are now available. We previously developed the S-AgarTrap method for easy genetic transformation of M. polymorpha using spores. In this study, we report production of a T-DNA insertion mutant library (approx. 10,000 lines) for M. polymorpha using the S-AgarTrap method. We further isolate and characterize a gain-of-function mutant that hyper-accumulates the flavonoid riccionidin A. The present study demonstrates that the S-AgarTrap-mediated production of a T-DNA insertion mutant library is a powerful tool for molecular biology in M. polymorpha.

Effect of the CLE14 polypeptide on GLABRA2 homolog gene expression in rice and tomato roots

Naoto Hayashi, Natthanon Rongkavilit, Takuya Tetsumura, Shinichiro Sawa, Takuji Wada, Rumi Tominaga-Wada . . . . . . . 205 [PDF]

The CLAVATA3/ESR (CLE) plant polypeptides act as peptide hormones in various physiological and developmental aspects in a diverse array of land plants. One of the CLE family of genes, CLE14, is reported to induce root hair formation in Arabidopsis thaliana roots. Previously, we demonstrated that the application of synthetic CLE14 polypeptide treatment induced excess root hairs, and reduced the expression level of the non-hair cell fate determinant gene, GLABRA2 (GL2) in Arabidopsis roots. In this study, we investigated the function of synthetic CLE14 polypeptide in rice (Oryza sativa) and tomato (Solanum lycopersicum) roots. We measured the expression levels of the OsGL2 and SlGL2 genes, i.e., homologs of the Arabidopsis GL2 gene, in rice and tomato seedlings, respectively. Although CLE14 polypeptide treatment induced excess root hair formation in rice roots, substantial root hair induction was not observed in tomato roots. However, the CLE14 polypeptide treatment significantly inhibited the expression of the GL2 homolog genes of rice (OsGL2) and tomato (SlGL2). Our findings thus indicated that CLE14 can inhibit the GL2 gene expression in both rice and tomato plants, similar to the effect seen in Arabidopsis.

Anise-cultured cells abolish 2,4-dichlorophenoxyacetic acid in culture medium

Kumi Tanida, Hajime Shiota . . . . . . . 209 [PDF] [Supplement]

In anise (Pimpinella anisum, family Apiaceae), callus-like embryogenic cells (embryogenic callus) are induced by culturing hypocotyl explants in 2,4-dichlorophenoxyacetic acid (2,4-D)-containing medium, and somatic embryos are formed from embryogenic callus transferred into 2,4-D-free medium. Anise somatic embryos are also induced even if embryogenic callus is continually cultured in 2,4-D-containing medium without subculturing. In this study, we aimed to clarify the dynamics of 2,4-D during anise cell culture. After culturing anise callus in 2,4-D-containing medium, 2,4-D in the medium was analyzed by thin-layer chromatography. In the medium, 2,4-D was decreased during anise callus culture, and fully abolished after 5-day culture. On the other hand, no decrease in 2,4-D was observed in the other Apiaceae species (carrot, fennel, dill, parsley, and coriander). After 7-day culture of anise callus, the medium was collected following removal of the cultured cells and 2,4-D was added to the collected medium. After 10 days of incubation and shaking, 2,4-D was markedly decreased in the medium. However, when the collected medium was heat-treated at 100°C, 2,4-D was detected after 20 days of incubation. Therefore, anise callus has a specific 2,4-D degradation system, in which heat-inactivated secreted molecules may participate.