Selection of a histidine auxotrophic Marchantia polymorpha strain with an auxotrophic selective marker
A histidine auxotrophic Marchantia polymorpha strain was generated using CRISPR/Cas9-mediated genome editing, which can be used for biocontainment. An auxotrophic selective marker gene applicable to the auxotrophic strain was also developed to select transformants without antibiotic selection. The cover photograph shows a regenerated thallus transformed with Agrobacterium tumefaciens harboring a binary vector pMpGWBhis03-Citrine-NLS that encodes a fluorescent protein and the auxotrophic selective marker protein. Green and red colors indicate fluorescence from the fluorescent protein and chloroplasts, respectively. (See Fukushima and Kodama, pp. 345–354)
This photograph was taken by Tatsushi Fukushima at Utsunomiya University (Tochigi, Japan) using an MZ16F stereo fluorescence microscope (Leica Microsystems) and a DP73 digital camera (Olympus).
Establishment of an efficient transformation method of garden stock using a callus formation chemical inducer
Garden stock (Matthiola incana) is an important floricultural plant that blooms from winter to spring. This study successfully obtained stable transgenic plants from M. incana (Tanahara et al., pp. 273–280). The leaf sections were cocultivated with the Agrobacterium tumefaciens GV3101 for 5 days, and then obtained hygromycin-resistant calli in a selection medium supplemented with 12.5 µM fipexide (FPX). Then, the transgenic calli were transferred to shoot formation medium. This protocol achieved a 0.7% transformation frequency. FPX is considered a breakthrough for establishing the transformation protocol of M. incana.
Location: Faculty of Agriculture, Shizuoka University (836 Ohya, Suruga-ku, Shizuoka, Japan)
Photography equipment: CASIO EXILM EX-ZR850
Comparison of constitutive promoter activities and development of maize ubiquitin promoter- and Gateway-based binary vectors for rice
Transgenic rice seeds segregating proUBQ:GFP (left) and pro35S:GFP (right) at T1 generation. Strong GFP signals were observed in proUBQ:GFP, indicating the activity of this promoter is stable in rice. On the other hand, pro35S:GFP was frequently silenced in rice. Using this ubiquitin promoter from maize, a simple series of binary vectors for rice with gateway system was developed (See Tsuda et al., pp. 139–146). Seeds were illuminated using a blue LED and the photograph was taken by a digital camera (Olympus TG-6) with a GFP filter.
A GSK3-like kinase coordinates the balance between cell proliferation and differentiation in Marchantia polymorpha
Glycogen synthase kinase 3 (GSK3) family has important roles in regulating cell proliferation and differentiation during various developmental processes in angiosperms. However, in the plant lineages except angiosperms, the function of GSK3s especially in the cell differentiation processes remains unknown. The model bryophyte Marchantia polymorpha has a single GSK3-like kinase MpGSK. Overexpression of MpGSK reduced the size of the meristem region, which consists of the stem cell zone (SCZ) including apical cells, and the dividing and differentiating cell zone (DDCZ). Conversely, Mpgsk loss-of-function mutants exhibited an undifferentiated cell mass phenotype. These findings indicate that MpGSK is involved in the regulation of cell differentiation. The cover shows the meristem region of M. polymorpha (proMpE2F:XVE>>MpGSK-L-1 cultured without β-estradiol) visualized by EdU assay (red) and cell wall staining (white). (See Furuya et al., pp. 65–72)
Photographed by Tomoyuki Furuya, Kobe University (Kobe, Japan) using the FLUOVIEW FV1000 (Olympus).
Environmental risk assessment of transgenic tomato in a confined field trial
The commercial use of genetically modified (GM) crops requires prior assessment of potential risks to the environment which may arise during open field production or distribution. In this study, we conducted confined field trials to assess environmental risks for Japan-based commercial production of GM tomato plants accumulating miraculin, a taste-altering protein that causes sour taste to be perceived as sweet (Hiwasa-Tanase et al., pp. 421–431). The cover shows photos of the confined field (bottom left) used for environmental risk assessment, GM and non-GM tomatoes that were cultivated for assessment of plant morphology and growth characteristics (bottom right), GM and non-GM tomato plants on the first day of overwintering ability test (top left), and tomato seedlings at 20 days post cold treatment during the early stages of growth (top right). In the photo showing cold tolerance tests (top right), the left and middle trays contain cold-treated non-GM and GM seedlings respectively, while the right tray contains seedlings without cold treatment.
Photographed and designed by Kyoko Hiwasa-Tanase. These pictures were taken using a digital camera (Canon SX280 HS) at the T-PIRC Gene Research Center, University of Tsukuba (Ibaraki, Japan) in 2018–2019.
“Tsukuba system” as a transient protein expression system in plants
The Tsukuba system is a powerful tool for transient protein expression in plants. The expression level of GFP with this system in Nicotiana benthamiana reached a yield of approximately 4 mg/g fresh mass within 3 days. No other expression systems reached this amount in 3 days. Thus, using our expression system, recombinant proteins can be obtained more rapidly and in large quantities. Furthermore, the Tsukuba system also enhances expression levels of recombinant proteins in several crops, such as tomatoes, eggplants, hot peppers, melons, orchids, soybeans, common beans, and radishes. (Nosaki et al., pp. 297–304)
Photographed by Kenji Miura (Univ. Tsukuba) in Univ. Tsukuba, Japan, 2018. Blue LED light was provided for GFP emission, which was observed with an ultraviolet-absorbing filter, Fujifilm SC-52.
Construction of transgenic Ipomoea obscura that exhibits new reddish leaf and flower colors due to introduction of β-carotene ketolase and hydroxylase genes
Ipomoea obscura, small white morning glory, is an ornamental plant belonging to the family Convolvulaceae, and generates white petals including a pale-yellow colored star-shaped center (left photo in the top). Transgenic I. obscura plants were constructed by introducing the crtW and crtZ genes for producing a red carotenoid astaxanthin. The transgenic plants generated bronze (reddish green) leaves (right pictures in the middle and bottom) and novel petals that exhibited a color change from pale-yellow to pale-orange in the star-shaped center part (right photo in the top). (See Otani et al., pp. 219–226)
Photographed by Motoyasu Otani (Ishikawa Pref. Univ.) at growth chamber room in Ishikawa Pref. Univ., Ishikawa, Japan. (Canon EOS 40D)
HIGS method controlled the gene expression of a parasitic plant, Cuscuta campestris
Stem parasitic plant, Cuscuta campestris, develops an attachment structure called “holdfast” on the surface of its stem contacting to the host plant. Epidermal cells of holdfasts showed outgrowth, in which epidermal cell-patterning genes of C. campestris were hypothesized to be involved. To prove this hypothesis, Host-Induced Gene Silencing (HIGS) method was employed (Sultana et al., pp. 47-56). Aritificial miRNA targeting one of the C. campestris epidermal cell-patterning genes was produced in the first host, Nicotiana tabacum (left), loaded to parasitizing C. campestris (center), and then the effect of HIGS was evaluated by letting C. campestris parasitize to the second host, Arabidopsis thaliana (right). This approach successfully silenced the target gene of C. campestris, and repressed the outgrowth of the holdfast epidermal cells.
Photographed by Daiki Fujiwara (Osaka Pref. Univ.). (PENTAX Optio WG-1)
Strategies of mechanical optimization in plants
This special issue contains articles dealing with new approaches and aspects in plant biomechanics that are developed in the MEXT KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas “Plant-Structure Optimization Strategy”. (Top Left) Four-dimensional observation of Arabidopsis cotyledon pavement cells (Higaki and Mizuno, pp. 429–435). (Top Right) Secondary cell wall deposition in Arabidopsis cotyledon pavement cells induced by the VND7-VP16-GR system (Kunieda et al., pp. 465–469). (Middle Left) Static stress simulation image for a cantilever beam of a low density polyethylene rod (Watanabe et al., pp. 485–488). (Middle Right) Image of passage of sorghum root (yellow) through metal mesh (cyan) buried in Toyoura sand (Yoshida et al., pp. 481–484). (Bottom) Maximum intensity projection of time-lapse images during gravitropism of the pea sprout (Nakaso et al., pp. 475–480).
Propagation of Polygonatum macranthum using a new sterilization procedure
Polygonatum macranthum is an economically important species owing to its nutritive and medicinal properties. However, the natural propagation of P. macranthum is very difficult to achieve since the species require a long seed germination time, and characterizes an extremely slow rhizome propagation. We developed an efficient protocol for the propagation of P. macranthum from immature seeds with seed coat. The surface sterilization procedure used a new surface sterilization protocol that utilized a low concentration of hypochlorite. Subsequent multiplication of microrizomes was efficiently achieved. (See D. Lekamge et al., pp. 353–357)
Photographed by Shinya Maki (Nagaoka University of Technology, Niigata Agricultural Research Institute Mountain Agricultural Technology Center) in Nagaoka, Niigata, Japan, (Olympus TG-5)
Technology in tissue culture toward horizon of plant biotechnology
- 1.Stable transformants of Japanese cedar (Sugi)
- Japanese cedar (Sugi, Cryptomeria japonica D Don.) is the most important afforestation coniferous tree in Japan. Since coniferous trees normally have a long juvenile period, the improvement of Sugi by a transgenic approach is effective in terms of shortening the breeding period. We describe a protocol for Agrobacterium-mediated transformation of Sugi using embryogenic tissues as explants isolated from immature seeds (pp. 147-156). The upper image shows fluorescent image of somatic embryos regenerated from the embryogenic tissue introduced with green fluorescent protein gene. The lower image shows transgenic Sugi plants grown in a semi-closed greenhouse.
This picture is taken by Ken-ichi Konagaya using MZ FLIII stereo fluorescence microscope (Leica Microsystems) and D60 digital camera (Nikon) at Forestry and Forest Products Research Institute (Ibaraki, Japan).
- 2.Regeneration of tobacco plastid transformants
- This picture shows regeneration shoots of tobacco under spectinomycin selection after bombardment of the plastid transformation vector including the GFP expression cassette. Regenerated shoots with green fluorescence contain transformed plastids, and with red fluorescence still contain wild type plastids. In shoots obtained on first round selection, several mixture patterns of wild type and transformed cells are seen (pp. 223-232).
This picture was taken used a fluorescence microscope (Leica MZ 16FA, Leica Microsystems, Germany) with a GFP2 filter (480-nm excitation filter/510-nm barrier filter) at NIAS, Ibaraki, Japan.
- 3.The culture room used for the production of microtubers
- Potato (Solanum tuberosum L.) crops are vegetatively propagated using tubers and facing many risks caused by pathogens. The production of microtubers under sterile conditions is a good way to produce disease-free materials for crop production. Bioreactors have been used for this purpose, but bioreactors are expensive and difficult to handle. We have developed a system using plastic culture bags with forced aeration system for both liquid medium and gaseous phase to produce microtubers of potato. Each bag was 300 mm X 490 mm (total volume 8 l). We were able to place 12 culture bags in each shelf space with dimensions of 120 cm width, 60 cm depth, and 65 cm height, and produced about 250,000 microtubers per year in a 66 m2 tissue culture room using the culture bag system (pp. 233-238).
Photographed by Noboru Ohnishi and Kanji Mamiya. This picture was taken using a digital camera (RICOH Caplio R7) at Tochigi, Japan.
- 4.Genome editing in wheat
- Due to the complex allohexaploid genome nature, genome editing is a promising approach in wheat, where developing a mutant is more challenging compared with diploid crops, especially for genetically recessive traits. Using Agrobacterium-delivered CRISPR/Cas9 system, wheat lines with loss-of-function of Qsd1, which controls seed dormancy in barley, was efficiently developed. The edited triple-recessive mutant of TaQsd1 showed longer seed dormancy and is expected to contribute to reducing pre-harvest sprouting of wheat in rain-prone areas. This result was achieved based on the improvement of wheat tissue culture (pp. 177-184) and the development of a method for detection of mutation (pp. 247-251).
Photographed by Fumitaka Abe, This picture was taken using a digital camera (Pentax K-7) at Institute of Crop Science, NARO (Ibaraki Japan).
- 5.Plant regeneration and rooting in transgenic maize
- Maize (Zea mays L.) is one of the three most important crops, and a tremendous effort for production of transformed maize has been made since the 1990s. We reviewed progress in maize tissue culture technology, which is a core component of gene transfer and editing processes, and presented typical protocols for creation of transgenic plants for both particle bombardment and Agrobacterium-mediated methods (pp. 121-128). Pictures are of regenerated plants of maize inbred A188, which is a model variety for experiments of tissue culture and transformation. Immature embryos of A188 were inoculated by Agrobacterium tumefaciens that had a bar gene and a GUS gene in its T-DNA. Transgenic cells grown from the immature embryos were selected and plants were regenerated on the media that contained phosphinothricin.
Photographed by Yuji Ishida. Both pictures were taken using a digital camera (Nikon Coolpix P310) at JT Plant Innovation Center (Shizuoka, Japan).
- 6.Genome edited potatoes showing SGA-reduced phenotype.
- Potato is one of the most important crops in the world. Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites found in potato that cause food poisoning issues. Using Agrobacterium-mediated transformation, transgenic potato harboring the TALEN expression vector targeting SSR2 gene, which is encoding a key enzyme for SGA biosynthesis with reduced SGA levels have been generated in previous research (Sawai et al., Plant Cell, 2014). In this issue, we report the transgene-free genome editing in potato through transient TALEN expression by Agrobacterium infection (pp. 205-211).
Photographed by Shuhei Yasumoto and Jekson Robertlee (Osaka University) in Osaka, Japan (Nikon D90).
- 7.Regenerated plants of Tricyrtis ‘Shinonome’ at the flowering stage
- The family Liliaceae (Cronquist system) contains various important ornamental plants such as Agapanthus, Hemerocallis, Hosta, Lilium, Muscari, Tricyrtis and Tulipa species. We have been examining the establishment of plant regeneration and genetic transformation systems in liliaceous ornamental plants for their biotechnological breeding and elucidation of the molecular mechanisms determining ornamental traits. Among liliaceous ornamental plants used, Tricyrtis spp. has several advantages for regeneration and transformation studies: efficient regeneration from embryogenic calli, higher transformation efficiency by an Agrobacterium-mediated method, relatively small plant size, ease of cultivation, and taking only 1 year from in vitro regeneration to flowering. We are now investigating the molecular mechanisms for determining plant form, flower color and flower form by using Tricyrtis spp. as liliaceous model plants (pp. 129-140).
Photographed by Masaru Nakano, Experimental greenhouse at Faculty of Horticulture, Niigata University (Niigata, Japan). Fujifilm FinePix S9000
- 8.The concept of in planta particle bombardment (iPB)
- The L2 layer cells within the SAM potentially develop into pollen and embryo sac. If genome editing occurs in these cells, the mutation can be inherited to the next generation. Gold particles can accommodate not only DNA but also RNA and protein including Cas9 and TALENs (pp. 171-176).
- 9.A GFP-expressing transgenic fruit of C. moschata
- Cucumber (Cucumis sativus L.) and Cucurbita species (squashes, pumpkins, and gourds), belonging to the Cucurbitaceae family, are among the major vegetable crops in the world. These pictures show GFP-positive fruits of C. moschata appears green under blue light owing to GFP fluorescence (Nanasato and Tabei, pp. 141-146).
Photographed by Yoshihiko Nanasato using a digital camera (Sony DSC-HX5V) and an epifluorescence stereomicroscope (Leica MZ16FA) at NIAS (Ibaraki, Japan).
- 10.Somatic embryogenesis of tea plant (Camellia sinensis) for transformation.
- Various kinds of tea beverage are produced from leaves of tea plant (Camellia sinensis). SHIZUOKA prefecture is as famous as a major tea plantation in Japan. Green tea is traditionally the most popular in Japan and it is a part of traditional culture and cuisine. Somatic embryos of tea plant are induced from cotyledons of immature seeds, and secondary embryogenesis occurs by transferring the medium containing auxin to hormone-free medium. Somatic embryos can develop to plantlets in vitro (pp. 195-203).
Photographed by Kazumi Furukawa using a digital camera (Olympus tough TG-5). Tea plantation area is at Fuji, Shizuoka, Japan. Somatic embryos and plantlets are cultured at KOSEN (Shizuoka, Japan)
- 11.Precocious flowering of transgenic apple (JM2)
- Apple MdTFL1 (an orthologue of TERMINAL FLOWER 1 from Arabidopsis) is essential to reproductive/vegetative growth and flower initiation in apple. The suppression of MdTFL1 expression caused strong induction to reproductive growth and set flowers on their shoot apexes. The picture represents flowers of MdTFL1 silencing transgenic JM2 of five years old, which bloomed at 12 month after transformation with Agrobacterium method (pp. 163-170).
Photographed by Masato Wada using a digital camera (Olympus SP-350) in NIFTS, NARO, Iwate, Japan.
- 12.Agrobacterium-mediated genetic transformation of ‘Shine Muscat’ grape
- The cultivation area and consumption of grape cultivar ‘Shine Muscat’ (Vitis labruscana), which was released by NARO, have rapidly increased. Based on our efficient method for embryogenic callus induction, we established Agrobacterium-mediated genetic transformation of ‘Shine Muscat’ (pp. 185-194). The picture shows the regenerated grape plants in vitro.
Photographed by Ikuko Nakajima (Institute of Fruit Tree and Tea Science, NARO) in Tsukuba, Ibaraki, Japan, and (SONY Cyber-shot).
Regeneration and flowering of kenaf (Hibiscus cannabinus)
Kenaf (Hibiscus cannabinu) is an annual plant belonging to Malvaceae. Because of its property of fast-growing and being enriched in fibers, kenaf is regarded as an important fiber crop. Genetic transformation is essential to improve the property of kenaf in short term, however, the callus induction and shoot regeneration has not been achieved 100% efficiency yet, and a setting for the flowering was lacking for this plant. In the research, efficient regeneration of kenaf was achieved by cultivating cotyledon explants on a medium containing a combination of plant hormones. Moreover, cultivation at 22°C achieved flowering and subsequent maturation of fruits of the regenerated plants.These findings would accelerate improvement of kenaf properties as materials. (See Odahara et al., pp. 9–14)
Photographed by Yoko Horii (RIKEN) in Wako, Saitama, Japan, (Canon PowerShot S90)
Soybean transformation system and effect of SYNC1 gene overexpression
The application of useful genes from model plants to crops is an important step to verify its agricultural usefulness. SYNC1, an asparaginyl-tRNA synthetase gene, was previously found through the FOX gene-hunting system of Arabidopsis cDNA by using super-growing root (SR) culture of Lotus corniculatus. In this study, we identified that SYNC1 gene has effects on plant morphology and amino acid contents of seed in soybean (Pages 233–240). The cover shows the Agrobacterium transformation system with GFP expression and plant morphology of SYNC1 overexpressed transgenic plant in soybean.
Photographed and designed by Takahiro Gondo. These pictures are taken using digital camera (Canon EOS Kiss X3) and fluorescence microscope (Olympus SZX12) at University of Miyazaki (Miyazaki, Japan).
The Lemma-like Sterile Lemmas in Rice (Oryza sativa L.)
Spikelet is a typical inflorescence structure of gramineous plants. The spikelet of rice consists of a top floret, a pair of rudimentary glumes and a pair of sterile lemmas. The floret includes a lemma, a palea, two lodicules, six stamens, and one pistil. The rudimentary glumes are generally regarded as severely reduced bract organs, but the origin of sterile lemmas has been widely debated. Recent studies suggested that the sterile lemmas are the vestigial lemmas of two lateral florets. In the research, the sterile lemma of nsg2 transformed into lemma-like organ provides evidence to support the “three-florets spikelet” hypothesis, which indicate that it is possible to cultivated a “three-florets spikelet” rice. If succeed, the number of grains per panicle would be dramatically increased and affect rice yield. (See Li et al., pp. 125–134)
Photographed by Yun-Feng Li (Southwest University) in Beibei, Chongqing, China, in summer and autum, 2016 (Hitachi SU3500, NIKON SMZ1500, Nikon E600).
Monitoring Autophagy in Rice Tapetum
Autophagy is crucial for post meiotic anther development in rice; it is required for programmed cell death-mediated degradation of the tapetum and pollen maturation. In this issue, Hanamata et al. (pp. 99–105 and the Supplementary Movie S1) introduces an in vivo imaging technique to analyze the dynamics of autophagy in rice tapetum by expressing GFPATG8, a marker for autophagosomes under the control of tapetum-specific promoters.The cover shows 3-dimensional visualization of autophagosomes/autophagy-related structures in rice tapetum at stage 10 during pollen maturation.This monitoring system offers a powerful tool to analyze the regulation of autophagy in rice tapetum.
Photographed by Shigeru Hanamata and Jumpei Sawada using the LSM5 EXCITER confocal fluorescence microscope (Carl Zeiss, Germany) at Tokyo University of Science. 3D-image processing was carried out using the 3D viewer of ImageJ software.
In this issue, Kinoshita et al (Pages 7 to 11) introduces a simplified technique to extract fluorescence signals in freely moving plant leaves. The cover shows fluorescent signals emitted from yellow fluorescent protein and autofluorescence from chlorophyll during a time lapse experiment. This protocol enables seamless image capturing to quantify fluorescent signals using standard microscope software. Photo and design by Natsuko Kinoshita and Aki Sugita, University of Tsukuba, using Leica LasX.
Photograph and design by Natsuko Kinoshita and Aki Sugita, University of Tsukuba, using DFC7000T color CCD camera and LasX (both from Leica Microsystems).
Albino cla1 mutant with normal tapetum and pollen coat
Plastidial isoprenoid defective cla1 mutants grown in soil show albino and a seedling lethal phenotype. However, when the seedlings were grown in a large agripot with MS agar supplemented with sucrose, only some that grew to the flower stage with albino phenotype were obtained. In cla1-1 mutant, the elaioplast, which is the tapetum cell specific organelle derived from plastid, showed normal phenotype (upper-left photograph), though the chloroplast in cla1-1 has been reported to be the abnormal phenotype previously. The pollen coat derived from tapetum cell also showed normal phenotype in cla1-1 (upper-right photograph). In this issue, Kobayashi et al. report that the plastidial isoprenoid biosynthesis pathway is not critical for the pollen coat formation (pp. 381–385).
Photographed by Rumi Aoyama using the digital camera and the JEM-1200 EX transmission electron microscope (Jeol, Tokyo, Japan) at Japan Woman’s University (Tokyo).
Botryococcus braunii showa colonies producing hydrocarbon oil
The precursors of triterpene hydrocarbons are synthesized in 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The light-dependent enzyme 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is considered to function as a key-regulatory enzyme in this pathway. In this issue, a docking model analysis suggesting the possible association of HDR with a photosynthetic ferredoxin in this organism is discussed (pp. 297–301).
The cover picture presents an image of algal colonies stained with BODIPY493/503 and DAPI. Hydrocarbon oil (stained in green) is observed around cells (red autofluorescence) including nucleus and organelle nucleoids (stained in blue).
This picture was taken by Hidenobu Uchida using Olympus fluorescence microscope BX51, which was equipped with ×20 objective lens, according to a previous report (Kuroiwa et al. 2012, Cytologia 77: 289–299). Photographing was technically assisted by Haruko Kuroiwa from Japan Women’s University.
A gemmaling transformed by AgarTrap method
AgarTrap (agar-utilized transformation with pouring solutions) is an easy and efficient Agrobacterium-mediated transformation technique for liverwort Marchantia polymorpha. It is a simplified protocol that can be completed solely on a single solid medium by pouring appropriate solutions. Three main procedures of AgarTrap includes: (1) planting plant tissue for pre-culture, (2) pouring transformation buffer for co-culture with Agrobacterium tumefaciens, (3) pouring selection buffer for selection of transformants. To date, we developed AgarTrap methods by using three tissue types of M. polymorpha namely, sporelings, gemmalings, and mature thallus pieces. Gemmaling is the most effective among all the tested tissue types as its transformation efficiency of the BC3-38 strain is nearly 100%. Our review article includes the protocols of the AgarTrap methods (pp. 93-99).
The cover picture presents a BC3-38 gemmaling transformed by AgarTrap with A. tumefaciens harbouring a binary vector, pMpGWB103-Citrine, that encodes yellow fluorescent protein (Citrine) and hygromycin B phosphotransferase. The AgarTrap was performed by 2 days pre-culture, and 2 days co-culture with A. tumefaciens and sealed by Parafilm under darkness. This picture was taken at 3 days after pouring selection buffer. Red (chlorophyll) and yellow-green (Citrine fluorescence) indicate non-transformed cells and transformed cells, respectively.
This picture is taken by Shoko Tsuboyama using MZ16F stereo fluorescence microscope (Leica Microsystems) and DP73 digital camera (Olympus) at Utsunomiya University (Tochigi, Japan).
Light-induced anthocyanin biosynthesis in Arabidopsis thaliana leaves
Plants synthesize the red/purple pigment anthocyanin upon environmental stimuli such as excess light exposure. An intact leaf of Arabidopsis thaliana exhibits green color, whereas a detached leaf after incubation on water under light illumination for four days accumulates anthocyanin (left). In this issue, Koyama and Sato document that the leaf of the double mutant of class II ETHYLENE RESPONSE FACTOR (ERF) genes decreased the rate and extent of the production of anthocyanin after the incubation under light illumination (right). This mutant also decreased the production of anthocyanin in the strong light condition and, thus, the authors propose the roles of these class II ERFs in the photoinhibition-mediated anthocyanin biosynthesis.
Photographed by Tomotsugu Koyama at Suntory Foundation for Life Sciences in Kyoto, Japan (PowerShot A2300 digital camera, Canon).
Nonhost resistance to Pyricularia oryzae in Arabidopsis thaliana
For a plant disease to occur, pathogen, plant and environmental conditions must interact. The susceptibility of host plant may change both with developmental stage and time of day. Rice blast caused by Pyricularia oryzae (syn. Magnaporthe oryzae) is a devastating disease of rice. The mechanisms of resistance to P. oryzae have been extensively studied, and the rice-P. oryzae pathosystem has become a model system in plant-microbe interaction studies. However, the mechanisms of resistance to P. oryzae in nonhost remain poorly understood. Yamauchi et al. have used the Arabidopsis-P. oryzae pathosystem to study nonhost resistance (NHR). In this issue, authors (pp. 207–210) reported that NHR to P. oryzae varies with time of inoculation under diurnal conditions in old leaves and old leaves become more susceptible to P. oryzae penetration after inoculation at dusk in pen2-1 plants (lower). These results suggested that leaf age and time of inoculation are involved in NHR to P. oryzae in Arabidopsis.
Photographed by Yuri Yamauchi (Fukui Prefectural University, FPU) in FPU, Japan, 2017 (AxioCam MRc, ZEISS).
Increasing plant resistance against powdery mildew by overexpression of active form of ARA6
GATA4 is a transcription factor that belongs to the GATA family. In this issue Shin et al., reports that the expression of the chimeric repressor for GATA4 (35S:GATA4-SRDX) results in tolerance to nitrogen deficiency in Arabidopsis (pp. 151–158). Roots, which are directly exposed to various environmental conditions in the soil, are important organs that determine plant nutritional balance. Morphological changes in roots can adapt to nutritional availability and increase nutrition uptake efficiency. In particular, an increase in root hair density increases the root surface area and increases the absorption of nutrients and water from the soil. 35S:GATA4-SRDX plants change in root structure and suppress root growth and increase root hair density both under nitrogen sufficient (left upper) and deficient conditions (left lower).
Photograph Location: National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan
Photograph Equipment: A fluorescence stereomicroscope (MZ16FA, Leica) equipped with a CCD camera (DFC300FX, Leica)
Increasing plant resistance against powdery mildew by overexpression of active form of ARA6
Powdery mildew is a popular disease caused by ascomycete fungi. As this disease affects near 10,000 plants and causes significant economic loss every year, a development of new tools to combat this disease is of great interest. In this issue, Inada et al. reported that overexpression of the active form of ARA6, a plant-specific member of RAB5 GTPase, impairs powdery mildew proliferation. Powdery mildew fungi form specialized infection hyphae called the haustorium in the apoplast of host epidermal cells. The haustorium is surrounded by the host-derived membrane called the extrahaustorial membrane. Previously, authors reported that host RAB5 GTPases, both plant-specific ARA6 and conventional ARA7, localized to the extrahaustorial membrane. In this issue, authors found that an overexpression of active form of ARA6, but not that of ARA7, suppresses powdery mildew proliferation. These results indicate a specific role of ARA6 in the plant-powdery mildew interaction. In addition, manipulation of the ARA6 activity was suggested to be a possible methodology to overcome this disease.
Photographed by Noriko Inada (Nara Institute of Science and Technology, NAIST) in NAIST, Japan, 2012 (Panasonic DMC-FX30).
Secretory cavities of Japanese pepper
Japanese pepper (Zanthoxylum piperitum), “Sansho” in Japanese, is a deciduous shrub, belonging to the family Rutaceae. In Japan, fresh young leaves are sometimes used as garnish and in sauces after mixed with miso (soybean paste) for some Japanese dishes because of its characteristic aroma. The fruits exhibit pungent taste together with strong aroma, and are also utilized as one of the seven components of Japanese blended spices called “Shichimi”. Oil secretory cavities are found at all sinuses of leaves, as shown in this photograph, as well as the surface of fruits and contain high amount of volatile compounds. The grade of Japanese pepper is sometimes determined according to the flavor of high quality of volatile mono- and sesqui-terpenes. In this issue, Fujita et al. have reported the formation of volatile terpenes and their biosynthetic enzymes in secretory cavities of Japanese pepper.
Photographed by Mitsuhiro Aida (Nara Institute of Science and Technology, NAIST) in NAIST, Japan, 2015 (ECLIPSE Ni-U microscope equipped with DIC optics, Nikon; VB-7010 cooled CCD camera, Keyence).
Homeotic floral organ changes of transgenic apple
Apple cultivars with homeotic changes, petals to sepals and stamens to carpels, set parthenocarpic fruits. The floral organ changes were caused by destruction of apple MdPISTILLATA (MdPI) gene, which is an orthologue of Arabidopsis class B gene PISTILLATA. The MdPI gene is able to recover the Arabidopsis pi mutant phenotype. The expression of MdPI localized at petals and stamens of apple flowers. The connection between the parthenocarpy and class B mutation was obscure, then transgenic apples suppressed MdPI function was produced. The cover picture represents a flower of antisense MdPI transgenic apple, which was analyzed about parthenocarpy in this article (pp. 395–401).
Location: Division of apple Research (Morioka), NIFTS, NARO
Camera: Olympus SP-350
Adaptive environmental responses of plants in synecoculture fields
Synecoculture is based on high-density mixed polyculture, which harbors a wide variety of edible plants and rich interactions between environment, flora and fauna: Leaf color change of no-fertilizer cabbage during winter. Tomato in Tokyo can extend its growth until december crawling over chestnut. Cruciferous vegetables (Brassicaceae) easily intercross and produce hybrid varieties with higher yield. Honey bees and butterflies are not only pollinators but also important enhancers of micronutrients profile of crops. Growth in various association of plants augments productivity of community, and enhanced ecological interactions stimulate the expression of health beneficial components such as secondary metabolites. The cooccurrence networks of relevant species from field observation are superimposed.
Location: Synecoculture fields in Ise (Mie), Oiso (Kanagawa) and Todoroki (Tokyo)
Camera: Panasonic DMC-LX5, Olympus TG-3
Cuticular nanoridges formation in floral organs
Floral organs are decorated by cuticle with nano-order structure called “nanoridges”, although leaves are covered by plane cuticle. Regulation of cuticle structure and thickness is important for stress tolerance to biotic and abiotic stress, plant growth and petal structural color. In this issue, Oshima and Mitsuda report that the expression of MIXTA-like MYB transcription factors using the promoters of cuticle biosynthesis genes successfully increased accumulation of cuticle in leaves and petals without growth defects in Arabidopsis thaliana (pp. 161–168). Increased nanoridges and ectopic outgrowth of epidermal cell of petal abaxial side, stamen and sepal (from top to bottom rows) in transgenic lines (middle and right columns) compared to those in wild type (left column) affected petal texture.
Photographed by Yoshimi Oshima (National Institute of Advanced Industrial Science and Technology, AIST) in AIST, Japan, 2015 (SEM, KEYENCE VE8800; stereomicroscopic camera, ZEISS AxioCam MRc5)
A transcriptomic analysis among max2, hxk1 and wild type seedlings in response to glucose
Strigolactones (SL) are plant hormone that play vital roles in plant architecture. Sugars have been known as important signal molecules to regulate plant growth and development. In this study (pp. 87–97), we found that SL mutants show hyposensitivity to sugar-induced early seedling development arrest. We performed a comparative transcriptomic analysis of max2, hxk1 and wild type (WT) seedlings grown with 2% glucose (G) or mannitol (C) as control. As shown in the figure, several groups of genes show different expression patterns among max2, hxk1 and WT in responses to glucose. These results indicate that SL signal shows crosstalk with sugar signal in HXK1 independent manner.
The transcriptomic data were obtained by short-read sequencing technology (Illumina) combined with a tag-based digital gene expression (DGE) system and analyzed by Pheatmap in R language program.
Agrobacterium-mediated transformation of switchgrass
Switchgrass (Panicum virgatum L.) is an important bioenergy crop. A reliable and efficient transformation method is required to assist with molecular breeding of this crop. After cocultivation with Agrobacterium under optimized conditions, caryopsis-derived Type I (embryogenic and compact) calli formed herbicide-resistant calli (1st row), which showed GFP-fluorescence (2nd row), and then regenerated transgenic shoots (3rd row). The present method was successfully applied to different genotypes, including a major lowland cultivar ‘Alamo’ (left), another lowland cultivar ‘Kanlow’ (middle) and a recalcitrant upland cultivar ‘Trailblazer’ (right) (pp. 19–26).
Photographed by Yoichi Ogawa (Honda Research Institute Japan) at Kazusa Unit (Kisarazu, Japan).
PEPCK promotes seedling growth in tomato
Phosphoenolpyruvate carboxykinase (PEPCK) is known as a key regulatory enzyme in the gluconeogenesis pathway in plants. However, its physiological function in whole plant development is not fully understood in plants with fresh berry-type fruit, including tomato.
In this study, in order to obtain more detailed information on the physiological function of PEPCK and the role of gluconeogenesis during development of tomato plant, we generated transgenic tomato plants overexpressing SlPEPCK gene and performed a detailed characterization of those plants. The results indicate multiple roles of PEPCK in the development of the tomato plant, especially in seedling growth, and the effect of the gluconeogenesis pathway on regulation of the sugar and organic acid contents in the fruit.
Photographed by Yong-Xing Huang (University of Tsukuba) in Tsukuba, Japan, 15 and 19 June, 2015.
‘Pinot Noir’ grape berries before and after ripening
‘Pinot Noir’ is a famous cultivar of grape (Vitis vinifera L.) for vine production. Grape accumulates important secondary metabolites, such as anthocyanin, tannin and resveratrol, in berry skin. The turning point of ripening of grape berry is called “veraison.” After veraison, properties of berry, including sugar and acid contents, color and firmness, change dramatically. In this issue, Suzuki et al. report the comparison of metabolomes of berry skin of ‘Pinot Noir’ before and aer veraison (pp.267-272).
Photographed by Katsuhiro Shiratake, Mami Suzuki and Masaki Ochiai (Nagoya University) in Azumino, Nagano, Japan, 23 July and 16 September 2010 (Canon EOS Kiss Digital N, Tamron SP 17–50 mm F/2.8 Di II) and 4 Augusta 2011 (Olympus E-620, Olympus Zuiko Digital ED 50 mm F2.0 Macro).
Color Modified Flowers by Anthocyanin Methyltransferase
The structures of B-ring of anthocyanins are known to change flower color greatly. The effect of 3′- and 5′-methoxylation of anthocyanins in flower color was studied by expressing S-adenosylmethionine: anthocyanin 3′,5′-O-methyltranferase (A3’5’OMT) gene from torenia in plants (pp. 109–117). he transgenic Nierembergia petals produced 3′- and 5′-methoxylated anthocyanins based on petunidin and malvidin in petals (the upper picture, the host flower is shown at the top). The paler color of the transgenics were due to the decrease of anthocyanin amount. Expression of a flavonoid 3′,5′-hydroxylase gene in a rose cultivar (the lower picture, left) resulted in delphinidin production and magenta color (center). Additional expression of the A3’5’OMT gene yielded more brilliant and attractive color (right) derived from malvidin accumulation than delphinidin alone. hese results indicate that the A3’5’OMT gene is a useful molecular tool to increase the varieties of flower color.
Nierembergia and roses were photographed by Noriko Nakamura (Suntory Global Innovation Center Ltd.) and Yukihisa Katsumoto (Suntory Global Innovation Center Ltd.), respectively, at Suntory Research Center (Osaka).
Effectiveness of CO2 Gas Feeding
Carbon fixation in the leaves and translocation to the fruits are the most fundamental functions for fruit productivity. In this issue, Yamazaki et al. report immediate and different responses of carbon fixation and translocation to enriched external CO2 concentrations in the same individuals of tomato (Solanum lycopersicum L. cv. ‘Micro-Tom’), by visualizing the movement of pulse-fed 11CO2 tracer in the test plants using a positron-emitting tracer imaging system (PETIS) (pp 31–37). Top: 400 ppm CO2. Middle: 1,500 ppm CO2. Bottom: 3,000 ppm CO2.
Bloom of Empress Tree
Paulownia tomentosa (empress tree), or Kiri in Japanese, is a fast-growing tree native to China in the family Paulowniaceae, producing tubular light-purple flowers. In Japan, the plant is adapted as a floral motif for a traditional emblem Kiri mon, or Paulownia crest, of which use by a Sengoku-era warlord Hideyoshi Toyotomi and also today by government (cabinet office) of Japan is known well. Including P. tomentosa, a number of species in the genera Paulownia, Sesamum (Pedaliaceae), and Phryma (Phrymaceae), all belonging to the order Lamiales, are known to produce specialized lignans of furofran type with methylenedioxy bridge, represented by sesamin from sesame seeds. In this issue, Noguchi et al. report that the formation of the methylenedioxy bridge is catalyzed by a conserved cytochrome P450 enzyme in the lignan-producing species (pp 493–504).
Photographed by Eiichiro Ono (Suntory Global Innovation Center Ltd.) in Hanamaki, Iwate, Japan, in spring, 2014 (SONY RX-100).
Christmas illumination in early winter
In Japan, Christmas illumination is common during winter and a variety of natural and artificial plants are decorated with colorful lights. In this issue, Sasaki et al. in National Agriculture and Food Research Organization (NARO) Institute of Floricultural Science (NIFS) have reported a generation of fluorescent Torenia flowers by combination of the highperformance fluorescent protein and the latest protein expression technologies (pp. 309–318). Their work may lead to a use of the fluorescent plants for Christmas illumination in the near future.
Photographed by Tsuyoshi Mizoguchi (International Christian University, ICU) in ICU, Japan, 5 Dec, 2014 (Ricoh GXR, Leica Super Angulon f4/21 mm).
Fruits of Citrus unshiu in mid autumn
In Japan, fruits of Citrus unshiu usually grow larger during summer and the color turns from green to orange in late autumn. e fruits are rich with the yellow pigment, -cryptoxanthin. -cryptoxanthin is a xanthophyll and possesses pro-vitamin A activity for mammals. In plants, xanthophyll cycles play important roles in the protection of photosynthetic apparatus from light-induced oxidative damage. In this issue, Kawabata & Takeda from Osaka Prefecture University report the eect of intense light on the pool size of the xanthophyll cycle in Arabidopsis thaliana (pp. 229–240).
Photographed by Tsuyoshi Mizoguchi (International Christian University, ICU) in ICU, Japan, 5 Sep, 2014 (Ricoh GXR, Leica Summicron f2/35 mm).
Flowers of Hydrangea macrophylla
Most parts of Japan experience a rainy season in early summer, usually from the beginning of June to mid July. Japanese hydrangea (Hydrangea macrophylla) blooms in June and the ower is one of the symbols of the rainy season in Japan. There is a dramatic change in humidity, from wet to dry condition, between the rainy season and mid summer in Japan. Most plants have the ability to adapt to such changes. Molecular mechanisms underlying the tolerance to the dry condition after the rainy season have not been fully understood. In this issue, Watanabe et al. in Hiroshima University have reported roles of purine metabolites in drought stress responses in Arabidopsis thaliana (pp. 173–178).
Photographed by Tsuyoshi Mizoguchi (International Christian University, ICU) in ICU, Japan, 30 May, 2014 (Ricoh GXR P10).
Flowers of Japanese apricot (Prunus mume)
We had record snowfalls over the last two weekends. Heavy snow in Tokyo damaged small plants and big trees alike including cherry and plum trees in Mitaka. There is a famous proverb “The fools who cut cherry trees, the fools who don’t cut plum trees.” Some plants including cherry trees are badly damaged by cutting and wounding. By contrast, other fruit trees including plum often require trimming for better growth. Molecular mechanisms underlying the tissue reunion occurring after the cutting and wounding have not been elucidated.In this issue, Pitaksaringkarn et al. in University of Tsukuba have reported a pivotal role of ARF6 and ARF8 of Arabidopsis thaliana in the tissue reunion process (pp. 49–53).
Photographed by Tsuyoshi Mizoguchi (International Christian University, ICU)
in ICU, Japan, 21 February, 2014 (Ricoh GXR P10).
Dimorphism in shoot
Juniperus chinesis is a very tough conifer tree. This species is easy to grow, has dense branches and has been propagated for making live fences of gardens in Japan. But trees of this species sometimes show unexpected changes in shoot morphology after triming. As shown in the center of this photograph, some shoots show vigorous growth and make spiny leaves, while shoots of this species usually make slow-growing branches with small, round, and densely arranged scaly leaves. Due to their vigorous growth, once they appear, the spiny type of shoots are not easy to control, which makes it difficult to maintain the beauty of the whole tree shape. Skills of gardeners is judged based on whether they can control this or not. This phenomenon is also interesting from viewpoint of switching between two types of organogenesis in shoot.
Photographed by Hirokazu Tsukaya (Univ. Tokyo) in Kamakura, Japan, 27 September, 2012
(Pentax Optio W80).
“Sui-fuyo” or Drunken Fuyo Hybiscus (Confederate rose) is a mutant of Hibiscus mutabilis L. (“Fuyo” in Japanese) that originally occurs in China. It has been widely cultivated in gardens in Japan as an easy-growing bush tree bearing many pink flowers even under the harsh summer high temperatures. There are two types of “Sui-fuyo”: a basic type with five petals and a double-flowered type. Both are characterized with a late pigmentation: the flower color is white just after the anthesis in the morning, and is gradually turned into pink or red in the midday to the evening. While wild-type flowers complete the synthesis of pink pigments before anthesis, the Sui-fuyo cannot start making pigments before the anthesis. We liken this gradual change in the flower’s color to a blush of a young women’s face after drinking Sake.
Photographed by Hirokazu Tsukaya (Univ. Tokyo) in Kamakura, Japan, 26 September, 2012 (Pentax Optio W80).
Left: Commercial tomato fruits with high sugar content which is popular in Japanese tomato market, and called as fruit tomato due to its sweetness and excelent flavor.
Right: Fruit of Earl’s Favourite-type melon, which is an unique melon genotype in Japan, and a breeding material for Japanese high quality melon cultivars.
Tomato and melon fruits were photographed by Kyoko Hiwasa-Tanase (University of Tsukuba) and Yoshiteru Sakata (National Agricultural and Food Research Organization), respectively.
A flower which likes wet environment
There are many caves in lime stone areas in rain tropics of Southeastern Asia. Such places are affluent in water supply, that is sometimes too much for some plants. Alocasia revera N.E.Br., shown here, is not such a weak weeds. It grows often in the entrance of caves where it is exposed to high-calcium, and alkali water supplied from the caves. A. reversa also enjoys rich nitrogen and phosphate, in the form of guano: excrements of bats that live in the caves. A. reversa is characteristic with its delicately variegated leaves with unequal distribution of intercellular spaces in the palisade tissue, and its lovely white bract with a tinge of pale purple on the edge. It’s tough epidermis with thickened wax is evident in both organs, which enables this species to tolerate high humidity.
Photographed by Hirokazu Tsukaya (Univ. Tokyo) in 2 Sept, 2010 (Pentax Optio W80).
A classic flower known from the 18th century
The 18th century was a peaceful era in Japan, during which a unique horticultural culture has developed. Boku-han, a cultivar of Camellia japonica, is a mutant in which stamens are transformed into white petaloids. Because pistils are complete and fertile, this cultivar has been often used as a parental line for breeding. This cultivar was formerly described in a catalog of horticultural plants in Edo (ancient Tokyo) in 1719. Owing to the strong contrast between the deep crimson red petals and the white petaloids, and in addition to its slender, delicate branching nature, Boku-han has been appreciated by many flower lovers and has been widely spread in Japan. Now Boku-han is also named as “Gakko” or moonlight, in the western part of Japan. Photographed by Hirokazu Tsukaya (Univ. Tokyo) in Kamakura, Japan, in 11 March, 2012 (Pentax Optio W80).
Photographed by Hirokazu Tsukaya (Univ. Tokyo) in Kamakura, Japan, in 11 March, 2012 (Pentax Optio W80).
Adaptation to floods
In rain tropics, river bank is exceptionally good place for plants in terms of the availability of sun light. If you enter in rain forests, you will find that sun light is mostly absorbed by multilayered tree leaves from 40-m-high to a few-m-high. River bank is different. It is an open place and plants can enjoy sun shine there, although frequent floods suffer the area. If some mutations enable a particular strain of plants to have narrow leaves and strong roots, such strain is expected to tolerate the floods and can occupy the open area. It seems that occurrence of such mutation is not rare. Here is such a variant of an orchid, Arundina graminifolia. This species originally has narrow leaves (as the latin name “graminifolia” means: “having rice-family-like leaves”), but the variant adapted to the flood environment has much narrower leaves (less than half in width) that typically shows the evolutionary trend of plants to live along river bank. What genes are involved in this type of evolution? Trials to solve it are now on going in several laboratories.
Photographed by Hirokazu Tsukaya (The University of Tokyo) in Betung-Kerihun National Park, West Kalimantan, Borneo, in 26 December, 2011 (Pentax Optio W80).
Fruits of transgenic papaya
The papaya is the fruit of Carica papaya L., a member of the family Caricaceae, which is widely grown in tropical and subtropical regions of the world. e yellow orange, ripe fruits are edible and rich in vitamins, minerals, and ber, while green unripe fruits are known to rich sources of papain, an enzyme used as a component of meat tenderizers and so on. In this issue Retuta et al. (Page 339) carried out the evaluation of selected transgenic papaya lines which are resistant to papaya ringspot virus. e hermaphrodite fruits are pyriform in shape (front row), while the female fruits are rounded in shape (second row).
Dispersal by an ocean current
Anagallis arvensis L., with its characteristic deep blue petals, distributes very widely in subtropics and tropics. Its preference of seashore ambient might have helped this species to distribute so widely. Although seeds are too small to be dispersed by an ocean current, capsules being rather tough and light in weight, may enable the seeds inside to dri without severe damages. A. arvensis is also well-known to accumulate the blue pigment, anthocyanin, in the petal cells, in a unique manner. If you examine these tiny petals under a microscope, there you will observe unexpectedly beautiful images.
Photographed by Hirokazu Tsukaya (The University of Tokyo) in Yakushima Island, in 16 May, 2010 (Pentax Optio W80).
The oil plant for future biodiesel production
A tree of Jatropha curcas L., with bunches of developing fruits. Matured fruit contains 2-4 grains of seeds, which serve as a rich source of triacylglycerol for biodiesel production.
Photographed at campus of the University of e Ryukyus by Prof Yoshinobu Kawamitsu.
Flushing of Zelcova serrata
Zelcova serrata (Thunb.) Makino is distributed in Japan, as well as in China, Korea, and Taiwan. Nevertheless, this species is often seen as a tree typically representing forest vegetation of Kanto Plain of Japan, partly because this species has been preferentially planted along streets and in parks. This tree shows a characteristic and beautiful branching pattern; a wonderful colorations in Spring, Summer and Autumn, respectively; and supplies us with good quality wood. Bonsai, a Japanese art of horticulture, also loves to try to reproduce the elegant tree figure on a small pot. For this species the sprout season is also the season of full bloom, although not obvious due to the greenish coloration of flowers.
Photographed by Hirokazu Tsukaya (Univ. Tokyo) in Hongo campus, the University of Tokyo, Japan, in 13 April, 2011 (Pentax Optio W10).