Plant Biotechnology Vol.35 No.4


Original Papers

Functional analyses of lipocalin proteins in tomato

Anung Wahyudi, Dinni Ariyani, Gang Ma, Ryosuke Inaba, Chikako Fukasawa, Ryohei Nakano, Reiko Motohashi . . . . . . . 303 [PDF] [Supplement]

In this study, two temperature-induced lipocalin genes SlTIL1 and SlTIL2, and a chloroplastic lipocalin gene SlCHL were isolated from ‘Micro-Tom’ tomato. The coding sequences of SlTIL1, SlTIL2 and SlCHL were 558, 558, and 1002 bp, respectively. By TargetP analysis, no characteristic transit peptides were predicted in the proteins of SlTIL1 and SlTIL2, while a chloroplastic transit peptide was predicted in the protein of SlCHL. The subcellular localization results indicated that SlTIL1 and SlTIL2 proteins were major localized in the plasma membrane, while SlCHL was localized in chloroplast. To understand the function of lipocalins, transgenic tomato over-expressed SlTIL1, SlTIL2 and SlCHL and their virus-induced gene silencing (VIGS) plants were generated. The phenotypes were significantly affected when the SlTIL1, SlTIL2 and SlCHL were over-expressed or silenced by VIGS, which suggested that the three lipocalins played important roles in regulating the growth and development of tomato. In addition, the level of ROS (O2 and H2O2) was low in SlTIL1, SlTIL2 and SlCHL over-expressed plants, while it was high in their silenced plants. The changes in the expression of SODs were consistent with the accumulations of ROS, which indicated that lipocalins might have an important role in abiotic oxidative stress tolerance in tomato plants. Especially SlTIL1 and SlTIL2 are localized around their membranes and protect them from ROS. The results will contribute to elucidating the functions of lipocalin in plants, and provide new strategies to improve the tolerance to abiotic stress in tomato plants.

Transcriptome-wide analysis of MADS-box family genes involved in aluminum and fluoride assimilation in Camellia sinensis

Junting Pan, Pinpin Chang, Xiaoli Ye, Jiaojiao Zhu, Dongqin Li, Chuanlei Cui, Bo Wen, Yuanchun Ma, Xujun Zhu, Wanping Fang, Yuhua Wang . . . . . . . 313 [PDF] [Supplement1] [Supplement2]

MADS-box transcription factors (TFs) are involved in a variety of processes in flowering plants ranging from root growth to flower and fruit development. However, studies of the tolerance-related functions of MADS-box genes are very limited, and to date no such studies have been conducted on Camellia sinensis. To gain insight into the functions of genes of this family and to elucidate the role they may play in tissue development and Al and F response, we identified 45 MADS-box genes through transcriptomic analysis of C. sinensis. Phylogenetic analysis of these CsMADS-box genes, along with their homologues in Arabidopsis thaliana, enabled us to classify them into distinct groups, including: M-type (Mα), MIKC* and MIKCc (which contains the SOC1, AGL12, AGL32, SEP, ANR1, SVP, and FLC subgroups). Conserved motif analysis of the CsMADS-box proteins revealed diverse motif compositions indicating a complex evolutionary relationship. Finally, we examined the expression patterns of CsMADS-box genes in various tissues and under different Al and F concentration treatments. Our qPCR results showed that these CsMADS-box genes were involved in Al and F accumulation and root growth in C. sinensis. These findings lay the foundation for future research on the function of CsMADS-box genes and their role in response to Al and F accumulation in root tissues.

Multiple shoot bud induction and plant regeneration studies of Pongamia pinnata

Si Nan Tan, Chong Siang Tee, Hann Ling Wong . . . . . . . 325 [PDF]

Pongamia pinnata is a legume plant which has great potential to be used as a biofuel feedstock. Conventional propagation of P. pinnata was found to be inefficient for mass propagation. Employing plant tissue culture techniques for micropropagation and further plant improvement of P. pinnata will be the right path to fulfill future challenges in biofuel production. This study aimed to establish a plant regeneration system for potential micropropagation and genetic manipulation of P. pinnata in future. In vitro nodal explants were used and Woody Plant Medium (WPM) containing 30 µM 6-benzylaminopurine (BAP) and 1 mM phloroglucinol (PG) was able to induce higher frequency of multiple shoot buds compared to other media investigated in this study. For shoot regeneration study, WPM containing 15 µM of zeatin and 1 mM PG was able to induce longer shoots while rooting of the regenerated shoots was enhanced by WPM supplemented with indole-3-butyric acid (IBA) in combination with silver thiosulphate (STS). A simple and effective acclimatisation protocol was established with very high survival frequency of regenerated plantlets. Root nodulation of the successfully acclimatised plants was also observed. In short, multiple shoot buds were successfully induced, regenerated and rooted in vitro. The rooted plantlets were successfully acclimatised and grown healthily. It was concluded that a successful plant regeneration protocol of P. pinnata was achieved for potential application in micropropagation and genetic manipulation.

Freeze-substitution transmission electron microscopy of gentian shoot tips cryopreserved at ultra low temperatures

Daisuke Tanaka, Takao Niino, Seizo Fujikawa, Matsuo Uemura . . . . . . . 335 [PDF]

Transmission electron microscopy (TEM) combined with freeze substitution was employed to examine the ultrastructure of cells of gentian shoot tips cooled to the ultra-low temperature of slush nitrogen and liquid nitrogen. When shoot tips were cooled in ultra-low temperature without plant vitrification solution 2 (PVS2) treatment, massive ice formation was observed throughout the cells, indicating that severe injury occurred during cooling. In contrast, when shoot tips were treated with PVS2 and subsequently cooled to ultra- low temperatures, no ice crystals were observed in the cells. In addition, the cells of PVS2-treated shoot tips exhibited considerable plasmolysis and formation of small vesicles in cytoplasm. These results clearly demonstrate that the PVS2 treatment is essential for preventing damage caused by ice formation and for successful cryopreservation of plant shoot tips.

Transcriptome sequencing and identification of cytochrome P450 monooxygenases involved in the biosynthesis of maslinic acid and corosolic acid in Avicennia marina

Mitsuki Nakamura, Tran My Linh, Le Quynh Lien, Hayato Suzuki, Nguyen Chi Mai, Vu Huong Giang, Keita Tamura, Nguyen Van Thanh, Hideyuki Suzuki, Ryo Misaki, Toshiya Muranaka, Ninh Khac Ban, Kazuhito Fujiyama, Hikaru Seki . . . . . . . 341 [PDF] [Supplement]

Grey mangrove (Avicennia marina) is a traditional medicine used for the treatment of various diseases, including rheumatism and ulcers; however, the compounds responsible for its curative effects remain largely unknown. Triterpenoids are a diverse group of plant-specialized metabolites derived from a common precursor, 2,3-oxidosqualene. Triterpenoids are potentially responsible for the beneficial effects of A. marina; however, the chemical profiles of triterpenoids in A. marina and their biosynthetic genes have not been identified. Cytochrome P450 monooxygenases (P450s) have key roles in the structural diversification of plant triterpenoids by catalyzing site-specific oxidation of triterpene scaffolds. Recent studies have revealed that the CYP716 family represents the most common clade of P450s involved in triterpenoid biosynthesis. In this study, we performed triterpenoid profiling and RNA sequencing of A. marina leaves. Mining of CYP716 family genes and enzymatic activity assays of encoded proteins revealed that CYP716A259 catalyzed oxidation at the C-28 position of the pentacyclic triterpene skeletons of β-amyrin, α-amyrin, and lupeol to produce oleanolic acid, ursolic acid, and betulinic acid, respectively. The other functionally defined P450, CYP716C53, catalyzed the C-2α hydroxylation of oleanolic acid and ursolic acid to produce maslinic acid and corosolic acid, respectively. The possible involvement of CYP716A259 and CYP716C53 in the biosynthesis of these health-benefiting compounds in A. marina leaves, and the possible contribution of the resulting compounds to the reported bioactivities of A. marina leaf extract, are discussed.

Cloning and characterization of a Mimulus lewisii NPR1 gene involved in regulating plant resistance to Rhizoctonia solani

Xia He, Yancai Shi . . . . . . . 349 [PDF]

The monkey flower Mimulus lewisii is a new emerging model plant for the study in corolla tube formation, pigmentation patterns and pollinator selection, etc. However, the cultivation and management of this plant are difficult due to its susceptibility to a wide range of pathogens and the lack of rigid varieties with high levels of resistance to pathogens. In this regard, genetic engineering is a promising tool that may possibly allow us to enhance the M. lewisii disease resistance against pathogens. Here, we reported the isolation and characterization of non-expressor of pathogenesis related gene 1 (NPR1) gene from M. lewisii. The phylogenetic tree constructed based on the deduced sequence of MlNPR1 with homologs from other species revealed that MlNPR1 grouped together with other known NPR1 proteins of Scrophulariaceae family, and was nearest to Mimulus guttatus. Furthermore, expression analysis showed that MlNPR1 was upregulated after SA treatment and fungal infection. To understand the defensive role of this gene, we overexpressed MlNPR1 in M. lewisii. The transgenic lines showed slight phenotypic abnormalities, but constitutive expression of MlNPR1 activates defense signaling pathways by priming the expression of antifungal PR genes. Moreover, MlNPR1 transgenic lines showed enhanced resistance to Rhizoctonia solani there was delay in symptoms and reduced disease severity than non-transgenic plants. Altogether, the present study suggests that increasing the expression level of MlNPR1 may be a promising approach for development of monkey flower cultivars with enhanced resistance to diseases.

AGL18-1 delays flowering time through affecting expression of flowering-related genes in Brassica juncea

Kai Yan, Chao-Chuang Li, Yu Wang, Xiao-Quan Wang, Zhi-Min Wang, Da-Yong Wei, Qing-Lin Tang . . . . . . . 357 [PDF] [Supplement]

Brassica juncea is an important vegetable and condiment crop widely grown in Asia, and the yield and quality of its product organs are affected by flowering time. AGAMOUS-LIKE18-1 (AGL18-1) belongs to a member of MADS-domain transcription factors, which play vital roles in flowering time control, but the biological role of AGL18-1 in B. juncea (BjuAGL18-1) has not been thoroughly revealed in flowering regulatory network. In this study, BjuAGL18-1 expressed highly in inflorescence and flower, but slightly in root, stem and leaf. The sense and anti-sense transgenic lines of BjuAGL18-1 were generated and showed that BjuAGL18-1 functioned as a flowering inhibitor and depressed growth of lateral branching. During the vegetative phase, BjuAGL18-1 induced another flowering repressor AGAMOUS-LIKE15 (BjuAGL15) but inhibited the flowering signal integrator of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (BjuSOC1) in Brassica juncea. Whereas, during the flower developmental phase, both SOC1 and AGAMOUS-LIKE24 (AGL24) were down-regulated by BjuAGL18-1. By contrast, AGL15 was promoted by BjuAGL18-1, while SHORT VEGETATIVE PHASE (SVP) was independent of BjuAGL18-1. Additionally, HISTONE DEACETYLASE 9 (HDA9) was highly induced by BjuAGL18-1. These results will provide valuable information for clarifying the molecular mechanism of BjuAGL18-1 in mediating flowering time.

Identification of 5′-untranslated regions that function as effective translational enhancers in monocotyledonous plant cells using a novel method of genome-wide analysis

Shotaro Yamasaki, Atsunobu Suzuki, Yasuaki Yamano, Harunori Kawabe, Daishin Ueno, Taku Demura, Ko Kato . . . . . . . 365 [PDF] [Supplement]

High expression of a transgene is often necessary to produce useful substances in plants. The efficiency of mRNA translation is an important determinant of the level of transgene expression. In dicotyledonous plants, the 5′UTR of certain mRNAs act as translational enhancers, dramatically improving transgene expression levels. On the other hand, translation enhancers derived from dicotyledonous plants are not so much effective in monocotyledonous plants, which are important as industrial crops and as hosts for production of useful substances. In this study, we evaluated the polysome association on a large scale with high resolution for each 5′UTR variant from multiple transcription start site in normal and heat-stressed Oryza sativa suspension cultures. Translational enhancer candidates were selected from the resultant large-scale data set, and their enhancer activities were evaluated by transient expression assay. In this manner, we obtained several translational enhancers with significantly higher activities than previously reported enhancers. Their activities were confirmed in a different monocotyledonous plant, Secale cereale, and using a different reporter gene. In addition, enhancer activities of tested 5′UTRs were different between monocotyledonous and dicotyledonous plants, suggesting that the enhancer activities were not compatible between them. Overall, we demonstrate these useful 5′UTRs as enhancer sequence for transgene expression in monocotyledonous plants.

Short Communication

Comparison of the N-glycosylation on recombinant miraculin expressed in tomato plants with native miraculin

Hiroyuki Kajiura, Kyoko Hiwasa-Tanase, Hiroshi Ezura, Kazuhito Fujiyama . . . . . . . 375 [PDF] [Supplement]

Miraculin is a promising protein with taste-modifying properties. Focusing on the unique function and potential of miraculin, recombinant miraculin production has been explored with the use of heterologous expression systems, but the activities of recombinant miraculins were much lower than those of native miraculin, probably due to the difference in post-translational modification, especially N-glycosylation. For practical use therefore, the differences between N-glycan of recombinant miraculin compared to that of native miraculin should be minimized. Here, to establish the platform for functional miraculin production, we expressed miraculin in tomato plants with the same taste-modifying activity as native miraculin purified from miracle fruit, and we compared the N-glycan structures with those of native miraculin. Our N-glycan structural analysis using purified miraculin, followed by hydrazynolysis, 2-pyridylamine (PA)-labeling, high-performance liquid chromatography, and a liquid chromatography tandem-mass spectrometry analysis revealed that both the native and recombinant miraculins carried an M3 structure as a predominant structure and that most of the N-glycan structures on the miraculins were pauci-mannosidic structures with a smaller amount of plant-specific α1,3-fucosylated and/or β1,2-xylosylated N-glycans and without a Lewis a epitope. These results indicate that the N-glycoform of native miraculin from miracle fruit and recombinant miraculin expressed in tomato plants are almost identical to each other with similar ratios and that, therefore, plant-specific N-glycans are essential for showing the full taste-modifying activity of miraculin.


The mevalonate pathway but not the methylerythritol phosphate pathway is critical for elaioplast and pollen coat development in Arabidopsis

Keiko Kobayashi, Masashi Suzuki, Toshiya Muranaka, Noriko Nagata . . . . . . . 381 [PDF] [Supplement]

Pollen coat components are derived from tapetum cells, which contain elaioplasts derived from plastids and tapetosome derived from the endoplasmic reticulum. In Brassica napus, the main neutral lipids in the elaioplast and tapetosome have been reported to be sterol ester and triacylglycerol, respectively. Isopentenyl pyrophosphate, the structural component of sterol, is produced via the cytosolic mevalonate (MVA) and plastidic methylerythritol phosphate (MEP) pathways. Although these two pathways are compartmentalized, partial cross-talk between them has been reported. To investigate the contribution of these two pathways in elaioplast formation, we characterized mutant pollen of these two pathways. We observed the anthers of male sterile hmg1-1 and atipi1 atipi2 mutants ultrastructurally, which were deficient in MVA pathway enzymes. hmg1-1 and atipi1atipi2 showed a shrunken elaioplast inner granule at the bicellular pollen stage. Conversely, in the cla1-1 mutant, which showed a defective MEP pathway, elaioplast development was normal. The pollen of hmg1-1 and atipi1atipi2 was coatless, whereas cla1-1 had a pollen coat. These results indicate that the MVA pathway but not the MEP pathway is critical for elaioplast development though the organelle is derived from plastids.

Use of a duckweed species, Wolffiella hyalina, for whole-plant observation of physiological behavior at the single-cell level

Minako Isoda, Tokitaka Oyama . . . . . . . 387 [PDF]

We developed a new model system to analyze physiological behavior at the single-cell level in whole plants. Wolffiella hyalina is a species of rootless duckweed, which has a thin and very small structure and can grow rapidly on the surface of culture medium. Epidermal and mesophyll cells were transfected with a reporter gene using particle bombardment and were observed at the single-cell level in the whole living plant. An EM-CCD camera system with a macro zoom microscope was used to capture time-lapse images of bioluminescence, and we successfully detected circadian rhythms in individual cells that expressed a luciferase gene under the control of a circadian promoter. We also detected individual S-phase cells in meristematic tissues of intact W. hyalina plants by using a 5-ethynyl-2′-deoxyuridine (EdU)-labeling assay. Our observations indicated that low-molecular-weight compounds could access the inside of the plant body. Thus, W. hyalina showed the experimental characteristics suitable for single-cell analyses that could be combined with whole-plant observations and/or pharmacological analyses/chemical biology.

Environmental risk assessment of impacts of transgenic Eucalyptus camaldulensis events highly expressing bacterial Choline Oxidase A gene

Ngoc-Ha Thi Tran, Taichi Oguchi, Etsuko Matsunaga, Akiyoshi Kawaoka, Kazuo N. Watanabe, Akira Kikuchi . . . . . . . 393 [PDF] [Supplement]

Under the Japanese biosafety regulatory framework for transgenic plants, data for assessing a transgenic plant’s impact on biodiversity must be submitted in order to obtain approval for a confined field trial. We recently reported the development of four novel transgenic Eucalyptus camaldulensis clones expressing the bacterial choline oxidase A (codA) gene, i.e., codAH-1, codAH-2, codAN-1, and codAN-2, and evaluated their abiotic tolerance by semiconfined screen house trial cultivation. Here we evaluated the impacts of the transgenic E. camaldulensis clones on productivities of harmful substances from those clones to affect soil microorganisms and/or other plants in the environment. A comparison of the assessment data between the transgenic trees and non-transgenic comparators showed no significant difference in potential impacts on biodiversity. The results contribute to sound-science evidence ensuring substantial equivalence between transgenic and non-transgenic E. camaldulensis.

Highly efficient G-AgarTrap-mediated transformation of the Marchantia polymorpha model strains Tak-1 and Tak-2

Shoko Tsuboyama, Yutaka Kodama . . . . . . . 399 [PDF]

The liverwort Marchantia polymorpha L. is an important model species for investigating land plant evolution. Effective genetic transformation techniques are crucial for plant molecular biology and simplified or improved techniques for specific cultivars or strains can accelerate research. Over the past several years, we developed a simple Agrobacterium-mediated transformation technique for M. polymorpha named AgarTrap (Agar-utilized transformation with pouring solutions). AgarTrap is an easy technique that involves pouring the appropriate solutions onto plant materials on a single solid plate of medium. We recently improved AgarTrap using gemmalings (G-AgarTrap) of the M. polymorpha female model strain BC3-38 and achieved a transformation efficiency of nearly 100%. Based on this improved technique, in the current study, we adopted two factors (sealing the Petri dish with Parafilm and dark treatment during co-cultivation) and optimized two factors (Agrobacterium strain and pre-culture period) of the improved G-AgarTrap for other model strains of M. polymorpha, the male strain Takaragaike-1 (Tak-1) and the female strain Takaragaike-2 (Tak-2). After optimization, the transformation efficiency of Tak-1 using G-AgarTrap was as high as 55% compared to approximately 30% using the previous protocol. Furthermore, using Tak-2, we achieved a transformation efficiency of nearly 100%. Our improved G-AgarTrap technique for Tak-1 and Tak-2 represents a promising tool for promoting the study of Marchantia.

The localization of rice prolamin species in protein body type I is determined by the temporal control of gene expression of the respective prolamin promoters

Ai Sasou, Takanari Shigemitsu, Shigeto Morita, Takehiro Masumura . . . . . . . 405 [PDF]

Rice prolamin species form a layered structure in the protein body type I (PB-I) storage organelle. Rice prolamins are classified as 10 kDa, 13a-1, 13a-2, 13b-1, 13b-2 and 16 kDa prolamin. Prolamin species form layer structure in PB-I in order of 10 kDa core, 13b-1 layer, 13a (13a-1 and 13a-2) and 16 kDa middle layer and 13b-2 outer-most layer. In a previous study, we showed that the fusion proteins in 13b-2 prolamin-GFP, 13a-1 prolamin-GFP and 10 kDa prolamin-GFP were localized in the same layer of PB-I as the native prolamin, when they were expressed by their respective native prolamin promoters. Our preliminary study suggested that the temporal control of the native prolamin promoters was responsible for the localization of the respective prolamins. The aim of this study was to determine whether the use of a prolamin promoter other than the native prolamin promoter would change the localization of prolamin-GFP fusion proteins. For this purpose, we generated transgenic lines expressing 13b-2 prolamin-GFP and 13a-1 prolamin-GFP fusion proteins driven by each prolamin promoter other than the native prolamin promoter. As a result, the localization of the fusion protein in PB-I was changed. Based on our results, foreign protein localization in PB-I can be achieved by the temporal control of the different prolamin promoters.